Monday. Symposium I: Cell Surface Receptors: Cell Proliferation and Differentiation (abstracts not available)
la
Monday. Symposium II: Cell Signaling in Development (1) 1 Molecular Genetics of Cell Signalling in the Mouse. A Bernstin, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada M5G 1X5. Mutations at the murine W and Steel (SI) loci result in defects in blood, pigment and germ cell development. Molecular analysis of these loci has shown that W encodes the Kit receptor tyrosine kinase (RTK) while Steel encodes its membrane-bound ligand. The complementary nature of the W and SI protein products is strikingly consistent with the identical phenotypes in these mice and the intrinsic vs. extrinsic nature of the cellular defects in W and SI mutants, respectively. I will discuss molecular and genetic aspects of these two loci, including the insights that have been learned concerning detailed molecular mechanisms of cell signalling and the possible role of the Kit signalling pathway in higher neural functions in the central nervous system. The mutational analysis of the Wand SI loci has also revealed the striking similarity in common developmental strategies and detailed molecular mechanisms used by evolutionarily widely divergent organisms to address common problems in development.
Minisymposium 1: Extracellular Matrix Structure and Function (2-5) 2
X
and
anfrom tenascin.
nstudies of the
L
W
ROD-containing
FN-II
L Aukhil. P.
,
P Eidkss.. Columbia University, Univ. of NC, and Duke Universty, Durham, NC. We have produced a bactal expsin proein o the dird FN-M domain of tenascn (Thfn3) and solved its struc to 1.8 A resoludon by x-ray crystallosraphy. The domain is 40x28x17 A, comnpisg two bet shoets of three and fur ands. mhe secdary struc is identcal to that of the extmclularadins of the grwth hononerceptor (hGOHR), the second domain of a4, and PapD, and is related to the Ig costant domain. The highly conserved resdues caacteristc of FN- domains (P W Y L Y) al projet into the centerof the beta sandwich, forming hydphobic contac with otherresidues. The N- and C-trmini are at opposte ends of the eonged domain. The RGD sequence of TNfn3 projec on an extnded type II', hairpin loop between the G and F stands, and is very close to the N-erminuL We have tested a number of cell tpes for adhesn to TNfn3 ad to largerrecombinnt segments containing it. Five diffaent lines ofendothl cells adheed to and spread on TNfn3 and to the ROD domain from fibmren, but fibroblasts and lioma cclls did not adhr to either of thesc single domains i
Antibody
blockdng
indic
that
endothei
ce
adhesion
was
3 Bloactive Peptides from Multiple Domains of Thrombpondin. W. A. Frazierl, M. T. Kosfeld" J. E. Murphy-UlIrich2 S. Gurusiddappa2, M. H1Ok2, S. S. Tolsma3, O.V. Volpert3, D. J. Good3, P. Polverlni3 and N. Boudcl3. 'Washington Uniersiy School of Medicine, St. Louis, MO 63110, 2 University of Albama at Birmingham 35294 and 3Northwestern Unversity Medical School, Chicago IL 60611. The thomsIndns (TS) are a family of modular gtycoproteins whose severl isoforms appear to modulate the migration, adhesion, growth and differentiation of many cell types. Cell binding sites have been In five separate domains of TS. We have attempted to identify ident bloactive peptide sequences from these domains. The N terminal heparin binding domnain disassembles focal adhesions of spread cells. This activity has been localized to a heparin binding 19 mer of TS1 and TS2 (residues 17-35). TS1, residues 74-95 is a potent adhesive factor for several cell types, binds hepain weakdy and does not labilize focal adhesions. 19mers from the type 1 repeats of TS containing the sequence CSVTCG inhibit anglogenesis in vivo and endothelial cell proliferation and
via
the avB3
integin. cndothelalcells adhered well to largersegments in which TNfn3 was exposed at the N-terminus, but the presence of TNfn2 appeared to inhibit adhesion. The proxmty of the ROD to the N-teminus of TNfh3 demonstrated in the cysal stucture suggests that the interface with TNfn2 may swically block access of the integrin. Noteably, the ROD oop in fibonectin is four amino acids longer, pemtting it to project up to 7 A farder from the protein surface, fclitating ineaction with the oa5Bl integrin.
4
Self-assembly and Calcium-biding Sites in Laminin: a Three Arm Interacton Model, P.D. Yurchenco and Y.-S. Chng. Robert Wood Johnson Medical School, Piscataway,N.J. 08854. Laminin, a four-arm glycoprotein, polymerizes to form a network in basement membranes. Assembly (critical concentraton of 0.1 MM) is entropy-driven and calcium-dependent. We have analyzed the lowaffinity interactions of defined fragments that contibute to polymerization by equilibrium gal filtration, equilibrium zonal velocity sedimentaton, rotary shadowing electron mkcroscopy and a calciumnitrocellulose binding assay. Fragment E4 (B1 domains IV-V) and El' (A/B2 short arm complex with globular domains) inhibit polymerization. E4 binds only to fragment El' in a caldum-dependent manner with a KD of -2 MM, and not to itself. El', however, also interacts with itself, but with less avidity. Eectron micrographs of El'-E4 mixtures reveal heterotrimers attached through all three chains while micrographs of El' alone reveal sparser homodimeric complexes attached through the A chain short arms. Fragment El (A/B2 short arm complex Intermediate In size between El' and P1' and lacking B2-VI) inhibits polymerzation, binds intact laminin and mixtures of El' + E4, but does not bind appreciably to isolated El' or E4. Intact laminin B chain, fragment El' and entactin, but not laminin A, E4, El or P1', possess significant calcium binding activity. We hypothesize that laminin polymerization requires the interaction of (at least) three lgands, one on each short arm, with the calcium-dependency residing principally in or near the outer B2 chain globule. (Funded by NIH grant DK36425).
mirtion in
tra
A 24-mer from the
procollagen-like domain of TS
with a homolog in FN, is an inhbitor of angiogenesis in vivo but has delayed effects on EC h vitro, suggesting an indirect action. The C terminal domain of TS1 binds cells in the absence of the upstream RGDA sequence of TS. This actity has been localized to 2 peptides with the sequences IRVVM and RFYVVMWK, both of which have homologs In lbminin. However, cells which do not attach to laminin bind well to these TS sequences, indicating distinct receptors for TS and laminin. TS contains 8 potential cell binding peptide sequences with a diverse array of biogical actities. The degree to which these bioactive peptides are conserved in TS isoforms will help to define similarities and differences in their bbgical activites. Supported by grants HD27712, HL1 4147 (WAF) HL44575 (JM-U) and CA52750 (NB).
5
Encoding For the Three Chains of the Anchoring Filament Protein Kalinin Show Similarity to the Laminin A. B1. and B2
cDNAS
Chains. D. Gerecke. D. Wagman. M. McDonough and R. Burg.tsag.n Dept. Dermatology, Mass. Gen. Hospital, Harvard Medical School, Boston, MA Kalinin is a protein whose tissue distribution is restricted to the epithelial-ECM junctional basement membrane zone of extemal tissues such as skin, cornea and sclera. Kalinin purified by immunoaffinity chromatography has three nonidentical chains 165, 155, and 140 kD in size (J.C.B. 114:567-576, 1991). Using anti-kalinin polyclonal serum, we have screened a human squamous carcinoma cell Xgtl 1 library and isolated a number of cDNAs corresponding to three chains of kalinin. Deduced amino acid sequences from the cDNAs encoding for the 140 kD chain Indicate it Is related to the previously described human laminin B1 chain. Deduced amino acid sequences from the cDNAs encoding for the 155 kD chain indicate it is related to the previously described human laminin B2 chain. This suggests that kalinin is a laminin isotype. Since molecules that have very similar amino acid sequences often have similar functions, we predict that kalinin and laminin will have similar functions. In fact, like laminin, kalinin is involved in attachment of epithelial cells to the basement membrane. In addition preliminary data indiQate that the 165 kD chain is a novel chain with similarity to the laminin A chain.
