Biomedical subjects
Publications and source records attributed to B J Schnapp.
Specific mRNAs achieve an asymmetric distribution in the cell by linking to molecular motors that walk along the cytoskeleton. Studies in S. cerevisiae have begun to define the nature of the RNA-motor linkage and identify She3p as an adaptor protein that links a type V myosin motor to specific ribonucleoproteins.
The cargo that the molecular motor kinesin moves along microtubules has been elusive. We searched for binding partners of the COOH terminus of kinesin light chain, which contains tetratricopeptide repeat (TPR) motifs. Three proteins were found, the c-jun NH(2)-terminal kinase (JNK)-interacting proteins (JIPs) JIP-1, JIP-2, and JIP-3, which are scaffolding proteins for the JNK signaling pathway. Concentration of JIPs in nerve terminals requires kinesin, as evident from the analysis of JIP COOH-terminal mutants and dominant negative kinesin constructs. Coprecipitation experiments suggest that kinesin carries the JIP scaffolds preloaded with cytoplasmic (dual leucine zipper-bearing kinase) and transmembrane signaling molecules (the Reelin receptor, ApoER2). These results demonstrate a direct interaction between conventional kinesin and a cargo, indicate that motor proteins are linked to their membranous cargo via scaffolding proteins, and support a role for motor proteins in spatial regulation of signal transduction pathways.
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.
Asymmetric mRNA localization within cells plays an important part in both development and physiology. Recent studies have provided a glimpse of the conserved molecular machinery that directs the localization of specific mRNAs.
Kinesin motors are presumed to transport various membrane compartments within neurons, but their specific in vivo functions, cargoes, and expression patterns in the brain are unclear. We have investigated the distribution of KIF3A, a member of the heteromeric family of kinesins, in the vertebrate retina. We find KIF3A at two distinct sites within photoreceptors: at the basal body of the connecting cilium axoneme and at the synaptic ribbon. Immunoelectron microscopy of the photoreceptor ribbon synapse shows KIF3A to be concentrated both at the ribbon matrix and on vesicles docked at the ribbon, a result that is consistent with the presence of both detergent-extractable and resistant KIF3A fractions at these synapses. KIF3A is also present in the inner plexiform layer, again at presynaptic ribbons. These findings suggest that within a single cell, the photoreceptor, one kinesin polypeptide, KIF3A, can serve two distinct functions, one specific for ribbon synapses.
We have investigated the mechanism by which conventional kinesin is prevented from binding to microtubules (MTs) when not transporting cargo. Kinesin heavy chain (HC) was expressed in COS cells either alone or with kinesin light chain (LC). Immunofluorescence microscopy and MT cosedimentation experiments demonstrate that the binding of HC to MTs is inhibited by coexpression of LC. Association between the chains involves the LC NH2-terminal domain, including the heptad repeats, and requires a region of HC that includes the conserved region of the stalk domain and the NH2 terminus of the tail domain. Inhibition of MT binding requires in addition the COOH-terminal 64 amino acids of HC. Interaction between the tail and the motor domains of HC is supported by sedimentation experiments that indicate that kinesin is in a folded conformation. A pH shift from 7.2 to 6.8 releases inhibition of kinesin without changing its sedimentation behavior. Endogenous kinesin in COS cells also shows pH-sensitive inhibition of MT binding. Taken together, our results provide evidence that a function of LC is to keep kinesin in an inactive ground state by inducing an interaction between the tail and motor domains of HC; activation for cargo transport may be triggered by a small conformational change that releases the inhibition of the motor domain for MT binding.
