Journal of Cell Biology
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Aurora-A kinase is required for centrosome maturation in <i>Caenorhabditis elegans </i> Centrosomes mature as cells enter mitosis, accumulating γ-tubulin and other pericentriolar material (PCM) components. This occurs concomitant with an increase in the number of centrosomally organized microtubules (MTs). Here, we use RNA-mediated interference (RNAi) to examine the role of the aurora-A kinase, AIR-1, during centrosome maturation in Caenorhabditis elegans. In air-1(RNAi) embryos, centrosomes separate normally, an event that occurs before maturation in C. elegans. After nuclear envelope breakdown, the separated centrosomes collapse together, and spindle assembly fails. In mitotic air-1(RNAi) embryos, centrosomal α-tubulin fluorescence intensity accumulates to only 40% of wild-type levels, suggesting a defect in the maturation process. Consistent with this hypothesis, we find that AIR-1 is required for the increase in centrosomal γ-tubulin and two other PCM components, ZYG-9 and CeGrip, as embryos enter mitosis. Furthermore, the AIR-1–dependent increase in centrosomal γ-tubulin does not require MTs. These results suggest that aurora-A kinases are required to execute a MT-independent pathway for the recruitment of PCM during centrosome maturation.
Journal of Cell Biology - Tập 155 Số 7 - Trang 1109-1116 - 2001
A dynamic ubiquitin equilibrium couples proteasomal activity to chromatin remodeling Protein degradation, chromatin remodeling, and membrane trafficking are critically regulated by ubiquitylation. The presence of several coexisting ubiquitin-dependent processes, each of crucial importance to the cell, is remarkable. This brings up questions on how the usage of this versatile regulator is negotiated between the different cellular processes. During proteotoxic stress, the accumulation of ubiquitylated substrates coincides with the depletion of ubiquitylated histone H2A and chromatin remodeling. We show that this redistribution of ubiquitin during proteotoxic stress is a direct consequence of competition for the limited pool of free ubiquitin. Thus, the ubiquitin cycle couples various ubiquitin-dependent processes because of a rate-limiting pool of free ubiquitin. We propose that this ubiquitin equilibrium may allow cells to sense proteotoxic stress in a genome-wide fashion.
Journal of Cell Biology - Tập 173 Số 1 - Trang 19-26 - 2006
<i>Drosophila</i> Aurora B Kinase Is Required for Histone H3 Phosphorylation and Condensin Recruitment during Chromosome Condensation and to Organize the Central Spindle during Cytokinesis Aurora/Ipl1-related kinases are a conserved family of enzymes that have multiple functions during mitotic progression. Although it has been possible to use conventional genetic analysis to dissect the function of aurora, the founding family member in Drosophila (Glover, D.M., M.H. Leibowitz, D.A. McLean, and H. Parry. 1995. Cell. 81:95–105), the lack of mutations in a second aurora-like kinase gene, aurora B, precluded this approach. We now show that depleting Aurora B kinase using double-stranded RNA interference in cultured Drosophila cells results in polyploidy. aurora B encodes a passenger protein that associates first with condensing chromatin, concentrates at centromeres, and then relocates onto the central spindle at anaphase. Cells depleted of the Aurora B kinase show only partial chromosome condensation at mitosis. This is associated with a reduction in levels of the serine 10 phosphorylated form of histone H3 and a failure to recruit the Barren condensin protein onto chromosomes. These defects are associated with abnormal segregation resulting from lagging chromatids and extensive chromatin bridging at anaphase, similar to the phenotype of barren mutants (Bhat, M.A., A.V. Philp, D.M. Glover, and H.J. Bellen. 1996. Cell. 87:1103–1114.). The majority of treated cells also fail to undertake cytokinesis and show a reduced density of microtubules in the central region of the spindle. This is accompanied by a failure to correctly localize the Pavarotti kinesin-like protein, essential for this process. We discuss these conserved functions of Aurora B kinase in chromosome transmission and cytokinesis.
Journal of Cell Biology - Tập 152 Số 4 - Trang 669-682 - 2001
Persistent activation of Nrf2 through p62 in hepatocellular carcinoma cells Suppression of autophagy is always accompanied by marked accumulation of p62, a selective autophagy substrate. Because p62 interacts with the Nrf2-binding site on Keap1, which is a Cullin 3–based ubiquitin ligase adapter protein, autophagy deficiency causes competitive inhibition of the Nrf2–Keap1 interaction, resulting in stabilization of Nrf2 followed by transcriptional activation of Nrf2 target genes. Herein, we show that liver-specific autophagy-deficient mice harbor adenomas linked to both the formation of p62- and Keap1-positive cellular aggregates and induction of Nrf2 targets. Importantly, similar aggregates were identified in more than 25% of human hepatocellular carcinomas (HCC), and induction of Nrf2 target genes was recognized in most of these tumors. Gene targeting of p62 in an HCC cell line markedly abrogates the anchorage-independent growth, whereas forced expression of p62, but not a Keap1 interaction-defective mutant, resulted in recovery of the growth defect. These results indicate the involvement of persistent activation of Nrf2 through the accumulation of p62 in hepatoma development.
Journal of Cell Biology - Tập 193 Số 2 - Trang 275-284 - 2011
Biogenesis of the multifunctional lipid droplet: Lipids, proteins, and sites Lipid droplets (LDs) are ubiquitous dynamic organelles that store and supply lipids in all eukaryotic and some prokaryotic cells for energy metabolism, membrane synthesis, and production of essential lipid-derived molecules. Interest in the organelle’s cell biology has exponentially increased over the last decade due to the link between LDs and prevalent human diseases and the discovery of new and unexpected functions of LDs. As a result, there has been significant recent progress toward understanding where and how LDs are formed, and the specific lipid pathways that coordinate LD biogenesis.
