Stable complexes of axoplasmic vesicles and microtubules: protein composition and ATPase activity

Fast transport of axonal vesicles and organelles is a microtubule-associated movement (Griffin, J. W., K. E. Fahnestock, L. Price, and P. N. Hoffman, 1983, J. Neuroscience, 3:557-566; Schnapp, B. J., R. D. Vale, M. P. Sheetz, and T. S. Reese, 1984, Cell, 40:455-462; Allen, R. D., D. G. Weiss, J. H. Hayden, D. T. Brown, H. Fujiwake, and M. Simpson, 1985, J. Cell Biol., 100:1736-1752). Proteins that mediate the interactions of axoplasmic vesicles and microtubules were studied using stable complexes of microtubules and vesicles (MtVC). These complexes formed spontaneously in vitro when taxol-stabilized microtubules were mixed with sonically disrupted axoplasm from the giant axon of the squid Loligo pealei. The isolated MtVCs contain a distinct subset of axoplasmic proteins, and are composed primarily of microtubules and attached membranous vesicles. The MtVC also contains nonmitochondrial ATPase activity. The binding of one high molecular mass polypeptide to the complex is significantly enhanced by ATP or adenyl imidodiphosphate. All of the axoplasmic proteins and ATPase activity that bind to microtubules are found in macromolecular complexes and appear to be vesicle-associated. These data allow the identification of several vesicle-associated proteins of the squid giant axon and suggest that one or more of these polypeptides mediates vesicle binding to microtubules.     

Identification of a MAP 2-like ATP-binding protein associated with axoplasmic vesicles that translocate on isolated microtubules

Axoplasmic vesicles were purified and observed to translocate on isolated microtubules in an ATP-dependent, trypsin-sensitive manner, implying that ATP-binding polypeptides essential for force generation were present on the vesicle surface. To identify these proteins [alpha 32P]8-azidoadenosine 5'-triphosphate ([alpha 32P]8-N3ATP), a photoaffinity analogue of ATP, was used. The results presented here identify and characterize a vesicle-associated polypeptide having a relative molecular mass of 292 kD that bound [alpha 32P]8-N3ATP. The incorporation of label is ultraviolet light-dependent and ATP-sensitive. Moreover, the 292-kD polypeptide could be isolated in association with vesicles or microtubules, depending on the conditions used, and the data indicate that the 292-kD polypeptide is similar to mammalian brain microtubule-associated protein 2 (MAP 2) for the following reasons: The 292-kD polypeptide isolated from either squid axoplasm or optic lobe cross-reacts with antiserum to porcine brain MAP 2. Furthermore, it purifies with taxol-stabilized microtubules and is released with salt. Based on these characteristics, the 292-kD polypeptide is distinct from the known force-generating molecules myosin and flagellar dynein, as well as the 110-130-kD kinesin-like polypeptides that have recently been described (Brady, S. T., 1985, Nature (Lond.), 317:73-75; Vale, R. D., T. S. Reese, and M. P. Sheetz, 1985b, Cell, 42:39-50; Scholey, J. M., M. E. Porter, P. M. Grissom, and J. R. McIntosh, 1985, Nature (Lond.), 318:483-486). Because the 292-kD polypeptide binds ATP and is associated with vesicles that translocate on purified MAP-free microtubules in an ATP-dependent fashion, it is therefore believed to be involved in vesicle-microtubule interactions that promote organelle motility.     

Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility

Axoplasm from the squid giant axon contains a soluble protein translocator that induces movement of microtubules on glass, latex beads on microtubules, and axoplasmic organelles on microtubules. We now report the partial purification of a protein from squid giant axons and optic lobes that induces these microtubule-based movements and show that there is a homologous protein in bovine brain. The purification of the translocator protein depended primarily on its unusual property of forming a high affinity complex with microtubules in the presence of a nonhydrolyzable ATP analog, adenylyl imidodiphosphate. The protein, once released from microtubules with ATP, migrates on gel filtration columns with an apparent molecular weight of 600 kilodaltons and contains 110-120 and 60-70 kilodalton polypeptides. This protein is distinct in molecular weight and enzymatic behavior from myosin or dynein, which suggests that it belongs to a novel class of force-generating molecules, for which we propose the name kinesin.     

