- Email: [email protected]
Structure, function and regulation of cytoplasmic dynein
Structure,
function
and regulation
of cytoplasmic
dynein
Trina A Schroer The Johns Hopkins
University,
Baltimore,
USA
Molecular cloning studies have provided valuable structural information about the different subunits of cytoplasmic dynein and their relationships to their axonemal dynein counterparts. Recent unexpected findings regarding the role of this enzyme in mitosis have emerged from mutational analyses and microinjection experiments, while studies of organelle transport in vivo have revealed clues to mechanisms for physiological regulation of dynein activity.
Current
Opinion
in Cell
Biology
Introduction
1994,
Structure
6:69-73
of cytoplasmic
dynein
subunits
Heavy chains
Cytoplasmic dynein, a minus-end directed, microtubule-based motor, is thought to drive the movement of intracellular structures as diverse as membranous organelles and chromosomes. Like the axonemal dyneins, the cytoplasmic species is a massive (greater than 1000000 Mr) multisubunit complex that consists of multiple polypeptides: two heavy chains of greater than 400 000 M,, three or four 74 000 Mr intermediate chains and four light chains of approximately 55 000 M, [1,2]. Ultrastructural studies indicate that each molecule has two large globular heads projecting from a common base, which comprises several smaller globular domains 131.The heavy chain, containing the sites for ATP hydrolysis and microtubule binding, probably makes up the bulk of each head, while the intermediate and light chains are believed to lie at the base of the molecule. The recent determination of the primary sequences of the three different subunits represents a significant advance towards a better understanding of dynein structure.
In late 1991, the report of full-length, primary sequences for the p heavy chain of sea urchin flagellar dynein 111,121 began a new era in studies of dynein structure and function. Structural predictions suggest that the molecule contains five nucleotidebinding sites, the phosphate-binding pockets of which are termed P-loops 1131(see Fig. 1). One P-loop is near the amino terminus, but the other four are clustered in a domain of approximately 100 000 M, located roughly halfway between the amino and carboxyl termini. The first P-loop (P-l) in the central cluster is thought to be the site of ATP hydrolysis 1111. An armament of molecular biological techniques (expression screening, hybridization screening and PCR) has enabled the determination of full-length cDNA sequences encoding cytoplasmic dynein heavy chains from Dictyostelium discoideum 114.1, Rattus norvegicus 115*,16.1, Saccharomyces cerevisiae 117**1, Caenorhabditis elegans u Lye, personal communication) and Drosophila melanogaster (T Hays, personal communication); a cytoplasmic heavy chain gene has also been identified in sea urchin 1181.The sequences predict proteins that range in size from 471305 M, (yeast) to 538482 M, (Dictyostelium), significantly larger than previous estimates from SDS-PAGE. As shown in Fig. 1, the cytoplasmic dynein heavy chains contain four centrally located P-loops, but lack the P-loop found near the amino terminus in sea urchin flagellar dynein. The axonemal and cytoplasmic proteins show some homology in the carboxy-terminal two-thirds of the molecule, with the greatest conservation occurring in the central P-loop cluster, particularly in the vicinity of the ATP hydrolytic site. The conserved carboxy-terminal region may represent the large globular head of the enzyme, while the divergent amino termini may correspond to the tail, a structure presumed to interact with other dynein subunits and cargo. The cytoplasmic
Because in vitro motility assays indicate that cytoplasmic dynein translocates membrane vesicles toward the minus ends of microtubules [4,51, the enzyme is assumed to participate in vivo in centripetal organelle movements and retrograde axonal transport. Immunofluorescence studies show that cytoplasmic dynein localizes to intracellular membranes 16,71 as well as to chromosome kinetochores and spindle poles 1891, suggesting an involvement in mitosis. Dynein-based motility undoubtedly involves a variety of intracellular components such as membrane receptors and cytosolic regulatory factors. The dynactin complex, an activator of dynein-based vesicle movements in vitro [6,1Ol, is the first identified candidate for a regulator of dynein activity. In this review we will discuss recent insights into possible functions of dynein as well as mechanisms for its regulation.
Abbreviations MAP-microtubule-associated
0 Current
protein;
Biology
PCR-polymerase
Ltd ISSN 0955-0674
chain
reaction. 69
70
Cvtoskeleton
Heavy
chain
Axonemal Ripneustes
gratilla
1 1
Cyfoplasmic Dictyosteliym
discoideum Rattus
Saccharomyces
Intermediate
1;
:,’
,. ,.)-. IL 1, .:L
norvegicus cerevisiae
1
I
.::
69 kDa
m
567
Cytoplasmic Rattus
7
b.
“lmJ1,
:I pqc.
il; IIF’
~1: H
n
.I’,II,
I.
I4725 [ 4644 1 4092
I!-‘”
chaii
Axonemal Chlamydomonas
‘_ 1. ~, -.;!-;1,
?!.,:. .: . . :,; ; .‘:ql
74 kDa
II1
643
r_1: m m 0 0
P-loop Possible coiled-coil Highly conserved Conserved Not conserved
I1994 Current Opinion in Cell Biology
heavy chains are homologous along their length, and are nearly identical in the region surrounding P-l (70 % identity between the Dictyostelium and human proteins; [19**1>. Although the cytoplasmic dynein heavy chain gene is part of a large superfamily 120,211 whose size probably reflects the diversity of the inner and outer arm heavy chains, it is not known whether multiple cytoplasmic species exist. Unlike their axonemal counterparts, which are composed of two (or three) different heavy chains, all known cytoplasmic dyneins contain two identical heavy chains. It is possible that multiple dynein isofomls, distinguished by their complement of intermediate and light chains, account for different types of intracellular dynein-based motility.
Intermediate
and light chains
A start has also been made on the other cytoplasmicdynein subunits. Although their precise functions have not yet been determined, the intermediate and light chains are believed to regulate enzyme activity and/or participate in cargo binding. The latter function was first suggested for the 78 000 M,. and 69 000 M, subunits of outer-arm dynein of the Chlamydomonas flagellum. These proteins are tightly associated with one another and lie at the base of the enzyme, where it is anchored to the A-subfiber microtubule 122,231. Antibody decoration experiments suggest that intermediate chains occupy a similar site on cytoplasmic dynein (ER Steuer, TA Schroer, JE Heuser and MP Sheetz, unpublished data); this region of dynein identified by ultrastructuna1 analysis is thought to bind cargo, i.e. membrane organelles, chromosomes or other intracellular components. The primary sequence of the rat cytoplasmic dynein intermediate chain, a molecule of 72 753 Mr [24'1, reveals some similarity to the 69000 Mr species from Chlamydomonas [251. The carboxy-terminal halves of the two molecules are approximately 26% identical and 48% similar in sequence, while the amino-terminal domains show no particular similarity to each other (see Fig. 1).
