Molecular Biology of the Cell最新文献

The type III intermediate filament protein vimentin plays an integral role in cell homeostasis and disease progression during fibrosis and cancer invasion. Previous work demonstrated that the pan-formin inhibitor small-molecule inhibitor of formin homology 2 domains (SMIFH2) induced a perinuclear collapse of the vimentin network, suggesting formins may regulate vimentin cytoskeleton organization. However, despite the designed function of SMIFH2 to inhibit formin homology 2 (FH2) domain-actin interactions, several major off-target effects of SMIFH2 have been reported, including inhibition of myosin family ATPase activity. SMIFH2 is also highly electrophilic, potentially reacting with nucleophilic residues within proteins other than formins. Therefore, we sought to determine the mechanism by which SMIFH2 disrupts the vimentin cytoskeleton. Depletion of specific formin proteins, targeted actin cytoskeleton disruption, or myosin family ATPase inhibition failed to replicate the SMIFH2 effect on the vimentin network. However, treatment with other electrophilic reagents, including prostaglandin A, reproduced the SMIFH2-mediated vimentin collapse, F-actin cytoskeletal changes, and activation of the NF-E2-related factor 2 stress sensory pathway. Additionally, fluorescence recovery after photobleaching analysis revealed that SMIFH2 inhibits vimentin filament dynamics, which was rescued by mutating the nucleophilic vimentin C328 residue. Thus, SMIFH2 disrupts the vimentin network due to its reactivity as an electrophilic species. This study reinforces the role of vimentin as a key stress sensor.

In eukaryotic organisms, the nucleus is remodeled to accommodate the space required for chromosome segregation. Remodeling strategies range from closed division, where the nuclear envelope remains intact, to open division, where the nuclear envelope is temporarily disassembled. While the budding yeast Saccharomyces cerevisiae (S. cerevisiae) undergoes closed mitosis, its meiotic nuclear division strategy is less understood. Here, we investigate nucleocytoplasmic compartmentalization during budding yeast meiosis and discover that meiosis II represents a semi-closed division marked by bidirectional mixing between the nucleus and cytoplasm. This includes nuclear entry of the Ran GTPase activating protein (RanGAP), typically cytoplasmic, although RanGAP relocalization appears to be a consequence, rather than a cause of permeability changes. This intercompartmental mixing occurs without nuclear envelope breakdown or dispersal of nucleoporins and is independent of known nuclear pore complex remodeling events. This phenomenon, termed virtual nuclear envelope breakdown (vNEBD), represents a unique mechanism distinct from other semi-closed divisions. We demonstrate that vNEBD is integrated into the meiotic program and regulated by the conserved meiotic kinase Ime2, and the meiosis-specific protein phosphatase 1 regulatory subunit, Gip1. Remarkably, the vNEBD event is conserved between S. cerevisiae and the distantly related Schizosaccharomyces pombe (S. pombe), indicating a fundamental role in meiosis.

The neurodegenerative disorder frontotemporal dementia (FTD) can be caused by a repeat expansion (GGGGCC; G4C2) in C9orf72. The function of wild-type C9orf72 and the mechanism by which the C9orf72-G4C2 expansion causes FTD, however, remain unresolved. Diverse disease models, including human brain samples and differentiated neurons from patient-derived induced pluripotent stem cells (iPSCs), identified some hallmarks associated with FTD, but these models have limitations, including biopsies capturing only a static snapshot of dynamic processes and differentiated neurons being labor-intensive, costly, and postmitotic. We find that patient-derived iPSCs, without being differentiated into neurons, exhibit established FTD hallmarks, including increased lysosome pH, decreased lysosomal cathepsin activity, cytosolic TDP-43 proteinopathy, and increased nuclear TFEB. Moreover, lowering lysosome pH in FTD iPSCs mitigates TDP-43 proteinopathy, suggesting a key role for lysosome dysfunction. RNA-seq reveals dysregulated transcripts in FTD iPSCs affecting calcium signaling, cell death, synaptic function, and neuronal development. We confirm differences in protein expression for some dysregulated genes not previously linked to FTD, including ciliary neurotrophic factor receptor (neuronal survival), Annexin A2 (anti-apoptotic), NANOG (neuronal development), and Moesin (cytoskeletal dynamics). Our findings underscore the potential of FTD iPSCs as a model for studying FTD cellular pathology and for drug screening to identify therapeutics.

