The small GTPase Rab5 regulates membrane docking and fusion in the early endocytic pathway. Here we reveal a new role for Rab5 in the regulation of endosome interactions with the microtubule network. ...Using Rab5 fused to green fluorescent protein we show that Rab5-positive endosomes move on microtubules in vivo. In vitro, Rab5 stimulates both association of early endosomes with microtubules and early-endosome motility towards the minus ends of microtubules. Moreover, similarly to endosome membrane docking and fusion, Rab5-dependent endosome movement depends on the phosphatidylinositol-3-OH kinase hVPS34. Thus, Rab5 functionally links regulation of membrane transport, motility and intracellular distribution of early endosomes.
Microtubules are dynamic polymers that interconvert between periods of slow growth and fast shrinkage. The energy driving this nonequilibrium behavior comes from the hydrolysis of GTP, which is ...required to destabilize the microtubule lattice. To understand the mechanism of this destabilization, cryo-electron microscopy was used to compare the structure of the ends of shrinking microtubules assembled in the presence of either GTP or the slowly hydrolyzable analogue guanylyl (α,β)methylenediphosphonate (GMPCPP). Depolymerization was induced by cold or addition of calcium. With either nucleotide, we have observed curled oligomers at the ends of shrinking microtubules. However, GDP oligomers were consistently more curved than GMPCPP oligomers. This difference in curvature between depolymerizing GDP and GMPCPP protofilaments suggests that GTP hydrolysis is accompanied by an increase in curvature of the protofilaments, thereby destabilizing the lateral interactions between tubulin subunits in the microtubule lattice.
It was recently shown that, in addition to the well-established microtubule-dependent mechanism, fast transport of organelles in squid giant axons also occurs in the presence of actin filaments ...Kuznetsov et al., 1992, Nature 356:722-725. The objectives of this study were to obtain direct evidence of axoplasmic organelle movement on actin filaments and to demonstrate that these organelles are able to move on skeletal muscle actin filaments. Organelles and actin filaments were visualized by video-enhanced contrast differential interference contrast (AVEC-DIC) microscopy and by video intensified fluorescence microscopy. Actin filaments, prepared by polymerization of monomeric actin purified from rabbit skeletal muscle, were stabilized with rhodamine-phalloidin and adsorbed to cover slips. When axoplasm was extruded on these cover slips in the buffer containing cytochalasin B that prevents the formation of endogenous axonal actin filaments, organelles were observed to move at the fast transport rate. Also, axoplasmic organelles were observed to move on bundles of actin filaments that were of sufficient thickness to be detected directly by AVEC-DIC microscopy. The range of average velocities of movement on the muscle actin filaments was not statistically different from that on axonal filaments. The level of motile activity (number of organelles moving/min/field) on the exogenous filaments was less than on endogenous filaments probably due to the entanglement of filaments on the cover slip surface. We also found that calmodulin (CaM) increased the level of motile activity of organelles on actin filaments. In addition, CaM stimulated the movement of elongated membranous organelles that appeared to be tubular elements of smooth endoplasmic reticulum or extensions of prelysosomes. These studies provide the first direct evidence that organelles from higher animal cells such as neurons move on biochemically defined actin filaments.
The molecular motor kinesin is an ATPase that mediates plus end-directed transport of organelles along microtubules. Although the biochemical properties of kinesin are extensively studied, conclusive ...data on regulation of kinesin-mediated transport are largely lacking. Previously, we showed that the proinflammatory cytokine tumor necrosis factor induces perinuclear clustering of mitochondria. Here, we show that tumor necrosis factor impairs kinesin motor activity and hyperphosphorylates kinesin light chain through activation of two putative kinesin light chain kinases. Inactivation of kinesin, hyperphosphorylation of kinesin light chain, and perinuclear clustering of mitochondria exhibit the same p38 mitogen-activated kinase dependence, indicating their functional relationship. These data provide evidence for direct regulation of kinesin-mediated organelle transport by extracellular stimuli via cytokine receptor signaling pathways.
At the metaphase to anaphase transition, chromosome segregation is initiated by the splitting of sister chromatids. Subsequently, spindles elongate, separating the sister chromosomes into two sets. ...Here, we investigate the cell cycle requirements for spindle elongation in budding yeast using mutants affecting sister chromatid cohesion or DNA replication. We show that separation of sister chromatids is not sufficient for proper spindle integrity during elongation. Rather, successful spindle elongation and stability require both sister chromatid separation and anaphase-promoting complex activation. Spindle integrity during elongation is dependent on proteolysis of the securin Pds1 but not on the activity of the separase Esp1. Our data suggest that stabilization of the elongating spindle at the metaphase to anaphase transition involves Pds1-dependent targets other than Esp1.
High-resolution atomic force microscopy (AFM) was applied to directly observe the dynamic assembly of single actin filaments in HeLa cell extracts in vitro. The F-actin filaments established a ...dynamic network and formed different types of junctions and branches. The connections of this network were X-, Y- or T-shaped. It was found that the actin filaments were densely covered by endosomes and vesicles from the cell extract, which are thought to stabilize their structures. Using time-lapse AFM, the growth, shrinkage, branching and the interaction of actin filaments with endosomes could be characterized. Our results indicate that the majority of F-actin filaments are static in HeLa extract and that only a minor fraction of filaments undergo dynamic changes. Furthermore, the AFM imaging approach not only provides unique insights into the assembly and dynamics of actin networks; it also builds an avenue to study in vitro assays of complex biological systems.
Thirteen Is the Lucky Number for Doublecortin Akhmanova, Anna; Severin, Fedor
Developmental Cell,
July 2004, 2004-Jul, 2004-07-00, 20040701, Volume:
7, Issue:
1
Book Review, Journal Article
Peer reviewed
Open access
Doublecortin is a microtubule-associated protein that is essential for normal brain development. A recent report published in Molecular Cell shows that doublecortin associates preferentially with ...microtubules made of 13 protofilaments, by recognizing a novel site between the protofilaments. These findings explain how doublecortin stabilizes microtubules and provide clues about its function during neuronal migration.
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