Fig. 1. Comparison of dynein polypeptide sequences. This schematic representation compares the primary sequences of axonemal and cytoplasmic dynein heavy [ll ,14*-l 6*,17**] and intermediate [24*,25] chains. The positions of predicted phosphate-binding P-loops and regions of alpha-helical coiled-coil are indicated for each polypeptide; the number of amino acids in each polypeptide is given on the right. The dynein heavy chains are aligned relative to the highly conserved P-loop, P-l (within the hatched box), and intermediate chains are aligned relative to the conserved carboxy-terminal domains.
It is proposed that the conserved carboxyl terminus associates with the heavy chain or another dynein subunit and that the amino terminus specifies what cargo the motor will bind 124’1. The 53-59 kDa cytoplasmic dynein light-chain family is fairly well characterized biochemically, if not functionally. The chicken light chains can be resolved by SDSPAGE into several Mr species, each of which is represented by multiple charge isoforms. Peptide mapping divides the light chains into two groups, the higher (57 000-59 000) and lower (53 000-55 000) M, isoforms (SR Gill, AF Salcedo, DW Cleveland, TA Schroer, abstract 936, Mol Biol Cell 1992, 3:161a). The sequence of a cDNA encoding the higher M, light chains predicts a globular protein that may contain a nucleotide-binding site near its amino terminus (SR Gill, DW Cleveland, TA Schroer, unpublished data) but otherwise displays no significant homology to other proteins. The lower M, light chain has yet to be cloned.
Functions Hela
of cytoplasmic
dynein
in mitosis
cells
Cytoplasmic dynein, which is located at kinetochores 18,9,261, may be a motor for poleward movements of chromosomes in prometaphase and anaphase A. In order to test this hypothesis, antibodies (either intact or Fab fragments) raised against a cytoplasmicdynein peptide from the highly conserved, proposed ATP-hydrolytic site (P-l; see Fig. 1) were injected into HeLa cells in interphase or at different stages of mitosis. Cells injected during or before prophase formed a monopolar spindle, but injections later in mitosis had no effect on chromosome motility or completion of mitosis 113”l. These results suggest that a major role of cytoplasmic dynein is the promotion and maintenance of centrosome separation at the very onset of mitosis, and that if the enzyme is involved in chromosome translocation, its function is redundant.
Structure, Yeast
Identification of genes encoding cytoplasmic dynein heavy chains in organisms tractable to genetic analysis makes it possible to study dynein function by analyzing the phenotypes of dynein mutants. Disnlptions of the dynein heavy-chain gene (DYNE) in yeast do not impair mitotic spindle formation or chromosome congression, but instead perturb the positioning of mitotic spindles within dividing cells [17**,27”1. Normally, the spindle bridges the junction between the mother cell and bud, so that one daughter nucleus lies in each cell after cytokinesis. In dynl mutants, the spindle can remain entirely within the mother cell, yielding an anucleate daughter, a phenotype somewhat similar to that of a tubulin mutation (tub2-410, 1281) that disrupts extranuclear microtubules. Spindle assembly, elongation and chromosome segregation are not impaired. It would appear that one function of dynein in yeast is to exert force on the cytoplasmic microtubules and thereby determine spindle position.
Regulation vitro
of cytoplasmic
dynein
activity
in
The in vitro motility assay, a powerful tool in the identification of microtubule motors, has enabled characterization of other factors that may influence microtubulebased motility in living cells. In vitro studies of vesicle transport by cytoplasmic dynein indicate that the system requires at least one additional component, the dynactin complex, a 20s multiprotein assembly composed of eight different proteins [6,101. Curiously, its major component is actin-related protein from vertebrates (also known as RPV or centractin), a protein in the actin superfamily [29”,3Ol. Although actinRPV shares only 55% sequence identity with authentic actins, it has retained the ability to form the 40nm filament that is the major structural domain of the dynactin complex (T Schroer, unpublished data). Another component of the complex, dynactin [61 (also known as pl5OGLUED t30,311>, is homologous to the Glued protein in Drosophila. Glued is essential during embryogenesis and may be involved in the development of the visual system [32l. In vertebrate cells, the dynactin complex can be detected in association with small cytoplasmic vesicles ([6,301; T Meads, TA Schroer, unpublished data), suggesting that it may participate in dynein-membrane interactions. In vitro motility studies also indicate that cytoplasmic dynein-based movements can be affected by certain microtubule-associated proteins 1331. MAP-2, but not tau protein, appears to stimulate detachment of motors from microtubules, causing a reduction in the frequency of microtubule and vesicle movements. A likely explanation is that the 10&200nm-long MAP-2 sidearm interferes with the binding of cytoplasmic dynein to the microtubule surface. In living cells,
function
and regulation
of cytoplasmic
dynein
Schroer
MAP-2 or other similarly acting proteins may well modulate dynein-based movements.
Physiological
regulation
of organelle
transport
Studies focused on the regulation of organelle transport in living cells have revealed that the process can be modulated by a variety of physiological effecters. In CV-1 kidney cells, the incidence of vesicle movements can be increased by addition of serum, CAMP and okadaic acid (a protein phosphatase inhibitor) i34.1, suggesting a role for reversible phosphorylation in intracellular motility. That centripetal and centrifugal movements are affected equally indicates coordinate regulation of cytoplasmic dynein- and kinesin-based vesicle movements. The finding that serum dramatically stimulates the association of cytoplasmic dynein with lysosomes [351 leads to the supposition that organelle motility may be controlled, at least in part, at the level of motor-membrane binding. Axonal transport is an example of microtubule-based vesicle motility taken to the extreme. To ensure continued viability, large amounts of membrane must be exported to the synapse (anterograde transport) and recycled to the cell body (retrograde transport). This process has long been studied in axoplasm extruded from the giant axon of squid, an in vitro system easily accessed by externally added agents. Using this system, Bloom et al. i36.1 have recently shown that in the presence of GTQ.5, a GTP analog that suppresses the hydrolytic cycle required for activity of many GTP-binding proteins, both anterograde and retrograde vesicle movements occur at reduced velocity; in contrast, no effects were seen with a variety of protein kinase and phosphatase inhibitors. Likely targets for GTPyS are the rab proteins, a family of small GTP-binding proteins that play key roles in many intracellular membrane traffic events t371. It would appear that the effector acts on a component of the transport machinery that is shared by cytoplasmic dynein and kinesin. Cultured sympathetic neurons support high levels of axonal transport. Particularly conspicuous is the bi-directional movement of large, refractile organelles that mediate membrane recycling to the cell body. Unlike the situation in other axoplasmic vesicles, the direction of movement of these membranes is governed by physiological stimuli. When axonal growth is prevented, the particles show a preference for retrograde movement [381; in contrast, increases in intracellular CAMP bias movement in the opposite direction i39’1. Careful analysis of the movement of individual particles revealed that preference for movement in the retrograde and anterograde directions arises in different ways. Retrograde preference involves a lengthening in duration of each retrograde movement, whereas anterograde preference results from increasing the probability that a particle moving in the anterograde direction will continue moving in the same direction after stopping (per-
71
72
Cvtoskeleton
sistence of movement). Duration and persistence may reflect overall motor activity or the affinity of motors for the particle surface.