Microtubule binding and translocation by inner dynein arm subtype I1

Structural, biochemical, and genetic evidence has demonstrated there are three inner dynein arm subforms, I1, I2, and I3, which differ in organization and composition (see Piperno et al.: J. Cell Biol. 110:379-389, 1990). Using dynein extracted from Chlamydomonas outer dynein armless mutant pf28, we have begun to define the structural and functional properties of isolated inner arm subforms. Inner dynein arm I1 was purified either by sucrose density gradient centrifugation or microtubule binding affinity. I1, composed of heavy chains 1 alpha and 1 beta, sedimented at 21S and selectively bound to and cross-linked purified microtubules in an ATP-sensitive manner. Deep etch electron microscopy revealed that the 21S sedimenting fraction contained two-headed structures in which large globular heads are connected by long, flexible-stem domains. In contrast, components derived from I2 and I3 sedimented as a mixture of 11S particles with single globular heads which did not bind to purified microtubules. Both the 21S and 11S sedimenting fractions supported microtubule translocation in in vitro motility assays. In 1 mM MgATP the I1-containing fraction produced very slow microtubule-gliding velocities (0.76 microns/sec) compared to the I2,I3-containing fraction (4.1 microns/sec).     

Identification and characterization of a novel microtubule-based motor associated with membranous organelles in tobacco pollen tubes

Pollen tube growth depends on the differential distribution of organelles and vesicles along the tube. The role of microtubules in organelle movement is uncertain, mainly because information at the molecular level is limited. In an effort to understand the molecular basis of microtubule-based movement, we isolated from tobacco pollen tubes polypeptides that cosediment with microtubules in an ATP-dependent manner. Major polypeptides released from microtubules by ATP (ATP-MAPs) had molecular masses of 90, 80, and 41 kD. Several findings indicate that the 90-kD ATP-MAP is a kinesin-related motor: binding of the polypeptide to microtubules was enhanced by the nonhydrolyzable ATP analog AMP-PNP; the 90-kD polypeptide reacted specifically with a peptide antibody directed against a highly conserved region in the motor domain of the kinesin superfamily; purified 90-kD ATP-MAP induced microtubules to glide in motility assays in vitro; and the 90-kD ATP-MAP cofractionated with microtubule-activated ATPase activity. Immunolocalization studies indicated that the 90-kD ATP-MAP binds to organelles associated with microtubules in the cortical region of the pollen tube. These findings suggest that the 90-kD ATP-MAP is a kinesin-related microtubule motor that moves organelles in the cortex of growing pollen tubes.     

A latent activity dynein-like cytoplasmic magnesium adenosine triphosphatase

A MgATPase has been isolated and characterized from unfertilized sea urchin eggs which is very similar, but not identical, to latent activity axonemal dynein. The cytoplasmic MgATPase activity sediments at 20 S, slightly slower than 21 S latent activity flagellar dynein. Activity is stimulated by nonionic detergent and is inhibited by sodium orthovanadate but is not as sensitive to vanadate as is 21 S flagellar dynein. The egg 20 S MgATPase is composed, at least in part, of three high molecular weight polypeptides. In addition, two intermediate-sized polypeptides appear to co-sediment with the 20 S MgATPase activity. A novel microtubule-affinity assay reveals that high molecular weight polypeptides 1 and 2 of the egg 20 S MgATPase can bind to reassembled microtubules and can be released from the microtubules with MgATP2-. Further, the apparent specific activity of the egg MgATPase is enriched 15-fold by a single microtubule binding step. The results suggest that the cytoplasmic 20 S MgATPase is a dynein-like microtubule translocator which resides in the unfertilized egg awaiting future incorporation onto microtubules in order to perform work. The egg 20 S enzyme might function in cytoplasmic microtubule-mediated movement or it might be a precursor of embryonic ciliary dynein.     

