Under stress, certain eukaryotic proteins and RNA assemble to form membraneless organelles known as stress granules. The most well-studied stress granule components are RNA-binding proteins that undergo liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by intrinsically disordered low-complexity domains (LCDs). Here we show that stress granules include proteasomal shuttle factor UBQLN2, an LCD-containing protein structurally and functionally distinct from RNA-binding proteins. In vitro, UBQLN2 exhibits LLPS at physiological conditions. Deletion studies correlate oligomerization with UBQLN2's ability to phase-separate and form stress-induced cytoplasmic puncta in cells. Using nuclear magnetic resonance (NMR) spectroscopy, we mapped weak, multivalent interactions that promote UBQLN2 oligomerization and LLPS. Ubiquitin or polyubiquitin binding, obligatory for UBQLN2's biological functions, eliminates UBQLN2 LLPS, thus serving as a switch between droplet and disperse phases. We postulate that UBQLN2 LLPS enables its recruitment to stress granules, where its interactions with ubiquitinated substrates reverse LLPS to enable shuttling of clients out of stress granules.
During interphase, Rab11-GTPase-containing endosomes recycle endocytic cargo. However, little is known about Rab11 and endosomes in mitosis. Here we show that Rab11 localizes to the mitotic spindle and regulates dynein-dependent endosome localization at poles. We found that mitotic recycling endosomes bind γ-TuRC components and associate with tubulin in vitro. Rab11-depletion or dominant-negative Rab11 expression disrupts astral microtubules, delays mitosis, and redistributes spindle pole proteins. Reciprocally, constitutively-active Rab11 increases astral microtubules, restores γ-tubulin spindle pole localization and generates robust spindles. This suggests a fundamental role for Rab11 activity in spindle pole maturation during mitosis. Rab11 depletion causes misorientation of the mitotic spindle and the plane of cell division. These findings suggest a molecular mechanism for the organization of astral microtubules and the mitotic spindle through Rab11-dependent control of spindle pole assembly and function. We propose that Rab11 and its associated endosomes co-contribute to these processes through retrograde transport to poles by dynein.
Summary The recycling endosome localizes to a pericentrosomal region via microtubule-dependent transport. We previously showed that Sec15, an effector of the recycling endosome component, Rab11-GTPase, interacts with the mother centriole appendage protein, centriolin, suggesting an interaction between endosomes and centrosomes (1, 2). Here we show that the recycling endosome associates with the appendages of the mother (older) centriole. We show that the mother centriole appendage proteins, centriolin and cenexin/ODF2, regulate association of the endosome components Rab11, the Rab11 GTP-activating protein Evi5, and the exocyst at the mother centriole. Development of an in vitro method for reconstituting endosome protein complexes onto isolated membrane-free centrosomes demonstrates that purified GTP-Rab11 but not GDP-Rab11 binds to mother centriole appendages in the absence of membranes. Moreover, centriolin depletion displaces the centrosomal Rab11 GAP, Evi5, and increases mother-centriole-associated Rab11; depletion of Evi5 also increases centrosomal Rab11. This indicates that centriolin localizes Evi5 to centriolar appendages to turn off centrosomal Rab11 activity. Finally, centriolin depletion disrupts recycling endosome organization and function suggesting a role for mother centriole proteins in the regulation of Rab11 localization and activity at the mother centriole.
Proteasomal shuttle factor UBQLN2 is recruited to stress granules and undergoes liquid-liquid phase separation (LLPS) into protein-containing droplets. Mutations to UBQLN2 have recently been shown to cause dominant X-linked inheritance of amyotrophic lateral sclerosis (ALS) and ALS/dementia. Interestingly, most of these UBQLN2 mutations reside in its proline-rich (Pxx) region, an important modulator of LLPS. Here, we demonstrated that ALS-linked Pxx mutations differentially affect UBQLN2 LLPS, depending on both amino acid substitution and sequence position. Using size-exclusion chromatography, analytical ultracentrifugation, microscopy, and NMR spectroscopy, we determined that those Pxx mutants that enhanced UBQLN2 oligomerization decreased saturation concentrations needed for LLPS and promoted solid-like and viscoelastic morphological changes to UBQLN2 liquid assemblies. Ubiquitin disassembled all LLPS-induced mutant UBQLN2 aggregates. We postulate that the changes in physical properties caused by ALS-linked Pxx mutations modify UBQLN2 behavior in vivo, possibly contributing to aberrant stress granule morphology and dynamics, leading to formation of inclusions, pathological characteristics of ALS.