PFARRCM, COUE M, GRK+OM PM, HAYS TS, PORTER ME, MCINI’OSHJR: Cytoplasmic Dynein Localizes to Rinctochores During Mitosis. Nature 1990, 345:263-265.
9.
STEUERER, SCHROERTA, WORDEMANL, SHEEX! MP: Cytoplasmic Dyncin Localixes to Mitotic Spindles and Rinetochores. Nature 1990, 345:266-268. SCHROERTA, SHEE’~Z MP: TWO Activators of MicrotubulcBased Vesicle Transport. / Cell Bfol 1991, 115:1389-1318. GIBBONS IR, GIBWNS BH, Mocz G, ASAI DJ: Multiple Nuclcotidc-Binding Sites in the Sequence of Dyncin B Heavy Chain. Nature 1991, 352640-643.
10.
Conclusions The past year has seen a number of significant advances in the areas of cytoplasmic dynein structure, function and regulation. The largest subunit of the enzyme, the so-called heavy chain, appears to be highly conserved, with particular homology near the active site. Primary sequence homologies are seen between analogous subunits of cytoplasmic and axonemal dyneins, suggesting conservation of the sequences of sites for subunit interaction. With the wealth of primary sequences now available, studies of the molecular details of dynein subunit interactions and dynein
References
8.
and recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as: . of special i&rest.. of outstanding interest 1.
LYE RJ, POHTERME, SCHOLEYJM, MCINXXH JR Identification of a Microtubule-Based Cytoplasmic Motor in the Nematode C. elegatts. Cell 1987, 51:309-318.
2.
PAXHAL BM, SHP~~NER HS, VALI.EE RB: MAP 1C is a Microtubulc-Activated ATPase which Translocatcs Micro tubules In v/trr, and has Dynein-Like Properties. / Cell Bfol 1987, 105:1273-X282.
3.
VALLEE RB, WALL JS, PASCHALBM, SHPETNERHS: MicrotubulcAssociated Protein 1C from Brain is a **Headed Cytosolic Dyncin. Nafnre 1988, 332:561-563.
4.
SCHNAPPBJ, REEX T’S: Dyncin is the Motor for Retrograde Axonal Transport of Organellcs. Proc Nat1 Acad Sci USA 1989, 86:154%1552.
5.
SCHROERTA, STEUEHER, SHEB~+ MP: Cytoplasmic Dynein is a Minus-End Direct Motor for Membranous Organellcs. Cell 1989, 56:937-946.
6.
GILI. SR, SCHROERTA, SZILAK I, STEUER ER, SHERZ MP, CLEVELAND DW: Dynactin, a Conserved, Ubiquitously Expressed Component of an Activator of Vesicle Motility Mediated by Cytoplasmic Dyncin. / Cell Efol 1391,
11.
12.
OCAWA K: Four ATPBiiding Sites in the Midregion of the f3 Heavy Chain of Dyncin. Nature 1991, 352643-645. SARAS~EM, SIBBALD PR, WITI~NGHOFEHA: The P-Loop - A 13. Common Motif in ATP- and GTPBinding Proteins. Trends Bfocbem Sci 1990, 153430-434. 14. K~XNCE MP, GRI%~M PM, MCINTOSHJR Dyncin from Dfc. tyostelh4m: Priiary Structure Comparisons Between a Cytoplasmic Motor Enzyme and Flagellar Dynein. / Cell Btol 1992, 119:1597-1604. This is the lirst reported sequence of a cytoplasmic dynein heavy chain, demonstrating significant homology, in the carboxy-terminal two-thirds of the protein, between cytoplasmic and axonemal dynein isoforms. 15. .
MIMMI A, PASCHALBM, MAZUMI~ARM, VAI.I.EERB: Molccular Cloning of the Retrograde Transport Motor Cytoplasmic Dyncin (MAP 1C). Narron 1993, 10:787-796. The sequence of this vcnebrate cytoplasmic dynein heavy chain shows significant overall homology to the Dic[wstelium protein and allows further comparison of the axonemal and cytoplasmic isoforms. 16. ZHANG ZZ, TANAKA Y, NONAKA S, ~=WA H, KAWASAKI H, . NAKATA T, HIROKAWAN: The Primary Structure of Rat Brain (Cytoplasmic) Dyncin Heavy Chain, a Cytoplasmic Motor Enzyme. Proc Nat1 Acad Scf USA 1993, 90:792&%7932. The sequence of this vertebrate cytoplasmic-dynein heavy chain shows significant overall homology to the Dfctyostelhun protein and allows further comparison of the axonemal and cytoplasmic isoforms. 17. ESHEI. D, URRESTARAZULA, VISSERSS, JAUNIA~JXJ-C, VAN .. VLI~REEDIJK JC, PLANTA KJ, GIBBONS IR: Cytoplasmic Dyncin is Required for Normal Nuclear Segregation in Yeast. Proc Natl Acad Sci USA 1993, 9O:in press. The full-length primary sequence of yeast dynein heavy chain shows similarity to Dtctyostelirrm and rdt cytoplasmic dyneios. Disruptions of the gene predominantly appear to affect nuclear positioning and not chromosome segregation. 18.
76:30>309.
VA~SBERGEA, KO~NCE MP, MCINTOSHJR Cytoplasmic Dyncin .. Plays a Role in Mammalian Mitotic Spindle Formation. / Cell Biol 1993, 123849858. Microinjection of antibodies directed to the highly conserved P-loop, P-l, disrupt formation of the mitotic spindle but not chromosome motility in Hela cells. Models are proposed to explain how dynein might participate in spindle pole separation. 20. R&~MUS.SONK, SERRM, GEPNERJ, GIBBONS I, HAYS TS: A Family of Dyncin Genes in Drvsopblla mekmogaster. Mol Btol Cell 1994, in press. 19.