Cytoplasmic dynein of the sea urchin egg. II. Purification, characterization and interactions with microtubules and Ca-calmodulin

Purification of cytoplasmic dynein from unfertilized sea urchin eggs was performed in the presence of protease inhibitors to avoid proteolysis throughout the purification procedure, which comprised several chromatographies, including a calmodulin-Sepharose 4B affinity column chromatography. This is the first report of the purification of cytoplasmic dynein to near homogeneity. The purified fraction was composed of a single high molecular weight polypeptide and some minor low molecular weight polypeptides. The high molecular weight polypeptide comigrated with flagellar dynein A beta chain from sperm on SDS-polyacrylamide gels. There was no polypeptide stainable with periodic acid-Schiff reagent (PAS) in the purified cytoplasmic dynein fraction. Cytoplasmic dynein showed characteristics quite similar to those of axonemal dynein, i.e. high substrate specificity for ATP and inhibition by low concentrations of vanadate, though its Ca-ATPase activity showed almost the same dependence on the concentration of either divalent cations or KC1 as the Mg-ATPase activity. The purified enzyme seemed to possess functional form as judged from its properties: 1) pH dependence of the ATPase activity, 2) dependence of the ATPase activity on MgCl2 and KCl concentration, 3) Km for Mg-ATP, and 4) binding to flagellar doublet microtubules. Cytoplasmic dynein bound to calmodulin-Sepharose 4B only in the presence of Ca2+, and was eluted with EGTA. Furthermore, the ATPase activity was enhanced 6-fold by calmodulin in a Ca2+-dependent manner. The activation by calmodulin was prevented by a stoichiometric amount of trifluoperazine.     

Plus-end motors override minus-end motors during transport of squid axon vesicles on microtubules

Plus- and minus-end vesicle populations from squid axoplasm were isolated from each other by selective extraction of the minus-end vesicle motor followed by 5'-adenylyl imidodiphosphate (AMP-PNP)- induced microtubule affinity purification of the plus-end vesicles. In the presence of cytosol containing both plus- and minus-end motors, the isolated populations moved strictly in opposite directions along microtubules in vitro. Remarkably, when treated with trypsin before incubation with cytosol, purified plus-end vesicles moved exclusively to microtubule minus ends instead of moving in the normal plus-end direction. This reversal in the direction of movement of trypsinized plus-end vesicles, in light of further observation that cytosol promotes primarily minus-end movement of liposomes, suggests that the machinery for cytoplasmic dynein-driven, minus-end vesicle movement can establish a functional interaction with the lipid bilayers of both vesicle populations. The additional finding that kinesin overrides cytoplasmic dynein when both are bound to bead surfaces indicates that the direction of vesicle movement could be regulated simply by the presence or absence of a tightly bound, plus-end kinesin motor; being processive and tightly bound, the kinesin motor would override the activity of cytoplasmic dynein because the latter is weakly bound to vesicles and less processive. In support of this model, it was found that (a) only plus-end vesicles copurified with tightly bound kinesin motors; and (b) both plus- and minus-end vesicles bound cytoplasmic dynein from cytosol.     

Vesikin, a vesicle associated ATPase from squid axoplasm and optic lobe, has characteristics in common with vertebrate brain MAP 1 and MAP 2

Vesikin, a protein that can associate with squid axoplasmic vesicles or optic lobe microtubules, has been implicated as a force-generating molecule involved in microtubule-dependent vesicle transport [Gilbert and Sloboda, 1986, 1988]. Because vesikin crossreacts with an antibody to porcine brain microtubule associated protein 2 (MAP 2), studies were conducted to compare squid vesikin and brain MAPs. When taxol stabilized microtubules containing vesikin as a microtubule associated protein were incubated in the presence of ATP, vesikin dissociated from the microtubule subunit lattice. This behavior would be expected for an ATP-dependent, force generating molecule that serves as a crossbridge between vesicles and microtubules. When chick brain microtubules were treated under the same conditions, MAP 2 remained bound to the microtubules while MAP 1 dissociated in a manner similar to vesikin. One dimensional peptide mapping procedures revealed that, although digestion of vesikin and MAP 2 generated several peptides common to both proteins, vesikin and MAP 2 are clearly not identical. Furthermore, the addition of vesikin or MAPS 1 and 2 to purified tubulin stimulated microtubule assembly in a manner dependent on the concentration of added protein. These findings demonstrate that brain MAPs share characteristics common to squid vesikin and support the suggestion that brain MAPs 1 and 2 might act as a force generating complex for vesicle transport in higher organisms.     