Cortical microtubules influence plant cell shape by guiding cellulose deposition. Epidermal hypocotyl cells in Arabidopsis thaliana create distinct cortical microtubule array patterns to enable axial cell growth. How these array patterns are created and maintained during cell wall formation is a critical and unsolved problem in cell biology. Previous work showed that arrays aligned longitudinally with the cell's growth axis have a "split bipolar" organization, with microtubules treadmilling toward the apical or basal ends of the cell from a region of antiparallel overlap at the cell's midzone. The underlying order or architecture of these coaligned arrays prompted us to ask whether microtubules oriented transversely to the cell's axis are organized to a similar degree. Creating new fluorescently tagged End-Binding Protein 1b (EB1b) probes to circumvent gain-of-function effects observed for GFP-EB1b, we found that transverse arrays form persistent, nearly unipolar domains of microtubules treadmilling around the short axis of the cell, independent of the EB1b probe used. Our findings reveal an organizational strategy for transverse arrays distinct from that of longitudinal arrays, with implications for the mechanisms of array pattern creation and maintenance.

The conserved phosphoinositide-dependent protein kinase PDK1 regulates cell growth and stress signaling in eukaryotes. In the fission yeast Schizosaccharomyces pombe, Pdk1 has been linked to cytokinesis, which could point to new functions for this kinase family. Here, we discovered that Pdk1 localizes to eisosomes, which create invaginations in the plasma membrane, in addition to the spindle pole body. Pdk1 promotes phosphorylation of the core eisosome protein Pil1 and regulates eisosome length. Dysregulated eisosomes are not responsible for cytokinesis defects previously observed in pdk1∆ cells. Instead, we found that Pdk1 regulates the localization of the anillin-like protein Mid1 and the protein kinase Sid2, which promotes cytokinesis as part of the septation initiation network. Our combined results provide insights into the role of Pdk1 in eisosomes and cytokinesis, which extend the functions of this conserved protein kinase family beyond canonical growth control pathways.

Lysosome exocytosis is one of the critical functions of lysosomes in maintaining cellular homeostasis and plasma membrane (PM) repair. At the basal level, the SNAREs (soluble-N-ethylmaleimide-sensitive-factor accessory-protein receptors) regulating the lysosome fusion with the cell surface have been poorly defined. Here, we identified a Qa-SNARE STX1A, localized majorly to lysosomes and a cohort to the PM in HeLa cells. Overexpression of GFP-STX1A in HeLa cells causes decreased lysosome number and their peripheral dispersion. However, STX1A knockdown in HeLa cells displayed an accumulation of lysosomes beneath the cell surface with reduced lysosome exocytosis. Consistently, TIRF imaging microscopy demonstrated an enhanced enrichment of LAMP1-positive vesicles at the cell surface in STX1A-depleted compared with control cells. Moreover, STX1A depletion reduces proteolytic activity without affecting the lysosome content or acidity. Additionally, these cells showed enhanced lysosome dispersion and autolysosome accumulation. Functionally, GFP-STX1A also localizes to LLOMe-induced GAL3-positive damaged lysosomes and reduces their number by enhancing exocytosis. Biochemically, STX1A forms a SNARE complex with SNAP23 or SNAP25 (Qbc) and VAMP2 (R), and their knockdown in HeLa cells mimics the STX1A-depletion phenotypes. Overall, these studies demonstrate a unique function of STX1A in regulating lysosomal exocytosis by localizing to these degradative organelles.

