Printed in Sweden Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/78/l 131~WJl$OZ.CO/O

Experimental Cell Research 113 (1978) 1-13

OCCURRENCE WITHIN

OF LOW DENSITY LARGE

FIBROBLASTS

LIPOPROTEIN

PITS ON THE SURFACE

AS DEMONSTRATED

RECEPTORS

OF HUMAN

BY FREEZE-ETCHING

LELIO ORCI, JEAN-LOUIS CARPENTIER, A. PERRELET, RICHARD G. W. ANDERSON, JOSEPH L. GOLDSTEIN and MICHAEL S. BROWN Institute

of Histology and Embryology, University of Geneva Medical School, 1211 Geneva 4, Switzerland, and Departments of Cell Biology and Internal Medicine, University of Texas Health Science Center at Dallas, Dallas, TX 75235, USA

SUMMARY The surface distribution of specific receptor sites for plasma low density lipoprotein (LDL) in cultured human fibroblasts was studied with the technique of freeze-fracturing/deep-etching. Through the use of LDL covalently linked to fenitin as a visual probe, the receptor sites were observed to be concentrated within large pits on the cell surface. These large pits corresponded to the coated nits nreviouslv identified as the sites of LDL-ferritin binding by transmission electron microscopy (TBM). On the true surface of the cell, these large pits appeared as shallow depressions of irregular contour. These large pits were also identified within the fracture plane of the membrane where they could be distinguished from a population of smaller pits that corresponded to small flask-shaped invaginations seen in TEM. The membrane that composed the large pits appeared to differ from the remainder of the plasma membrane in that it contained twice the number of intramembrane particles per pm2 of surface and in that these intramembrane particles were of larger mean diameter. The current data lend support to the hypothesis that the large coated pits of the plasma membrane represent discrete regions where the membrane is structurally adapted to carry out the adsorptive endocytosis of receptor-bound macromolecules.

Normal human tibroblasts in monolayer culture derive their membrane cholesterol through the action of a high affinity cell surface receptor that specifically binds the major cholesterol-carrying lipoprotein in human plasma, low density lipoprotein (LDL) (see [ 1, 21 for reviews). Binding of LDL to the receptor is followed within minutes by the internalization of the lipoprotein through endocytosis. The lipoprotein is delivered to cellular lysosomes where its large component of cholesterol esters is hydrolysed, and the resultant free

cholesterol is then available to be utilized for metabolic purposes by the cell. Recent ultrastructural studies using LDL coupled to ferritin (LDGferritin) have demonstrated that in fibroblasts the LDL receptor sites are preferentially located over indented, thickened regions of plasma membrane that often exhibit a fuzzy coat on both the external and cytoplasmic surfaces [3-51. When normal fibroblasts have bound LDL-ferritin at 4°C and are then warmed to 37”C, these so-called coated pits [w] can be seen to invaginate into the cell to form Exp Cdl Res 113 (1978)

2

Orci et al.

Figs I and 2. Detail of thin sections showing the peripheral cytoplasm of normal tibroblasts incubated with LDL-fenitin. The cell membrane (CM) whose typical trilaminar aspect can be resolved in suitably oriented areas bends to a variable extent so as to form shallow pits (fig. 1) or fully formed sacs (fig. 2, asterisk). These indented regions of the membrane are

underlined by a fuzzy coat and covered with a thick cell coat (glycocalyx). LDL-ferritin (arrows) binds specifically to such membrane areas especially at the edge of the invaginated membrane. In both cells, part of the rough endoplasmic reticulum cistema (RER) and elements of the microtilamentous cell web (CW) are illustrated. Bar, 0.2 pm. x82000.

coated endocytic vesicles that eventually fuse with lysosomes [4, 51. The above conclusions were derived from electron microscopic examination of thin sections of fibroblasts, a technique that reveals only the cross-sectional appearance of a thin strip of membrane. In the current studies, we have used the technique of freeze-fracturing/deep-etching to examine the localization of LDL receptors on the true surface of fibroblasts. In addition, we have studied the relation of these binding sites to the structure of the interior of the bilayer membrane as revealed by the freezefracture technique. The results show that the LDL receptor sites are localized within large pits on the cell surface. Besides being indented, the membrane in these pits differs from adjacent membrane regions in that it contains an increased concentration of intramembrane particles. These large pits, which correspond to the coated pits observed by transmission electron microscopy (TEM), appear to represent discrete regions of the cell surface in which the plasma mem-

brane is specialized for carrying out adsorptive endocytosis.