Isolation of microtubules and a dynein-like MgATPase from unfertilized sea urchin eggs

Taxol was used to prepare microtubules from unfertilized eggs of sea urchins Lytechinus pictus, Strongylocentrotus droebachiensis , and Strongylocentrotus purpuratus. By electron microscopy, these microtubules possessed normal morphology and were decorated with projections. The polypeptides present were tubulin plus microtubule-associated proteins (MAPs) which included various high molecular weight polypeptides, and a Mr = 80,000 polypeptide. These MAPs were extracted from the microtubules by differential centrifugation in high ionic strength buffers, yielding a pellet of microtubules which were not decorated with projections. The Mr = 80,000 and high molecular weight MAPs were separated using Bio-Gel A-1.5 m chromatography, and shown to bind taxol-stabilized microtubules assembled from purified bovine brain tubulin. A dynein-like MgATPase activity is present in sea urchin egg extracts. 10-20% of this MgATPase co-pelleted with the taxol-assembled microtubules, under conditions where greater than 90% of the tubulin pelleted. During subsequent fractionation of the microtubules, by (i) high salt extraction followed by gel filtration or sucrose density gradient fractionation or (ii) ATP extraction, the MgATpase co-purified with high Mr MAPs. The MgATPase which remained in the microtubule-depleted egg extract was partially purified by (NH4)2SO4 fractionation, followed by Bio-Gel A-5 m and hydroxylapatite chromatography. The high Mr MAP MgATPase and the hydroxylapatite MgATPase both contained a prominent polypeptide (Mr approximately 350,000), which co-migrated on sodium dodecyl sulfate gels with the major heavy chain of dynein extracted from sperm axonemes. Our data suggest that this Mr approximately 350,000 polypeptide is cytoplasmic dynein.     

Quantitation of the dynein pool in unfertilized sea urchin eggs

A dynein-like ATPase activity has been isolated previously from soluble extracts of unfertilized sea urchin eggs. However, the use of non-quantitative isolation techniques, in particular affinity for microtubules or Ca2+/calmodulin, has precluded accurate estimates of dynein pool size. We have taken the unique approach of using dynein-like ATPase activity to quantitate the egg dynein pool. This approach is based on the isolation by anion-exchange chromatography on DEAE-Sephacel of a peak of dynein-like ATPase activity comprising 65% of soluble ATPase activity in the cytosolic extract. Identification of cytoplasmic dynein was based on dose-dependent inhibition by erythro-9-[3-(2-hydroxynonyl)]adenine and orthovanadate, low GTPase activity and a sedimentation coefficient of 12 S. Two high molecular weight polypeptides corresponding to the A- and D-bands of axonemal dynein were shown to copurify with dynein-like ATPase activity and to undergo specific photocrosslinking with [alpha-32P]ATP, suggesting that they were egg dynein catalytic polypeptides. The specific ATPase activity of these putative catalytic polypeptides was determined to be 1.2 mumol.min-1.mg-1. The specific dynein-like ATPase activity of the crude soluble extract of unfertilized sea urchin eggs was determined to be 0.004 mumol.min-1.mg-1. The concentration of putative dynein catalytic polypeptides was therefore determined from the ratio of the specific activities of crude to pure cytoplasmic dynein catalytic polypeptide to be 0.33% of soluble protein, or 99 pg per egg. This is approximately 3-fold greater than the mass of dynein catalytic polypeptides estimated to be present in cilia at the blastula stage of sea urchin embryonic development. The large amount of cytoplasmic dynein in unfertilized eggs suggests that it could act as a precursor of embryonic ciliary dynein. Three minor peaks of ATPase activity were also resolved from cytosolic extracts and shown to be dynein-like. However, their GTPase activities were 2-4-fold higher than that of cytoplasmic dynein, raising the possibility that egg cytoplasm may contain several isoforms of dynein.     