Journal of Cell Biology - Tập 204 Số 5 - Trang 635-646 - 2014
p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and coimmunoprecipitated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutant huntingtin. We suggest that p62 may, via LC3, be involved in linking polyubiquitinated protein aggregates to the autophagy machinery.
Journal of Cell Biology - Tập 171 Số 4 - Trang 603-614 - 2005
Tiam–Rac signaling mediates trans-endocytosis of ephrin receptor EphB2 and is important for cell repulsion Ephrin receptors interact with membrane-bound ephrin ligands to regulate contact-mediated attraction or repulsion between opposing cells, thereby influencing tissue morphogenesis. Cell repulsion requires bidirectional trans-endocytosis of clustered Eph–ephrin complexes at cell interfaces, but the mechanisms underlying this process are poorly understood. Here, we identified an actin-regulating pathway allowing ephrinB+ cells to trans-endocytose EphB receptors from opposing cells. Live imaging revealed Rac-dependent F-actin enrichment at sites of EphB2 internalization, but not during vesicle trafficking. Systematic depletion of Rho family GTPases and their regulatory proteins identified the Rac subfamily and the Rac-specific guanine nucleotide exchange factor Tiam2 as key components of EphB2 trans-endocytosis, a pathway previously implicated in Eph forward signaling, in which ephrins act as in trans ligands of Eph receptors. However, unlike in Eph signaling, this pathway is not required for uptake of soluble ligands in ephrinB+ cells. We also show that this pathway is required for EphB2-stimulated contact repulsion. These results support the existence of a conserved pathway for EphB trans-endocytosis that removes the physical tether between cells, thereby enabling cell repulsion.
Journal of Cell Biology - Tập 214 Số 6 - Trang 735-752 - 2016
Exosomes mediate cell contact–independent ephrin-Eph signaling during axon guidance The cellular release of membranous vesicles known as extracellular vesicles (EVs) or exosomes represents a novel mode of intercellular communication. Eph receptor tyrosine kinases and their membrane-tethered ephrin ligands have very important roles in such biologically diverse processes as neuronal development, plasticity, and pathological diseases. Until now, it was thought that ephrin-Eph signaling requires direct cell contact. Although the biological functions of ephrin-Eph signaling are well understood, our mechanistic understanding remains modest. Here we report the release of EVs containing Ephs and ephrins by different cell types, a process requiring endosomal sorting complex required for transport (ESCRT) activity and regulated by neuronal activity. Treatment of cells with purified EphB2+ EVs induces ephrinB1 reverse signaling and causes neuronal axon repulsion. These results indicate a novel mechanism of ephrin-Eph signaling independent of direct cell contact and proteolytic cleavage and suggest the participation of EphB2+ EVs in neural development and synapse physiology.
Journal of Cell Biology - Tập 214 Số 1 - Trang 35-44 - 2016
Myosin 1b functions as an effector of EphB signaling to control cell repulsion Eph receptors and their membrane-tethered ligands, the ephrins, have important functions in embryo morphogenesis and in adult tissue homeostasis. Eph/ephrin signaling is essential for cell segregation and cell repulsion. This process is accompanied by morphological changes and actin remodeling that drives cell segregation and tissue patterning. The actin cortex must be mechanically coupled to the plasma membrane to orchestrate the cell morphology changes. Here, we demonstrate that myosin 1b that can mechanically link the membrane to the actin cytoskeleton interacts with EphB2 receptors via its tail and is tyrosine phosphorylated on its tail in an EphB2-dependent manner. Myosin 1b regulates the redistribution of myosin II in actomyosin fibers and the formation of filopodia at the interface of ephrinB1 and EphB2 cells, which are two processes mediated by EphB2 signaling that contribute to cell repulsion. Together, our results provide the first evidence that a myosin 1 functions as an effector of EphB2/ephrinB signaling, controls cell morphology, and thereby cell repulsion.
Journal of Cell Biology - Tập 210 Số 2 - Trang 347-361 - 2015
Gulp1 controls Eph/ephrin trogocytosis and is important for cell rearrangements during development Trogocytosis, in which cells nibble away parts of neighboring cells, is an intercellular cannibalism process conserved from protozoa to mammals. Its underlying molecular mechanisms are not well understood and are likely distinct from phagocytosis, a process that clears entire cells. Bi-directional contact repulsion induced by Eph/ephrin signaling involves transfer of membrane patches and full-length Eph/ephrin protein complexes between opposing cells, resembling trogocytosis. Here, we show that the phagocytic adaptor protein Gulp1 regulates EphB/ephrinB trogocytosis to achieve efficient cell rearrangements of cultured cells and during embryonic development. Gulp1 mediates trogocytosis bi-directionally by dynamic engagement with EphB/ephrinB protein clusters in cooperation with the Rac-specific guanine nucleotide exchange factor Tiam2. Ultimately, Gulp1’s presence at the Eph/ephrin cluster is a prerequisite for recruiting the endocytic GTPase dynamin. These results suggest that EphB/ephrinB trogocytosis, unlike other trogocytosis events, uses a phagocytosis-like mechanism to achieve efficient membrane scission and engulfment.
Journal of Cell Biology - Tập 218 Số 10 - Trang 3455-3471 - 2019
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