2a
Minisymposium 1: Extracellular Matrix Structure and Function (6-7). Monday
6 Molecular and Genetic Analyses of Type IV Collagen Function in C elegans lIM Kramer. and M.Hk Sibley. Department of Cell, Molecular and Structural Biology, Northwestern University Medical School, Chicago, IL 60611. Mutations In genes encoding extracellular matrix (ECM) components provide valuable tools for studying the functions of ECM In vivo. We have shown that the basement membrane-specific al and ct2(iV) collagen chains of C. elegans are encoded by the genetic loci emb-9 and let-2, respectively. Most mutations In these two genes cause arrest during the morphogenetic phase of embryogenesis, demonstrating a requirement for normal basement membrane structure for embryonic development. The sequence alterations in 3 al and 17 a2(iV) mutant alleles have been determined. 18 of these mutations cause replacement of Gly residues with Glu, Asp, or Arg, within the (Gly-X-Y), triple-helical regions of the collagens. Thus, although type IV collagens normally contain numerous interruptions of the triple-helix, these aberrant 'Interruptions' are not tolerated. Most of these mutations are temperature-sensitive and may disrupt triple-helix assembly. Different Gly replacements cause different phenotypic severities indicating that some Glys are more crucial than others for normal type IV coliagen function. One allele Is a splice acceptor mutation and displays a strict embryonic lethal phenotype. The final allele Is an Ala to Thr change (X positlon) and has a temperature-sensitive lethal phenotype. This allele is not likely to interfere with triple-helix assembly, but could disrupt higher order assembly of type IV collagen or Interaction with other ECM components. Mutatlons In the al chain are semidominant, such that heterozygotes varlably arrest during larval development, or are sterile or subfertile. The distrlbution of mutations In these genes Is clearly non-random, possibly because some mutations would result in strict dominant lethality and could not be maintained. The a2 chain Is present in two alternatively spliced forms, one is the major embryonic form, the other is the major larval/adult form. The functional importance of these alternate versions of the a2 chain is under investigation.
7
Kinetic and Chimeric Analysis of Ligand and Divalent Cation Selection by The B3. Integrins. J,W. Smith and J.C. Loftus. The Committee for Vascular Biology, The Scripps Research Institute. La Jolla, Ca 92037. One of the major unresolved issues in integrin biochemistry is how integrins distinguish between ligands. Of particular interest is the role of divalent cations because they clearly influence ligand selection. Integrins a,133 and alIbD33 are excellent comparative models to decipher this puzzle because they have considerable sequence homology, but have different ligand binding properties and divalent ion requirements. We have begun to define the ligand association rate constants for these integrins in the presence of different divalent ions. Our studies reveal that both receptors have a higher affinity for Mn2+ than Ca2+. However the complex between alIbB3 and Ca2+ binds fibrinogen 20 to 50fold faster than the Mn2+ species. In stark contrast, fibrinogen can only bind to in the presence of Mn2+. Thus, ligand binding by these highly related aC03 integrins is markedly influenced by different divalent ions at the level of kl. These results explain the inability of Mn2+ to support platelet aggregation and the lack of tumor cell adhesion to fibrinogen in the absence of Mn +. To probe the structural basis of ligand selection and divalent cation preference we have genetically engineered chimeric aIIb/Ci, subunits and expressed these with wild-type 13. The chimeras consist of the divalent ion binding sites of aIlb substituted into the a, backbone. To date we have generated chimeras where the cation binding loop and intervening sequence of sites #1-4, #1-2, #2-3, and #3-4 have been substituted. Each of the chimeras is expressed on the cell surface and is capable of binding RGD. It appears that substitution of divalent ion binding sites #2-3 or #3-4 of alIb into the a, sequence is not sufficient to alter the ligand binding phenotype. However, substitution of all four sites or sites #1-2 apparently alters the ligand phenotype of the receptor. It remains to be seen if this correlates with a change in divalent ion preference.
Minisymposium 2: Mechanisms of Motility (8-11) 8
Molecular Cloning of a Cytoplassnic Dynein Heavy Chain Gene from Dictoteliwu. M.P. Koonce, P.M. Grissom, and J.R MeIntosh. Dept. MCD Biology, Univ. Colorado, Boulder, CO. 80309 Dynein is a high molecular weight, microtubule-associated mechanochemical enzyme. In axonemes, dynein slides adjacent doublet microtubules against one another. Cytoplasmic forms of dynein have been identified in a wide variety of cell types, and are believed to have a motile role in intracellular vesicle transport. Dynein has also been localized to spindle microtubules and to the idnetochore region of mitotic chromosomes, suggesting that it may contribute to chromosome or spindle movments during cell division. We have cloned a dynein heavy chain gene from the cellular slime mold Dictyosteliwn. Using an antiserum against the heavy chain, we screened a cDNA expression library to isolate the initial clone. Additional, overlapping clones were isolated using restriction fragments of clone #1 to reprobe the library, and by adapting a PCR-based strategy to walk in the 3' direction along genomic DNA. In total, 14,318 bp were cloned, which contain an open reading frame of 4725 aa. The deduced amino acid sequence shows regions of strong homology with the sea urchin flagelar B-dynein heavy chain, especially in and around the ATP-binding consensus sequence GPAGTGKT. This is one of four consensus sequences in the Dictyostelium gene and, because of the homology with the flagellar gene, is likely to be the principle ATP-hydrolytic site (see also Gibbons et al, Nature 352;640). Overall, the cytoplasmic and flagellar sequences are 51% similar and 28% identical along their entire length. The region of least homology occurs at the amino terminal end of the gene. We are beginning to map functional domains of the heavy chain (see abstract by Vaisberg et al) and are pursuing gene knockout expeiments by homologous recombination. Supported by the NIH and the Damon Runyon-Walter Winchell Cancer Research Foundation.
10
Acin-RPV: The MaJor Component of the Dynacrin Complex. Activator of I D JM H zan and.LP Cytoplasmic ne_in TA. ShL4 EHeusr2 Ls-Millerk3 Department of Biology, Johns Hopkins University, Baltimore, MD 212181, Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 631102 and Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 117243. The dynactin complex is a heteromultimer composed of at least 8 distinct polypeptides that cosediments with cytoplasmic dynein at 20S and activates the enzyme to transport vesides on microtubules in vitro. Its major component is a 45kD protein, pI-6.8, that is present in a stoichiometry of at least eight copies per dynactin complex. The complex also contains actin, the actin-cpping protein capZ and dynactin (the vertebrate homolog of the Gluedprotein in Drosophila). By immunofluorescence, dynactin localizes to intracellular membranes induding endosomes and lysosomes. Actin-RPV is a human actin-related protein (identified by doning and sequencing) that shares 54% sequence identity with authentic actin. The actin-RPV sequence predicts a 43kD polypeptide with pI-6.2. Antibodies to actin-RPV react strongly with the 45WD component of the dynactin complex and protein sequencing confirms that this polypeptide is the chicken homolog of actinRPV. Dynactin complexes contain 40 nm long helical filaments that resemble f-actin, suggesting that actin-RPV has the capacity to polymerize, perhaps as a copolymer with actin. Unlike actin, actin-RPV and capZ are present in only trace amounts in brain and both are highly enriched in the dynactin comple Actin-RPV is not present in the stable f-actin complexes associated with the erythrocyte plasma membrane.