Fractionation of Tetrahymena ciliary membranes with triton X-114 and the identification of a ciliary membrane ATPase

Cilia were isolated from Tetrahymena thermophila, extracted with Triton X-114, and the detergent-soluble membrane + matrix proteins separated into Triton X-114 aqueous and detergent phases. The aqueous phase polypeptides include a high molecular mass polypeptide previously identified as a membrane dynein, detergent-soluble alpha and beta tubulins, and numerous polypeptides distinct from those found in axonemes. Integral membrane proteins partition into the detergent phase and include two major polypeptides of 58 and 50 kD, a 49-kD polypeptide, and 5 polypeptides in relatively minor amounts. The major detergent phase polypeptides are PAS-positive and are phosphorylated in vivo. A membrane-associated ATPase, distinct from the dynein-like protein, partitions into the Triton X-114 detergent phase and contains nearly 20% of the total ciliary ATPase activity. The ATPase requires Mg++ or Ca++ and is not inhibited by ouabain or vanadate. This procedure provides a gentle and rapid technique to separate integral membrane proteins from those that may be peripherally associated with the matrix or membrane.     

Flagellar adenosine triphosphatases from annelid spermatozoa: electrophoretic identification of dyneins

Axonemes of sperm flagella were prepared from the annelid, Tylorrhynchus heterochaetus. Dialysis of the axonemes against 1 mm Tris-HCl buffer (pH 8.3)-0.1 mm EDTA-0.1 mm dithiothreitol (Tris-EDTA solution) caused disintegration of typical 9 + 2 microtubules into each doublet, resulting in extraction of one-third of the protein and almost all ATPase activity. Agarose polyacrylamide gel electrophoresis of the extract showed the presence of three kinds of dyneins actively stained for ATPase (designated as bands I, II, and III) and two non-ATPase proteins (bands IV, V). The polypeptide components of each dynein molecule and intact axoneme were analyzed by subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis to obtain the following results: (1) In the highmolecular-weight region, the intact axonemes yield two major polypeptides with molecular weights of 365,000 and 345,000 (designated as bands A and B, respectively) and three minor polypeptides, 310,000, 290,00, and 270,00 (C1, C2, C3). (2) All three dyneins contain A-band polypeptide as a common polypeptide component. In addition, band I dynein and band II dynein also contain B and C1 polypeptides, and C3 polypeptide, respectively, as high-molecular-weight components. (3) Band III dynein also contains four polypeptides in the lower molecular-weight region, which migrate similarly with those of 21 S dynein from sea urchin sperm flagella or 18 S dynein from Chlamydomonas.     

Quantitative changes in maize cytoplasmic proteins induced by aluminium

Three-day-old maize seedlings were subjected to 100 μM AlCl3 for 24 h. Cytoplasmic proteins were isolated from root tips, root base and from coleoptiles. After fractionation of cytoplasmic proteins on anion chromatography column Bio-Scale Q2 sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was used to monitor Al-induced changes in polypeptide composition of particular fractions. Four (root) and 7 (coleoptile) fractions were eluted from the column with linear 0 - 1.0 M NaCl gradient. In fraction 1 of cytoplasmic proteins from root tips Al induced accumulation of polypeptide with molecular mass of 16 kD and simultaneous reduction of two polypeptides (67.5 and 60 kD). In fraction 1 isolated from mature zone of maize roots Al-induced accumulation of 22 kD polypeptide and reduction of 67.5, 60, and 14 kD polypeptides. Most pronounced changes were revealed in coleoptile. In three protein fractions increased accumulation of polypeptides with molecular mass of 14, 17.5, 20, 24.5, 28, 30, and 37.5 kD were observed. In the remaining three root or four coleoptile fractions of cytoplasmic proteins, no differences were found between Al-treated and control maize seedlings. This revised version was published online in July 2006 with corrections to the Cover Date.     