21.
GIBBONS BH, Aslu DJ, TANG W-J, HAYS TS, GIBBONS IR: Expression of Axoncmrd and Cytoplasmic Dyncin Genes in Sea Urchin Embryos. Mol Bfol Cell 1994, in press.
22.
KING SM, WITMAN GB: Localization of an Intermediate Chain of Outer Arm Dynein by lmmunoelcctron Microscopy. / Bfol Chem 1990, 265:19807-19811. KING SM, WII.KERSONCG, WITMAN GB: The Mr 78,000 Intermediate Chain of Cblumydomonas Outer Arm Dyncin
115:1639-1650.
7.
LIN SXH, COLLINS CA: Immunolocalization of Cytoplasmic Dynein to Lysosomcs in Cultured Cells. / Cell Scf 1992. 101:12%137.
GIBBONS IR, Ashl DJ, TANG WJ, GII~BONSBH: A Cytoplasmic Dynein Heavy Chain in Sea Urchin Embryos. Btol Cell 1992,
23.
Structure, Interacts with Alpha-Tubtdin 26684018407.
in Situ. J Biol Chem 1991,
24. .
PASCHALBM, MIKAMI A, PFI.SI~RKK, VALLEE RB: Homology of the 74kD Cytopiasmic Dyncin Subunit with a Fiagcllar Dyncin PoIypcptidc Suggests an IntraccIIuIar Targeting Function. J Cell Biol 1992, 118:113+1143. The primary sequence of the intermediate chain of cytopiasmic dynein is shown to be partially homologous to the 69COO Mr subunit of Chhmydomonus outer-arm dynein. 25.
26.
MITCHELL DR, KANG Y: Identification of oda6 as a Cblamydomonas Dyncin IMutant by Rescue with the Wild-Type Gene. J Cell Biol 1991, 113:835842. WOHDEMAN L, SXUER E, SHE!ZIZMP, M~TCHISONTJ: ChcmicaI Subdomains within the Kinctochorc Domain of Isolated CHO Mitotic Chromosomes. J Cell Biol 1991, 114:28>294.
27. ..
LI Y-Y, YEH E, HAYS T, BLOOM K: Disruption of Mitotic Spindle Orientation in a Yeast Dyncin Mutant. Proc Nut1 Acud Scl USA 1993, 90:10096-10100. Yeast dynein heavy chain is found to bc homologous to other cytoplasmic dynein heavy chains and highly homologous to cytopiasmic and axonemal proteins within the region of the central P-loop cluster. Gene disruptions appear to perturb positioning of the mitotic spindle but not spindle assembly, elongation, or chromosome segregation. SULLIVANDS, HUFFAKERTC: Astral Microtubules Arc Not Rc28. quit-cd for Anaphasc B In Saccharomyces cenrvfsfae. J Cell Bfol 1992, 119:37+388. LEES-MILLER JP, HELFMAN DM, SCHHOI% TA: A Vertebrate 29. .. Actin-Related Protein is a Component of a Multisubunit Complex Involved in MicrotubuleBased Vesicle Motility. Nature 1992, 359:244-246. The dynactin complex, an activator of cytoplasmic dynein in uitro, contains eight different poiypcptides. The most abundant of these is shown to be actin-RF’V, a protein whose primary sequence is 55% identical to actin and whose structure is predicted to be higNy similar to actin. PASCHAL BM. HO~ZDAUR ELF, PFISIXR KK, CLARK S. MEYER 30. DI, VALLEE RB: Characterization of a 5@kDa Polypcptidc in Cytoplasmic Dyncin Preparations Reveals a Complex with ~15Ootu~ and a Novel Actin. J Bfol Cbem 1993, 268:15318-15323. 31.
HOL~BALIR ELF, HAMMARBACKJA, PASCHALBM, KRAVII’ NG, PFISTERKK, VALLEE RB: Homology of a 150K Cytoplasmic Dyncin-Associated Polypcptidc with the Lhvsopbika Gene Glued. Nature 1991, 351:573-583.
function
and regulation
of cytoplasmic
dynein
Schroer
HARTE PJ, MNKEL DR: Gcnctic Analysis of Mutations at the Glued Locus and Interacting Loci in Lhsopbiha melanogastet. Genetics 1982, 101,:477-501. LOPEZ LA, SHEEXZ MP: Stcric Inhibition of Cytoplasmic 33. Dyncin and Kincsin Motility by MAP2. Cell Motfl Cytcxkeleton 1993, 24:1-16. HAMM-ALVAREZSF, KIM PY, SHE~Z MP: Regulation of Vesicle 34. . Transport in CV-1 Cells and Extracts. J Cell Scf 1993, 106:in press. This report demonstrates that the incidence of vesicle motility in living cells can be altered by a variety of agents that alter protein phosphoryiation, providing great support to the widely assumed (but never proven) model that microtubule-based motor activity is regulated by reversible phosphorylation. LIN SXH, COLLINS CA: Regulation of the IntraccIhdar Dis35. tribution of CytopIasmic Dyncin by Serum Factors and Calcium. J Cell Sci 1993, 105:57%588. 36. BLOOM GS, RICHARDSSW, LEOPOLD PL, RITCHEYDM, BRADY . ST: GTpys Inhibits OrgancUe Transport Along Axonal Microtubulcs. J Cell Biol 1993, 120:467-476. Addition of a non-hydrolyzable GTP analog to extruded squid axoplasm inhibits the velocity of fast axonal transport, indicating that microtubule-based vesicle transport should be included in the ranks of membrane traffic events that are mediated by GTP-binding proteins. PFEFFERS: GTP-Binding Proteins in IntraccIIular Transport. 37. Trends Cell Blol 1992, 2:41-46. HOLLENBECK PJ, BRAY D: Rapidly Transported OrgancIIcs 38. Containing Membrane and Cytoskclctal Components: Their Relation to Axonal Growth. J Cell Bioll987, 1052827-2835. HOLLENBECKPJ: Products of Endocytosis and Autophagy arc 39. . Retrieved from Axons by Regulated Retrograde Organcllc Transport. J Cell Bfol 1993, 121305-315. Analysis of the bidirectional movement of an axonal organeIIe that participates in membrane recycling indicates that direction preference can be infiuenced in at least two separate ways. Net retrograde movement is favored by increasing the duration of each movement, whereas antcrograde movement is favored by increasing the probability that a non-moving vesicle will resume movement in the anterograde direction. 32.