Molecular cloning and domain analysis of the Mr7S,OOO intermediate chain of Chlamydomonas outer arm dynein. S CLGiug, C.G. Wilkers & Qh L Witman, Worcester Foundation for Experimental Biology, Shrewsbury, MA. Chiamydomonas outer arm dynein consists of 3 heavy chains, 2 intermediate chains (IC78 and IC69) and 10 light chains (LCs). The ICs and several LCs form a discrete complex located at the base of the dynein (JBC 2, 19807). Recently, we demonstrated that IC78 interacts directly with a-tubulin in situ (JBC 2X, 8401). We have now extended our analysis of IC78 by molecular cloning. Peptide sequence from a CnBr fragment was used to design an oligonucleotide with which to probe a cDNA library made in XZapIl. A clone for IC78 was obtained and confirmed by immunoprecipitation of the translated product with monoclonal antibody 1878A (specific for IC78), and by matching the predicted N-terminal sequence with that determined by protein sequencing (17/17 residues correct). The clone recognizes a mRNA of -2.7 kb and encodes a protein of 683 amino acids with a predicted mass of 76,524 Da. IC78 has no overt sequence similarity with other microtubule (MT)-binding proteins but is homologous with IC69 (Mitchell & Kang, JCB 113, 835), indicating that these two chains evolved from a common ancestor. To investigate the MT-binding properties of IC78, we constructed and translated in xism a series of N- and C-terminally deleted versions of the polypeptide and assessed their ability to bind to axonemes lacking outer arms and to taxol-stabilized brain MTs. Some of the constructs bound avidly, and delimit two regions of IC78 involved in dynein-tubulin interactions. Interestingly, intact IC78 and constructs containing both regions bound relatively poorly or not at all. This is consistent with the observation that, by themselves, neither the outer arm's csp dimer (which contains IC78) nor its y subunit will bind to axonemes. It is possible that association of the y subunit with the ox dimer induces a conformational change in IC78 which leads to binding of outer arm dynein to the axoneme.
11
Dynamics of the
Neuronal Microtubules and Intermediate Filaments Laser ?Iuorescence lnotobleacn Kecovery Nethoai ants
DY rluorescence Uv knotoactlvatlon Metnoai s UKaDe anat caagea B. niro DeDawa)et. or Anat. ant cell 1ol0., Sch o Mea., Univ. btuaiea
Or TOKyo, JAP The co,ponents of the neuronal cytoskeleton travel slowly down the long axon of the neuron after their synthesis in the cell body. Concerning the mechanism of the slow axonal transport a major question has been whether the transport of tubulin or neurofilament proteins down the axon is in the form of assembled polymers or free oligoseric proteins. In order to address this question we microInjected labelled proteins into mouse dorsal root ganglion cells (DRG) and Xenopus embryos whose nerve cells were isolated and cultured at later stage. Then we illuminated a narrow region of the axon with laser or UV (365nm) microbeam and analysed by fluorescence-photobleach recovery or caged-fluorescence photoactivation methods. In mouse DRG photobleached or photoactivated microtubule (MT) and neurofilament (NF) segments were stationary regardless of the position ofillumination. However, in the Xenopus axon photoactivated or photobleached (NT) segments frequently moved towards the axon tip without significant spreading. Then we analysed the behaviour of the axonal plasma membrane in both systems. Analysis of the position of polystyrene beads adhering to the neurites of Xenopus neurons revealed anterograde movement of the beads at the rate similar to the rate of MTs or NF movement. In contrast no movement of the beads was observed in mouse DRG. Observation of the neurite outgrowth of the neuron suggested that the Xenopus neurites are passively dragged by the actively advancing owth cones. Thus our data strongly suggest that the forwar1 movement of photobleached or photoactivated ITs is due to passive pulling of the whole axonal structure (including MTs and the Plasma membrane) by the__growth cone. Therefore, both in mouse and Xenopus neurons most MTs and NFs in the axon are stationary relative to the axonal membrane and the actively moving elements could not be MT or NF bundles but possibly small oligomers.
Monday. Minisymposium 2: Mechanisms of Motility (12-14)
3a
12
13
Actin polymer dynamics in the propulsion of the intracellular bacterial parasite Listeria monocytgogenes. L. A. Theriot and LL Mitchison*, Departments of Biochemistry and Biophysics and *Pharmacology, University of California, San Francisco, CA 94143.
Dictyostelium Cells Devoid of Two Unconventional Myosins Show Defects in Growth. K. D. Novak & M. A. Titus Dept. of Cell Biology, Duke Univ. Med. Ctr., Durham, NC 27710. We have generated strains of Dictyostelium that are missing two unconventional myosin genes using homologous recombination. The myoB and myoC genes encode typical protozoan myosin Is while the myoA genes codes for a "short myosin I" that lacks the ATPinsensitive actin-binding region found in the tail of protozoan myosin Is. Single mutants were first generated using the THY1 gene as a selectable marker (kind gift of J. Hadwiger & R.A. Firtel) and the second gene was then eliminated using neomycin resistance. The following pairwise combination of mutants have been generated: myoA-/myoB- and myoB-/myoC-. The behavior of the double mutant strains has been compared to that of the wild type and single mutant strains (myoA-, myoB- and myoC-). Initial analysis shows that the double mutants have slower growth rates in liquid media (either in suspension or a surface) when compared to either wild type or single mutant strains (doubling times of >24 hours versus 8 hours) and take longer to complete development on starvation plates (36 - 40 hours versus 24 hours for the wild type strain). Generation of a third double mutant, myoA-/myoC- and further phenotypic analysis of mutant strains is currently in progress and the results will be presented. This work is supported by grants from the American Cancer Society
Listeria monocytogenes, a gram positive bacterium, is a facultative intracellular pathogen which grows directly in the cytoplasm of host cells and exploits a host actin-based motility mechanism to allow its intracellular spread. Moving intracellular L. monocytogenes display a "comet tail" made of host actin filaments. Fluorescence photoactivation of labeled actin filaments in the tail indicates that the filaments remain stationary in the cytoplasm as the bacterium moves. The apparent length of the tail is linearly proportional to the rate of movement. The bacterium appears to induce assembly of actin filaments near its surface, which are released and crosslinked into the stationary tail structure. One host cell protein likely to be involved in this process is profilin, which is known to promote exchange of ATP for ADP bound to actin monomer and is localized to the surface of intracellular L. monocytogenes. L. monocytogenes motility can be reconstituted in cytoplasmic extracts. Manipulation of profilin content in these extracts has striking effects on tail morphology and bacterium movement.
14
M I.
CL _ Brain M.K. O'Shee E.M. E anC. R.E. io- LL Woleni*. P.onschoe R.E. L osoa. and M.S. Moosle . 'Biology Dept, Yale Univ, New Haven, CT ad Biochm. Dept, Univ. de Sao Paulo, Ribeirao Preto, Bazil. Chicken myoein-V is a member of a newly recpized clas of myoeins that also includes the gene products of the mouse diute locus and the yeat MY02 Sen. Chiken bain myosin-V is the first member of thi clm to be puified as a protein, and we tport bero some of its properties. Electron micrcopy (peformd by J. Heuser) of myosin-V has smh n that it is two-headed aod contains a rod-like segment of -30 am tht tenats in a globula domein. Unlike myoun-l1, myoein-V does not appea to form filsuet. Myosin-V cosedim t with F-actin in an ATP-msitive fashion, has actinactiv Mg-ATPas activity, and is a babed-ed directed motor that can utmsocate acti filamets at rat of up to 400 nm/sec. Myosin-V cosediments with liposomew conisting of the acidic phospholipid phophatidylglycrol but not with liposomes cosidting of pbophatidykholine. Myosin-V's Mg-ATPss activity and motility are both reuated by calcium in the rnge of 0-10 uM, and motility in the presence of calcium is completely ihibited by the calodulin anagonist W-7 at 100 uM. Baed on scnnig deneitomatry w esaiste that each myonin-V heavy chai is ssociated with at lst 4 +/- 1 calmodulin light chains. Two additional low moleuar weight proteins copurify with myon-V and mey epreset additional light chios or modified forms of calhduln. Fuso protein consucts demonstrae tht the myoein-V nk domein, which consist of six tedomly rped IQ-motifs, provides the calnodulin binding sites detectable by tho '11-cahmodulin overlay techiqus. Similar motifs are prseat in the neck domains of aU myons in rgions known to be important for light chain bindg. A doetild coepeison of the bhad sequence from 30 myosins indicates that p90, dilute, and MY02 prnet the fifth clas of myosins to be discovered so far, and that the myosinm sa whole cm be divided into swven distinct classes. This analysis indicaes the likely evolutionary rebtionships among the myosm head sces and may provide a umful system for the clssification of newly discovered unconventionl myosins. (Supported by gamts from the MDA, NIH, ACS, FAPESP, CNPq and CAPES)
Minisymposium 3: Endocytosis and Exocytosis (15-16) 15
&
A.F. Obeflh8 1..