Localization of kinesin in cultured cells

Kinesin was isolated from bovine brain and used to elicit polyclonal antibodies in rabbits. The specificities of the resulting antibodies were evaluated by immunoblotting. Antibodies purified from these sera by their affinity for brain kinesin react with a polypeptide of approximately 120 kD in extracts from bovine brain, PtK1 cells, and mouse neuroblastoma cells. They bind to a pair of polypeptides of approximately 120 kD present in crude kinesin prepared from Xenopus eggs and with a single polypeptide of approximately 115 kD in extracts from Drosophila embryos. Antibodies raised against kinesin prepared from fruit fly embryos (by W. M. Saxton, Indiana University, Bloomington, IN) and from neural tissues of the squid (by M. P. Sheetz, Washington University, St. Louis, MO) cross react with the mammalian, the fly, and the frog polypeptides. Kinesin antigen was localized in cultured cells by indirect immunofluorescence. PtK1 cells in interphase showed dim background staining of cytoplasmic membranous components and bright staining of a small, fibrous, juxtanuclear structure. Double staining with antibodies to microtubules showed that the fibrous object was usually located near the centrosome. On the basis of shape, size, and location, we identify the kinesin-positive structure as a primary cilium. PtK1 cells in mitosis are stained at their poles during all stages of division. The structure stained is approximately spherical, but wisps of faint fluorescence also extend into the body of the spindle. Antibodies to squid or fruit fly kinesin produce identical patterns in PtK1 cells. Controls with preimmune and preabsorbed sera show that the centrosome staining is not due simply to the common tendency of rabbit antisera to stain this structure. Similar centrosome and spindle pole staining was visible when antibodies to bovine brain or squid kinesin were applied to the A6 cell line (kidney epithelial cells from Xenopus laevis). Some possible functions of kinesin localized at the spindle poles are discussed.     

Dynein isoforms in sea urchin eggs

Biochemical and immunological analysis of unfertilized sea urchin eggs has revealed the presence of at least two distinct isoforms of cytoplasmic dyneins, one soluble and the other microtubule-associated. The soluble enzyme is a 20 S particle with a MgATPase activity that can be activated 5-fold by nonionic detergents. It contains heavy chain polypeptides that 1) comigrate with the dynein heavy chains of embryonic cilia; 2) cross-react with antibodies against flagellar dynein; and 3) are cleaved by UV irradiation in the presence of MgATP and sodium vanadate into specific peptide fragments. The soluble egg dynein is, therefore, closely related to axonemal dynein and may be a ciliary precursor. Egg microtubule preparations contain a distinct dynein-like polypeptide, previously designated HMr-3 (Scholey, J.M., Neighbors, B., McIntosh, J.R., and Salmon, E.D. (1984) J. Biol Chem. 259, 6516-6525). HMr-3 binds microtubules in an ATP-sensitive fashion; it sediments at 20 S on sucrose density gradients, and it is susceptible to vanadate-sensitized UV cleavage. However, HMr-3 can be distinguished from the soluble cytoplasmic dynein on the basis of its weak cross-reactivity with antiflagellar dynein antibodies, its heavy chain composition on high resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis, its low specific ATPase activity, and the molecular weight of its vanadate-induced UV cleavage fragments. HMr-3 may represent a dynein-like polypeptide that is distinct from the pool of ciliary dynein precursors.     

Synthesis and Posttranslational Activation of Sulfhydryl-Endopeptidase in Cotyledons of Germinating Vigna mungo Seeds