TA Schrocr, Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA.
73
function
and regulation
of cytoplasmic
dynein
Trina A Schroer The Johns Hopkins
University,
Baltimore,
USA
Molecular cloning studies have provided valuable structural information about the different subunits of cytoplasmic dynein and their relationships to their axonemal dynein counterparts. Recent unexpected findings regarding the role of this enzyme in mitosis have emerged from mutational analyses and microinjection experiments, while studies of organelle transport in vivo have revealed clues to mechanisms for physiological regulation of dynein activity.
Current
Opinion
in Cell
Biology
Introduction
1994,
Structure
6:69-73
of cytoplasmic
dynein
subunits
Heavy chains
Cytoplasmic dynein, a minus-end directed, microtubule-based motor, is thought to drive the movement of intracellular structures as diverse as membranous organelles and chromosomes. Like the axonemal dyneins, the cytoplasmic species is a massive (greater than 1000000 Mr) multisubunit complex that consists of multiple polypeptides: two heavy chains of greater than 400 000 M,, three or four 74 000 Mr intermediate chains and four light chains of approximately 55 000 M, [1,2]. Ultrastructural studies indicate that each molecule has two large globular heads projecting from a common base, which comprises several smaller globular domains 131.The heavy chain, containing the sites for ATP hydrolysis and microtubule binding, probably makes up the bulk of each head, while the intermediate and light chains are believed to lie at the base of the molecule. The recent determination of the primary sequences of the three different subunits represents a significant advance towards a better understanding of dynein structure.
In late 1991, the report of full-length, primary sequences for the p heavy chain of sea urchin flagellar dynein 111,121 began a new era in studies of dynein structure and function. Structural predictions suggest that the molecule contains five nucleotidebinding sites, the phosphate-binding pockets of which are termed P-loops 1131(see Fig. 1). One P-loop is near the amino terminus, but the other four are clustered in a domain of approximately 100 000 M, located roughly halfway between the amino and carboxyl termini. The first P-loop (P-l) in the central cluster is thought to be the site of ATP hydrolysis 1111. An armament of molecular biological techniques (expression screening, hybridization screening and PCR) has enabled the determination of full-length cDNA sequences encoding cytoplasmic dynein heavy chains from Dictyostelium discoideum 114.1, Rattus norvegicus 115*,16.1, Saccharomyces cerevisiae 117**1, Caenorhabditis elegans u Lye, personal communication) and Drosophila melanogaster (T Hays, personal communication); a cytoplasmic heavy chain gene has also been identified in sea urchin 1181.The sequences predict proteins that range in size from 471305 M, (yeast) to 538482 M, (Dictyostelium), significantly larger than previous estimates from SDS-PAGE. As shown in Fig. 1, the cytoplasmic dynein heavy chains contain four centrally located P-loops, but lack the P-loop found near the amino terminus in sea urchin flagellar dynein. The axonemal and cytoplasmic proteins show some homology in the carboxy-terminal two-thirds of the molecule, with the greatest conservation occurring in the central P-loop cluster, particularly in the vicinity of the ATP hydrolytic site. The conserved carboxy-terminal region may represent the large globular head of the enzyme, while the divergent amino termini may correspond to the tail, a structure presumed to interact with other dynein subunits and cargo. The cytoplasmic
Because in vitro motility assays indicate that cytoplasmic dynein translocates membrane vesicles toward the minus ends of microtubules [4,51, the enzyme is assumed to participate in vivo in centripetal organelle movements and retrograde axonal transport. Immunofluorescence studies show that cytoplasmic dynein localizes to intracellular membranes 16,71 as well as to chromosome kinetochores and spindle poles 1891, suggesting an involvement in mitosis. Dynein-based motility undoubtedly involves a variety of intracellular components such as membrane receptors and cytosolic regulatory factors. The dynactin complex, an activator of dynein-based vesicle movements in vitro [6,1Ol, is the first identified candidate for a regulator of dynein activity. In this review we will discuss recent insights into possible functions of dynein as well as mechanisms for its regulation.
Abbreviations MAP-microtubule-associated
0 Current
protein;
Biology
PCR-polymerase
Ltd ISSN 0955-0674
chain
reaction. 69
70
Cvtoskeleton
Heavy
chain
Axonemal Ripneustes
gratilla
1 1
Cyfoplasmic Dictyosteliym
discoideum Rattus
Saccharomyces
Intermediate
1;
:,’
,. ,.)-. IL 1, .:L
norvegicus cerevisiae
1
I
.::
69 kDa
m
567
Cytoplasmic Rattus
7
b.
“lmJ1,
:I pqc.
il; IIF’
~1: H
n
.I’,II,
I.
I4725 [ 4644 1 4092
I!-‘”
chaii
Axonemal Chlamydomonas
‘_ 1. ~, -.;!-;1,
?!.,:. .: . . :,; ; .‘:ql
74 kDa
II1
643
r_1: m m 0 0
P-loop Possible coiled-coil Highly conserved Conserved Not conserved
I1994 Current Opinion in Cell Biology
heavy chains are homologous along their length, and are nearly identical in the region surrounding P-l (70 % identity between the Dictyostelium and human proteins; [19**1>. Although the cytoplasmic dynein heavy chain gene is part of a large superfamily 120,211 whose size probably reflects the diversity of the inner and outer arm heavy chains, it is not known whether multiple cytoplasmic species exist. Unlike their axonemal counterparts, which are composed of two (or three) different heavy chains, all known cytoplasmic dyneins contain two identical heavy chains. It is possible that multiple dynein isofomls, distinguished by their complement of intermediate and light chains, account for different types of intracellular dynein-based motility.
Intermediate
and light chains
A start has also been made on the other cytoplasmicdynein subunits. Although their precise functions have not yet been determined, the intermediate and light chains are believed to regulate enzyme activity and/or participate in cargo binding. The latter function was first suggested for the 78 000 M,. and 69 000 M, subunits of outer-arm dynein of the Chlamydomonas flagellum. These proteins are tightly associated with one another and lie at the base of the enzyme, where it is anchored to the A-subfiber microtubule 122,231. Antibody decoration experiments suggest that intermediate chains occupy a similar site on cytoplasmic dynein (ER Steuer, TA Schroer, JE Heuser and MP Sheetz, unpublished data); this region of dynein identified by ultrastructuna1 analysis is thought to bind cargo, i.e. membrane organelles, chromosomes or other intracellular components. The primary sequence of the rat cytoplasmic dynein intermediate chain, a molecule of 72 753 Mr [24'1, reveals some similarity to the 69000 Mr species from Chlamydomonas [251. The carboxy-terminal halves of the two molecules are approximately 26% identical and 48% similar in sequence, while the amino-terminal domains show no particular similarity to each other (see Fig. 1).