is
BaPAlch and J.M. Fernanez. Dept.
l: W.E
of Physiol. and Biophys., Mayo Clinic, Rochester MN 55905. Recent patch clamp sudies of the exocytotic fuaion pore have led us to propose that fusion pore formation is directed by a macromolecular protein scaold. The regulation of this scaffold can be studied in patch-clamped mast ceUls by using the cel membrane capacitance as an assay of fuswon pore formation, while at the same time perfuisig the cell interior. Inclusion of the hydrolysis resistant GTP analogue, GPyS, p the pipette solution causes compIet deg aion at low (30nM) [Ca'1. Not onl is GT?yS a sufffcient stmulu but the response does not washout: flash photolysis of caged GTPyS, after s long as 17min of cell perfusion, is still able to induce complete deganulation indicating that the target GUP binding protein is closely aociated with the scaold regulating exocytgsis. In contrast to UTPy, does not stimulate exocytosis at low Ca=+, and is able to competitively inhibit the simulation by GTPVS. These results suggest that a wstained ctivation of a GTP binding protein is necessary for exocytosis in mast cells and that GTP is ineffective because itjis rapidly hydrolyzed. When GTP is used in combination with elevated [Caz+tI (0.5-2,) exocytosis is simulated. It appears that under conditions of elevated Ca'+ a trans nt activation of the GTP binding protein is now sufficient or that Ca+ modulates the GTPase cycle to aflow GTP to cause a sustained activation. Rab3AL, a synthetic ol' tde, corresponding to the effector domain of rab3a, a ra-like small UT? binding protein found in secretory vesicles, is also able to simlate complete degranulaton of mast cells. RablAL and ral, peptides based on othgr small UT? binding proteins, were ineffective. The response requires Mg+ and ATP and can be acceerated by GDPPS, an inacvtor of UTP binding proteins. These results suggest that rab3AL competes with an endogenous rab protein for acdvation of an effector protein. We conclude tht a small GTP binding protein of the rab family forms part of the exocytotic scaffold responsible for direcN membrane fusion.
16 Syntaxin: A Synaptic Protein Implicated in Docking of Synaptic Vesicles at Presynaptic Active Zones, M. K. Bennett, N. Calakos,
K. G. Miller, and R. H. Scheller, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305. Synaptic vesicles are responsible for the storage and calcium-regulated exocytosis of neurotransmitter. The temporal and spatial specificity of neurotransmitter release requires the localization of both synaptic vesicles and calcium channels to a region of the presynaptic nerve terminal known as the active zone. We have identified a pair of 35 kD proteins, which we have named syntaxins, that directly interact with the synaptic vesicle protein p65 (synaptotagmin). These nervous system specific proteins are 84% identical, each with a carboxyl-terminal membrane anchor. Syntaxin is localized to the plasma membrane in cultured hippocampal neurons, with a high concentration at synaptic sites. An antibody generated against syntaxin is capable of immunoprecipitating 22% of solubilized c-conotoxin binding activity, indicating an interaction with the N-type voltage-gated calcium channel. We propose that syntaxin plays a role in docking synaptic vesicles near calcium channels at presynaptic active zones. Both p65 and syntaxin are in vitr substrates for casein kinase 11, an enzyme that is coimmunoprecipitated with a monoclonal antibody against p65. The physiological functions of p65 and syntaxin, as well as possible regulatory effects of their phosphorylation, are currently under investigation.
Minisymposium 3: Endocytosis and Exocytosis (17-20). Monday
4a 17 Interactions of Dynamin with Isolated Plasma Membranes.
Burress
and
Vallee.
Cell
Biology Group,
18
H,L Shpetner, Q,
Worcester
Foundation
for
Experimental Biology. Shrewsbury, MA 01545. ¶ynamin is a GTPase which exhibits high turnover rates (0.04-0.20 sec ) in Xitro that are accelerated up to 75-fold by microtubules (Shpetner and Vallee, Nature = 733, 1992). Genetic studies have implicated dynamin in the budding of clathrin-coated and possibly non-clathrin-coated vesicles from the plasma membrane (Chen et al., Nature, 35.: 583, 1991; van der Bliek and Meyerowitz, Nature, 351: 411, 1991). To clarify the role of dynamin in these processes, we have begun to study the interactions of dynamin with isolated plasma membranes, prepared by sonication of cultured cells pre-adsorbed to polylysine-coated coverslips (Moore et al., Science, 2, 558, 1987). Membranes were prepared from COS-7 cells transiently transfected with either wild-type or mutant dynamin constructs (see abst. by Burgess et al.). The mutant constructs either contained a point mutation in the first GTP-binding consensus element, or lacked the entire GTP-binding domain. Membranes from untransfected cells did not contain detectable dynamin as assayed by immunofluorescence microscopy. In contrast, membranes prepared from cells transfected with the wild-type or either mutant construct exhibited bright, punctate dynamin staining that partially co-localized with clathrin. We have also examined the binding of exogenous brain dynamin to membranes prepared from untransfected cells. In the presence of GMP-PNP, the membranes exhibited bright, punctate staining that also co-localized partially with clathrin. In contrast, only very faint dynamin staining was detected in the presence of GTP. These findings provide direct evidence for a specific interaction of dynamin with the plasma membrane. They suggest that this interaction involves the GTP-bound form of dynamin, and that, in the presence of GTP, dynamin exists primarily in a GDP-bound state. Supported by the NIH to RBV.
Miorosoess. and Segregates from the SCAs after Insulin urie.2 G.E. LilnhArd1 and ajisz±on- C C Cain.1 S .M. astla,2 Dartmouth Medical School, Hanover, N.H.1 and University of Virginia, Charlottesville, VA2 Two components of regulated secretory vesicles and secretory granules, the VAMPs (17-18 kD) and the SCAMPs (3539 kD), are components of GluT4-containing vesicles isanunopurified from low density microsomes (LDM) of rat adipocytes. Imunoisolation of vesicles with antibodies against either GluT4 or the SCAMPs resulted in the quantitative depletion of GluT4, SCAMPs and VAMPs from the LDM. The SCAMPs have the same electrophoretic mobilities as two of the most abundant GluT4 vesicle proteins, as assayed by protein staining. Subcellular fractionation showed that the highest concentrations of the VAMPs and SCAMPs are in the LDM, with very low concentrations in the plasma membrane (PM) and high density microsomes in basal (non-stimulated) cells. Vesicle subpopulations within the LDM which differ widely in the relative densities of GluT4, VAMPs, and SCAMPs were not detected either by isolation of vesicles with limiting amounts of anti-SCAMP or by EM imnmunogold staining of isolated vesicles. Insulin induced a substantial redistribution of GluT4 and the VAMPs, but not the SCAMPs, from the LDM to the PM. The VAMPs and GluT4 remaining in the LDM after insulin stimulation were colocalized with the SCAMPS. Differential trafficking of these components may underly the partial segregation of GluT4 and the VAMPs from the SCAMPs in response to insulin.
