Biochimica et Biophysica Acta, 457 (1976) 57-108 © Elsevier Scientific Publishing Company, A m s t e r d a m - Printed in The Netherlands BBA 85158

T R A N S M E M B R A N E C O N T R O L OF T H E RECEPTORS ON N O R M A L A N D T U M O R CELLS I. C Y T O P L A S M I C I N F L U E N C E OVER CELL S U R F A C E C O M P O N E N T S * GARTH L NICOLSON

Department of Cancer Btology, The Salk Institute for Biological Studtes, San Dtego, Cahf 92112 and Department of Developmental and Cell Btology, Umverstty o] Californta, Irvlne, lrvine, Caltf 92664 (US.A ) (Received December 30th, 1975)

CONTENTS I.

Introduction

58

II_ Orgamzation of the cell surface

_ . _

A. Fluid mosaic m e m b r a n e model B

M e m b r a n e restraints on lateral mobility

62

1 Planar association of m e m b r a n e c o m p o n e n t s .

62

2 Protem-hpld m e m b r a n e domains

65

3_ Peripheral m e m b r a n e restraints.

66

4 Membrane-associated (cytoskeletal) restraints_ IlL Transrnembrane control mechanisms

67

. . .

70

A_ Erythrocyte m e m b r a n e

70

B

76

L y m p h o i d cell surfaces 1 L y m p h o i d cells

76

2 M e m b r a n e isolation and characterization

76

3. Receptor redlstrlbuhon and capping

78

4 Cytoskeletal trartsmernbrane control of lymphoid surface receptors

83

C_ Fibroblast cell surfaces.

88

1. Plasma m e m b r a n e ~solatlon and characterization

88

2 Receptor redistribution o n fibroblasts _ IV

58 58

89

Final c o m m e n t

. .

Acknowledgments . References

. . _ .

96 96 97

Abbreviations B cells, bone marrow-derived cells; lg, l m m u n o g l o b u h n , surface lg, cell surface-bound l m m u n o g l o b u h n ; LETS, large-external, transformation°sensitive protein; SF antigen, fibroblast surface antigen; T cells, thymus-derived cells * Dedicated to the m e m o r y of the late Professor G o r d o n M T o m k m s

58 I. INTRODUCTION An overwhelming amount of data has imphcated the cell surface as the primary s~te for control of cell growth, dwlslon, development, commumcatlon, differentiation and death. Cells must react to their envxronments and to extracellular stlmuh through their surfaces, particularly their plasma membranes, so this organelle has invoked the interest of a large number of investigators in many fields, especially over the last 70 years [1] In the field of cancer biology the tumor cell surface and plasma membrane have long been recogmzed as critical for the control of wayward cell muluplication, escape from host immunological surveillance and metastasis to secondary sites [2] Many other factors are undoubtedly involved in these complex processes, but a thorough understanding of the composltton, synthesis, degradaUon, orgamzatlon, activities and display and dynamics of cell membrane components will aid in our understanding of the neoplastic state [3-10] This article and its companion review [549] will expand upon current ideas and knowledge of the structure, organization and dynamics of cell membranes and their surface receptors, including recent information on transmembrane hnkages and control mechanisms. Many parts of the reviews wdl, of course, be incomplete due to the lack of data and/or statable systems to approach the problems It is hoped that the reader wdl understand these hm~tat~ons m the formulation of models that attempt to clarify certain aspects of cell surface behavior and stimulate further experimental investigation_ II ORGANIZATION OF THE CELL SURFACE IIA. Fired mosaw membrane model

An understanding of phenomena such as cell surface receptor dynamics is dependent on a thorough knowledge of the organization of the plasma membrane and its associated structures Since this is not feasible at the present time, the interpretation of most cell surface data has been based on models, which for the present serve as useful conceptions to explain an enormous variety of physical, chemical and biological data on membrane structure [3,10-33]_ The early plasma membrane models of Danlelh and Davson [34] and Davson and Danielli [35] endeavored to explain the physical and chemical aspects of membrane organization and were based, in part, on the characteristics of hpid films The proposal that membrane hpids are formed into a bllayer configuration [36] seemed sound at the time, and it followed that the more hydrophlhc proteins must coat each side of the lipid bilayer This proposal was later advanced as the "unit membrane model" by Robertson [37] based on electron microscopic observations of O s O : s t a m e d membranes, where a typical railroad track appearance was seen of approximately the correct d~menslons for biological membranes estimated by physical techniques. However, these membrane models were unable to explain a large number of experiments, which indicated that the hydrophdlc portions of membrane phosphohpids are susceptible to enzymattc attack and are not complexed to any great degree to membrane proteins [38-42]. In fact, most membrane protems are globular and not fl-structures or "random coils"

59 [5,11,38--45] as originally proposed. Rather than being hydrophilic, these proteins are quite insoluble m low ionic strength neutral solutions [46-48] but can be stabilized m low polarity solvents or m solunons containing chaotroplc agents or detergents (reviewed in ref 48). Some proteins span the membrane and are accessible to chemmal and enzymatic modification at either membrane surface [49-57]. In certain cases proteins and glycoprotems are probably arranged into oligomenc complexes [58] which serve important functions in transport and permeability phenomena [59-63], whde in other cases these components are probably noncovalently linked to other membrane constituents. Membrane components are highly asymmetric with regard to their hydrophihc and hydrophoblc portions of their structures. To explain the fact that the hydrophllic parts of liplds and proteins are probably exposed to the external aqueous phase, and their hydrophobm regions sequestered from the aqueous phase and stabilized by an apolar environment, Green et al. [64] and Benson [65] proposed that membranes are constructed of globular hpoproteln subunits with the hpld acyl tails intercalated into the membrane protein complexes This proposal was seemingly supported by electron microscopic observations, which suggested the presence of large globular repeating umts in membranes (for example, see Sjostrand [66]). However, these latter experiments have been criticized on technical grounds, and the hpoproteln subunit proposal does not fit with the preponderance of physical evidence lndmatmg that the bulk membrane liplds are organized into a fired bilayer configuranon. The evidence for a hpld bilayer structure m membranes is overwhelming" X-ray diffraction [67-70], differential scanning calorimetry [71-74], electron spin resonance [75-83] and nuclear magnetic resonance [44,84,85] measurements indicate that the majority of the phosphohplds are in fluid bilayer state at 37 °C These data cannot distinguish between an interrupted and a continuous bllayer and do not indicate the exact percentages of hplds that are associated with proteins (boundary lipid [86]), tightly bound to proteins [20,87] or "frozen" in less fluid or non-fluid lipid domains [88] The merger of these opposing schools of thought came with the development of the "hquid crystalline" [19,89] and "fired mosaic" [20, 31] models of membrane structure. In the latter model the structure and thermodynamics of membrane components have been considered [ll], along with all the available evidence on hpld and protein structure, organization and lateral mobility [20,28-31] For example, proteins that are tightly associated with the membrane (integral membrane proteins) have been found to be structurally amphipathlc or asymmetric with regard to their hydrophihc and hydrophoblc pornons of their structures, as are membrane llplds [54,90-92]. Integral membrane proteins interact strongly with hydrophoblc molecules such as lipids and qmte often contain some tightly bound phospholipid (not to be confused with "boundary" lipid) when isolated and characterized [20,28]. Integral proteins are globular, containing appreciable amounts of a-hehcal structure [38,43~5], but in their overall amino acid compositions do not slgmficantly differ from soluble globular proteins [6,13,28] Their interactions m two phases, aqueous and hydro-

60 carbon, require that their structures be asymmetric (just as the membrane phospholiplds), and it is believed that portions of their structures are stabilized by hydrophoblc forces within the membrane hydrophobic zone bounded by acyl regions of the phospholipid bilayer (Segrest and Feldmann [93] examined the ratio of eight hydrophobic to hydrophihc amino acids in peptldes isolated from human erythrocyte M N (integral) glycoprotein and could easily identify the portions of the molecule that penetrated into the hydrophoblc z o n e ) Some integral membrane glycoprotelns clearly span the membrane, indicating that their structures are probably tnmodal or hydrophllic-hydrophobic-hydrophIhc [49-51,54,55,94]. These components are proposed to be arranged in ohgomeric transmembrane complexes [13,58] Once inserted into the membrane, integral bi- and trlmodal glycoproteins or their complexes appear to be stable with respect to their transmembrane orientation; that is, they do not appear to "tumble" or rotate from one side of the membrane to the other at an appreoable rate. This has been demonstrated by chemical analysis [95,96], chemical labeling [97,98] and ultrastructural locahzatlon of membrane oligosaccharides [99-102], which indicate that the overwhelming proportion of membrane ohgosaccharldes is present at the outer plasma membrane surface; essentially none can be detected at the inner membrane surface. Thus, there appears to be little tendency for glycoproteins and glycolipids to undergo transmembrane rotations. Similarly, phospholipids have been found to undergo transmembrane "flip-flop" or rotations at very slow rates This has been demonstrated using hposomes of dlstearoyl phosphatidylcholine bearing spin labels attached to polar head groups [80] or by using a purified phospholipld exchange protein to remove outer bllayer [32p]phospholiplds from mixed llposomes [103] Flip-flop of phosphohplds across native blologmal membranes appears to be faster; for example, the half-time for flipping is approximately 20-30 min in erythrocyte membranes [104]. Freeze-cleavage electron microscopy has proven to be a useful tool for visualizing discontinuities in the hydrophobic regions of membranes [104a]. Membranes are freeze-fractured down a plane formed by the halves of the lipid bllayer [105-107], and in almost every biological membrane studied numerous structural dlscontinuitms or particles are present, which can be visualized by replica and heavy metal shadowing techniques. That these particles are due to intercalated proteins in a lipid bilayer matrix has been demonstrated with artificial lipid membrane systems to which membrane proteins have been introduced [108-112]. In the human erythrocyte membrane the membrane-Intercalated particles are approximately 85 A in diameter and are known to contain transmembrane integral glycoprotelns [58,113,114]. (Estimates of particle size, shape and molecular weight are difficult to determine because of the possibilities that conformational and shape differences, inclusion of "boundary lipid", thickness of the platinum-carbon replica and depth of penetration of an intercalated structure may affect these measurements [58].) These membrane intercalated particles are capable of rapid lateral mobility under certain conditions [114-116] (see Section IIIA) There is now strong evidence indicating that most plasma membrane com-

61

T2

T~

0

Fig 1 Modified version of the Fluid Mosaic Model of m e m b r a n e structure T1 and T2 represent different points m tame Certain hypothettcal integral m e m b r a n e glycoprotem components are

uncoupled and free to diffuse laterally m the membrane plane formed by a fired bdayer matrix, whale others such as the integral glycoprotem complex GP~ may be "anchored" or relatavely ampeded by a mlcrofilament-mlcrotubule cytoskeletal assemblage (M) Under certain condmons some antegral membrane complexes (GP2) can be displaced by membrane-assocmted cytoskeletal components an an energy-dependent process (from Nlcolson [29]) ponents are capable of lateral moblhty in the m e m b r a n e plane, even t h o u g h their rates of mobility may differ widely (see reviews, refs 10,20,27,29,30 and 117). For example, electron paramagnetlc and nuclear magnetic resonance data indicate that membrane phospholiplds are in rapid lateral motion (D ~ - 1 0 -s cm 2 s -1) [79,83,118-123]. Other methods ~uch as fluorescence polarization [124-127], fluorescent exlmer formation [128] and partltionmg of isotope-labeled h y d r o p h o b i c c o m p o u n d s [129] have yielded similar results Estimates of m e m b r a n e protein movements are much lower, as expected, but can still be quite significant For example, Edldin and F a m b r o u g h [130] used fluorescent antibodies and estimated the lateral diffusion o f antigens on muscle fiber surfaces to be approximately D ~ 10 -9 cm 2 s 1 Poo and Cone [131] utilized the natural c h r o m o p h o r e retinal m r h o d o p s m to measure ItS lateral diffusion in rod outer segment membranes and calculated D ~ 3 10 -9 c m 2 s -1 Different rates of antigen and lectln receptor moblhty have been noted on different cells (discussed in detail in Section Ill and c o m p a n i o n review, ref. 549), and there is a considerable a m o u n t o f evidence indicating that structural restraints are operating in some situations which impede the mobilities of certain m e m b r a n e components and probably determine long range surface t o p o g r a p h y to a certain degree. In some cells the display of surface receptors ts n o n - r a n d o m [ 132], and serious proposals for membrane structure and organization must take this fact into account The Fluid Mosaic M e m b r a n e Model prcooses that membranes are c o m p o s e d of a fluid lipid bllayer matrix which allows lateral movements of m e m b r a n e components through a somewhat viscous (depending on lipid composition, temperature, etc ) environment (Fig l) The viscosity of the m e m b r a n e lipid matrix has been estimated by several techniques. S o l o m o n measured the diffusion coefficient o f methanol in