BACKGROUND: Cytoplasmic mRNA localization is a widespread mechanism for restricting the translation of specific mRNAs to distinct regions of eucaryotic cells. This process involves specific interactions between cellular factors and localization signals in the 3' untranslated regions of the localized mRNA. Because only a few of these cellular factors have been identified, it is not known whether common factors are utilized for the localization of different mRNAs. We recently discovered Vera, a protein that binds specifically to the Vg1 localization element and is involved in the localization of Vg1 mRNA in Xenopus oocytes. RESULTS: To characterize further the role of Vera in the localization of Vg1 mRNA, we have purified the Vera protein and cloned its gene. Vera is homologous to chicken zip-code-binding protein (ZBP), which binds to a short RNA sequence required for localization of beta-actin mRNA in chick embryo fibroblasts. The predicted amino-acid sequences of Vera and ZBP contain five RNA-binding domains and putative signals for nuclear localization and export. Like the binding of ZBP to beta-actin mRNA, Vera specifically binds to a repeated sequence motif in the Vg1 localization element that is required for Vg1 mRNA localization in Xenopus oocytes. CONCLUSIONS: Vera, a highly conserved component of the mRNA localization machinery, participates in localizing different mRNAs in different cell types. Thus, Vera appears to be a general factor for mRNA localization, and additional factors may be required to specify diverse patterns of RNA localization.
We have cloned from rat brain the cDNA encoding an 89,828-Da kinesin-related polypeptide KIF3C that is enriched in brain, retina, and lung. Immunocytochemistry of hippocampal neurons in culture shows that KIF3C is localized to cell bodies, dendrites, and, in lesser amounts, to axons. In subcellular fractionation experiments, KIF3C cofractionates with a distinct population of membrane vesicles. Native KIF3C binds to microtubules in a kinesin-like, nucleotide-dependent manner. KIF3C is most similar to mouse KIF3B and KIF3A, two closely related kinesins that are normally present as a heteromer. In sucrose density gradients, KIF3C sediments at two distinct densities, suggesting that it may be part of two different multimolecular complexes. Immunoprecipitation experiments show that KIF3C is in part associated with KIF3A, but not with KIF3B. Unlike KIF3B, a significant portion of KIF3C is not associated with KIF3A. Consistent with these biochemical properties, the distribution of KIF3C in the CNS has both similarities and differences compared with KIF3A and KIF3B. These results suggest that KIF3C is a vesicle-associated motor that functions both independently and in association with KIF3A.
In many organisms, pattern formation in the embryo develops from the polarized distributions of messenger RNAs (mRNAs) in the egg. In Xenopus, the mRNA encoding Vg1, a growth factor involved in mesoderm induction, is localized to the vegetal cortex of oocytes. A protein named Vera was shown to be involved in Vg1 mRNA localization. Vera cofractionates with endoplasmic reticulum (ER) membranes, and endogenous Vg1 mRNA is associated with a subcompartment of the ER. Vera may promote mRNA localization in Xenopus oocytes by mediating an interaction between the Vg1 3' untranslated region and the ER subcompartment.
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.
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In the budding yeast Saccharomyces cerevisiae, the spindle pole body (SPB) serves as the microtubule-organizing center and is the functional analog of the centrosome of higher organisms. By expressing a fusion of a yeast SPB-associated protein to the Aequorea victoria green fluorescent protein, the movement of the SPBs in living yeast cells undergoing mitosis was observed by fluorescence microscopy. The ability to visualize SPBs in vivo has revealed previously unidentified mitotic events. During anaphase, the mitotic spindle has four sequential activities: alignment at the mother-daughter junction, fast elongation, translocation into the bud, and slow elongation. These results indicate that distinct forces act upon the spindle at different times during anaphase.
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Do biological motors move with regular steps? To address this question, we constructed instrumentation with the spatial and temporal sensitivity to resolve movement on a molecular scale. We deposited silica beads carrying single molecules of the motor protein kinesin on microtubules using optical tweezers and analysed their motion under controlled loads by interferometry. We find that kinesin moves with 8-nm steps.
