CLIP-associating protein (CLASP) 1 and CLASP2 are mammalian microtubule (MT) plus-end binding proteins, which associate with CLIP-170 and CLIP-115. Using RNA interference in HeLa cells, we show that the two CLASPs play redundant roles in regulating the density, length distribution and stability of interphase MTs. In HeLa cells, both CLASPs concentrate on the distal MT ends in a narrow region at the cell margin. CLASPs stabilize MTs by promoting pauses and restricting MT growth and shortening episodes to this peripheral cell region. We demonstrate that the middle part of CLASPs binds directly to EB1 and to MTs. Furthermore, we show that the association of CLASP2 with the cell cortex is MT independent and relies on its COOH-terminal domain. Both EB1- and cortex-binding domains of CLASP are required to promote MT stability. We propose that CLASPs can mediate interactions between MT plus ends and the cell cortex and act as local rescue factors, possibly through forming a complex with EB1 at MT tips.
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.
Chromosome segregation depends on kinetochores, the structures that mediate chromosome attachment to the mitotic spindle. We isolated mutants in IPL1, which encodes a protein kinase, in a screen for budding yeast mutants that have defects in sister chromatid separation and segregation. Cytological tests show that ipl1 mutants can separate sister chromatids but are defective in chromosome segregation. Kinetochores assembled in extracts from ipl1 mutants show altered binding to microtubules. Ipl1p phosphorylates the kinetochore component Ndc10p in vitro and we propose that Ipl1p regulates kinetochore function via Ndc10p phosphorylation. Ipl1p localizes to the mitotic spindle and its levels are regulated during the cell cycle. This pattern of localization and regulation is similar to that of Ipl1p homologs in higher eukaryotes, such as the human aurora2 protein. Because aurora2 has been implicated in oncogenesis, defects in kinetochore function may contribute to genetic instability in human tumors.
Different classes of endosomes exhibit a characteristic intracellular steady-state distribution governed by interactions with the cytoskeleton. We found a kinesin-3, KIF16B, that transports early endosomes to the plus end of microtubules in a process regulated by the small GTPase Rab5 and its effector, the phosphatidylinositol-3-OH kinase hVPS34. In vivo, KIF16B overexpression relocated early endosomes to the cell periphery and inhibited transport to the degradative pathway. Conversely, expression of dominant-negative mutants or ablation of KIF16B by RNAi caused the clustering of early endosomes to the perinuclear region, delayed receptor recycling to the plasma membrane, and accelerated degradation. These results suggest that KIF16B, by regulating the plus end motility of early endosomes, modulates the intracellular localization of early endosomes and the balance between receptor recycling and degradation. We propose that this mechanism could have important implications for signaling.
Although programmed cell death (PCD) is extensively studied in multicellular organisms, in recent years it has been shown that a unicellular organism, yeast Saccharomyces cerevisiae, also possesses death program(s). In particular, we have found that a high doses of yeast pheromone is a natural stimulus inducing PCD. Here, we show that the death cascades triggered by pheromone and by a drug amiodarone are very similar. We focused on the role of mitochondria during the pheromone/amiodarone-induced PCD. For the first time, a functional chain of the mitochondria-related events required for a particular case of yeast PCD has been revealed: an enhancement of mitochondrial respiration and of its energy coupling, a strong increase of mitochondrial membrane potential, both events triggered by the rise of cytoplasmic [Ca2+], a burst in generation of reactive oxygen species in center o of the respiratory chain complex III, mitochondrial thread-grain transition, and cytochrome c release from mitochondria. A novel mitochondrial protein required for thread-grain transition is identified.
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 (␣,)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.
Microtubules facilitate the maturation of phagosomes by favoring their interactions with endocytic compartments. Here, we show that phagosomes move within cells along tracks of several microns centrifugally and centripetally in a pH- and microtubuledependent manner. Phagosome movement was reconstituted in vitro and required energy, cytosol and membrane proteins of this organelle. The activity or presence of these phagosome proteins was regulated as the organelle matured, with “late” phagosomes moving threefold more frequently than “early” ones. The majority of moving phagosomes were minus-end directed; the remainder moved towards microtubule plus-ends and a small subset moved bi-directionally. Minus-end movement showed pharmacological characteristics expected for dyneins, was inhibited by immunodepletion of cytoplasmic dynein and could be restored by addition of cytoplasmic dynein. Plus-end movement displayed pharmacological properties of kinesin, was inhibited partially by immunodepletion of kinesin and fully by addition of an anti-kinesin IgG. Immunodepletion of dynactin, a dynein-activating complex, inhibited only minus-end directed motility. Evidence is provided for a dynactin-associated kinase required for dyneinmediated vesicle transport. Movement in both directions was inhibited by peptide fragments from kinectin (a putative kinesin membrane receptor), derived from the region to which a motility-blocking antibody binds. Polypeptide subunits from these microtubule-based motility factors were detected on phagosomes by immunoblotting or immunoelectron microscopy. This is the first study using a single in vitro system that describes the roles played by kinesin, kinectin, cytoplasmic dynein, and dynactin in the microtubule-mediated movement of a purified membrane organelle.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.