19
20
Intracellular Sorting Signals of the Insulin-Regulatable Glucose Transporter (GLUT-4). R.C. Piper. C. Tai. D. E. James. Dept. Cell Biology and Physiology, Washington University, St. Louis, MO. 631110 The insulin-regulatable glucose transporter (GLUT-4) is an intracellular membrane protein which can be induced to move to the plasma membrane in response to insulin in muscle and fat cells. GLUT-4 is efficiently sequestered (
la
Monday. Symposium II: Cell Signaling in Development (1) 1 Molecular Genetics of Cell Signalling in the Mouse. A Bernstin, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada M5G 1X5. Mutations at the murine W and Steel (SI) loci result in defects in blood, pigment and germ cell development. Molecular analysis of these loci has shown that W encodes the Kit receptor tyrosine kinase (RTK) while Steel encodes its membrane-bound ligand. The complementary nature of the W and SI protein products is strikingly consistent with the identical phenotypes in these mice and the intrinsic vs. extrinsic nature of the cellular defects in W and SI mutants, respectively. I will discuss molecular and genetic aspects of these two loci, including the insights that have been learned concerning detailed molecular mechanisms of cell signalling and the possible role of the Kit signalling pathway in higher neural functions in the central nervous system. The mutational analysis of the Wand SI loci has also revealed the striking similarity in common developmental strategies and detailed molecular mechanisms used by evolutionarily widely divergent organisms to address common problems in development.
Minisymposium 1: Extracellular Matrix Structure and Function (2-5) 2
X
and
anfrom tenascin.
nstudies of the
L
W
ROD-containing
FN-II
L Aukhil. P.
,
P Eidkss.. Columbia University, Univ. of NC, and Duke Universty, Durham, NC. We have produced a bactal expsin proein o the dird FN-M domain of tenascn (Thfn3) and solved its struc to 1.8 A resoludon by x-ray crystallosraphy. The domain is 40x28x17 A, comnpisg two bet shoets of three and fur ands. mhe secdary struc is identcal to that of the extmclularadins of the grwth hononerceptor (hGOHR), the second domain of a4, and PapD, and is related to the Ig costant domain. The highly conserved resdues caacteristc of FN- domains (P W Y L Y) al projet into the centerof the beta sandwich, forming hydphobic contac with otherresidues. The N- and C-trmini are at opposte ends of the eonged domain. The RGD sequence of TNfn3 projec on an extnded type II', hairpin loop between the G and F stands, and is very close to the N-erminuL We have tested a number of cell tpes for adhesn to TNfn3 ad to largerrecombinnt segments containing it. Five diffaent lines ofendothl cells adheed to and spread on TNfn3 and to the ROD domain from fibmren, but fibroblasts and lioma cclls did not adhr to either of thesc single domains i
Antibody
blockdng
indic
that
endothei
ce
adhesion
was
3 Bloactive Peptides from Multiple Domains of Thrombpondin. W. A. Frazierl, M. T. Kosfeld" J. E. Murphy-UlIrich2 S. Gurusiddappa2, M. H1Ok2, S. S. Tolsma3, O.V. Volpert3, D. J. Good3, P. Polverlni3 and N. Boudcl3. 'Washington Uniersiy School of Medicine, St. Louis, MO 63110, 2 University of Albama at Birmingham 35294 and 3Northwestern Unversity Medical School, Chicago IL 60611. The thomsIndns (TS) are a family of modular gtycoproteins whose severl isoforms appear to modulate the migration, adhesion, growth and differentiation of many cell types. Cell binding sites have been In five separate domains of TS. We have attempted to identify ident bloactive peptide sequences from these domains. The N terminal heparin binding domnain disassembles focal adhesions of spread cells. This activity has been localized to a heparin binding 19 mer of TS1 and TS2 (residues 17-35). TS1, residues 74-95 is a potent adhesive factor for several cell types, binds hepain weakdy and does not labilize focal adhesions. 19mers from the type 1 repeats of TS containing the sequence CSVTCG inhibit anglogenesis in vivo and endothelial cell proliferation and
via
the avB3
integin. cndothelalcells adhered well to largersegments in which TNfn3 was exposed at the N-terminus, but the presence of TNfn2 appeared to inhibit adhesion. The proxmty of the ROD to the N-teminus of TNfh3 demonstrated in the cysal stucture suggests that the interface with TNfn2 may swically block access of the integrin. Noteably, the ROD oop in fibonectin is four amino acids longer, pemtting it to project up to 7 A farder from the protein surface, fclitating ineaction with the oa5Bl integrin.
4
Self-assembly and Calcium-biding Sites in Laminin: a Three Arm Interacton Model, P.D. Yurchenco and Y.-S. Chng. Robert Wood Johnson Medical School, Piscataway,N.J. 08854. Laminin, a four-arm glycoprotein, polymerizes to form a network in basement membranes. Assembly (critical concentraton of 0.1 MM) is entropy-driven and calcium-dependent. We have analyzed the lowaffinity interactions of defined fragments that contibute to polymerization by equilibrium gal filtration, equilibrium zonal velocity sedimentaton, rotary shadowing electron mkcroscopy and a calciumnitrocellulose binding assay. Fragment E4 (B1 domains IV-V) and El' (A/B2 short arm complex with globular domains) inhibit polymerization. E4 binds only to fragment El' in a caldum-dependent manner with a KD of -2 MM, and not to itself. El', however, also interacts with itself, but with less avidity. Eectron micrographs of El'-E4 mixtures reveal heterotrimers attached through all three chains while micrographs of El' alone reveal sparser homodimeric complexes attached through the A chain short arms. Fragment El (A/B2 short arm complex Intermediate In size between El' and P1' and lacking B2-VI) inhibits polymerzation, binds intact laminin and mixtures of El' + E4, but does not bind appreciably to isolated El' or E4. Intact laminin B chain, fragment El' and entactin, but not laminin A, E4, El or P1', possess significant calcium binding activity. We hypothesize that laminin polymerization requires the interaction of (at least) three lgands, one on each short arm, with the calcium-dependency residing principally in or near the outer B2 chain globule. (Funded by NIH grant DK36425).
mirtion in
tra
A 24-mer from the
procollagen-like domain of TS
with a homolog in FN, is an inhbitor of angiogenesis in vivo but has delayed effects on EC h vitro, suggesting an indirect action. The C terminal domain of TS1 binds cells in the absence of the upstream RGDA sequence of TS. This actity has been localized to 2 peptides with the sequences IRVVM and RFYVVMWK, both of which have homologs In lbminin. However, cells which do not attach to laminin bind well to these TS sequences, indicating distinct receptors for TS and laminin. TS contains 8 potential cell binding peptide sequences with a diverse array of biogical actities. The degree to which these bioactive peptides are conserved in TS isoforms will help to define similarities and differences in their bbgical activites. Supported by grants HD27712, HL1 4147 (WAF) HL44575 (JM-U) and CA52750 (NB).
5
Encoding For the Three Chains of the Anchoring Filament Protein Kalinin Show Similarity to the Laminin A. B1. and B2
cDNAS
Chains. D. Gerecke. D. Wagman. M. McDonough and R. Burg.tsag.n Dept. Dermatology, Mass. Gen. Hospital, Harvard Medical School, Boston, MA Kalinin is a protein whose tissue distribution is restricted to the epithelial-ECM junctional basement membrane zone of extemal tissues such as skin, cornea and sclera. Kalinin purified by immunoaffinity chromatography has three nonidentical chains 165, 155, and 140 kD in size (J.C.B. 114:567-576, 1991). Using anti-kalinin polyclonal serum, we have screened a human squamous carcinoma cell Xgtl 1 library and isolated a number of cDNAs corresponding to three chains of kalinin. Deduced amino acid sequences from the cDNAs encoding for the 140 kD chain Indicate it Is related to the previously described human laminin B1 chain. Deduced amino acid sequences from the cDNAs encoding for the 155 kD chain indicate it is related to the previously described human laminin B2 chain. This suggests that kalinin is a laminin isotype. Since molecules that have very similar amino acid sequences often have similar functions, we predict that kalinin and laminin will have similar functions. In fact, like laminin, kalinin is involved in attachment of epithelial cells to the basement membrane. In addition preliminary data indiQate that the 165 kD chain is a novel chain with similarity to the laminin A chain.