Exp Cdl Rrs II3 (1978)

MATERIALS

AND METHODS

Cells Cultured tibroblasts were derived from skin biopsies obtained from either a normal subject or a patient with the receptor-negative form of homozygous familial hypercholesterolemia (FH) [9]. Cells were grown in monolayer as previously described [4]. All experiments were done in a similar format: On day 0, confluent monolavers of cells from stock flasks were dissociated with -0.05% trypsin, 0.02 % EDTA solution, and 1~10~ cells were seeded into each 60x15 mm Petri dish containing 3 ml of growth medium with 10% fetal calf serum (FCS). On day 3, the medium was replaced with 3 ml of fresh growth medium containing 10% FCS. On day 5, each monolayer was washed with 3 ml of phosphate-buffered saline, after which 2 ml of fresh medium containing 5 % (v/v) human lipoproteindeficient serum (LPDS) was added (final protein cont., 2.5 mglml). Experiments were initiated on day 7 after the cells had been incubated for 48 h in the presence of LPDS.

Lipoproteins Human LDL (density l.Ol!Xl.O63 g/ml) and human LPDS (density >1.215 g/ml) were obtained from the plasma of individual healthy subjects and prepared by differential ultracentrifugation [lo]. Ferritin was coupled to LDL (LDL-ferritin) as previously described [3, 41. More than 90% of the LDL particles

Study of LDL receptors by freeze-etching

Fig. 3. Replica of a normal fibroblast showing the characteristic freeze-fracture appearance of large and small invaginations in the outer leaflet (E-face) of the membrane. Large invaginations or pits can be identiBed as polymorphous bulges (asterisk) in the membrane face, while small-sized rings signal the necks of

3

small invaginations (i). Each type of invagination occupies a discrete area of the cell membrane with only a small degree of spatial mixing between the two types. The inset shows the counterparts of the two types of invaginations as seen in thin section. Bar, 1 pm; inset, 0.2 pm. X 17000; inset: x65000.

Exp Cd Res I13 (1978)

4

Orci et al.

Fig. 4. Typical morphology of small imaginations (i) on the P fracture face of the flbroblast membrane. On this face (cytoplasmic leaflet), the small invaninations appear as small roundish depressions that &e much

smaller than the large pits (asterisk). Intramembrane particles are scattered randomly on the smooth phospholipid background. Bar, 0.1 pm. x 101000.

were labeled with ferritin, and on the average there were two ferritin molecules coupled to each LDL particle, as revealed by negative staining [3, 41.

fixed pellets were assigned a code number, and mailed from Dallas to Geneva, where they were further processed as described below.

Incubation of fibrobias ts

Thin section electron microscopy

Monolayers of Iibroblasts were placed in a 4°C cold room for 30 min, after which the medium was removed and replaced with 2 ml of ice-cold growth medium containing 10% LPDS. Where indicated, the medium was supplemented with either native fenitin (100 pg/ml) or LDL-ferritin at a concentration corresponding to 60 pg/ml of LDL-protein. After incubation at 4°C for 2 h, each monolayer was washed five times with ice-cold nhosohate-buffered saline and fixed in the cold for i h with 2% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.3. After fixation, the fibroblasts were scraped off the dish. The cells from 2 dishes were pooled together and centrifuged in a Beckman microfuge (12000 rpm, 10 min, 4”C), the

Each glutaraldehyde-fixed pellet was post-fixed in 2% 0~0, in 0.1 M phosphate buffer, pH 7.4, dehydrated in graded ethanol, and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate.

E;p Cell Res 113 (1978)

Freeze-fracture After fixation in glutaraldehvde, the pellet was immersed in 30 % glycerol buffered with O:l M phosphate buffer, pH 7.4, for at least 30 min, rapidly frozen in Freon 22, cooled in liquid nitrogen, then fractured and shadowed in a Balzers BAF 301 apparatus (Balzers Liechtenstein) according to Moor & Mtihlethaler [ 111.

Study of LDL receptors

Fig. 5. Typical morphology of large pits on the cyto-

by freeze-etching

5

plasmic leaflet (P-face) of the fibroblast membrane. These pits appear as polymorphous identations (as-

terisk) in the fracture face. The pits contain a distinct population of large intramembrane particles. Bar, 0.1 Wm. X 105000.