Kinesin is a cytoplasmic motor protein that moves along microtubules and can induce microtubule bundling and sliding in vitro. To determine how kinesin mediates microtubule interactions, we determined the shapes and mass distributions of squid brain kinesin, taxol-stabilized microtubules (squid and bovine), and adenosine 5'-[beta, gamma-imido]triphosphate-stabilized kinesin-microtubule complexes by high-resolution metal replication and by low-temperature, low-dose dark-field scanning transmission electron microscopy of unfixed, directly frozen preparations. Mass mapping by electron microscopy revealed kinesins loosely attached to the carbon support as asymmetrical dumbbell-shaped molecules, 40-52 nm long, with a mass of 379 +/- 15 kDa. The mass distribution and shape of these molecules suggest that these images represent kinesin in a shortened conformation. Kinesin-microtubule complexes were organized as bundles of linearly arrayed microtubules, stitched together at irregular intervals by cross-bridges typically < or = 25 nm long. The crossbridges had a mass of 360 +/- 15 kDa, consistent with one kinesin per crossbridge. These results suggest that kinesin has a second microtubule binding site in addition to the known site on the motor domain of the heavy chain; this second site may be located near the C terminus of the heavy chains or on the associated light chains. Thus, kinesin could play a role in either crosslinking or sliding microtubules.
This paper addresses the question of whether microtubule-directed transport of vesicular organelles depends on the presence of a pool of cytosolic factors, including soluble motor proteins and accessory factors. Earlier studies with squid axon organelles (Schroer et al., 1988) suggested that the presence of cytosol induces a > 20-fold increase in the number of organelles moving per unit time on microtubules in vitro. These earlier studies, however, did not consider that cytosol might nonspecifically increase the numbers of moving organelles, i.e., by blocking adsorption of organelles to the coverglass. Here we report that treatment of the coverglass with casein, in the absence of cytosol, blocks adsorption of organelles to the coverglass and results in vigorous movement of vesicular organelles in the complete absence of soluble proteins. This technical improvement makes it possible, for the first time, to perform quantitative studies of organelle movement in the absence of cytosol. These new studies show that organelle movement activity (numbers of moving organelles/min/micron microtubule) of unextracted organelles is not increased by cytosol. Unextracted organelles move in single directions, approximately two thirds toward the plus-end and one third toward the minus-end of microtubules. Extraction of organelles with 600 mM KI completely inhibits minus-end, but not plus-end directed organelle movement. Upon addition of cytosol, minus-end directed movement of KI organelles is restored, while plus--end directed movement is unaffected. Biochemical studies indicate that KI-extracted organelles attach to microtubules in the presence of AMP-PNP and copurify with tightly bound kinesin. The bound kinesin is not extracted from organelles by 1 M KI, 1 M NaCl or carbonate (pH 11.3). These results suggest that kinesin is irreversibly bound to organelles that move to the plus-end of microtubules and that the presence of soluble kinesin and accessory factors is not required for movement of plus-end organelles in squid axons.
Kinesin, a mechanoenzyme that couples ATP hydrolysis to movement along microtubules, is thought to power vesicle transport and other forms of microtubule-based motility. Here, microscopic silica beads were precoated with carrier protein, exposed to low concentrations of kinesin, and individually manipulated with a single-beam gradient-force optical particle trap ('optical tweezers') directly onto microtubules. Optical tweezers greatly improved the efficiency of the bead assay, particularly at the lowest kinesin concentrations (corresponding to approximately 1 molecule per bead). Beads incubated with excess kinesin moved smoothly along a microtubule for many micrometres, but beads carrying from 0.17-3 kinesin molecules per bead, moved, on average, only about 1.4 microns and then spontaneously released from the microtubule. Application of the optical trap directly behind such moving beads often pulled them off the microtubule and back into the centre of the trap. This did not occur when a bead was bound by an AMP.PNP-induced rigor linkage, or when beads were propelled by several kinesin molecules. Our results are consistent with a model in which kinesin detaches briefly from the microtubule during a part of each mechanochemical cycle, rather than a model in which kinesin remains bound at all times.