62 human erythrocyte membranes and calculated from the permeability coefficient an apparent membrane viscosity of approximately ~ -- 2 :~ 1.7 P (21 °C). Poo and Cone [131 ] used flash photolysls to measure rhodopsln lateral diffusion in rod outer segment membranes and calculated a value of ~t -- 2 P (20 °C) Rudy and Gitler [125] utilized fluorescence polarization measurements to study the fluorescence anlsotropy of perylene in erythrocyte ghosts and a value of ~/ 1 7 P (20 °C) can be calculated from their data. Lange et al [133] computed a viscosity of rt - - 0.9 P from electron spin resonance and data of Scandella et al. [83]. These measurements indicate that biological membranes have the apparent viscosity of light machine oil [27] Although the bulk membrane liplds are postulated to be freely intermixing, certain hpld-lipid interactions may favor long-range sorting out. This means that specific lipid domains or "islands" are possible (particularly in the presence of divalent cations such as Ca 2+) where the lipid composition may differ from the average lipid composition of the membrane [134-137] Also, it is known in at least one membrane system that the lipid bflayer is asymmetric with respect to the composition of its two halves [22]. Chemical labeling techniques [138] and enzymatic hydrolysis of phosphohplds [41,42] have demonstrated that in the human erythrocyte membrane, sphlngomyelin, phosphatldylethanolamlne and phosphatldylserine are predomlnately in the inner half of the bilayer, with phosphatldylcholine in the outer half The role of lipid asymmetry in maintaining certain physiological functions of membranes is not clear, but asymmetric hpid c o m p o s m o n could regulate differential membrane fluidity in each half of the bllayer [123,139] Experimental evidence suggests that the saturated fatty acids of membrane phosphohplds [529] and cholesterol [530,531] may be asymmetrically distributed in the outer bllayer leaflets of a variety of plasma membranes This lipid asymmetry could lead to differential flmdity of the inner and outer bilayer leaflets. Alternatively, the higher proportion of anionic phosphollpids on the inner portion of the bllayer may facilitate ionic interactions (through, say, Ca 2+) with membraneassociated and peripheral membrane components. l i B . M e m b r a n e r e s t r a m t s on l a t e r a l m o b i h t y

Although it Js well established that plasma membrane components such as proteins and glycoprotelns are mobile in the membrane plane, restraining mechanisms can impede or prevent dramatic rearrangements. Lateral movements can be restricted by several mechanisms. (a) association or aggregation of membrane components through horizontal interactions; (b) sequestration or exclusion of membrane components to (or from) specific lipid domains, (c) restraint by peripheral membrane components at the inner or outer membrane surfaces, or (d) restraining by membraneassociated (cytoskeletal) components at the inner membrane surface (Fig. 2) I 1 B - I . P l a n a r a s s o c i a t i o n o f m e m b r a n e c o m p o n e n t s . Membrane components can associate by non-covalent forces in the membrane plane leading to the formation of aggregates or ordered paracrystalline arrays. This phenomenon has been well studied in the membranes of certain bacteria [140], but is occasionally detected in mammalian cells as well An example is the formation of cell junctions. There are

63

MOBILITY

PLANAR

RESTRAINT

MECHANISMS

AGGREGATION OR ASSOCIATION

PROTEIN ASSOCIATIONS DOMAIN

LIPID ASSOCIATIONS

FORMATION

RANDOM PERIPHERAL

MEMBRANE

INNER SURFACE MEMBRANE-ASSOCIATED

UNCOUPLED (STATE I)

SEQUESTRATION COMPONENTS

UNCOUPLED

EXCLUSION ,

~

OUTER SURFACE

(CYTOSKELETAL) COMPONENTS

TRANSLOCAT ION IMPEDANCE

UNCOUPLED (STATE 2)

Fig. 2 Some possible restraining mechamsms on the lateral mobd~ty of cell surface receptors many types of cell junctions (gap junctions, septate junctions, desmasomes, tight junctions (zona occludens) [141-142a]), but they are all characterized by a planar association or aggregation of a small fraction of specialized plasma membrane components in specific regions of the plasma membrane These are regions of cell-tocell association or contact, and they usually account for only a small percentage of

64 the total cell surface area. Cell junctions do not appear to be completely static structures The entire junctional complex may have slight lateral mobility, perhaps as "frozen" protelnous domains in a fluid lipid matrix Because the junctional components interact with presumably complementary junctional components on adjacent cells, the position of one cell relative to another may be the most important factor in maintaining junctional location and lateral mobility. Once cell contact is broken, the complexes appear to be capable of greater lateral mobility, and ultimately they can be degraded or broken down [142-144]. Another example of lateral associations is the lateral interactions of viral envelope components after insertion into the plasma membrane Although ostensibly random in distribution at first, the viral components may laterally associate to eventually form a viral "bud" [145,146]. However, this process may also require inner membrane surface interactions with virus nucleoprotein, etc [147,148] (see peripheral membrane restraint, Section lIB-3) The forces generating stable lateral associations could be of various types Integral proteins could associate by interactions in the membrane hydrophobic zone if these interactions are favored over proteln-hpld interactions. Associations could also be driven by interactions involving hydrophihc structures such as glycoproteln or glycohpid ohgosaccharldes Saccharlde-saccharide interactions leading to stable polymeric complexes are cooperative processes in some systems [149]. Ionic bridges between charged groups, hydrogen bonds, etc. may stabilize lateral association, and conditions could exist where disulfide cross bridges are Important in complex stabilization [150]. In model phospholipld systems and in membranes from Mycoplasma or bacterial fatty acid auxotrophs, it has been demonstrated that lipid phase separation and lateral segregation or "sorting out" can occur [72,110,119,123,137,151-157]. Simple binary hpld mixtures when cooled below specific phase transition temperatures segregate into d omalns of solid lipid "icebergs" in a fluid lipid "sea" [88,119,122,151,155, 156,158-160]. Cooling further results In a solid matrix with trapped fluid domains [134,156,158]. At first glance these experiments might not seem pertinent to mammallan cell membranes; however, temperature is relative and in certain cell membranes transitions near 37 °C are apparently common [139,161], leading some investigators to suggest that cells may grow under conditions where their membranes fall within the range of a major lipid phase transition [122,155,162-164] Lipid phase transitions are affected by the presence of cholesterol [126,165-170], and divalent cations such as Ca 2+ [135-137,171,172] In homeothermic animals the cholesterol content apparently determines the fluidity of the bulk lipid in membranes and provides a mixture of fluid and solid domains at physiological temperatures [122] When membrane cholesterol content exceeds approximately 30 m o l ~ , membrane hplds are prevented from undergoing major phase transitions [152,167,173,174]. This is thought to be due to the different effects cholesterol has on fluid and solid phase lipids In the fluid phase, cholesterol decreases lateral molecular separation and bdayer fluidity, while in the solid phase cholesterol increases lateral molecular spacing, and hence it increases solid phase lipid fluidity [175-178] At physiological temperatures

65 cholesterol can condense and rigldlfy membranes without sohdlfylng them [168,179, 180] Cholesterol rich membrane domains exist in model systems and have been proposed or found in biological membranes [181,182]; components in these domains would be expected to have lowered mobility and rotational freedom [76,168]. It is conceivable that in most plasma membranes under physiological conditions a fraction of the total lipid could exist in a state unlike the majority [88], particularly if lipid-lipid associations [183] led to segregation and "mlcroheterogeneity" It would be difficult to detect such microheterogenelty using physical techniques, which sample the average state of the membrane hplds. HB-2. Proteln-hptd membrane domains As discussed above, protein-protein and lipid-lipid associations can occur leading to the formation of protein or lipid domains When lipids, glycoliplds, proteins and glycoproteins are present as In biological membranes, this situation becomes more complex because membrane protein interactions with solid-phase or fluid-phase lipid appear to be different [110,123,164,184-187] These differences could be due to certain structural requirements necessary for protein-lipid associations such as lipid btlayer thickness [188], lipid acyl chain distortion, bending or rotation [44,75,82,191,192] and lipid lateral compressibility [123,164,193], all of which are known to change between fluid and solid lipid phases_ In the fluid state the degree of chain distortion or bending from the molecular axis formed by the fatty acid methylene chain increases with increasing distance from the polar head groups producing a gradient of fluidity which is highest in the interior regions of the lipid bilayer [76,189,190] Alteration in lipid phase could determine more favorable or less favorable protein interactions (reviewed in ref. 194), which in turn could result in selective sequestration or exclusion of proteins into specific domains. If proteins were sequestered into solid domains, their lateral mobility would be limited to that of the domain, and if excluded from extensive solid domains, their display would be limited to those areas of fluid lipid. In bacterial membranes from fatty acid auxotrophs where the lipid composition can be manipulated for experimental purposes, Kleemann et al [157] and Kleemann and McConnell [184] have shown that fluid-solid lateral separations can occur at critical transition temperatures leading to the formation of "fluid" and "solid" membrane domains. When they examined the membranes from auxotroph bacteria (grown on two phosphohplds with widely differing melting points) at temperatures above the upper transition point for the membrane liplds, the membranes were In a completely fluid state, as determined by electron spin resonance measurements after incorporation of paramagnetlc probes Into the outer membrane Freeze-fracture electron microscopy revealed a random array of membrane-intercalated particles across the entire membrane Below the upper transition temperature for the binary lipid mixture (similar for both biological membranes and isolated membrane hplds) where "fluid" and "solid" lipid domains exist, membrane-intercalated particles were excluded from regions identified as "solid" by their characteristic freeze-fracture morphology. Aldehyde fixation prevented particle redistribution into the fluid lipid domains Similar results have been obtained with other systems [110,153,185].

66

/ / G%~ Y

MF

2~ MT

....

Fig 3. Transmembrane control over the distribution and mobflay of cell surface receptors by peripheral and membrane-associated cytoskeletal components In this example the moblhty of integral glycoprotem complexes GP3 and GP4 is controlled by outer surface peripheral components and also transmembrane linkages to membrane-associated cytoskeletal elements In addition, complexes GP~ and GP.~ could be sequestered into a specific hpld domain indicated by the shaded area, while complex GP2 exists in a free or "'unanchored" state and is capable of lateral motion MF, microfilaments, MT, mlcrotubules. These results indicate that the membrane-Intercalated particles selectively partition into a "fluid" lipid environment and are excluded from solid phase hptd domains. Of course, the converse situation m~ght also be true for some systems The sequestration o f certain components into "solid" hpld domains would slow their lateral mobility to the moblhty of the entire domain (Fig 3) IIB-3 Peripheralmembrane restraints. Peripheral membrane components can be distinguished from integral membrane components by their location in the m e m b r a n e and by the types of forces that hold them in or to the membrane. Integral membrane components are overwhelmingly stabilized by hydrophoblc forces and interact strongly with the acyl groups of the membrane hplds, while peripheral membrane components are generally stabilized by forces other than lipid hydrophobic interactions and lie predomlnately or completely outside the integral membrane zone formed by the lipid bllayer These latter components probably interact with integral membrane components by iomc and hydrogen bonds and other interactions, and some are loosely associated and can be removed from the membrane without subsequent membrane disruption [20, 30] At the outer membrane surface these components could be proteoglycans complexed to integral glycoprotelns and glycohpids, and at the inner surface they could be proteins b o u n d by lomc lnteracUons (e.g requiring Ca 2+) to lipid head groups or integral membrane proteins and transmembrane glycoproteins. The control over human erythrocyte integral membrane glycoprotelns by the inner surface peripheral protein spectrm is discussed in Section IIIC In these systems the restraining forces are probably due to the formation of peNpheral lattices across large portions of the membrane inner or outer surfaces These lattices or networks may link specific components or an array of different components and impede their lateral mobilitles_

67

Fig 4_ Orgamzation of the membrane-associated cytoskeletal systems in cultured 3T3 cells. PM, plasma membrane; MF, microfilaments, MT, mlcrotubules X 54000