Fig. 1. Comparison of dynein polypeptide sequences. This schematic representation compares the primary sequences of axonemal and cytoplasmic dynein heavy [ll ,14*-l 6*,17**] and intermediate [24*,25] chains. The positions of predicted phosphate-binding P-loops and regions of alpha-helical coiled-coil are indicated for each polypeptide; the number of amino acids in each polypeptide is given on the right. The dynein heavy chains are aligned relative to the highly conserved P-loop, P-l (within the hatched box), and intermediate chains are aligned relative to the conserved carboxy-terminal domains.
It is proposed that the conserved carboxyl terminus associates with the heavy chain or another dynein subunit and that the amino terminus specifies what cargo the motor will bind 124’1. The 53-59 kDa cytoplasmic dynein light-chain family is fairly well characterized biochemically, if not functionally. The chicken light chains can be resolved by SDSPAGE into several Mr species, each of which is represented by multiple charge isoforms. Peptide mapping divides the light chains into two groups, the higher (57 000-59 000) and lower (53 000-55 000) M, isoforms (SR Gill, AF Salcedo, DW Cleveland, TA Schroer, abstract 936, Mol Biol Cell 1992, 3:161a). The sequence of a cDNA encoding the higher M, light chains predicts a globular protein that may contain a nucleotide-binding site near its amino terminus (SR Gill, DW Cleveland, TA Schroer, unpublished data) but otherwise displays no significant homology to other proteins. The lower M, light chain has yet to be cloned.
Functions Hela
of cytoplasmic
dynein
in mitosis
cells
Cytoplasmic dynein, which is located at kinetochores 18,9,261, may be a motor for poleward movements of chromosomes in prometaphase and anaphase A. In order to test this hypothesis, antibodies (either intact or Fab fragments) raised against a cytoplasmicdynein peptide from the highly conserved, proposed ATP-hydrolytic site (P-l; see Fig. 1) were injected into HeLa cells in interphase or at different stages of mitosis. Cells injected during or before prophase formed a monopolar spindle, but injections later in mitosis had no effect on chromosome motility or completion of mitosis 113”l. These results suggest that a major role of cytoplasmic dynein is the promotion and maintenance of centrosome separation at the very onset of mitosis, and that if the enzyme is involved in chromosome translocation, its function is redundant.
Structure, Yeast
Identification of genes encoding cytoplasmic dynein heavy chains in organisms tractable to genetic analysis makes it possible to study dynein function by analyzing the phenotypes of dynein mutants. Disnlptions of the dynein heavy-chain gene (DYNE) in yeast do not impair mitotic spindle formation or chromosome congression, but instead perturb the positioning of mitotic spindles within dividing cells [17**,27”1. Normally, the spindle bridges the junction between the mother cell and bud, so that one daughter nucleus lies in each cell after cytokinesis. In dynl mutants, the spindle can remain entirely within the mother cell, yielding an anucleate daughter, a phenotype somewhat similar to that of a tubulin mutation (tub2-410, 1281) that disrupts extranuclear microtubules. Spindle assembly, elongation and chromosome segregation are not impaired. It would appear that one function of dynein in yeast is to exert force on the cytoplasmic microtubules and thereby determine spindle position.
Regulation vitro
of cytoplasmic
dynein
activity
in
The in vitro motility assay, a powerful tool in the identification of microtubule motors, has enabled characterization of other factors that may influence microtubulebased motility in living cells. In vitro studies of vesicle transport by cytoplasmic dynein indicate that the system requires at least one additional component, the dynactin complex, a 20s multiprotein assembly composed of eight different proteins [6,101. Curiously, its major component is actin-related protein from vertebrates (also known as RPV or centractin), a protein in the actin superfamily [29”,3Ol. Although actinRPV shares only 55% sequence identity with authentic actins, it has retained the ability to form the 40nm filament that is the major structural domain of the dynactin complex (T Schroer, unpublished data). Another component of the complex, dynactin [61 (also known as pl5OGLUED t30,311>, is homologous to the Glued protein in Drosophila. Glued is essential during embryogenesis and may be involved in the development of the visual system [32l. In vertebrate cells, the dynactin complex can be detected in association with small cytoplasmic vesicles ([6,301; T Meads, TA Schroer, unpublished data), suggesting that it may participate in dynein-membrane interactions. In vitro motility studies also indicate that cytoplasmic dynein-based movements can be affected by certain microtubule-associated proteins 1331. MAP-2, but not tau protein, appears to stimulate detachment of motors from microtubules, causing a reduction in the frequency of microtubule and vesicle movements. A likely explanation is that the 10&200nm-long MAP-2 sidearm interferes with the binding of cytoplasmic dynein to the microtubule surface. In living cells,
function
and regulation
of cytoplasmic
dynein
Schroer
MAP-2 or other similarly acting proteins may well modulate dynein-based movements.
Physiological
regulation
of organelle
transport
Studies focused on the regulation of organelle transport in living cells have revealed that the process can be modulated by a variety of physiological effecters. In CV-1 kidney cells, the incidence of vesicle movements can be increased by addition of serum, CAMP and okadaic acid (a protein phosphatase inhibitor) i34.1, suggesting a role for reversible phosphorylation in intracellular motility. That centripetal and centrifugal movements are affected equally indicates coordinate regulation of cytoplasmic dynein- and kinesin-based vesicle movements. The finding that serum dramatically stimulates the association of cytoplasmic dynein with lysosomes [351 leads to the supposition that organelle motility may be controlled, at least in part, at the level of motor-membrane binding. Axonal transport is an example of microtubule-based vesicle motility taken to the extreme. To ensure continued viability, large amounts of membrane must be exported to the synapse (anterograde transport) and recycled to the cell body (retrograde transport). This process has long been studied in axoplasm extruded from the giant axon of squid, an in vitro system easily accessed by externally added agents. Using this system, Bloom et al. i36.1 have recently shown that in the presence of GTQ.5, a GTP analog that suppresses the hydrolytic cycle required for activity of many GTP-binding proteins, both anterograde and retrograde vesicle movements occur at reduced velocity; in contrast, no effects were seen with a variety of protein kinase and phosphatase inhibitors. Likely targets for GTPyS are the rab proteins, a family of small GTP-binding proteins that play key roles in many intracellular membrane traffic events t371. It would appear that the effector acts on a component of the transport machinery that is shared by cytoplasmic dynein and kinesin. Cultured sympathetic neurons support high levels of axonal transport. Particularly conspicuous is the bi-directional movement of large, refractile organelles that mediate membrane recycling to the cell body. Unlike the situation in other axoplasmic vesicles, the direction of movement of these membranes is governed by physiological stimuli. When axonal growth is prevented, the particles show a preference for retrograde movement [381; in contrast, increases in intracellular CAMP bias movement in the opposite direction i39’1. Careful analysis of the movement of individual particles revealed that preference for movement in the retrograde and anterograde directions arises in different ways. Retrograde preference involves a lengthening in duration of each retrograde movement, whereas anterograde preference results from increasing the probability that a particle moving in the anterograde direction will continue moving in the same direction after stopping (per-
71
72
Cvtoskeleton
sistence of movement). Duration and persistence may reflect overall motor activity or the affinity of motors for the particle surface.