A sulfhydryl-endopeptidase was purified as a 33 kilodalton (kD) mass polypeptide from cotyledons of Vigna mungo seedlings. Immunoblot analysis with antiserum made against the purified enzyme showed that the sulfhydryl-endopeptidase was synthesized only in the cotyledons during germination and that the amount of the enzyme increased until 4 days after imbibition and decreased thereafter. Next, an RNA fraction was prepared from cotyledons of 3 day old seedlings and translated in a wheat germ system. The synthesis of a 45 kD polypeptide was shown by the analysis of its translation products by immunoprecipitation with the antiserum to the endopeptidase and gel electrophoresis. When the RNA fraction was translated in the presence of canine microsomal membranes, a smaller polypeptide, having a 43 kD molecular mass, was detected as the translation product. When membrane-bound polysomes, but not free polysomes, prepared from cotyledons were used for translation in the wheat germ system, both the 43 and 45 kD polypeptides were synthesized. By incubation of a crude enzyme extract from cotyledons at 5 ± 1°C at neutral pH, the 43 kD polypeptide was sequentially cleaved to the 33 kD polypeptide via 39 and 36 kD intermediate polypeptides. The endopeptidase was activated simultaneously with the processing. Two-dimensional polyacrylamide gel electrophoresis showed that the 33 kD polypeptide was the fully activated form of the enzyme, whereas little or no activity was detected in other forms. From the present results, we postulate that the sulfhydryl-endopeptidase is first synthesized as the 45 kD precursor with a 2 kD signal peptide being cleaved, and that the 43 kD polypeptide is further cleaved to give the 33kD mature enzyme.     

Cytoplasmic dynein is a vesicle protein.

Microtubule-based organelle transport is thought to be mediated by the force-generating proteins cytoplasmic dynein and kinesin. These motor proteins have been characterized based on their ability to associate with and translocate microtubules. We show here that cytoplasmic dynein is also present as a peripheral membrane protein of purified synaptic vesicles. The vesicle-associated cytoplasmic dynein is identified by its photo-induced cleavage in the presence of ATP and vanadate. Purified, soluble cytoplasmic dynein is competent to bind to vesicle membranes stripped of endogenous peripheral membrane proteins by alkaline pH. Dynein binding to membranes is saturable at a concentration of 1.00 +/- 0.15 pmol/micrograms vesicle protein and has a dissociation constant of 22.3 +/- 2.4 nM. The association of cytoplasmic dynein with the membrane cannot be reversed by incubation with ATP. Furthermore, following binding to membranes, dynein retains its ability to bind ATP and to be photo-cleaved in the presence of vanadate. The presence of cytoplasmic dynein on synaptic vesicles and its ability to bind to extracted membranes supports current models of microtubule-based organelle translocation.     

No Conventional Function for the Conventional Kinesin?

A paper by DeGiorgis et al. (DeGiorgis JA, Petukhova TA, Evans TA, Reese TS. Kinesin-3 is an organelle motor in the squid giant axon. Traffic 2008; DOI: 10.1111/j.1600-0854.2008.00809.x) in this issue of Traffic reports on the identification and function of a second squid kinesin, a kinesin-3 motor. As expected, the newly discovered motor associates with axoplasmic organelles in situ and powers motility along microtubules of vesicles isolated from squid axoplasm. Less expected was the finding that kinesin-3 may be the predominant motor for anterograde organelle movement in the squid axon, which challenges the so far undisputed view that this function is fulfilled by the conventional kinesin, kinesin-1. These novel findings let us wonder what the real function of kinesin-1--the most abundant motor in squid axons--actually is.     

Dynactin-dependent, dynein-driven vesicle transport in the absence of membrane proteins: a role for spectrin and acidic phospholipids

We reconstituted dynein-driven, dynactin-dependent vesicle transport using protein-free liposomes and soluble components from squid axoplasm. Dynein and dynactin, while necessary, are not the only essential cytosolic factors; axonal spectrin is also required. Spectrin is resident on axonal vesicles, and rebinds from cytosol to liposomes or proteolysed vesicles, concomitant with their dynein-dynactin-dependent motility. Binding of purified axonal spectrin to liposomes requires acidic phospholipids, as does motility. Using dominant negative spectrin polypeptides and a drug that releases PH domains from membranes, we show that spectrin is required for linking dynactin, and thereby dynein, to acidic phospholipids in the membrane. We verify this model in the context of liposomes, isolated axonal vesicles, and whole axoplasm. We conclude that spectrin has an essential role in retrograde axonal transport.