IIB-4. Membrane-assoctated (cytoskeletal) restraints. Another type of restraining system which has provoked wide attention recently [29,117,195-204] is constraint by membrane-associated cytoskeletal components These components differ operationally (defined in refs. 29 and 117) from peripheral membrane components due to (a) their transient nature (the ability to change organization and associations rapidly depending on a variety of cellular factors), (b) dependency on cell energy systems for maintenance, (c) sensitivity to drugs that disrupt cytoskeletal components, and (d) their structural linkage to other cell organelles and electron microscopic similarity to cytoskeletal filaments and tubules such as mlcrofilaments and mlcrotubules There are basically three types of cytoskeletal component that are at times assooated with cell membranes thin filaments or microfilaments, thick filaments and small tubules or microtubules (Figs_ 4 and 5) Microfilaments are cytoplasmic protein polymers arranged in double helical filamentous structures of diameter approximately 6-8 nm and variable length up to several #m. They often occur in parallel groups, bundles or sheets closely apposed to the plasma membranes of a variety of cells [205-210] Microfilaments are probably actin-containing filaments, although Pollard and Welhing [210] point out that the evidence is mainly circumstantial. Non-

m

C~

C) r-

3

o_

m

q

;z-

f-

X_m Oo

69 muscle actin has been identified m microfilament regions by the heavy meromyosin binding [211,212] and fluorescent antibody techniques [213-215] Actin monomers are single polypeptide proteins of approx 43 000 molecular weight which contain Ca 2+ or Mg 2+ and ATP [216-218]. Non-muscle actin is capable of polymerlzaUon to high molecular weight thin filaments m the presence of 0.1 M KC1 or 1 mM MgC12 [210, 219]. Upon polymerization the bound ATP is rapidly hydrolyzed to A D P and free phosphate [216]_ Actin filaments are involved in a variety of cellular processes such as cell motlhty and morphogenetic movements, cytokinesls, secretion, cytoplasmic streaming, endocytosis, etc (revxewed in ref. 210) These processes and the state of actin polymerization or microfilament organization can be disrupted by a related group of mold metabohtes called cytochalaslns, the most widely used being cytochalasln B (reviewed m refs. 220-222). Cytochalasin B disrupts mlcrofilament organization in a variety of cell systems [220,223-226]. Although useful, its effects may not be universal [227], and It has the undesirable side effect of inhibiting some cellular transport systems [228-23l]_ Thick filaments similar to in vRro polymerized myosin have been observed m a wide variety of cell types, although myosin in vlvo has been chiefly associated with thin filaments that brad fluorescent anti-myosin [232] or fall to bind heavy meromyosin [233,234]. Myosin has also been locahzed outside the mlcrofilament regions m a less structured organization which could be synonymous with thick filaments [235] Thick filaments of diameter approx 10 nm are often found deep in the cell cytoplasm with a few extending to the plasma membrane cortex in certain regions (Fig 5) [236,237] Myosin has also been detected on the outer surfaces of cells by a variety of techniques [238-241] Non-muscle myosms are complex proteins that bind reversibly to actin filaments forming actlnomyosm complexes, and they possess an actin-actlvated ATPase In contrast to cytoplasmic actin, myosms appear to vary considerably in their physical, chemical and enzymatic propertxes [210]. Cytoplasmic myosins have sedimentation coefficients of about 6S, large Stokes radn (indicating molecular asymmetry) and molecular weights of approximately 500000-540000 [240] Myosin is composed of heavy protein chains of approx 200000 molecular weight and usually two classes of light chains of molecular weights approx 16000 and approx_ 19000 [242,243]. The molecular shape of myosin ~s somewhat that of a hammer with the hght chains complexed with parts of the two heavy chains to form two globular heads and the remainder of the heavy chains intertwined to form the "handle" of approximate length 140 nm [244]. Microtubules are polymeric complexes of the protein tubulin arranged into long tubular structures of outside diameter approx 25 nm and inside diameter approx. 15 nm [245-247] Tubuhn dimers of molecular weight approx. 110000 probably assemble to form the mlcrotubular structures [248,249]. The assembly and disassembly of microtubules are sensmve to temperature, Ca 2+ concentration, pH, pressure and other blocking agents Low temperature and high Ca 2+ concentration (approx 10-s M) favor depolymerlzatlon of microtubules to tubuhn subunlt aggregates [245,246,248,250,251]_ Mlcrotubular structures are Involved in nuclear events during mitosis, but they are also present in the cytoplasm of most non-d~v~dmg

70 cells [236,252] (Fig 4) Mlcrotubules have been found m association with microfilaments and with cell membranes [206,208,253,254] (Fig 4), and they appear to be involved in the control and display of certain cell surface components (see Sections IIIB and IIIC). Alkaloid drugs such as colchlclne, colcimld, vlnblastlne sulfate and vmcnstlne disrupt mlcrotubules and modify a variety of cell surface phenomena, for example lectin-mediated cell agglutination [255,256], fibroblast aggregation [257], lymphocyte mltogemc transformation [258,258a], phagocytosls [259,260] and movement of surface receptors [195-198] Mlcrofilaments and mlcrotubules have been proposed to play interconnected but opposite roles in maintaining the distribution and mobihty of some cell surface receptors [203,204]. This theory will be discussed m detail (see Section III), it suffices here to indicate that membrane-assocmted components perform ~mportant functions m the regulaUon and display of certain receptors In conclusion, there appears to be a variety of ways m which a cell can control the mobility and topographic display of its surface receptors. In this section control by lateral assocmt~ons, sequestration or exclusion from membrane domains, membrane peripheral interactions and membrane-assocmted cytoskeletal systems were discussed, but other yet to be discovered mechanisms of surface control probably exist Of course, a cell may use one, some or all of these mechanisms, plus others, to exert transmembrane control over the dynamics and organization of its plasma membrane (Figs 2 and 3) Ill TRANSMEMBRANE CONTROL MECHANISMS The concept of a cell having transmembrane control over its cell surface is relatively new, but there exists a body of evldence which makes this control system a reality that must be dealt with m any proposal for regulation of cell surface organizatmn, The response of a cell to its external enwronment and the interactions of metabohtes, hormones, growth factors, etc. with the cell surface, resulting in an allosteric event or a signal being transferred or transduced across the plasma membrane, is an example of transmembrane control originating outside the cell. The mechanisms involved m external slgnahng across the membrane have been covered m other reviews [31,261] and will not be considered in detail here except where such signaling may occur via the cell's membrane-associated cytoskeletal system What structural reformation suggests that peripheral and membrane-associated elements are directly hnked to and control outer surface plasma membrane components? in most cases ewdence for transmembrane control is indirect, and is based on drug effects and other studies where these systems are ~mpaired, but Jt is clear in a few systems that transmembrane structural hnkages are real and play an important role m determining surface receptor dynamics.

IliA_ Erythrocyte membrane The human erythrocyte has been an essential workhorse for membrane data

71 and the development of new techniques and theories. Owing to ease of isolation, purity and quantity of material available, the erythrocyte has been utilized as a plasma membrane source by almost every membrane researcher at one time or another [13,24,32,262] Compared to most plasma membranes the erythrocyte ghost membrane is relatively simple_ It is composed of approximately 20 proteins and glycoproteins and several types of liplds and glycohplds [13,14,32,42,52,263-266]. The majority of its proteins and glycoprotelns are considered to be integral and are not solubllized without the aid of detergents, chaotropic agents, bile salts, organic solvents or harsh pH treatments [20,30-32,266-268] Approximately 20-25 ~ of the membrane protein can be eluted rather easily from membrane ghosts in low ionic strength buffers [20,269-272]; hence it is considered peripheral. The peripheral membrane components removed by this treatment are mainly: (a) two polypeptldes of 210000-240000 molecular weight (bands I and lI on sodium dodecyl sulfatepolyacrylamide gels) which associate to form a protein that has been called spectrin [273] or tecktln A [271], and (b) a component of molecular weight approx. 43000 (band V on sodium dodecyl sulfate-polyacrylamlde gels) which appears to be an actln [13,24,32,274] Spectrin has been shown to be an inner surface peripheral protein by localization studies using ferrltln-conjugated antl-spectrin [100,275], in intact cells it cannot be detected by lactoperoxidase labeling techniques [55,276,277]. Band V actin-hke component is probably located on the inner face of the membrane as well, because it is also not labeled in intact cells [49,55,276,277] and is eluted from ghosts along with spectrln in low ionic strength neutral buffers [32,52,278] Of the other protein components In the membrane, two integral glycoprotelns are of special interest. These are (a) an approx 90000-100000 molecular weight glycoproteln(s) (band III) which IS called Component a [50] or Component IIl (Flndlay [279] has estimated the molecular weight of a Component llI that binds cancanavalin A to be approx 150000 by gel filtration in detergent solution) and (b) the major slaloglycoprotein of 30000-50000 apparent molecular weight [280] which is also called the MN-glycoprotein [90] or glycophorin [54]. Component Ill and glocophorln are the major proteins exposed to the external environment [49,55,276,277] and are in addition the receptors for a variety of viruses, lectins and specific antibodies. Glycophorin has been well characterized and is composed of a single polypeptide chain of approximately 206 amino acids [54,94] It contains about 6 0 ~ carbohydrate by weight, mainly in the form of two principal types of oligosaccharides [281,282] which bear a variety of antigens (ABO, MN, etc.), and receptors for several lectlns (wheat germ agglutlnln, Phaseolus vulgaris and Lens cuhnaris phytohemagglutinlns) and viruses (influenza, NDV) [90,94,282-284]. Glycophorin is proposed to be a trimodal amphlpathlc molecule with a hydrophdic-hydrophoblc-hydrophilic structure [54], and it Js reported to span the membrane exposing its N-terminal glycopeptIde region to the extracellular environment, an internal hydrophoblc region to the lipid hydrophobic zone of the membrane, and its C-terminal hydrophilic region to the cell interior [54,94]. The other major outer surface glycoprotein(s), collectively called Component III, contains less carbohydrate (6-8 ~ ) than glycophorln and is believed

72 to be the component(s) involved m anion transport [13,60-62,285]_ This glycoprotein(s) bears receptors for concanavahn A [279] and Ricmu~ commums 1 agglutinm [286] and it, hke glycophorm, has been reported to span the membrane [50,51,55] Recent evidence that Component(s) III is not a single species and is composed of more than glycoproteln has been obtained by Roses [532] SolubihzatJon of Component(s) III m I ~, deoxycholate followed by affimty chromatography on concanavalin A Sepharose columns separated a minor Component Ill concanavahn A-blndmg glycoproteln_ Using the endogenous erythrocyte protein klnase which phosphorylates a Component Ill molecule [533], Roses [532] was able to separate a [7,-32p]ATPphosphorylated glycoprotem from non-phosphorylated Component(s) Ill glycoproteins by concanavahn A/Sepharose chromatography_ Component(s) II! appears to be complexed w~th other integral membrane protein and maintains these associations after extraction into solutions containing Trmon X-100 [I 50]. In the presence of this nomomc detergent Component(s) IIl remains a dlmer hnked together by disulfide bridges [97] complexed to Component V1 (glyceraldehyde-3-phosphate dehydrogenase, a tetramer) [287-289] m the ratio of one tetramer of Component VI per Component I11 dlmer [150] It has been suggested that Component(s) II1 [50], glycophorm [I 13,114] or both [13,58,290] make up the membrane-intercalated particle revealed by freezefracture_ Using freeze-fracture deep-etch electron microscopy, Tlllack et al [114] and MarchesI et al. [94] found that the glycophorln receptors for P vulgarts phytohemagglutlnin and wheat germ agglutmln were associated with the membraneintercalated particles S~mllarly, Pinto da Silva et al. [I 13] found that ABO antigens (borne by glycophorm but also by glycohplds) and low pK, amonlc groups of glycophorm [290] were also associated with the particles Gmdottl [13] suggested that glycophorm alone, due to its molecular weight and number of copies per cell, could not be the sole component of the 85 A freeze-fracture particle and that Component Ill was a logical partner for glycophorin m an ohgomenc complex This suggestion has received support from the findings of Pinto da Silva and Nicolson [58] that concanavahn A receptors (borne by Component II1, but not by glycophorm [286,291]) are exclusively associated with the membrane-intercalated particles Other inner surface membrane components are probably present m this complex, such as Component VI (mentioned above), Component IVa (a protein kmase responsible for phosphorylatmg Component Ill [292]), Component Vii [293] and Component IVb [97,150] The role of this ohgomerlc complex m transport processes [59,60] and water movement [63] led to the proposal that this structure be called a "permeaphore" [58] In the human erythrocyte there is good evidence that a peripheral protein network is responsible for the characteristic biconcave cell shape, resistance to deformation and other physical properties of the intact cell [32,294]. The evidence that a network of spectrln (probably in conjunction with actin-hke Component V) controls these properties and the mobdlty and topography of permeaphore complexes (or components which make up the membrane-intercalated particles) is the following.