2a
Minisymposium 1: Extracellular Matrix Structure and Function (6-7). Monday
6 Molecular and Genetic Analyses of Type IV Collagen Function in C elegans lIM Kramer. and M.Hk Sibley. Department of Cell, Molecular and Structural Biology, Northwestern University Medical School, Chicago, IL 60611. Mutations In genes encoding extracellular matrix (ECM) components provide valuable tools for studying the functions of ECM In vivo. We have shown that the basement membrane-specific al and ct2(iV) collagen chains of C. elegans are encoded by the genetic loci emb-9 and let-2, respectively. Most mutations In these two genes cause arrest during the morphogenetic phase of embryogenesis, demonstrating a requirement for normal basement membrane structure for embryonic development. The sequence alterations in 3 al and 17 a2(iV) mutant alleles have been determined. 18 of these mutations cause replacement of Gly residues with Glu, Asp, or Arg, within the (Gly-X-Y), triple-helical regions of the collagens. Thus, although type IV collagens normally contain numerous interruptions of the triple-helix, these aberrant 'Interruptions' are not tolerated. Most of these mutations are temperature-sensitive and may disrupt triple-helix assembly. Different Gly replacements cause different phenotypic severities indicating that some Glys are more crucial than others for normal type IV coliagen function. One allele Is a splice acceptor mutation and displays a strict embryonic lethal phenotype. The final allele Is an Ala to Thr change (X positlon) and has a temperature-sensitive lethal phenotype. This allele is not likely to interfere with triple-helix assembly, but could disrupt higher order assembly of type IV collagen or Interaction with other ECM components. Mutatlons In the al chain are semidominant, such that heterozygotes varlably arrest during larval development, or are sterile or subfertile. The distrlbution of mutations In these genes Is clearly non-random, possibly because some mutations would result in strict dominant lethality and could not be maintained. The a2 chain Is present in two alternatively spliced forms, one is the major embryonic form, the other is the major larval/adult form. The functional importance of these alternate versions of the a2 chain is under investigation.
7
Kinetic and Chimeric Analysis of Ligand and Divalent Cation Selection by The B3. Integrins. J,W. Smith and J.C. Loftus. The Committee for Vascular Biology, The Scripps Research Institute. La Jolla, Ca 92037. One of the major unresolved issues in integrin biochemistry is how integrins distinguish between ligands. Of particular interest is the role of divalent cations because they clearly influence ligand selection. Integrins a,133 and alIbD33 are excellent comparative models to decipher this puzzle because they have considerable sequence homology, but have different ligand binding properties and divalent ion requirements. We have begun to define the ligand association rate constants for these integrins in the presence of different divalent ions. Our studies reveal that both receptors have a higher affinity for Mn2+ than Ca2+. However the complex between alIbB3 and Ca2+ binds fibrinogen 20 to 50fold faster than the Mn2+ species. In stark contrast, fibrinogen can only bind to in the presence of Mn2+. Thus, ligand binding by these highly related aC03 integrins is markedly influenced by different divalent ions at the level of kl. These results explain the inability of Mn2+ to support platelet aggregation and the lack of tumor cell adhesion to fibrinogen in the absence of Mn +. To probe the structural basis of ligand selection and divalent cation preference we have genetically engineered chimeric aIIb/Ci, subunits and expressed these with wild-type 13. The chimeras consist of the divalent ion binding sites of aIlb substituted into the a, backbone. To date we have generated chimeras where the cation binding loop and intervening sequence of sites #1-4, #1-2, #2-3, and #3-4 have been substituted. Each of the chimeras is expressed on the cell surface and is capable of binding RGD. It appears that substitution of divalent ion binding sites #2-3 or #3-4 of alIb into the a, sequence is not sufficient to alter the ligand binding phenotype. However, substitution of all four sites or sites #1-2 apparently alters the ligand phenotype of the receptor. It remains to be seen if this correlates with a change in divalent ion preference.
Minisymposium 2: Mechanisms of Motility (8-11) 8
Molecular Cloning of a Cytoplassnic Dynein Heavy Chain Gene from Dictoteliwu. M.P. Koonce, P.M. Grissom, and J.R MeIntosh. Dept. MCD Biology, Univ. Colorado, Boulder, CO. 80309 Dynein is a high molecular weight, microtubule-associated mechanochemical enzyme. In axonemes, dynein slides adjacent doublet microtubules against one another. Cytoplasmic forms of dynein have been identified in a wide variety of cell types, and are believed to have a motile role in intracellular vesicle transport. Dynein has also been localized to spindle microtubules and to the idnetochore region of mitotic chromosomes, suggesting that it may contribute to chromosome or spindle movments during cell division. We have cloned a dynein heavy chain gene from the cellular slime mold Dictyosteliwn. Using an antiserum against the heavy chain, we screened a cDNA expression library to isolate the initial clone. Additional, overlapping clones were isolated using restriction fragments of clone #1 to reprobe the library, and by adapting a PCR-based strategy to walk in the 3' direction along genomic DNA. In total, 14,318 bp were cloned, which contain an open reading frame of 4725 aa. The deduced amino acid sequence shows regions of strong homology with the sea urchin flagelar B-dynein heavy chain, especially in and around the ATP-binding consensus sequence GPAGTGKT. This is one of four consensus sequences in the Dictyostelium gene and, because of the homology with the flagellar gene, is likely to be the principle ATP-hydrolytic site (see also Gibbons et al, Nature 352;640). Overall, the cytoplasmic and flagellar sequences are 51% similar and 28% identical along their entire length. The region of least homology occurs at the amino terminal end of the gene. We are beginning to map functional domains of the heavy chain (see abstract by Vaisberg et al) and are pursuing gene knockout expeiments by homologous recombination. Supported by the NIH and the Damon Runyon-Walter Winchell Cancer Research Foundation.
10
Acin-RPV: The MaJor Component of the Dynacrin Complex. Activator of I D JM H zan and.LP Cytoplasmic ne_in TA. ShL4 EHeusr2 Ls-Millerk3 Department of Biology, Johns Hopkins University, Baltimore, MD 212181, Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 631102 and Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 117243. The dynactin complex is a heteromultimer composed of at least 8 distinct polypeptides that cosediments with cytoplasmic dynein at 20S and activates the enzyme to transport vesides on microtubules in vitro. Its major component is a 45kD protein, pI-6.8, that is present in a stoichiometry of at least eight copies per dynactin complex. The complex also contains actin, the actin-cpping protein capZ and dynactin (the vertebrate homolog of the Gluedprotein in Drosophila). By immunofluorescence, dynactin localizes to intracellular membranes induding endosomes and lysosomes. Actin-RPV is a human actin-related protein (identified by doning and sequencing) that shares 54% sequence identity with authentic actin. The actin-RPV sequence predicts a 43kD polypeptide with pI-6.2. Antibodies to actin-RPV react strongly with the 45WD component of the dynactin complex and protein sequencing confirms that this polypeptide is the chicken homolog of actinRPV. Dynactin complexes contain 40 nm long helical filaments that resemble f-actin, suggesting that actin-RPV has the capacity to polymerize, perhaps as a copolymer with actin. Unlike actin, actin-RPV and capZ are present in only trace amounts in brain and both are highly enriched in the dynactin comple Actin-RPV is not present in the stable f-actin complexes associated with the erythrocyte plasma membrane.