The freeze-fracture replicas were then cleaned in a sodium hypochlorite solution for 2 h, rinsed in distilled water, and mounted on copper grids.

microscope. Magnifications were calibrated with a reference grid (Fullam, Inc., 2160 lines/mm).

Deep-etching This procedure was the same as that described for freeze-fracture except for two steps. (1) The fixed pellet was immersed in distilled water (in place of glycerol) for a period of 30 min; (2) the freezefractured specimen was kept at -100°C for 5 min before making the platinum replica, allowing water to sublimate from the fractured tissue. This procedure permits the uncovering of the outer surface of the cell membranes, in contrast to freeze-fracture alone which reveals only the membrane inner structure. Thin section and replicas of freeze-fractured/deep etched material were examined in a Philips EM300 electron

Quantitative evaluation One freeze-fracture renlica was nrepared from each pellet of fibroblasts. For each rephca, nhotogranhs of the surface of 50 different cells were iakenat i6 300 magnification for determination of the number of membrane nits. On the same realica. two random pictures of 12 different membrane faces (6 P-faces and 6 E-faces) were taken at X 35 000 maaniftcation for determination of the number and size of intramembrane particles. Quantitation was performed on x3 enlarged positive prints by a person who was unaware of the experimental conditions. The number of large and small pits on the exposed faces of membrane was counted, Freeze-fracture.

Exp Cell Res 113 (1978)

6

Orci et al.

Table 1. Number of small and large pits on the membrane offibroblasts as determined by freeze-fracture Membrane pits Cell strain

Small (count per pm*)

Large (count perpm2)

Normal FH Homozygote

1.92kO.37 3.30f0.29

0.52f0.05 0.63f0.06

Monolayers were incubated at 4°C in the absence of LDL-fenitin as described in Materials and Methods. For each cell strain, 4 pellets were prepared, each of which contained the cells from 2 Petri dishes. For each pellet, the number of small and large membrane pits in 50 cells was determined as described in Materials and Methods. The values given represent the mean f 1 S.E.M. for data obtained on 4 pellets from the same cell strain.

and the total area of the exposed faces was determined by planimetry. The number of intramembrane particles was counted inside a rectangular grid calibrated so as to represent 0.25 pm2. The number obtained was multiplied by 4 in order to obtain the result per pm*. Measurement of the size of intramembrane particles was carried out with the aid of an ~8 magnifier containing a reticle calibrated in tenths of a millimeter. For each pellet, 10 different cell membranes showing clearly both their inner (fracture face) and outer (etched face) surface were assessed. The etched membrane face was photographed at a fixed magnification of x27500. The number of bound ferritin particles and the area of exposed surface (determined by planimetry) was assessed for each of the 10 etched faces on ~3 enlarged positive prints. In addition, the number of ferritin particles associated with large pits was recorded.

Deep-etching.

RESULTS Thin section The general morphology of the fibroblast surface was similar to that previously reported [3-51. Characteristically, the cell membrane presents at intervals large in&nations or pits covered by a prominent external glycocalyx on the extracellular side and by a fuzzy coat on the cytoplasmic side of the plasma membrane (figs 1 and 2). The length of these coated regions varies from 0.1 to 0.5 Wm. In addition, we obExp Cell Res 113 (1978)

served smaller non-coated, flask-shaped invaginations measuring about 0.05 pm at their opening in the membrane. Fibroblasts from a subject with the homozygous form of familial hypercholesterolemia (FH) were indistinguishable from normal cells in these ultrastructural characteristics. In particular, the FH homozygote cells contained both small and large pits. In normal fibroblasts incubated with LDL-ferritin, ferritin molecules were observed to bind to the coated, indented regions of the plasma membrane (figs 1 and 2). The binding frequently occurred at the edge of the invaginated segment of membrane (fig. 2). By contrast, the FH homozygote fibroblasts, which lack LDL receptors as determined biochemically [ 1, 21, showed no ferritin binding at any site on the plasma membrane when incubated under the same conditions with LDL-ferritin. These observations are in agreement with the previously reported ultrastructural findings of Anderson et al. [3,4]. Freeze-fracture During freeze-fracture, the plasma membrane is split in the middle of its phospho-

Table 2. Number of intramembrane particles on fracture faces in regions where the plasma membrane is not invaginated Intramembrane particles Cell strain

P-Face E-Face (count/pm*) (count/fim’)

P+E Faces

Normal

851+40

431f20

1 282k48

FH Homozygote

637f53

363f52

1000+94

(count/Pm7

The replicas examined were the same as those in table 1. The number of intramembrane particles per pm2 of non-invaginated membrane was determined as described in Materials and Methods. The values given represent the mean + S.E.M. for data obtained on 4 pellets from the same cell strain.