73 First, in intact erythrocytes relatively free diffusion of surface proteins [295] and ligand-lnduced movements of antigens [296-298] does not occur to an appreciable extent Similarly, the mobility of the membrane-intercalated particles is impeded [115,116], and their dispersed distribution appears to be under cellular control; that is, the particles are not freely diffusing to any great degree in intact erythrocytes because they cannot be clustered by multlvalent hgands, proteolytlc enzymes or low p H treatments. However, when membrane ghosts are formed by hypotonlc lysis, these particles show a dramatic enhancement of mobility in response to certain treatments such as low pH ( < 5 8) IllS,116] or trypsin treatment I114, 299]. An explanation for these observations is that a peripheral membrane spectrln-Component V network controls the mobility of permeaphore complexes in intact cells When cells are hypotonlcally lysed, resulting in release or reorganization of some spectrin and Component V, this control IS apparently lost [116], even though a large number of spectrln molecules remains bound to the tuner membrane surface and can be localized there with ferrltln antibodies (unless Ca 2+ chelators are added) [275] Integral membrane components are not affected by these treatments [267] When spectrln and Component V are thoroughly depleted from erythrocyte ghosts, the membranes tend to break up and form small rounded vesicles without the characteristic erythrocyte biconcave shape [46,116,273,275], indicating that peripheral components are important in maintaining the shape and size characteristics of erythrocytes Second, chemical crosslinklng agents such as the imldoester dlmethylmalonlmldate, which reacts with z-amino groups, can be used to examine neighboring proteins, and these blfunctional reagents have demonstrated association between integral membrane components and spectrln. H u m a n erythrocyte ghosts crosshnked with dlmethylmalonimidate (4.9 ,~ between functional lmldoester groups) and solublhzed with sodium dodecyl sulfate do not show appreciable changes in protein staining patterns on sodium dodecyl sulfate-polyacrylamlde gels. A minor periodateSchlff band GP-A does appear, however This new band contains the glycoprotelns PAS-1, PAS-2 (glycophorin) and some lipid [300,301] Therefore, under control conditions, membrane proteins are not significantly crosslinked, otherwise, some bands would disappear or shift tO higher molecular weight, indicating the presence of lntermolecular crosshnks [300] However, if lectlns are added to ghost suspensions in situations where certain membrane glycoproteins are known to be aggregated on the outer membrane surface, spectrln is crosslinked at the inner surface by dlmethylmalommldate into high molecular weight complexes Under these conditions of lectln-induced enhancement in spectrin crosshnklng, integral membrane glycoprotelns are not appreciably crosslinked by dlmethylmalonlmidate and can be recovered upon solubllization of the membrane [293] Lectins which bind to Component(s) IlI such as R c o m m u m s I agglutinin [293] or to glycophorln such as P i,ulgarls phytohemagglutinin (Jl, T. H , personal communication) enhance dlmethylmalonlmidate-crosslinking of spectrln when lectln-lnduced redistribution occurs If lectln-induced rearrangements on ghost membranes are not favored (1 e at low temperature) or lectin molecules have been blocked or later removed by specific saccharide lnhibitors,

74 dlmethylmalonlmidate crosslinking of spectrln is not enhanced. These lectlns bind only to outer membrane surface components [100,101] and do not Interact with isolated spectrin, so their effects must be transmembrane, mediated by the membrane glycoprotelns Interestingly, a much longer imldoester, dimethylsuberlmldate (l 1 A between functional groups), will crosshnk spectrln without lectln-lnduced perturbation of the outer membrane surface. Thus, the perturbation of permeaphore transmembrane glycoprotelns and their lateral movement into aggregates allows dJmethylmalonlmldate crosslinking of spectrln (and to a lesser degree other inner surface components known to be associated with component(s) III such as protein kinase (band IVa) and Component VII) [293] That this was a specific lectln-lnduced transmembrane aggregation event due to spectrin association with components in the permeaphore complex and not the consequence of nonspecific membrane rearrangements was demonstrated by prevention of spectrln crosshnklng upon removal of the permeaphore-bound lectm molecules by specific hapten. Thus, spectrin appears to be linked directly or indirectly to a transmembrane structure Wang and Rlchards [302] have used another type of lmldate crossllnklng reagent (dlmethyldithloblsproplonlmldate) and have demonstrated that some Component Ili (s) molecules (or molecules of sxmllar molecular weight) can be crosshnked directly to spectrln. Third, the transmembrane aggregation of outer surface components can be performed from the cytoplasmic side of the erythrocyte ghost membrane by use of highly purified antl-spectrln IgG [303,304] Affinity-purified anti-spectrln lgG was sequestered reside erythrocyte ghosts by osmotic shock, and the ghosts were incubated for several minutes at 37 °C and then fixed with glutaraldehyde When the distribution of slalic acid receptors (almost all slalic acid is on glycoprotein [94]) was determined after mounting the ghost membranes on thin films and observing them by electron microscopy, it was observed that antl-spectrin caused transmembrane aggregation of slaloreceptors borne by glycophorln (Fig. 6) The transmembrane effects of antlspectrin on staloreceptor distribution were concentration, time and temperature dependent and reqmred crosshnklng antibodies (monovalent Fab would not substitute) [304] The linkage of spectrln to integral membrane glycoproteins was thus confirmed, and the low mobility of permeaphore complexes in intact cells probably reflects linkage (direct or indirect) to a spectrin-Component V peripheral network [305], which forms a tough and elastic peripheral support for the more easily deformable lipid bllayer The forces revolved in spectrln-Component V (actm) interactions are unknown, but these molecules can form assooatlons in wtro [305]. Lipid molecules at the inner half of the lipid bllayer, such as phosphatldylserine, may be additionally linked to spectrln through Ca 2+ bridges (ref 306 and McConnell, H_ M , personal communication) to add extra strength in what Steck [32] has called a "two-ply" membrane structure_ A possible model for the permeaphore with complexed perlpherall proteins in shown In Fig 7. The main problem in extrapolating the above findings to other more complicated cells IS that the human erythrocyte membrane, although interesting, is probably an inaccurate model for most plasma membranes. The physical properties of the

75

+ ANTISPECTRIN

(~) + BIFUNCTIONAL REAGENT

Fig 6 Transmembrane control over the glycoprotem complex m human erythrocyte membranes Sequestration of ant~-spectrln inside resealed erythrocyte ghosts or exterior treatment with lectins results in transmembrane aggregation of outer or tuner surface membrane components, respectwely See text for explanation.

"v

rzb

/

"v'r

-~o Fig 7. Hypothetical model of the human erythrocyte "permeaphore"_ Colored hpld head groups indicate asymmetric dlstrlbuhon of phosphatldylserine and phosphatldylglycerol GP, glycophorm, Sp, spectrm; III, component 11I or component a; IVa, component IVa, IVb, component IVb, V, component V or actln; VI, component VI or GP-3-D, VII, component VII_

76 erythrocyte membrane are qmte distinct from other membranes [307-313], and spectrm or even immunologlcally cross-reacting molecules have not been identified m other ceils [274]. This indicates that the erythrocyte membrane may be umque In its organization and structure_ Nonetheless, the orgamzanon of the erythrocyte membrane into glycoprotem complexes and their control by peripheral protein components were important to estabhsh in eukaryotlc cells in many cell membranes which have been isolated and purified, actm or actm-hke components (reviewed in ref 210) and also myosin-hke molecules [314,315] are common. These elements are probably revolved with other cytoske[etal elements such as mlcrotubules in transmembrane regulation of the cell surface

IlIB Lymphoid cell surJaces IIIB-I. Lymphoid cells. The next most studied of plasma membranes are those of lymphoid cells. Unfortunately, lymphocytes, macrophages, monocytes, etc are not homogeneous populations of identical or almost identical cells, like erythrocytes, so their characterization has not progressed to a s~mllar degree Two of the main flmCtlonal classes of lymphoid cells revolved jointly In the immune response, thymlc-derived lymphocytes or T cells and bone marrow-derived lymphocytes or B cells, are not homogeneous as once thought, but occur as heterogeneous mixtures of ceils [316 320]. However, these classes of lymphoid cells are still valuable as tools for studying plasma membrane control mechamsms, because of their capacity to be mJtogenically stimulated toward proliferation and differentiation into effector (T cells) of cell-mediated immune reactions, as well as cells which cooperate with B cells in the antibody response and antibody secreting B cells [321,322] The mltogenlc stimulus can be triggered with antigens, lectlns or antibodies, but once initiated these lymphoid cells follow dzstlnct pathways depending upon their cell type The most useful triggering agents for mltogenesJs have been specific lectlns (reviewed in refs. 29 and 323-325) In certain cases these mltogens need not enter the lymphoid cells to trigger them, because lectlns such as concanavahn A and P. 1,ulgaris phytohemagglutmm attached to solid supports are capable of (B cell) lymphocyte stimulation [324,326] IllB-2 Membrane tsolatton and charactert~atton Purification and characterlzatmn of lymphoid cell plasma membranes has been accomplished by a variety of different techniques (reviewed in refs 327-329). These methods involve shear, osmonc shock, nitrogen cawtatlon or gas-bubble nucleation, and they umversally suffer from similar problems of cell rupture with release of hydrolytic enzymes Following cell disruption, plasma membrane fragments or vesicles are usually purified by differentml centrifugatJon, dlfferentml or lsopycmc density gradient centnfugatlon, centrifugal density perturbation, two-phase aqueous polymer partmon and/or electrophoresls [328,329] Using nitrogen cavitation-disruption and purification of membranes by d~fferential and Jsopycnlc density gradient cenmfugatmn m dextrans, Schmldt-Ullrlch et al, [330] fractmnated calf and rabbit thymocyte (considered to be T cell precursors) plasma membrane vesicles into light and heavy (density) fractions,

77 PM1 and PM2- Plasma membrane fractions PMI and PM2 may represent mixtures of different membrane domains, alternatively, they may arise artlfactually during the isolation procedures and have no relationship to native membrane orgamzatlon. Polyacrylamlde gel electrophoresis of purified thymocyte plasma membrane vesicles in sodium dodecyl sulfate yielded approximately 10-15 Coomassie blue-staining bands varying In (apparent) molecular weight from 17000 to 280000 Fraction PM~ contained more of the heavier (apparent molecular wieght range 60000-280000) glycoprotelns compared to PM2, which contained relatively more of the hghter (apparent molecular weight range 17000-45000) proteins In a subsequent publication [331] these authors discovered that binding of the mltogen concanavahn A did not change the protein composition of fractions PMt and PM2, but augmented turnover and release from the cell of certain high molecular weight components, in particular a glycoprotem of molecular weight approx 55000. Lactoperoxldase-catalyzed surface iodlnatlon has been used to identify the exposed components of thymocytes and bone marrow ceils [332,333] Trowbridge et al [332] found that normal and activated peripheral T cells possess at least two additional high molecular weight components approx. (170000-190000), which are missing on B cells, while B cells had instead a protein of apparent molecular weight around 220000 These components were found on a variety of lymphoid tumors and could be used to categorize tumors as to T or B cell origin. Unique T cell surface proteins were present on several Thy-l positive lymphomas [332,334], while myeloma or lymphosarcoma lines expressed neither T nor B cell components. Lymphoid cells are characterized by their surface display of immunoglobuhn molecules. Experiments on the mltogemc stimulation of lymphocytes by anti-lmmunoglobulin first suggested the presence of Ig at the cell surface [335,336]. This was later confirmed by complement-mediated kdllng of lymphocytes sensitized wtth antl-~ light chain [337], surface localization of fluorescent-labeled anti-lg [338-340], antl-Ig autoradlography [338-342], lmmunocyte adherence [343] and lactoperoxidase labeling of surface proteins followed by solubdizatlon and lmmunopreopitation of labeled Ig [344,345] Surface Ig (cell surface-bound immunoglobulin) seems to be predomlnately 8 S monomeric [346-348] IgM from anti-chain precipitation experiments [337,346,349,350], although substantial amounts of lgG have also been found on lymphoid cell surfaces [333,339]. In this case a "switch" from the synthesis of IgM to IgG in highly dtfferentlated plasma cells may account for the cell surface presence of both molecules in these mixed lymphocyte populations [351]. Another surface Ig which has surface properties similar to IgG has been documented recently on highly differentiated B cells of older mice [352,353], and in humans it appears that a large population of lymphocytes carries both surface IgM and IgD [354] There seems to be general agreement that B-lymphocytes express surface Ig [346,347,350,355-358]; however there is some controversy concerning T-lymphocytes' surface expression of Ig. Hammerhng and Rajewsky [350], Feldmann et al. [348] and Smith et al [333] have found T-cell surface IgM, but Vltetta et al, [358] and Grey et al. [359] failed to demonstrate IgM on T cells. This discrepancy may be due to the different methodo-