PFARRCM, COUE M, GRK+OM PM, HAYS TS, PORTER ME, MCINI’OSHJR: Cytoplasmic Dynein Localizes to Rinctochores During Mitosis. Nature 1990, 345:263-265.
9.
STEUERER, SCHROERTA, WORDEMANL, SHEEX! MP: Cytoplasmic Dyncin Localixes to Mitotic Spindles and Rinetochores. Nature 1990, 345:266-268. SCHROERTA, SHEE’~Z MP: TWO Activators of MicrotubulcBased Vesicle Transport. / Cell Bfol 1991, 115:1389-1318. GIBBONS IR, GIBWNS BH, Mocz G, ASAI DJ: Multiple Nuclcotidc-Binding Sites in the Sequence of Dyncin B Heavy Chain. Nature 1991, 352640-643.
10.
Conclusions The past year has seen a number of significant advances in the areas of cytoplasmic dynein structure, function and regulation. The largest subunit of the enzyme, the so-called heavy chain, appears to be highly conserved, with particular homology near the active site. Primary sequence homologies are seen between analogous subunits of cytoplasmic and axonemal dyneins, suggesting conservation of the sequences of sites for subunit interaction. With the wealth of primary sequences now available, studies of the molecular details of dynein subunit interactions and dynein
References
8.
and recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as: . of special i&rest.. of outstanding interest 1.
LYE RJ, POHTERME, SCHOLEYJM, MCINXXH JR Identification of a Microtubule-Based Cytoplasmic Motor in the Nematode C. elegatts. Cell 1987, 51:309-318.
2.
PAXHAL BM, SHP~~NER HS, VALI.EE RB: MAP 1C is a Microtubulc-Activated ATPase which Translocatcs Micro tubules In v/trr, and has Dynein-Like Properties. / Cell Bfol 1987, 105:1273-X282.
3.
VALLEE RB, WALL JS, PASCHALBM, SHPETNERHS: MicrotubulcAssociated Protein 1C from Brain is a **Headed Cytosolic Dyncin. Nafnre 1988, 332:561-563.
4.
SCHNAPPBJ, REEX T’S: Dyncin is the Motor for Retrograde Axonal Transport of Organellcs. Proc Nat1 Acad Sci USA 1989, 86:154%1552.
5.
SCHROERTA, STEUEHER, SHEB~+ MP: Cytoplasmic Dynein is a Minus-End Direct Motor for Membranous Organellcs. Cell 1989, 56:937-946.
6.
GILI. SR, SCHROERTA, SZILAK I, STEUER ER, SHERZ MP, CLEVELAND DW: Dynactin, a Conserved, Ubiquitously Expressed Component of an Activator of Vesicle Motility Mediated by Cytoplasmic Dyncin. / Cell Efol 1391,
11.
12.
OCAWA K: Four ATPBiiding Sites in the Midregion of the f3 Heavy Chain of Dyncin. Nature 1991, 352643-645. SARAS~EM, SIBBALD PR, WITI~NGHOFEHA: The P-Loop - A 13. Common Motif in ATP- and GTPBinding Proteins. Trends Bfocbem Sci 1990, 153430-434. 14. K~XNCE MP, GRI%~M PM, MCINTOSHJR Dyncin from Dfc. tyostelh4m: Priiary Structure Comparisons Between a Cytoplasmic Motor Enzyme and Flagellar Dynein. / Cell Btol 1992, 119:1597-1604. This is the lirst reported sequence of a cytoplasmic dynein heavy chain, demonstrating significant homology, in the carboxy-terminal two-thirds of the protein, between cytoplasmic and axonemal dynein isoforms. 15. .
MIMMI A, PASCHALBM, MAZUMI~ARM, VAI.I.EERB: Molccular Cloning of the Retrograde Transport Motor Cytoplasmic Dyncin (MAP 1C). Narron 1993, 10:787-796. The sequence of this vcnebrate cytoplasmic dynein heavy chain shows significant overall homology to the Dic[wstelium protein and allows further comparison of the axonemal and cytoplasmic isoforms. 16. ZHANG ZZ, TANAKA Y, NONAKA S, ~=WA H, KAWASAKI H, . NAKATA T, HIROKAWAN: The Primary Structure of Rat Brain (Cytoplasmic) Dyncin Heavy Chain, a Cytoplasmic Motor Enzyme. Proc Nat1 Acad Scf USA 1993, 90:792&%7932. The sequence of this vertebrate cytoplasmic-dynein heavy chain shows significant overall homology to the Dfctyostelhun protein and allows further comparison of the axonemal and cytoplasmic isoforms. 17. ESHEI. D, URRESTARAZULA, VISSERSS, JAUNIA~JXJ-C, VAN .. VLI~REEDIJK JC, PLANTA KJ, GIBBONS IR: Cytoplasmic Dyncin is Required for Normal Nuclear Segregation in Yeast. Proc Natl Acad Sci USA 1993, 9O:in press. The full-length primary sequence of yeast dynein heavy chain shows similarity to Dtctyostelirrm and rdt cytoplasmic dyneios. Disruptions of the gene predominantly appear to affect nuclear positioning and not chromosome segregation. 18.
76:30>309.
VA~SBERGEA, KO~NCE MP, MCINTOSHJR Cytoplasmic Dyncin .. Plays a Role in Mammalian Mitotic Spindle Formation. / Cell Biol 1993, 123849858. Microinjection of antibodies directed to the highly conserved P-loop, P-l, disrupt formation of the mitotic spindle but not chromosome motility in Hela cells. Models are proposed to explain how dynein might participate in spindle pole separation. 20. R&~MUS.SONK, SERRM, GEPNERJ, GIBBONS I, HAYS TS: A Family of Dyncin Genes in Drvsopblla mekmogaster. Mol Btol Cell 1994, in press. 19.