Study of LDL receptors by freeze-etching

Table 3. Comparison

of the number of intramembrane particles on the P-face of the plasma membrane in non-invaginated regions and in large pits Intramembrane particles

Cell strain

Non-invaginated regions (count per pm*)

Large pits (count perpm2)

Normal FH Homozygote

85lf40 637fS3

1 434f72 142lf81

The replicas examined were the same as those used in table 1. The number of intramembrane particles on the P-face in non-invaginated areas and within large pits was determined as described in Materials and Methods. The values represent the mean k S.E.M. for data obtained on 4 pellets from the same cell strain.

lipid matrix and yields two complementary halves or fracture faces [12]. One corresponds to the cytoplasmic or inner leaflet of the membrane and is designated the Pface [13]; the other represents the outer leaflet and is called the E-face [13]. Structurally, both faces appear as smooth surfaces containing randomly dispersed protrusions. The smooth areas are thought to represent the phospholipid matrix, while the protrusions, called intramembrane particles, represent, at least in part, membrane proteins [ 14-171. Freeze-fracture replicas of fibroblasts reveal exact counterparts of both small and large pits seen in thin sections. On the P-faces, both types of pits appear as depressions-large or small-in the fracture plane (figs 4 and 5), while on Efaces small pits are seen as small elevated craters (fig. 3) and large ones as irregular bulges (fig. 3). The enface view of the membrane in the freeze-fracture preparations reveals that small and large pits tend to occur in separate regions with little intermixing. As

7

measured on replicas, the large pits average 0.2 pm in diameter and the small pits approx. 5-fold less. This clear size distinction permitted separate enumeration of the number of large and small pits per pm2 of membrane. In normal Iibroblasts, the number of small pits was about 5-fold greater than the number of large pits (table 1). In cells from an FH homozygote, the number of small and large pits was similar to that in the normal cells. Moreover, the distribution of the pits on the cell membrane was similar in the two cell strains. Tables 2 and 3 present a quantitative analysis of intramembrane particles as visualized by freeze-fracture. In non-invaginated areas of plasma membrane, the number of intramembrane particles was about 2-fold greater on the P-face as compared with the E-face, a finding consistent with previous observations in other membranes [14]. The data were similar in the normal and FH homozygote cells (table 2).

30

20

iC

c 0

40

60

izo

160

200

40

60

120

160

200

Fig. 6. Abscissa: diameter of particles (A); ordinate: frequency distribution (% of total). Frequency distribution of the diameter of the intramembrane particles located in non-invaginated regions of the membrane as compared with those located in large pits. The replicas examined were the same as those used in table 1. The diameter of the intramembrane particles of the P-face was measured as described in Materials and Methods. The values represent the data obtained on 4 pellets from each cell stain. A, normal fibroblasts; B, FH homozygote fibroblasts. Exp Cell Rev 113 (1978)

8

Orci et al.

Figs 7 to 10. Freeze-fractured/deep-etched prepara-

tions of fibroblasts incubated with LDL-ferritin at 4°C. In each figure, the step delimiting the fracture face (P) from the etched cell surface and representing the thickness of the fractured outer leatlet of the membrane is indicated by a dotted line. Fig. 7. Normal fibroblast. Detail of the tibroblast surface showing parts of the inner leaflet (fracture face P) and of the outer surface (cell surface) of the plasma

The number of intramembrane particles per pm2 of membrane surface was about twice as large within the large pits as in the noninvaginated regions of membrane (table 3). Fig. 6 shows the frequency distribution of the diameter of intramembrane particles in non-invaginated membrane regions and in large pits. In normal fibroblasts, the intramembrane particles located within the large pits exhibited a significantly greater diameter than did the intramembrane particles in non-invaginated regions of the membrane (90.6k2.1 8, vs 113.3k2.9 A, p

Occurrence of low density lipoprotein receptors within large pits on the surface of human fibroblasts as demonstrated by freeze-etching.

Printed in Sweden Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/78/l 131~WJl$OZ.CO/O Experimenta...
11MB Sizes 0 Downloads 0 Views