78 log~es used to solubJlize and lmmunopreclpitate surface lg [333,360]. Although surface lg has been difficult to detect directly on T cells, a variety of T cell functions can be blocked by anti-Ig, suggesting its functional presence [361-363]. Little is known concerning the bond(s) between lg molecules and the cell surface Antlsera to /z (heavy) chain brads less successfully to surface lgM than antlsera to L (light) chain [364], and Fu and Kunkel [365] found that antlbodtes directed against the carboxyl-termmal end of the # chain failed to react w~th surface lgM on human leukemia cells. These experiments suggest that surface Ig is bound through its heavy chains m the F~ region, but it ~s not known whether surface-lg Js attached to an integral membrane protein receptor or whether the carboxyl-termlnal end of the heavy chains can unfold to reveal a hydrophobic amino acid sequence which interacts with the bydrophobic zone of the membrane [366] As discussed by Vitetta and Uhr [366] the properties of the cell surface/~ chain could prowde the answer to cell surface lg binding, because secreted/~ chains possess different electrophoretlc properties when compared to surface bt chains [367] The pathway(s) of lg secretion m lymphoid cells has been extensively investigated (see reviews by Perms et al. [351], Parkhouse [368], Melchers and Andersson [369]. Vltetta and Uhr [366,370], Melchers and K n o p f [371] and K n o p f [372]) In brief summary, L and H chains are synthesized m the rough endoplasmlc retlculum where assembly of L-H dlmer and tetramer molecules occurs [373,374] Imtml sacchande moieties such as D-Man and o-GIcNAc are addedat this point, and the lg molecules are transported to the Golg~ complex where additional sacchandes such as D-Gal and L-Fuc are added by membrane-bound glycosyltransferases [371,375-379]. In the Golgl, Ig is "packaged" into membrane vesicles which presumably travel to the plasma membrane where fusion occurs (slmdar to Palade's proposal [380] for secretion), and secreted lg is released in the process [377,378] During this process the Ig molecules are thought to be membrane-assocmted to faohtate addition of saccharide umts. Once fusion with the plasma membrane occurs, most of the lg is released as secreted lg, whde some remains bound to the porHon of reverted membrane derived from the fusion vesicle and becomes nascent surface lg HIB-3 Receptor redtstrlbutton and capping The distributions and dynamics of surface Ig on lymphoid cells have been investigated using a variety of methods (reviewed by Unanue and Karnovsky [381]) revolving lmmunofluorescence [298, 338-340,356,382], lmmunoautoradiography [383,384] and immunoelectron microscopy [356,384-389]. The inherent distribution of surface lg molecules seen on most mouse lymphoid cells (after labehng prefixed cells at room temperature or unfixed cells at lo~v temperatures) is not entirely random or disperse, but rather surface lg appears to be displayed in small random dusters [386], which form an interconnected lacy network across the entire cell surface [385]. On human B lymphocytes labeling with antx-lg at low temperature (4 "' C) y~elds a more aggregated pattern of surface lg [400] compared to surface Ig on mouse B lymphocytes, e~ther because of some antibody-induced surface aggregation at these temperatures or concentration of the surface Ig determinants into particular membrane domains. Unanue and Karnovsky

79 +

OPO,o,- -O-D- o VALENT ^ ~

~'%,,~_

'DISPERSED'

'CLUSTERS'

'PATCHES'

'CAP'

Fig_ 8. Schematic pathway to lymphoid cell capping

[381] favor the latter explanation because the surface distribution at 4 °C remains constant at this temperature for several hours. Recently Abbas et al. [401] investigated the inherent distribution of mouse B cell surface Ig using monovalent Fab antibodies coupled to ferritin. Labeling paraformaldehyde-fixed cells at room temperature or unfixed cells at 4 °C with ferritm-anti-lg (Fab) followed by freezefracturing and deep etching to reveal the outer cell surface demonstrated that the small clusters of surface lg formed interconnecting networks across the cell surface leaving large surface regions without Ig expression It is hkely that this may represent an accurate picture of the inherent distribution of surface Ig on at least mouse B cells. U p o n binding anti-Ig at physiological temperatures, hgand-lnduced redlstributton of surface Ig occurs on most, tf not all, lymphoid cells. It was first noted by Taylor et al. [356] that after B cell binding of anti-Ig the surface Ig receptors aggregated into "clusters" and "patches" and subsequently into "caps" at one end of the cell (Figs. 8 and 9) Capping is thought to result from a countercurrent flow of membrane components in which the crosshnked antibody-receptor clusters grow to patches that are transported toward one end of the cell while the remaining components flow in the opposite direction [386,402] The idea of countercurrent flow of membrane components away from the capping region and the concept that capping was a form of membrane flow associated with cell movement [386] has been disputed by Ryan et al. [403]. These latters authors found that immoblhzed cells can form caps, although the positions of the caps on lmmobdized cells were different, forming over nuclear instead of "tall" regions Thus, a moving cell may sweep receptor clusters and patches to the tall region in the course of locomotion Usually the capped end of the cell has many finger-like projections and appears to be in the uropod [404] region over the Golgi [386,387], although the location of the cap can vary depending on receptor and cell type [399,405] Karnovsky et al. [385] examined mouse B lymphocytes labeled with hemocyanln-anti-Ig by freeze-cleavage-deep-etch electron microscopy and observed that the topographic distribution of surface Ig changed rapidly from the usual small clusters or loose lacy network to patches and then caps in less than 2 rain on some cells. The antl-lg-Ig caps once established are not stable, endocytosis of antl-Ig-Ig complexes occurs [356,385-387,389,406,407], and some of the anti-Ig-surface Ig complexes appear to be shed into the media [385,408-411]. After the antx-lg-surface Ig complexes are endocytosed or shed, it takes from 6 to 20 h to resyntheslze and redlsplay surface Ig [298], consistent with

80

"

3

Fig 9_ Morphology of a lymphocyte cap. The cell shown is a thymocyte capped with antl-lg The cap has been vlsuahzed with hybrid antibody to lgG and Southern Bean Mosaic Virus The virus particles are locahzed over the Golgl region (courtesy of Dr C Stackpole)_ Its turnover rate d e t e r m i n e d by biochemical labehng [412]. C a p p i n g o f surface lg p r o d u c e d by anti-Ig has been r e p o r t e d on both T and B lymphocytes for a variety o f species [298,350,356,357,381,384,386,387,399,400,405,413-416] a n d certain alterations in c a p p i n g p h e n o m e n a are k n o w n to occur after neoplastic t r a n s f o r m a t i o n F o r example, a n t i - l g induced c a p p i n g o f surface lg on lymphocytes from patients with c h r o m c l y m p h o c y t i c leukemia occurs rarely [533]. These will be discussed in m o r e detad in a c o m p a n i o n review [549] In contrast to the r a p i d a n t i - l g - m d u c e d surface lg m o v e m e n t into caps, some of the cell surface antigens on routine l y m p h o i d cells are redistributed at much lower rates. T a y l o r et al [356] noted that H-2 and Thy-1 (0) alloantlgens on mouse lymphocytes were not c a p p e d directly b y a l l o a n t i b o d y , a sandwich of a n t l - m o u s e - I g

81 was required to effect capping. These observations were confirmed by others [357, 399], although Neauport-Sautes et al. [417] were able to demonstrate independent capping of different classes of H-2 (H-2 k and H-2 d) coded by different alleles on the same genetic loci using alloantibody alone. Simdar results were also found for different HL-A antigen classes [418,419], and Ig classes (IgM and IgD) [420] on human cells Redistribution of H-2 antigens on transformed fibroblasts into caps by alloantibody alone was attained by Edldm and Weiss [421] It is reasonable to assume that different restraining systems in d~fferent cell types may control antigen mobdlty and abihty of antigens to be redistributed; thus, tt is not unreasonable to obtain apparently conflicting results using different experimental systems. Independent capping or co-capping of lymphoid cell surface lectm receptors has been investigated recently by de Petns [422] and Yahara and Edelman [198]. Capping of concanavalin A receptors resulted in co-capping of surface Ig or H-2, Thy-I (0) and other alloantlgens under certain conditions [198,422]. However, Karnovsky and Unanue [423] and Loor [424] reported that surface lg and concanavalin A-concanavalin A receptor complexes move independently on mouse B cell surfaces This ostensible discrepancy might be explained by considering the possible multitude of different receptor classes for concanavalin A [425,426] and their heterogeneous affinities [427]. At low concanavahn A concentrations (usually < 10 #g/ml), certain high-affinity lectm receptors may be labeled and redistributed independently from surface lg, but at high lectin concentrations (usually > 2 5 #g/ml) concanavalin A could bind directly to the surface Ig molecule or its receptor [422]. Extensive crosslinkmg of a heterogeneous receptor population might prevent redistribution as discussed by Nicolson [117] and Loor [424] Alternatively, Yahara and Edelman [197] have found that the expression of these seemingly antagonistic concanavahn A properties depends upon the temperature at which binding occurs. Incubation of lymphocytes in concanavahn A solutions at 37 °C did not result in lectm-induced capping of concanavahn A receptors (and it inhibited antl-Ig-induced capping), but incubation at 4 °C followed by removal of unbound concanavalin A molecules and elevation of the incubation temperature to 37 °C resulted in lectin receptor capping. Explanations for this curious result revolve around temperature-dependent disruption of cytoskeletal elements and will be discussed in the next section (IIIB-4) Other lymphoid cells such as human polymorphonuclear leukocytes do not show an impediment of capping when treated with high concentrations of concanavalin A, Although leukocyte movement is stopped upon binding the lectm, concanavalin A caps are rapidly established over the nuclear region [403] Several surface receptors have been found to undergo patching and capping on lymphoid cells (Table I) (also reviewed m Unanue and Karnovsky [381]) These include, in addition to concanavahn A [357,385,387,399,424,428-434] and surface Ig [195,298,356,357,385-387,405,411,424,432], a variety of other cell surface antigens and receptors (Table I). A comprehensive study of murme lymphocyte surface receptor and alloantlgen redistribution has been undertaken by Stackpole et al [415]. These investigators determined the requirements for capping of H-2 b, Thy-l.2

82 TABLE I REDISTRIBUTION OF LYMPHOID CELL SURFACE RECEPTORS INTO 'CAPS' Species Mouse

Cell Splemc lymphocytes

Receptor lmmunoglobulm Concanavalm A

Thymlc lymphocytes

Polymorphonuclearleukocytes Macrophage

H-2 C3 F~ Thy-1 ((9) TL H-2 Poly-e-lysme: Phytohemagglutlnln Concanavalm A Concanavahn A Phytohemagglutmm, lmmunoglobulm, Concanavahn A

References 298 405 196 387 440 395 536 538 356 392 399 407

356, 357, 384-386, 407, 408, 415, 535 198, 202, 357, 385399, 415, 422, 430, 441,531 405, 415, 417 537 539 399, 405, 415 399, 405, 415 405, 415 424, 540

357, 387, 399, 433 444 432

Rabbit

Splenic lymphocytes

/52 immunoglobuhn

413

Guinea Pig

Lymph node lymphocytes

immunoglobuhn

389

Chicken

Thymlc lymphocytes Bursal lymphocytes

Concanavahn A Concanavahn A lmmunoglobuhn

431 431 541,542

Human

Peripheral blood lymphocytes

HL-A fl2-Mlcroglobuhn Immunoglobuhn Concanavahn A Concanavahn A

418,419, 543 448, 544 400, 540. 545, 546 547 403

Polymorphonuclear leukocytes

(0-C3H) and T L 1,2,3 alloantlgens and surface lg and c o n c a n a v a h n A receptors on mouse l y m p h o i d cells by lmmunofluorescence and evtdenced that alloantlgens and concanavalin A receptors c a p p e d slowly and incompletely c o m p a r e d to surface Ig_ F o r example, surface a n t M g e h o t e d f o r m a t i o n o f approx. 80%; caps in a b o u t 5 rain, whde anti-Thy-I 2 evoked a p p r o x 20~o caps m a b o u t 20 rain at 37 °C Caps f o r m e d were of two different types, the usual u r o p o d - a s s o c i a t e d caps over the Golgi regxon (93 ~ o f the surface Ig and 83 ~, o f the concanavalin A caps on spleen cells), and caps which f o r m e d o p p o s i t e the Golgl region (75 ~,; of the Thy-I and 72°o of the T L caps on thymus cells) Occasionally the same antigen capped m a different m a n n e r on unhke cells. Thy-I caps f o r m e d p r e d o m m a t e l y over the Golgl on spleen cells [405] but were observed largely opposite the Golgl on thymus cells [399,405]_ The surface receptors present in relatively large numbers on the cell surface were found to be less efficiently c a p p e d than c o m p o n e n t s represented Infrequently (with the exception o f