Yeast cell fusion is equivalent to fertilization. Cell fusion requires the removal of the intervening cell wall, regulated by Fus2 and other cell fusion proteins. Fus2 is an amphiphysin-like protein that forms a complex with the BAR protein Rvs161 and the highly conserved Rho-like GTPase Cdc42 at the zone of cell fusion (ZCF); however, the function of these proteins in cell fusion has been unclear. Here, we show that the Fus2-Rvs161-Cdc42 complex regulates a mating-specific secretion event to mediate cell fusion via cell wall removal. Use of fluorogen-activated protein fusions demonstrated that the secretion of cell wall remodeling enzymes Scw4 and Gas1 is dependent on Fus2, whereas the secretion of Scw10 is independent of Fus2. We found that Cdc42 is not required for secretion per se, but instead functions to focus Fus2 at the ZCF, thereby allowing concentrated release of cell wall remodeling enzymes. Localized secretion of cell wall remodeling enzymes would overcome cell wall repair pathways. Additionally, Prm1, required for efficient membrane fusion, colocalizes with Fus2 at the ZCF. Localization of Prm1 at the ZCF is dependent on Fus2 and Rvs161. We propose that the Fus2-regulated vesicle population includes membrane fusion proteins as well as cell wall remodeling factors.

Motor-driven transport on microtubules is critical for distributing organelles throughout the cell. Most commonly, organelle movement is mediated by cargo adaptors, proteins on the surface of an organelle that directly recruit microtubule-based motors. An alternative mechanism called hitchhiking was recently discovered: some organelles move, not by recruiting the motors directly, but instead by using membrane contact sites (MCS) to attach to motor-driven vesicles and hitchhike along microtubules. Organelle hitchhiking is observed across fungi and animals. In filamentous fungi, nearly all peroxisomes move by hitchhiking on early endosomes (EE). In the fungus Aspergillus nidulans, EE-associated linker proteins PxdA and DipA are critical for establishing EE-peroxisome MCS required for peroxisome movement. Whether peroxisome-membrane proteins exist that regulate peroxisome hitchhiking on EEs is not known. Through a forward mutagenesis screen, we discovered an acyl-CoA binding (ACB) domain-containing protein AcbdA/AN1062 that localizes to peroxisomes via its tail-anchored transmembrane domain (TMD). Deleting the AcbdA gene or only its N-terminal ACB domain perturbs the movement and distribution of peroxisomes. Importantly, AcbdA is not required for the movement of EEs or for the recruitment of PxdA and DipA on EEs. Fatty acid (FA)-induced increases in peroxisome movement require AcbdA, suggesting that peroxisome hitchhiking on EEs is coupled to FA metabolism. Mutating a conserved FFAT motif, predicted to interact with the endoplasmic reticulum (ER), has no effect on peroxisome movement. Taken together, our data indicate that AcbdA is a peroxisome-membrane protein required for peroxisome hitchhiking on EEs. AcbdA's involvement in peroxisome hitchhiking represents a divergence from known functions of Acbd4/5 proteins and adds layers to our understanding of the functionality of the Acbd4/5 family of proteins.

During embryonic development, neural crest-derived melanoblasts, which are precursors of pigment-producing melanocytes, disperse throughout the skin by long-range cell migration that requires adhesion to the ECM. Members of the integrin family of cell-ECM adhesion receptors are thought to contribute to melanocyte migration in vitro. However, due to the functional redundancy between different integrin heterodimers, the precise role of integrins in melanoblast migration, as well as the mechanisms that regulate them in this process, especially in in vivo contexts, remain poorly understood. To address this, we utilize the existing transcriptomic databases to identify different integrin subunits that are specifically expressed in melanoblasts, melanocytes, and melanoma cancer cell lines. We then use mouse embryonic skin explants combined with drug and small-molecule-based perturbations to target different integrins as well as specific mechanisms that modulate integrin activity. Individual melanoblasts from live imaging movies are tracked using high-resolution, quantitative, automated analysis, and cell morphology, cell migration, and actin-based protrusions are analyzed. Overall, we uncover the nonredundant roles of different integrin heterodimers and elucidate the function of outside-in integrin activation in melanoblasts. Finally, we describe the function played, in vivo, by integrin-mediated adhesion to specific ECM ligands during melanoblast migration.