Molecular cloning and domain analysis of the Mr7S,OOO intermediate chain of Chlamydomonas outer arm dynein. S CLGiug, C.G. Wilkers & Qh L Witman, Worcester Foundation for Experimental Biology, Shrewsbury, MA. Chiamydomonas outer arm dynein consists of 3 heavy chains, 2 intermediate chains (IC78 and IC69) and 10 light chains (LCs). The ICs and several LCs form a discrete complex located at the base of the dynein (JBC 2, 19807). Recently, we demonstrated that IC78 interacts directly with a-tubulin in situ (JBC 2X, 8401). We have now extended our analysis of IC78 by molecular cloning. Peptide sequence from a CnBr fragment was used to design an oligonucleotide with which to probe a cDNA library made in XZapIl. A clone for IC78 was obtained and confirmed by immunoprecipitation of the translated product with monoclonal antibody 1878A (specific for IC78), and by matching the predicted N-terminal sequence with that determined by protein sequencing (17/17 residues correct). The clone recognizes a mRNA of -2.7 kb and encodes a protein of 683 amino acids with a predicted mass of 76,524 Da. IC78 has no overt sequence similarity with other microtubule (MT)-binding proteins but is homologous with IC69 (Mitchell & Kang, JCB 113, 835), indicating that these two chains evolved from a common ancestor. To investigate the MT-binding properties of IC78, we constructed and translated in xism a series of N- and C-terminally deleted versions of the polypeptide and assessed their ability to bind to axonemes lacking outer arms and to taxol-stabilized brain MTs. Some of the constructs bound avidly, and delimit two regions of IC78 involved in dynein-tubulin interactions. Interestingly, intact IC78 and constructs containing both regions bound relatively poorly or not at all. This is consistent with the observation that, by themselves, neither the outer arm's csp dimer (which contains IC78) nor its y subunit will bind to axonemes. It is possible that association of the y subunit with the ox dimer induces a conformational change in IC78 which leads to binding of outer arm dynein to the axoneme.
11
Dynamics of the
Neuronal Microtubules and Intermediate Filaments Laser ?Iuorescence lnotobleacn Kecovery Nethoai ants
DY rluorescence Uv knotoactlvatlon Metnoai s UKaDe anat caagea B. niro DeDawa)et. or Anat. ant cell 1ol0., Sch o Mea., Univ. btuaiea
Or TOKyo, JAP The co,ponents of the neuronal cytoskeleton travel slowly down the long axon of the neuron after their synthesis in the cell body. Concerning the mechanism of the slow axonal transport a major question has been whether the transport of tubulin or neurofilament proteins down the axon is in the form of assembled polymers or free oligoseric proteins. In order to address this question we microInjected labelled proteins into mouse dorsal root ganglion cells (DRG) and Xenopus embryos whose nerve cells were isolated and cultured at later stage. Then we illuminated a narrow region of the axon with laser or UV (365nm) microbeam and analysed by fluorescence-photobleach recovery or caged-fluorescence photoactivation methods. In mouse DRG photobleached or photoactivated microtubule (MT) and neurofilament (NF) segments were stationary regardless of the position ofillumination. However, in the Xenopus axon photoactivated or photobleached (NT) segments frequently moved towards the axon tip without significant spreading. Then we analysed the behaviour of the axonal plasma membrane in both systems. Analysis of the position of polystyrene beads adhering to the neurites of Xenopus neurons revealed anterograde movement of the beads at the rate similar to the rate of MTs or NF movement. In contrast no movement of the beads was observed in mouse DRG. Observation of the neurite outgrowth of the neuron suggested that the Xenopus neurites are passively dragged by the actively advancing owth cones. Thus our data strongly suggest that the forwar1 movement of photobleached or photoactivated ITs is due to passive pulling of the whole axonal structure (including MTs and the Plasma membrane) by the__growth cone. Therefore, both in mouse and Xenopus neurons most MTs and NFs in the axon are stationary relative to the axonal membrane and the actively moving elements could not be MT or NF bundles but possibly small oligomers.
Monday. Minisymposium 2: Mechanisms of Motility (12-14)
3a
12
13
Actin polymer dynamics in the propulsion of the intracellular bacterial parasite Listeria monocytgogenes. L. A. Theriot and LL Mitchison*, Departments of Biochemistry and Biophysics and *Pharmacology, University of California, San Francisco, CA 94143.
Dictyostelium Cells Devoid of Two Unconventional Myosins Show Defects in Growth. K. D. Novak & M. A. Titus Dept. of Cell Biology, Duke Univ. Med. Ctr., Durham, NC 27710. We have generated strains of Dictyostelium that are missing two unconventional myosin genes using homologous recombination. The myoB and myoC genes encode typical protozoan myosin Is while the myoA genes codes for a "short myosin I" that lacks the ATPinsensitive actin-binding region found in the tail of protozoan myosin Is. Single mutants were first generated using the THY1 gene as a selectable marker (kind gift of J. Hadwiger & R.A. Firtel) and the second gene was then eliminated using neomycin resistance. The following pairwise combination of mutants have been generated: myoA-/myoB- and myoB-/myoC-. The behavior of the double mutant strains has been compared to that of the wild type and single mutant strains (myoA-, myoB- and myoC-). Initial analysis shows that the double mutants have slower growth rates in liquid media (either in suspension or a surface) when compared to either wild type or single mutant strains (doubling times of >24 hours versus 8 hours) and take longer to complete development on starvation plates (36 - 40 hours versus 24 hours for the wild type strain). Generation of a third double mutant, myoA-/myoC- and further phenotypic analysis of mutant strains is currently in progress and the results will be presented. This work is supported by grants from the American Cancer Society
Listeria monocytogenes, a gram positive bacterium, is a facultative intracellular pathogen which grows directly in the cytoplasm of host cells and exploits a host actin-based motility mechanism to allow its intracellular spread. Moving intracellular L. monocytogenes display a "comet tail" made of host actin filaments. Fluorescence photoactivation of labeled actin filaments in the tail indicates that the filaments remain stationary in the cytoplasm as the bacterium moves. The apparent length of the tail is linearly proportional to the rate of movement. The bacterium appears to induce assembly of actin filaments near its surface, which are released and crosslinked into the stationary tail structure. One host cell protein likely to be involved in this process is profilin, which is known to promote exchange of ATP for ADP bound to actin monomer and is localized to the surface of intracellular L. monocytogenes. L. monocytogenes motility can be reconstituted in cytoplasmic extracts. Manipulation of profilin content in these extracts has striking effects on tail morphology and bacterium movement.
14
M I.
CL _ Brain M.K. O'Shee E.M. E anC. R.E. io- LL Woleni*. P.onschoe R.E. L osoa. and M.S. Moosle . 'Biology Dept, Yale Univ, New Haven, CT ad Biochm. Dept, Univ. de Sao Paulo, Ribeirao Preto, Bazil. Chicken myoein-V is a member of a newly recpized clas of myoeins that also includes the gene products of the mouse diute locus and the yeat MY02 Sen. Chiken bain myosin-V is the first member of thi clm to be puified as a protein, and we tport bero some of its properties. Electron micrcopy (peformd by J. Heuser) of myosin-V has smh n that it is two-headed aod contains a rod-like segment of -30 am tht tenats in a globula domein. Unlike myoun-l1, myoein-V does not appea to form filsuet. Myosin-V cosedim t with F-actin in an ATP-msitive fashion, has actinactiv Mg-ATPas activity, and is a babed-ed directed motor that can utmsocate acti filamets at rat of up to 400 nm/sec. Myosin-V cosediments with liposomew conisting of the acidic phospholipid phophatidylglycrol but not with liposomes cosidting of pbophatidykholine. Myosin-V's Mg-ATPss activity and motility are both reuated by calcium in the rnge of 0-10 uM, and motility in the presence of calcium is completely ihibited by the calodulin anagonist W-7 at 100 uM. Baed on scnnig deneitomatry w esaiste that each myonin-V heavy chai is ssociated with at lst 4 +/- 1 calmodulin light chains. Two additional low moleuar weight proteins copurify with myon-V and mey epreset additional light chios or modified forms of calhduln. Fuso protein consucts demonstrae tht the myoein-V nk domein, which consist of six tedomly rped IQ-motifs, provides the calnodulin binding sites detectable by tho '11-cahmodulin overlay techiqus. Similar motifs are prseat in the neck domains of aU myons in rgions known to be important for light chain bindg. A doetild coepeison of the bhad sequence from 30 myosins indicates that p90, dilute, and MY02 prnet the fifth clas of myosins to be discovered so far, and that the myosinm sa whole cm be divided into swven distinct classes. This analysis indicaes the likely evolutionary rebtionships among the myosm head sces and may provide a umful system for the clssification of newly discovered unconventionl myosins. (Supported by gamts from the MDA, NIH, ACS, FAPESP, CNPq and CAPES)
Minisymposium 3: Endocytosis and Exocytosis (15-16) 15
&
A.F. Obeflh8 1..