83 H-2 alloantlgens on thymus cells), suggesting that an inverse relationship exists between the proximity of analogous surface components and capping efficiency. This, of course, assumes a uniform distribution of receptors over the entire cell surface. Alloantlgens and other receptors were once thought to be inherently distributed in large patches localized m certain regions on lymphoid cells [392,393,435,436], but they are now believed to be essentially dispersed or present m small clusters and/or networks [385,395-397,399] (Previous studies on the ultrastructural distribution of surface receptors utilizing indirect antibody or hybrid antibody techniques with markers such as ferntin or plant viruses [390-394] have proven to be inaccurate owing to antibody- and marker-reduced redistribution [395-399].) Stackpole et al. [415] noted that TL alloantlgens capped the most effectively and rapidly of the alloantlgens tested in dilute alloantlsera TL is also the most expediently modulated alloantlgen [437,438]. Cell surface receptor modulation and escape from Immunological destruction will be discussed in the companion review [549]; it sut~ces here to mention that when cells are treated with antisera against certain surface receptors at 37 °C, they rapidly lose complement sensitivity [437,439] There are a variety of environmental factors and drugs which block capping, but do not affect the formation of receptor clusters or patches, for example inhibition of cell metabolism by specific drugs can stop cap formation without affecting the dispositions of clusters and patches in a variety of systems [298,356,408] (Table II) These results indicate that capping reqmres a metabohcally more active cell than does the smaller-scale patch formation. Low temperature inhibits both patch and cap formation [298, 356,357,408], possibly both by restraining cell metabolism and to a lesser degree, by increasing plasma membrane viscosity Drugs which influence macromolecular synthesis (Table 11) have no apparent effect on cap formation Capping is sometimes inhibited by the cytoskeletal-dlsruptlng drug cytochalasm B, which is known to act on microfilaments [220,223-226]. The direct impact of cytochalasin B on capping ranges from negligible [403,408] to shght [ 197,198,298,356,440] to moderate [204]. de Petrls [202] has found that preformed concanavahn A caps can be dismantled or abohshed by cytochalasm B eLther in the presence or in the absence of vinblastine sulfate. Taken with previous findings of Sallstrom and Alm [431] that preformed caps can be reversed by metabolic inhlbltors such as K C N or lodoacetlc acid, these results strongly implicate the cytoskeletal system in cap formation and maintenance. IlIB-4 Cytoskeletal transmembrane control of lymphoid surface receptors. The Involvement, in part, of a membrane-associated cytoskeletal system in controlling the topography and mobility of lymphoid cell surface receptors has been demonstrated by experiments where cell energy or cytoskeletal systems are impaired during receptor redistribution (Table lI) Drugs that disrupt microfilament orgamzatlon (the cytochalasms) inhibit lymphocyte capping to variable degrees [196,202,204,298,422,440], while drugs that disrupt microtubules have little effect [204,408] or enhance capping somewhat [195,202,422,440] When cytochalasm B plus colchiclne or vinblastine is administered simultaneously to lymphocytes [202,204,422] or polymorphonuclear

84 TABLE 1I EFFECTS OF LIGANDS, DRUGS AND ENVIRONMENT ON CAP FORMATION References Treatment

Inhibition of caps

Low temperature (0 °-4 °C)

298, 356, 357, 386 408, 431 298, 356, 357, 387 195, 198, 298,408, 431,440 298,408 408 403,408

Monovalent hgand Sodmm aztde (10-3-10 z M) 2,4-Dmltrophenol (5 10-3 M) Ohgomycm (100 pg/ml) Sodmm cyamde (10-¢ M) Carbonyl cyamde m-chlorophenylhydrazone (10-'t-10-3 M) [odoacetamJde (10-3 M) Cytochalasm B (10-30/~g/ml) Vmblastme or colchlcme (10-50/~g/ml) Cytochalasm B (10 pg/ml) ~ colchlcme (10-s M) Puromycm (0 5/lg/mll Cyclohexlmide (0 4/tg/ml) Cychc AMP (10-~ M) Local anesthetics (10-s 10 3 M)

298 403,408,431 198",202", 204,298", 356*, 422", 440* 421,422"

No inhibition of caps

403, 408 196"*, 198"*, 202"*, 356, 403, 408, 440, 548**

202, 204, 403, 548 421 *** 548 203, 20-~, 521

408 408 408

* Some inhibition of capping, but not complete ** Increase in capping *** lnhlbltmn only after 1-6 h.

leukocytes [403], capping ts s t n k m g l y hmdered These results, considered m the light of previously m e n t i o n e d data on inhibition of antt-lg reduced surface lg capping by metabohc mhxNtors such as N a N s [195,298,381,403,408] lmphcate the e n t a i l m e n t of both microfilaments a n d m~crotubules m t r a n s m e m b r a n e processes which lead to cap formation Lectms such as c o n c a n a v a h n A have a dramatic effect on lymphocyte capping of both mouse B and T cell surface receptors [195-198,298,422,440] Patching and capping of receptors such as lg were f o u n d to be reversibly mhxblted by the prior b i n d i n g of c o n c a n a v a l i n A (usually 50-100 ~g/ml) to lymphocyte surfaces [195,197, 298,424], however, the m h i N t i o n of receptor patching and capping by c o n c a n a v a l i n A could be blocked or reversed by colchzcme, vlnblastme or related drugs [196,440]_ A divalent derivative of c o n c a n a v a h n A, succmyl-concanavahn A [442], was unable to immobd~ze surface Ig and prevent its patching and capping, unless antl-concanavahn A was added to facdttate crosshnkmg of surface-bound succmyl-concanavahn A molecules [440]. This suggests the involvement of c o n c a n a v a h n A receptor cross-

85 linking and clustering in the surface receptor immoblhzatlon process. As previously mentioned, under certain conditions (low temperature pretreatment, or low ( < 5 #g/ml) lectin concentrations) concanavalln A reduced capping of its own receptors on lymphocytes [196,197,424,440], but if added at 37 °C the concanavalin A receptors remain dispersed [198]. Since low temperature is known to disrupt mlcrotubules [245, 248,442,442a,443], these results could be interpreted as follows, concanavahn A binding to lymphocytes results m local concanavalin A-receptor clustering or other membrane alterations(s) which trigger a transmembrane event, possibly a rearrangement of inner membrane surface peripheral components that are linked to the cytoskeletal system through a colchicme-sensitlve component (microtubules) Not all lymphoid cells react in the same way as lymphocytes to lectm-induced capping or Inhibition of capping. On other types of ceils, such as human polymorphonuclear leukocytes, concanavalin A induced caps when reacted with cells at 37 °C or after lectin pretreatment at 4 °C followed by washing and shifting to 37 °C, although the cellular location of the resulting caps was dissimilar [403] Interestingly, mouse polymorphonuclear leukocytes do not cap well w~th concanavalin A unless the cells are pretreated with colchlcine Ohver et al [444] found that colchlclne treatment of mouse leukocytes dramatically increased capping induced by 5 #g/ml concanavalin A. Lectlnreduced capping of colchiclne-treated cells was antagomzed by cyclic G M P or agents which stimulate its production. Leukocytes from beige CH mice (CH (C57BL/6J bg/bg), a mouse analog of the Chediak-Higashl syndrome of man is characterized by the presence of giant cellular granules, Impaired leukocyte chemotaxis and defective lysosomal degranulatlon possibly due to granule membrane defects [445-447]) capped at high frequency with concanavahn A (approx 44% cells capped at 5 #g/ml), and capping on these cells was blocked by cyclic GMP. These authors suggested that cyclic G M P promoted mlcrotubule polymerization or Interaction with the plasma membrane resulting in increased "anchorage" of surface receptors Yahara and Edelman [197,198,441] favor the existence of a lymphocyte colchiclne-sensitlve microtubule system that links concanavalin A receptors to other surface receptors [441] resulting in transmembrane control [29,302]. Of course it could be argued [422] that the lectln-immoblhzing effects were due solely to cross linking of cell surface receptors that also share concanavahn A-binding sites. This latter explanation is not supported by earlier capping experiments of Karnovsky and Unanue [423] and Karnovsky et al. [385], where co-capping of surface Ig and concanavahn A receptors was not observed, but de Petns [422] and Loor [424] have recently challenged the lmprobabihty of lectin receptor-surface lg co-capping and have presented evidence that it occurs at higher ( > 2 5 #g/ml) concanavahn A concentrations In their extenswe studies Yahara and Edelman [196, 197, 441] observed that concanavahn A induced caps, but only in colchlcme-treated lymphocytes, and co-capping with concanvahn A receptors was demonstrated for surface lg, Thy-I (0), H-2, Fc receptors, fl2-microglobulin and several other lectin receptors [441] Lower concentrations of concanavahn A ( < 1 0 g/ml) caused some co-capping, but most of the other receptors they examined did not co-cap and remained dispersed across the lymphocyte surface.

86 (Although fl2-mlcroglobuhn and HL-A antigens on human lymphocytes appear to co-cap after treatment with anti-~2-microglobulin [448], this may merely reflect the fact that HL-A antigens are hnked to fl2-microglobulin [449,450].) The converse experiment (capping of surface lg) did not lead to co-capping of concanavalin A receptors. To rule out simple lectm crosshnkmg of relatwely immobile surface receptors to other components bearing lectin-bmdmg sites as a part of their structures lectms were coupled to "sohd'" supports which can attach locally to only a small percentage of the lymphocyte's lectin-bmdmg s~tes, and modulation of surface receptor moblhty was analysed When concanavahn A was denvatized to nylon fibers, latex spheres or other cells, these supports were capable of binding cells [451,452] Lymphocytes bound to concanavahn A-nylon fibers and treated with antl-lg d~d not form surface lg caps, m spite of the fact that the majority of the concanavahn A receptors (and others as well) cannot possibly react with the derivatized nylon surface [452] Apparently, ~mmoblhzmg only a small fraction of the surface concanavalin A receptors blocks capping of the surface lg Similarly the concanavahn A-denvatlzed latex spheres or concanavahn A-labeled platelets upon interaction with murine splemc lymphocytes (m exlzenments where ~.~ 10 partlcles were bound per cell) prevented subsequent anti-Ig, anti-F~- or anti-H-2-induced patching and capping, moreover without slgmficantly altering the distribution of the majority of concanavahn A receptors The inhibition of ant~-Ig capping was abohshed by pretreatlng the lymphocytes with colchicine, and under these condlt~ons the concanavahn A-platetets and-latex beads capped to one pole of the cell whde the majority of concanavahn A receptors remained d~spersed Sodmm azlde prevented redistribution of lectin-denvatlzed platelets and beads, identical to its effects on redistribution of the bound native lectm [195,408,424, 440] When Yahara and Edelman [441] examined the colchlcine-treated cells for capping of surface lg, H-2 and Fc receptors in the presence of several cell-bound concanavahn A-platelets or beads, they observed maximal capping [441] m addition to co-capping with antl-lg [424,441]. Although unlikely, co-capping could have been due to sequestration or non-specific trapping of the surface lg receptors during cluster or patch formation or, more likely, it occurred because adjacent receptors are actually crosslinked during the estabhshment of clusters and patches [198]. This is probably favored when transmembrane restraints are removed. Comparing the inhibition of antl-lg-induced capping by concanavahn A-platelets and -beads wlth concanavahn A-fibers [452], Jt was noted that antl-lg capped on concanavahn Afiber-bound lymphoc3,tes at random locations around the cell surface Yahara and Edelman [441] proposed that the concanavahn A-fibers were less efficient, because they could not attach at multiple points on the lymphocyte surface in contrast to the more dramatic inhibition of antl-lg-mduced capping by the binding of less than ten concanavahn A-platelets or -beads per lymphocyte. Current theories on the transmembrane control of surface receptor mobdlty and topography on lymphoid cells have evolved in a number of laboratories Berhn and his collaborators gathered extensive ewdence indicating that the microtubule

87 DIRECT A slale

o ,,,oe

INDIRECT Aslole

F state

Estate

~ stole

3--CBP A stole

2

F slate

F stale

A

slate

Outside

rn~croldoments

Inside

CBP

'

CBP

Fig 10 Transmembrane control by membrane-assocmted cytoskeletal elements as proposed by Yahara and Edelman [197,198,440,441,453]_ Receptors are assumed to interact with colchlcmebinding proteins (CBP) m an equilibrium consisting of two states, anchored (A) and free (F) As indicated m (1), receptors may interact directly or indirectly via the membrane-intercalated particles Alternatively, as shown m (2), only certain glycoprotem receptors may interact with the CBP and the CBP may interact with membrane assooated mlcrofilament assemblages (from Edelman [453])

system can exert t r a n s m e m b r a n e c o n t r o l over a variety of surface receptors and functions [199,255,256,259,260]. E d e l m a n a n d Y a h a r a [198,440,441,453] p r o p o s e d a m o r e e l a b o r a t e m o d e l for the m o d u l a t i o n o f l y m p h o c y t e surface receptors by a t r a n s m e m b r a n e c o n t r o l system involving m i c r o t u b u l e - m l c r o f i l a m e n t systems. In this m o d e l different classes o f surface receptors at any one time are relatively i m m o b i l i z e d a n d linked (so called " A - s t a t e " or a n c h o r e d state) or they are mobile a n d unlinked ( " F - s t a t e " or free state) to the cell's m i c r o t u b u l e - m i c r o f i l a m e n t systems [440,453] (Fig 10). T r a n s m e m b r a n e receptor-cytoskeleton association is hypothesized to be an e q u i h b n u m process where some receptors are linked to b o t h mlcrotubules and mlcrofilaments AssoclaUon with m i c r o f l l a m e n t s a n d microtubules could explain the inhibition o f c a p p i n g by cell energy poisons a n d by combinations o f cytochalasin B and colchicine (see T a b l e 11) W h e n certain multlvalent ligands such as c o n c a n a v a l l n A brad to the l y m p h o c y t e cell surface, local clustering occurs, which could faclhtate r e c e p t o r redistribution Alternatively, the binding o f larger a m o u n t s o f c o n c a n a v a h n A or multiple local binding o f derlvatlzed c o n c a n a v a h n A might shift the equilibrium t o w a r d the A-state, resulting in an inhibition o f redistribution and capping. Finally, the i n d e p e n d e n t b u t Interconnected roles o f microfilaments and m i c r o t u b u l e s [203, 204] wdl be considered in the next section.