21.
GIBBONS BH, Aslu DJ, TANG W-J, HAYS TS, GIBBONS IR: Expression of Axoncmrd and Cytoplasmic Dyncin Genes in Sea Urchin Embryos. Mol Bfol Cell 1994, in press.
22.
KING SM, WITMAN GB: Localization of an Intermediate Chain of Outer Arm Dynein by lmmunoelcctron Microscopy. / Bfol Chem 1990, 265:19807-19811. KING SM, WII.KERSONCG, WITMAN GB: The Mr 78,000 Intermediate Chain of Cblumydomonas Outer Arm Dyncin
115:1639-1650.
7.
LIN SXH, COLLINS CA: Immunolocalization of Cytoplasmic Dynein to Lysosomcs in Cultured Cells. / Cell Scf 1992. 101:12%137.
GIBBONS IR, Ashl DJ, TANG WJ, GII~BONSBH: A Cytoplasmic Dynein Heavy Chain in Sea Urchin Embryos. Btol Cell 1992,
23.
Structure, Interacts with Alpha-Tubtdin 26684018407.
in Situ. J Biol Chem 1991,
24. .
PASCHALBM, MIKAMI A, PFI.SI~RKK, VALLEE RB: Homology of the 74kD Cytopiasmic Dyncin Subunit with a Fiagcllar Dyncin PoIypcptidc Suggests an IntraccIIuIar Targeting Function. J Cell Biol 1992, 118:113+1143. The primary sequence of the intermediate chain of cytopiasmic dynein is shown to be partially homologous to the 69COO Mr subunit of Chhmydomonus outer-arm dynein. 25.
26.
MITCHELL DR, KANG Y: Identification of oda6 as a Cblamydomonas Dyncin IMutant by Rescue with the Wild-Type Gene. J Cell Biol 1991, 113:835842. WOHDEMAN L, SXUER E, SHE!ZIZMP, M~TCHISONTJ: ChcmicaI Subdomains within the Kinctochorc Domain of Isolated CHO Mitotic Chromosomes. J Cell Biol 1991, 114:28>294.
27. ..
LI Y-Y, YEH E, HAYS T, BLOOM K: Disruption of Mitotic Spindle Orientation in a Yeast Dyncin Mutant. Proc Nut1 Acud Scl USA 1993, 90:10096-10100. Yeast dynein heavy chain is found to bc homologous to other cytoplasmic dynein heavy chains and highly homologous to cytopiasmic and axonemal proteins within the region of the central P-loop cluster. Gene disruptions appear to perturb positioning of the mitotic spindle but not spindle assembly, elongation, or chromosome segregation. SULLIVANDS, HUFFAKERTC: Astral Microtubules Arc Not Rc28. quit-cd for Anaphasc B In Saccharomyces cenrvfsfae. J Cell Bfol 1992, 119:37+388. LEES-MILLER JP, HELFMAN DM, SCHHOI% TA: A Vertebrate 29. .. Actin-Related Protein is a Component of a Multisubunit Complex Involved in MicrotubuleBased Vesicle Motility. Nature 1992, 359:244-246. The dynactin complex, an activator of cytoplasmic dynein in uitro, contains eight different poiypcptides. The most abundant of these is shown to be actin-RF’V, a protein whose primary sequence is 55% identical to actin and whose structure is predicted to be higNy similar to actin. PASCHAL BM. HO~ZDAUR ELF, PFISIXR KK, CLARK S. MEYER 30. DI, VALLEE RB: Characterization of a 5@kDa Polypcptidc in Cytoplasmic Dyncin Preparations Reveals a Complex with ~15Ootu~ and a Novel Actin. J Bfol Cbem 1993, 268:15318-15323. 31.
HOL~BALIR ELF, HAMMARBACKJA, PASCHALBM, KRAVII’ NG, PFISTERKK, VALLEE RB: Homology of a 150K Cytoplasmic Dyncin-Associated Polypcptidc with the Lhvsopbika Gene Glued. Nature 1991, 351:573-583.
function
and regulation
of cytoplasmic
dynein
Schroer
HARTE PJ, MNKEL DR: Gcnctic Analysis of Mutations at the Glued Locus and Interacting Loci in Lhsopbiha melanogastet. Genetics 1982, 101,:477-501. LOPEZ LA, SHEEXZ MP: Stcric Inhibition of Cytoplasmic 33. Dyncin and Kincsin Motility by MAP2. Cell Motfl Cytcxkeleton 1993, 24:1-16. HAMM-ALVAREZSF, KIM PY, SHE~Z MP: Regulation of Vesicle 34. . Transport in CV-1 Cells and Extracts. J Cell Scf 1993, 106:in press. This report demonstrates that the incidence of vesicle motility in living cells can be altered by a variety of agents that alter protein phosphoryiation, providing great support to the widely assumed (but never proven) model that microtubule-based motor activity is regulated by reversible phosphorylation. LIN SXH, COLLINS CA: Regulation of the IntraccIhdar Dis35. tribution of CytopIasmic Dyncin by Serum Factors and Calcium. J Cell Sci 1993, 105:57%588. 36. BLOOM GS, RICHARDSSW, LEOPOLD PL, RITCHEYDM, BRADY . ST: GTpys Inhibits OrgancUe Transport Along Axonal Microtubulcs. J Cell Biol 1993, 120:467-476. Addition of a non-hydrolyzable GTP analog to extruded squid axoplasm inhibits the velocity of fast axonal transport, indicating that microtubule-based vesicle transport should be included in the ranks of membrane traffic events that are mediated by GTP-binding proteins. PFEFFERS: GTP-Binding Proteins in IntraccIIular Transport. 37. Trends Cell Blol 1992, 2:41-46. HOLLENBECK PJ, BRAY D: Rapidly Transported OrgancIIcs 38. Containing Membrane and Cytoskclctal Components: Their Relation to Axonal Growth. J Cell Bioll987, 1052827-2835. HOLLENBECKPJ: Products of Endocytosis and Autophagy arc 39. . Retrieved from Axons by Regulated Retrograde Organcllc Transport. J Cell Bfol 1993, 121305-315. Analysis of the bidirectional movement of an axonal organeIIe that participates in membrane recycling indicates that direction preference can be infiuenced in at least two separate ways. Net retrograde movement is favored by increasing the duration of each movement, whereas antcrograde movement is favored by increasing the probability that a non-moving vesicle will resume movement in the anterograde direction. 32.
TA Schrocr, Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA.
73