is
BaPAlch and J.M. Fernanez. Dept.
l: W.E
of Physiol. and Biophys., Mayo Clinic, Rochester MN 55905. Recent patch clamp sudies of the exocytotic fuaion pore have led us to propose that fusion pore formation is directed by a macromolecular protein scaold. The regulation of this scaffold can be studied in patch-clamped mast ceUls by using the cel membrane capacitance as an assay of fuswon pore formation, while at the same time perfuisig the cell interior. Inclusion of the hydrolysis resistant GTP analogue, GPyS, p the pipette solution causes compIet deg aion at low (30nM) [Ca'1. Not onl is GT?yS a sufffcient stmulu but the response does not washout: flash photolysis of caged GTPyS, after s long as 17min of cell perfusion, is still able to induce complete deganulation indicating that the target GUP binding protein is closely aociated with the scaold regulating exocytgsis. In contrast to UTPy, does not stimulate exocytosis at low Ca=+, and is able to competitively inhibit the simulation by GTPVS. These results suggest that a wstained ctivation of a GTP binding protein is necessary for exocytosis in mast cells and that GTP is ineffective because itjis rapidly hydrolyzed. When GTP is used in combination with elevated [Caz+tI (0.5-2,) exocytosis is simulated. It appears that under conditions of elevated Ca'+ a trans nt activation of the GTP binding protein is now sufficient or that Ca+ modulates the GTPase cycle to aflow GTP to cause a sustained activation. Rab3AL, a synthetic ol' tde, corresponding to the effector domain of rab3a, a ra-like small UT? binding protein found in secretory vesicles, is also able to simlate complete degranulaton of mast cells. RablAL and ral, peptides based on othgr small UT? binding proteins, were ineffective. The response requires Mg+ and ATP and can be acceerated by GDPPS, an inacvtor of UTP binding proteins. These results suggest that rab3AL competes with an endogenous rab protein for acdvation of an effector protein. We conclude tht a small GTP binding protein of the rab family forms part of the exocytotic scaffold responsible for direcN membrane fusion.
16 Syntaxin: A Synaptic Protein Implicated in Docking of Synaptic Vesicles at Presynaptic Active Zones, M. K. Bennett, N. Calakos,
K. G. Miller, and R. H. Scheller, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305. Synaptic vesicles are responsible for the storage and calcium-regulated exocytosis of neurotransmitter. The temporal and spatial specificity of neurotransmitter release requires the localization of both synaptic vesicles and calcium channels to a region of the presynaptic nerve terminal known as the active zone. We have identified a pair of 35 kD proteins, which we have named syntaxins, that directly interact with the synaptic vesicle protein p65 (synaptotagmin). These nervous system specific proteins are 84% identical, each with a carboxyl-terminal membrane anchor. Syntaxin is localized to the plasma membrane in cultured hippocampal neurons, with a high concentration at synaptic sites. An antibody generated against syntaxin is capable of immunoprecipitating 22% of solubilized c-conotoxin binding activity, indicating an interaction with the N-type voltage-gated calcium channel. We propose that syntaxin plays a role in docking synaptic vesicles near calcium channels at presynaptic active zones. Both p65 and syntaxin are in vitr substrates for casein kinase 11, an enzyme that is coimmunoprecipitated with a monoclonal antibody against p65. The physiological functions of p65 and syntaxin, as well as possible regulatory effects of their phosphorylation, are currently under investigation.
Minisymposium 3: Endocytosis and Exocytosis (17-20). Monday
4a 17 Interactions of Dynamin with Isolated Plasma Membranes.
Burress
and
Vallee.
Cell
Biology Group,
18
H,L Shpetner, Q,
Worcester
Foundation
for
Experimental Biology. Shrewsbury, MA 01545. ¶ynamin is a GTPase which exhibits high turnover rates (0.04-0.20 sec ) in Xitro that are accelerated up to 75-fold by microtubules (Shpetner and Vallee, Nature = 733, 1992). Genetic studies have implicated dynamin in the budding of clathrin-coated and possibly non-clathrin-coated vesicles from the plasma membrane (Chen et al., Nature, 35.: 583, 1991; van der Bliek and Meyerowitz, Nature, 351: 411, 1991). To clarify the role of dynamin in these processes, we have begun to study the interactions of dynamin with isolated plasma membranes, prepared by sonication of cultured cells pre-adsorbed to polylysine-coated coverslips (Moore et al., Science, 2, 558, 1987). Membranes were prepared from COS-7 cells transiently transfected with either wild-type or mutant dynamin constructs (see abst. by Burgess et al.). The mutant constructs either contained a point mutation in the first GTP-binding consensus element, or lacked the entire GTP-binding domain. Membranes from untransfected cells did not contain detectable dynamin as assayed by immunofluorescence microscopy. In contrast, membranes prepared from cells transfected with the wild-type or either mutant construct exhibited bright, punctate dynamin staining that partially co-localized with clathrin. We have also examined the binding of exogenous brain dynamin to membranes prepared from untransfected cells. In the presence of GMP-PNP, the membranes exhibited bright, punctate staining that also co-localized partially with clathrin. In contrast, only very faint dynamin staining was detected in the presence of GTP. These findings provide direct evidence for a specific interaction of dynamin with the plasma membrane. They suggest that this interaction involves the GTP-bound form of dynamin, and that, in the presence of GTP, dynamin exists primarily in a GDP-bound state. Supported by the NIH to RBV.
Miorosoess. and Segregates from the SCAs after Insulin urie.2 G.E. LilnhArd1 and ajisz±on- C C Cain.1 S .M. astla,2 Dartmouth Medical School, Hanover, N.H.1 and University of Virginia, Charlottesville, VA2 Two components of regulated secretory vesicles and secretory granules, the VAMPs (17-18 kD) and the SCAMPs (3539 kD), are components of GluT4-containing vesicles isanunopurified from low density microsomes (LDM) of rat adipocytes. Imunoisolation of vesicles with antibodies against either GluT4 or the SCAMPs resulted in the quantitative depletion of GluT4, SCAMPs and VAMPs from the LDM. The SCAMPs have the same electrophoretic mobilities as two of the most abundant GluT4 vesicle proteins, as assayed by protein staining. Subcellular fractionation showed that the highest concentrations of the VAMPs and SCAMPs are in the LDM, with very low concentrations in the plasma membrane (PM) and high density microsomes in basal (non-stimulated) cells. Vesicle subpopulations within the LDM which differ widely in the relative densities of GluT4, VAMPs, and SCAMPs were not detected either by isolation of vesicles with limiting amounts of anti-SCAMP or by EM imnmunogold staining of isolated vesicles. Insulin induced a substantial redistribution of GluT4 and the VAMPs, but not the SCAMPs, from the LDM to the PM. The VAMPs and GluT4 remaining in the LDM after insulin stimulation were colocalized with the SCAMPS. Differential trafficking of these components may underly the partial segregation of GluT4 and the VAMPs from the SCAMPs in response to insulin.
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Intracellular Sorting Signals of the Insulin-Regulatable Glucose Transporter (GLUT-4). R.C. Piper. C. Tai. D. E. James. Dept. Cell Biology and Physiology, Washington University, St. Louis, MO. 631110 The insulin-regulatable glucose transporter (GLUT-4) is an intracellular membrane protein which can be induced to move to the plasma membrane in response to insulin in muscle and fat cells. GLUT-4 is efficiently sequestered (