88 IIIC_ Fibroblast Cell Surfaces HIC-1 Plasma membrane isolation and characterlzatton Isolation and purification

of fibroblast and other cell plasma membranes have taken a slightly different course from lymphoid cells [327]. The methods of Warren and Gllck [454,455] have proven to be popular where fluoresceln mercuric acetate is used to '"strengthen" the plasma membrane, so that during cell disruption it breaks to form large sheets instead of small vesmles Purification of the large membrane fragments by differential and isopycmc cenmfuganon m sucrose or glycerol gradients yielded plasma membranes virtually free of internal membrane contamination, although some internal components may be "'fixed" to the plasma membrane by these treatments. One troublesome aspect of the Warren technique is that plasma membrane marker enzymes are inactivated, leaving only phase and electron m~croscopy and composmon as evidence for pumy Perdue and Snelder [456] modified the Warren techmques by using Zn 2+ fixation, whmh can be partially reversed by chelating agents After chelation and removal of Zn 2+, the membrane sheets veslculate and were purified further w~th the aid of active plasma membrane marker enzymes [457] Alternatively, Atkinson and Summers [458] used hypotonlc swelhng of cells followed by rapid Dounce homogemzatlon with the subsequent addmon of MgCI2 The large membrane fragments and ~esicles were ultimately purified by sucrose density gradmnt techniques Brunette and Till [459] have developed a phase separation technique using aqueous solutions of mixed polymers to purify membrane fragments and vesicles_ ZnClg-treated, homogenized plasma membranes were first partmlly puNfied by centrlfugatlon and then further purified by partltiomng during cenmfugation m a two-phase (dextran and polyethyleneglycol) polymer mixture After several partitions purified plasma membrane sheets were obtained These three techmques for membrane purificatmn have been compared for the plasma membrane of sarcoma 180 cells [460] Shin and Carraway [461] observed that inclusion of Zn z+ m the homogemzatlon med~a resulted in a greater degree of membrane integrity and better retennon of large molecular weight polypeptldes. Otherwise, there appeared to be httle difference between the purified membranes obtained by these varied techmques, with the exception that Tess contaminating lntracellular membrane material remained after two-phase partltlonmg Examinations of the total protem profiles of purified untransformed and transformed fibroblast plasma membranes by polyacrylamlde gel electrophoresIs In sodium dodecyl sulfate have revealed few major differences [462-465] When Sakiyama and Burge [463] examined 3T3 and transformed 3T3 fibroblast plasma membranes, they found almost identical proteins and glycoprotelns on polyacrylamlde gels One difference they d~d note, however, was the secretion by untransformed cells of a high molecular wmght glycoprotem, which was missing from supernatants of transformed cells One component present m purified untransformed chick fibroblast plasma membranes ~s an approx. 45000 molecular weight protein that is absent or greatly diminished after Rous sarcoma virus transformation [465] This protein has been called J by Robbins [466], and it appears to correlate with the transformed

89 phenotype in temperature-sensitwe virus-transformed cells When cells are shifted from the temperature non-permissive for transformation to the permissive temperature, the synthesis of this component drops to 10-25 ~ of the normal level within 36 h. Its disappearance is not due to increased degradatton m cells displaying the transformed phenotype. The most promising approach for obtaining cell surface differences between untransformed and transformed fibroblast plasma membranes has been surface labehng utilizing lactoperoxidase-catalyzed iodmation [276] or galactose oxldase-catalyzed tntiation [97,467] Untransformed fibroblast plasma membranes are characterized by the presence of a high molecular wexght protein (approx 220000-250000), which is accesstble to surface labeling techniques [468-471] (for a more detatled discussion see Hynes [472]). This component bears strong relationship to a fibroblast surface antigen isolated and purified by Ruoslahti and coworkers [473,474] and will be discussed m more detail in ref 549. HIC-2 Receptor redlstrlbunon on fibroblasts. The binding of multivalent hgands to fibroblast cell surfaces generally results m receptor redistribution from a dispersed state or diffusely distributed small clusters to larger clusters and patches. This phenomenon has been repeatedly documented with surface antigens and lectln receptors Fibroblasts attached to or grown on glass or plastic substrates have surface antigens non-randomly displayed across their entire surface [421,475]_ Edidm and Weiss [421] used indirect fluorescent antibody techmques to study receptor redxstrlbutlon and capping on mouse C1 1d (an L cell derivative) and human VA-2 fibroblasts The inherent dlstrtbutlon (labehng at 0 °C) of antigens on these cells (at this level of resolution) appeared to be essentially random, but h~gher antigen concentrations appeared to be present at the tips of pseudopods and other peripheral surface structures (see also Weiss and Subjeck [476]). Approximately half of the mouse cells incubated with anti-H-2 for 35 rain at 37 °C and subsequently with fluorescent antllmmunoglobuhn at 0 °C underwent capping over the permuclear region of their cytoplasms (Fig. 11). Similar to lymphoid cells cap formation, but not cluster or patch formation, was inhibited by temperatures below 15 °C, dimtrophenol, N a C N , N a F and cytochalasin B, but the latter to only a certain degree In contrast to lymphoid cells, cycloheximide (10 #g/ml) and colcemld (0.4 #g/ml) were effectwe in blocking capping, but only after they were present in the culture medxa for ~ 16 h. However, this is consistent with the findings of Ray et al. [477] m which membrane proteins arrive at the plasma membrane from a precursor pool that takes several hours to deplete. The sensitivity of capping to microtubule-disruptmg drugs may be due to quantitative d~fferences in the microtubules of fibroblastic cells Most fibroblasts have an extenswe cytoplasmic microtubule system [206-208], while mlcrotubules in lymphoid cells are rarely seen [198,422]. Llgand-induced redistribution occurs at different rates on different fibroblast cell surfaces. Edidm and Weiss [421] determined that alloantlbody alone was able to reduce capping of H-2 receptors on transformed CI ld cells, but the rate of cap formation was less than when indirect antibody sandwich techniques were used, contrasting the, previously mentioned findings on lymphoid cells where indirect

90

I'

'CAP'~

'PATCHES'

+ POLYVALENT LIGAND ~

D ' ISPERSED'

~+ LIGAND

REMAINS 'DISPERSED'

/

l ~

'CLUSTERS'

If / /TIME

l

COALESCENCE OF C ' LUSTERS'

Fag I l Alternate pathways of hgand-mduced redistribution on fibroblastic cells

antibodies are necessary for capping of most antigens [356,357,385,386] On the other hand, VA-2 human fibroblasts did not ehc~t caps when treated with antisera followed by fluorescent anti-lmmunoglobuhn. These cells formed instead small clusters of anugen-anttbody complexes which eventually coalesced and were internahzed by endocytosls These quite different series of events have been described separately by Phllhps and Perdue [478] as "capping'" and "coalescence of focl or clusters" (F~g. l 1). The latter process differs from the former in that the small antigenantibody clusters come together m vamous regions on the cell surface, and endocytoszs eventually occurs at these multiple sites. Cell motility affects these processes and probably modifies or determines the rate and direction of movement of the aggregated hgand-receptor complexes [209,479]. Proteolys[s of chick [473] or human [474] fibroblasts releases a cell type-specific surface antigen (SF) which ~s also secreted or released from cells m wvo and can be detected m serum. SF antigen Js a major surface component(s) of fibroblasts (up to 0 5 o~, of the total protein of normal cultured fibroblasts) and is now thought to be a complex of at least three polypept~des (molecular weights approximately 210000, 145000 and 45000) (ref 480 and Vaher~, A., personal commumcatlon)_ One of these components, SF 210 (approx 210000 molecular weight), is probably the high molecular weight surface glycoprotem labeled by lactoperoxldase-catalyzed lodlnataon [468,471,481-483] and galactose ox~dase-catalyzed trot]at]on [467,484,485]. Interestingly, the approx 45000 molecular weight component (SF 45) electrophoretically comzgrates with purified fibroblast actxn suggesting the possibility that SF antigen

91 molecules may be related to membrane actln [486] Immunodiffusion data indicate that SF 210 and SF 145 carry similar antigens, however, the biochemical characteristics, metabolism and turnover of SF 210 and SF 145 are distinctly different. SF 210 is glycosylated, accessible to surface labeling and trypsin-sensitive, whereas SF 145 is characterized by a rapid turnover rate during pulse-chase studies [480,487,488]. The distribution of SF antigen in cultured fibroblasts has been examined by fluorescence microscopy followed by scanning electron microscopy of the same ceils [489]. Before antl-SF labeling, fibroblasts were fixed in acetone or glutaraldehyde to prevent hgand-mduced redistribution Cells were next treated in situ with anti-SF, washed and incubated in a fluorescent-antx-immunoglobulin solution. Samples were washed, evaluated for immunofluorescence, critical point dried and then shadowed with gold for scanning electron microscopic observation Four different forms or location of SF antigen were detectable by these procedures : (a) intracellular location in the cytoplasm, (b) cell surface location m association with fibrdlar structures such as microfilaments, surface ridges and other surface appendages, (c) localization on the growth substrate as a fibrous network extending outward from the cell body, and (d) m a soluble form m the extracellular m e d m m [486]. The cell surface appearance of SF antigenic sites indicated a non-random net-like or streak-hke distribution Many surface regions lacked detectable SF antigens, while others displayed dense SF antigen concentrations, which were particularly p r o m m a n t on structures that were subsequently identified by scanning electron microscopy as surface ridges and mlcrovdh. Brief trypsimzatlon removed SF antigens, and their reappearance correlated with cell flattening and the extension of cell processes characteristic of fibroblastlc morphology [490-492]. All available evidence indicates that SF 210 is probably the same component as that which G r a h a m et al [493] have recently isolated and purified from untransformed hamster fibroblasts, and Yamada and Weston [483] have isolated from ch~ck fibroblasts. A variety of drugs that act o n cytoskeletal systems are known to affect the agglutination of fibroblasts by lectins [29,117]. Yin et al. [256] found that treatment of concanavahn A-labeled SV3T3 fibroblasts with colchicine lowered erythrocyte adherence to the SV3T3 cell surface. Subsequent experiments determined that treatment of SV3T3 cells with the alkaloid drug resulted in the formation of a caplike aggregation of concanavahn A receptors near the cell nuclear area [200], instead of the usual multiple clusters of concanavalin A receptors [433,494 A96]. Ukena et al. [200] have described the effect of colchlcme on concanavahn A-reduced redistribution as an effect on the secondary organization of surface receptors, the primary organization being the lectin-mduced formation of small multiple receptor clusters The inhibition of cell agglutination by colchlcme was proposed to occur by concentration of receptors to a limited cell surface region reducing the probabdity of cell-cell interaction, consistent with the findings of Rutishauser and Sachs [497, 498] for nylon fiber-bound cells. Other drugs such as dibutyryl 3', 5'-cychc A M P and 3',5'-cychc A M P reduce the agglutmabdity of hamster ovary cells by wheat germ agglutlnln and convert these ceils to a more fibroblastic morphology [499] The

