Molecular Biology of the Cell最新文献

Cell-surface glypicans distribute several extracellular ligands, including the Wnts, which are secreted to function at short and long range in a tissue. The Drosophila glypican Dally-like protein (Dlp) interacts with Wnts to inhibit short-range Wnt signaling and promote long-range signaling by the Drosophila Wnt1, Wingless (Wg). Dlp-dependent long-range Wg distribution in the fly ovary is attenuated by metalloproteinase 2 (Mmp2). Here, we report that Mmp2 destabilizes cell-surface Dlp, causing it to be internalized. Further, after Mmp2 cleavage, Dlp sequesters more Wg, suggesting that cleaved Dlp removes Wg from the extracellular space to limit its availability for signaling. Based on these and our previous results, we propose that coordinated activities of uncleaved and cleaved Dlp regulate proper extracellular Wg distribution. Overall, this study identifies the molecular basis of protease-mediated inhibition of a cell-surface glypican to modulate ligand distribution and function.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of rare but severe autoimmune diseases characterized by necrotizing inflammation of small blood vessels, leading to organ damage, particularly in the kidneys and respiratory tract. The current understanding of AAV pathogenesis has moved beyond a simple model of autoantibody-mediated damage to recognize a complex, self-sustaining inflammatory circuit. Central to this circuit is a dysregulated triad between neutrophils, macrophages, and the vascular endothelium. This review synthesizes our current understanding of this innate immune axis, detailing the pathogenic sequence from the initial loss of tolerance to the subsequent inflammatory priming event that triggers the pathogenic activation of neutrophils. The chronicity of AAV arises from powerful feed-forward amplification loops that sustain inflammation, which are cemented by the active suppression of the body's intrinsic resolution pathways. Finally, we discuss how advanced bioengineered platforms, such as vasculitis-on-a-chip models, are essential for deconstructing this complex pathology and are poised to accelerate the development of a new generation of targeted, pro-resolution therapies. This review provides a comprehensive framework for understanding the central role of neutrophil-macrophage cross-talk in the perpetuation of AAV.

RNA molecules are localized to subcellular regions through interactions between localization-regulatory cis-elements and trans-acting RNA binding proteins (RBPs). However, the identities of RNAs whose localization is regulated by a specific RBP, as well as the impacts of that RNA localization on cell function, have generally remained unknown. Here, we demonstrate that the RBP HNRNPA2B1 acts to keep specific RNAs out of neuronal projections. Using subcellular fractionation, high-throughput sequencing, and single-molecule RNA FISH, we find that hundreds of RNAs demonstrate markedly increased abundance in neurites in HNRNPA2B1 knockout cells. These RNAs often encode motor proteins and are enriched for known HNRNPA2B1 binding sites and motifs in their 3' UTRs. The speed and processivity of microtubule-based transport are impaired in these cells. HNRNPA2B1 point mutations that increase its cytoplasmic abundance relative to wildtype lead to stronger suppression of RNA mislocalization defects than seen with wildtype HNRNPA2B1. We further find that the subcellular localizations of HNRNPA2B1 target RNAs are sensitive to perturbations of RNA decay machinery, suggesting that HNRNPA2B1's known role in regulating cytoplasmic RNA stability may explain these observations. These findings establish HNRNPA2B1 as a negative regulator of neurite RNA abundance and a necessary factor for efficient motor-dependent cargo transport.

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.

Septins are hetero-oligomeric cytoskeletal proteins that assemble into filaments and scaffolds on the plasma membrane to aid cytokinesis, morphogenesis, and other cellular processes. Epitope tagging is widely used to study septin localization and function. However, it is technically challenging to test the functionality of tagged septins outside fungi. Fission yeast provides an ideal genetic system to test functionalities and localizations of tagged septins. mEGFP/mYFP-tagged septins Spn1 and Spn4 localize to the division site as double rings during cytokinesis, but tdTomato tagged septins also localize to puncta or short linear structures across the plasma membrane. It was proposed that these additional septin structures serve as diffusion barriers and are important for the localizations and functions of several proteins, including the NDR-kinase Sid2 and active Cdc42 GTPase. By analyzing cell morphology, cytokinesis defects, and genetic interactions between tagged septins and three mutations, we find that septins are less functional with tdTomato or 3HA than with other tags. Additionally, Sid2 appearance at the division site is after septins and delayed in septin deletions, contrary to previous reports. Our data re-emphasize the need for rigorous functional tests of tagged septins and for caution in interpreting function and localization data when using epitope-tagged septins.

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.