SUMMARY Majewski Osteodysplastic Primordial Dwarfism type II (MOPDII) is caused by mutations in the centrosome gene pericentrin (PCNT) which lead to severe pre- and post-natal growth retardation[1]. As in MOPDII patients, disruption of pericentrin (Pcnt) in mice caused a number of abnormalities including microcephaly, aberrant hemodynamics analyzed by in utero echocardiography and cardiovascular anomalies; the latter being associated with mortality, as in the human condition[1]. To identify the mechanisms underlying these defects, we tested for changes in cell and molecular function. All Pcnt−/− mouse tissues and cells examined showed spindle misorientation. This mouse phenotype was associated with misdirected ventricular septal growth in the heart, decreased proliferative symmetric divisions in brain neural progenitors and increased misoriented divisions in fibroblasts; the same phenotype was seen in fibroblasts from three MOPDII individuals. Misoriented spindles were associated with disrupted astral microtubules and near complete loss of a unique set of centrosome proteins from spindle poles (ninein, Cep215, centriolin). All these proteins appear to be crucial for microtubule anchoring and all interacted with Pcnt, suggesting that Pcnt serves as a molecular scaffold for this functionally-linked set of spindle pole proteins. Importantly, Pcnt disruption had no detectable effect on localization of proteins involved in the cortical polarity pathway (NuMA, p150glued, aPKC). Not only do these data reveal a spindle-pole-localized complex for spindle orientation, but they identify key spindle symmetry proteins involved in the pathogenesis of MOPDII.
During vesicular transport, the assembly of the coat complexes and the selection of cargo proteins must be coordinated with the subsequent translocation of vesicles from the donor to an acceptor compartment. Here, we review recent progress toward uncovering the molecular mechanisms that connect transport vesicles to the protein machinery responsible for cytoskeleton-mediated motility. An emerging theme is that vesicle cargo proteins, either directly or through binding interactions with coat proteins, are able to influence cytoskeletal dynamics and motor protein function. Hence, a vesicle's cargo composition may help direct its intracellular motility and targeting.
Virus-host interactions drive a remarkable diversity of immune responses and countermeasures. We found that two RNA viruses with broad host ranges, vesicular stomatitis virus (VSV) and Sindbis virus (SINV), are completely restricted in their replication after entry into Lepidopteran cells. This restriction is overcome when cells are co-infected with vaccinia virus (VACV), a vertebrate DNA virus. Using RNAi screening, we show that Lepidopteran RNAi, Nuclear Factor-κB, and ubiquitin-proteasome pathways restrict RNA virus infection. Surprisingly, a highly conserved, uncharacterized VACV protein, A51R, can partially overcome this virus restriction. We show that A51R is also critical for VACV replication in vertebrate cells and for pathogenesis in mice. Interestingly, A51R colocalizes with, and stabilizes, host microtubules and also associates with ubiquitin. We show that A51R promotes viral protein stability, possibly by preventing ubiquitin-dependent targeting of viral proteins for destruction. Importantly, our studies reveal exciting new opportunities to study virus-host interactions in experimentally-tractable Lepidopteran systems.DOI: http://dx.doi.org/10.7554/eLife.02910.001
The centrosome acts as a microtubule-organizing center (MTOC) from the G to G phases of the cell cycle; it can mature into a spindle pole during mitosis and/or transition into a cilium by elongating microtubules (MTs) from the basal body on cell differentiation or cell cycle arrest. New studies hint that the centrosome functions in more than MT organization. For instance, it has recently been shown that a specific substructure of the centrosome-the mother centriole appendages-are required for the recycling of endosomes back to the plasma membrane. This alone could have important implications for a renaissance in our understanding of the development of primary cilia, endosome recycling, and the immune response. Here, we review newly identified roles for the centrosome in directing membrane traffic, the immunological synapse, and the stress response.
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