92 dtbutyryl cychc AMP-induced fibroblast-hke conversion of the hamster ovary cells was reversed by colcemid and vlnblastlne sulfate [500]. Sheppard [501] studied the effects of dlbutyryl cyclic AMP on fibroblast cell cultures and discovered that addition of dibutyryl cyclic A M P to transformed fibroblast cells lowered their saturation density, modified their morphology and decreased their lectin agglutmabIlity An reverse relationship between lntracellular cychc A M P levels and concanavalm Areduced agglutination of mouse fibroblasts has been found recently by Wllhngham and Pastan [502] Using a temperature-sensitive mutant of 3T3 cells /3T3cAMP '~) in which the cellular concentration of cychc A M P falls rapidly after a temperature shift, these mvestzgators were able to show that high levels of cychc A M P correlated with decreased lectm agglutinabdlty, and low cychc AMP levels correlated with increased agglutmab~hty Prior treatment of cells with a cychc AMP phosphodzesterase inhibitor or dJbutyryl cychc A M P blocked the increase m lectm agglutmabihty of 3T3cAMP re' reduced by temperature shift. In an extension of these studies Wilhngham et al [503] recently proposed that the major effect of cyclic A M P on cell agglutination is vm the cytoskeletal elements (mlcrofilaments) within the microwlh. They observed that, when cellular cyclic nucleotJde levels drop, drastic rearrangements in the cytoskeletal system occur w~th an increase m the density of surface structures such as microvilh and pseudopodia They have additionally claimed that these cell appendages solely control lectm-medmted agglutination of fibroblastic cells Although their model is intriguing and conceptually simple, this is undoubtedly not the only phenomenon which controls cell agglutination (see Nlcolson [29, 549] and Rapin and Burger [533] for discussions on cell agglutination by lectlns)_ Finally, Kaneko et al [504] reported that mlcrofilament-dlsruptmg cytochalasm B inhibited concanavahn A- and R t c t m t s ~ ' o m m u n t s agglutmin-medmted agglutination, but did not affect cell surface lectm binding. Local anesthetics such as the tertiary amine series (cocaine, dlbucame, tetracaine, procaine, meplvacame, hdocame, etc ) dramatically enhance the lectln agglutmabihty of fibroblasts [203,204,505] The cellular s~te of action of local anesthetics is generally thought to be the cell membrane, where the abdity of these drugs to associate w~th and modify the organization of membrane acidic phosphohp~ds correlates well wLth their biological activities (reviewed recently by Seeman [509] and Papahadjopoulos [516]) In addition to their anesthetic properties with excitable membranes [506-508], local anesthetics produce a variety of effects on cells and membranes including bllayer and cell expansion [509], altered osmotic fragility [510] and mhlbitlon of cell spreading [511], movement [512,513], adhesion [514,515] and fusion [512]. Local anesthetics are considered to associate with acidic phospholiplds by both hydrophic and electrostatic interactions [508,517-519], and they produce dramatic dmordering m lipid bilayers and enhance the flmdity of phosphohpids in membranes [519,520] However, these latter effects occur only at higher anesthetic concentrations than are required to produce anesthesia or modafy lectm-mediated cell agglutination. Poste et al [203,204] have found that the agglutination of anesthetic-treated untransformed mouse and hamster fibroblasts is accompanied by an enhancement in lectm-mduced

93 (o)

(b)

unlreatecl

colchlcme

C;

cytochalasmB

dlbucalneor colchlcmeplus cytochalasmB

[

~ // ,,,,,

Fig. 12 Schematic representation of the effect of colchlcme, cytochalasin B and dlbucame hydrochloride on the distribution of concanavahn A receptors on untransformed mouse 3T3 cells (a) Segment of plasma membrane showing integral membrane proteins carrying concanavahn A receptors embedded within a hpld bllayer and linked on the inner membrane face to microtubules and mlcrofilaments_ (b) Representation of the planar view of the distribution of concanavahn A receptors over the upper surface of the entire cell (1) Untreated 3T3 cells (a) the integral proteins are hnked to both mlcrotubules and mlcrofilaments; and (b) concanavahn A receptors remain randomly dispersed after binding of concanavahn A to the cell surface. (2) 3T3 cells treated with colchicme. (a) disruption of mlcrotubules by colchicine allows concanavahn A-induced aggregation of integral proteins which are still hnked to an intact microfilament system resulting in redistribution of concanavalln A receptors into a single large "cap" as shown in (b) (3) 3T3 cells treated with cytochalasin B: (a) despite disruption of mlcrofilaments by cytochalasin B, integral, membrane proteins remain "anchored" by their connection to mlcrotubules and binding of concanavalin A to the cell surface does not induce redistribution of concanavahn A receptors which remain randomly dispersed as shown m (b) (4) 3T3 cells after treatment with dlbucame or combined treatment with colchlclne and cytochalasln B: (a) disruption of the linkage of integral membrane proteins to both microtubules and m~crofilaments allows concanavalln A-mediated redistribution of concanavalin A receptors ~o form multiple "clusters" and "patches" distributed over the entire cell as shown in (h) (from Poste et al [203])

94 redistribution of concanavalin A receptors to form multiple clusters and patches. Fluorescence polarization measurements on the rotational relaxation of a probe (dlphenylhexatrlene) that partitions into the plasma membrane hydrophobic zone indicated that local anesthetics used at concentrations which produce maximum enhancement in cell agglutination have only a very small effect on lipid fluidity [203], so enhancement in agglutination cannot be solely explained by increases in membrane fluidity produced by these drugs Local anesthetics also reversed the inhibition of concanavahn A-mediated agglutination of transformed fibroblasts by colchlcine and vinblastine sulfate and prevented the mobilization of clustered lectin receptor complexes to caps [203,204]. The effects of local anesthetics such as dibucame or tetracame in enhancing lectln-medmted cell agglutination and lectin-lnduced redistribution of concanavahn A receptors on 3T3 cells and, alternatively, in inh~biting surface-immunoglobuhn [204,521] and concanavahn A [550] receptor capping on splenic lymphocytes [204,521] were duplicated by treating ceils with colchiclne (or Vmca alkaloids) together with cytochalasin B [204] These results lmphcate involvement of both membrane-assocmted microtubules and microfilaments in the transmembrane control of receptor moblhty and topography (Fig 12) Direct evidence that low concentrations of local anesthetics modify cytoskeletal elements or their plasma membrane attachment points was obtained using electron microscopy where the reversible disruption or dissolution of membrane-associated mlcrofilaments and mlcrotubules by dlbucame, tetracaine or procaine resulted m 3T3 cell rounding and surface blebmg [254] The mechamsm by which local anesthetics disrupt mlcrotubule-microfilament systems is unknown. The ability of local anesthetics to displace membrane Ca 2÷ may disturb the linkage of cytoskeletal elements to components located on the inner face of the plasma membrane. For example, the erythrocyte inner surface peripheral membrane protein spectrin (Section IliA) which exerts transmembrane control over a variety of surface receptors through glycophorln and component(s) IlI, is d~splaced from membranes by Ca2+-chelatlng agents [270,273,275] and will only reassociate with lipid membranes in the presence of Ca 2÷ [306]. Mlcrotubule integrity is also controlled by Ca 2÷ concentration [246,247,251] In support of this possibihty Poste and N~colson [534] found that an experimentally reduced Increase in lntracellular Ca 2+ concentration due to exposure of cells to the calcium ionophores A23187 or X537A produced identical effects to colchlcine or vinblastine sulfate treatment on cell surface receptor mobility in the presence of cytochalasin B. Local anesthetics might increase cellular Ca 2÷ concentrations to levels sufficient to reduce mlcrotubule depolymerlzation [246,251], or they could competitwely affect Ca2÷-sensitlve functions necessary for mlcrotubule maintenance [522]. The effects of local anesthetics on mlcrofilaments is obscure, but changes in cellular Ca 2÷ could affect adenylcyclase, Ca2+-reqmrlng ATPases or actomyosin complexes [210] (in addmon to a multitude of other processes [523]), or local anesthetics could directly interact with these membrane-bound enzymes or the cytoskeletal system itself. The apparently contradictory results of receptor redistribution on local anesthetic-treated fibroblast and lymphocyte cell surfaces can be explained by proposing

95

ACTIN FILAMENTS ~,[

MYOSIN MOLECULES

~

MICROTUBULES

Fig 13 Hypothetical interactions between membrane-assocmted mlcrotubule and m~crofilament systems revolved in transmembrane control over cell surface receptor mobdlty and distribution This model envisages opposite roles for m~crofilaments (contracnle) and mlcrotubules (skeletal) and suggests that they are hnked to one another or to the same plasma membrane tuner surface components at spectfic "nucleation" points In addition peripheral membrane components at the tuner or outer membrane surface may extend this control over specific membrane domains The uncoupled ollgomenc transmembrane glycoprotem complex to the left reprecents a membraneintercalated partiole revealed by freeze cleavage. dual and opposing roles for mlcrofilaments and mtcrotubules in transmembrane receptor control [204]. Taken with the theories developed by Berlin and coworkers [199,255,256,260] and Edelman and his collaborators [196-198,441,453,524], this further modification o f existing models envisages opposite roles for microfilaments (contractile) and microtubules (skeletal) and suggests that they are linked to one another or to the same plasma m e m b r a n e inner surface components. In this hypothetical model for cytoskeletal control over surface receptor mobility and t o p o g r a p h y (Fig. 13), transmembrane surface hnkages are proposed to exist, and these are amplified mainly t h r o u g h inner or outer surface peripheral components (Fig. 3) as suggested by Hynes [469], hmitmg the actual number of transmembrane structures needed to communicate across the m e m b r a n e These transmembrane structures could be components in membrane-Intercalated parncles, but available evidence suggests that they are p r o b a b l y independent in most cells of at least the majority o f these large o h g o m e n c complexes [385,423,525-527]. Of course, structures too small or thin (say, less than 20 A) to be vlsuahzed by heavy metal shadowing, or larger structures linked together at the interface between the halves of the bllayer, would

96 not be visuahzed by freeze-cleavage replica techniques. The independent hukage of both a microtubule system and a microfilament system with several hypothetical "nu cleation" or "anchorage" points would allow, for example, mlcrofilament function via contractile activity even if cellular microtubules were disrupted by drugs or low temperature. Functional dislocation of receptors from anchoring components stabilized by a microtubules skeletal system due to colchlcine treatment would favor hgand-mduced receptor redistribution, but as long as the receptors remained coupled to a membraneassociated microfilament system redistribution of receptor clusters (secondary orgamzatlon) could occur leading eventually to caps (Fig. 12 2) (for example, as in Ukena et al [200]). When a cell's microfilament system zs impaired (say, by cytochalasm B) but receptor redistribution into clusters occurs normally, hnkage of some components m the cluster to a transmembrane structure anchored by microtubules would be expected to prevent capping (Fig 12 3) Destruction of both the mlcrofilament and mlcrotubule systems (with local anesthetics or a combination of colchmme and cytochalasm B) would result in the formation of randomly displayed multiple clusters (Fig 12 4) [203,204] Finally, consistent with recent findings that myosin is a component of the outer cell surface, contractile systems could exist on both sides of the plasma membrane (Fig 13) and conceivably be involved in opposite contractile motions serving as a fine control over cell surface movements. These contractile structures would also be expected to exist in a coupled state occasionally interacting with anchorage components transmembrane hnked to cytoplasmic microtubulemlcrofilament systems. Outer surface myosin transmembrane hnked to a microfilament assemblage could aid in the formation and higher than average density display of certain surface components [476]. IV FINAL COMMENT It is becoming increasingly apparent that the plasma membrane is not an autonomous cell organelle Its couphng to various cytoplasmic structures and other organelles suggest that the cell surface is under strict and coordinated cytoplasmic control. Complicated events such as capping, adhesion, locomotion, endocytosls, etc. seem to require transmembrane communication and structural linkages with cytoskeletal components, m particular the mzcrofilament/mlcrotubule system. The roles played by its mare constituents mlcrotubules (skeletal-anchorage) and microfilaments (contractde-translocate) appear to be responslle for a variety of cell surface phenomena requiring specific, well controlled membrane movements_ In the second, compamon part of thls review [549] cell surface alterations caused by neoplastJc transformation will be discussed, and the altered state of transmembrane control over the surface receptors on tumor cells evaluated_ ACKNOWLEDGMENTS The excellent and untlrlng assistance of P. Delmonte, G. Beattie and especially A. Brodgmskl is gratefully acknowledged I thank my colleagues Drs J. C Robblns

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Transmembrane control of the receptors on normal and tumor cells. I. Cytoplasmic influence over surface components.

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