Molecular Biology of the Cell最新文献

The cell wall (CW) protects fungal cells from various challenges, making its integrity essential for cell survival. CW integrity is monitored by transmembrane sensors that activate effectors to promote CW synthesis in response to injuries. Sensors of the WSC (Wall Surface Component) family are found in most fungi, and share a conserved architecture, with a cytoplasmic tail, a single transmembrane domain, and a long Serine Threonine Rich domain (STR) prolonged by a WSC domain, both embedded in the CW. These extracellular domains promote force detection in the CW, sensor clustering and cell survival. Interestingly, Wsc sensors exhibit variations in domain sequence and size among fungal species. To understand how these variations impact force detection, we expressed Wsc sensors taken from Saccharomyces cerevisiae and Candida albicans, in the fission yeast Schizosaccharomyces pombe. Remarkably, we found that a subset of these foreign sensors cluster at sites of CW compression, but that others failed, suggesting divergences in mechanosensing abilities. By swapping sensor domains, we demonstrate that both the cytoplasmic tail and STR domain influence sensor relocalization to sites of CW compression. These findings reveal a high level of functional plasticity in fungal sensors and identify tuneable modules that may regulate mechanosensing of various CWs.

In yeast, early adaptation to hyperosmotic stress involves organelle-based mechanisms, including synthesis of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂) in the endolysosomal system. This low-level signaling lipid drives vacuolar fragmentation and activates the V-ATPase, which acidifies the vacuole and promotes salt sequestration. Under NaCl stress, PI(3,5)P₂ rapidly accumulates, triggering increased V-ATPase activity and vacuolar remodeling; these responses are impaired by deficient PI(3,5)P₂ synthesis. We visualized movements of a GFP fusion protein with the cytosolic domain of V-ATPase subunit Vph1 (Vph1NT-GFP) in a microfluidic system during salt stress. Upon NaCl addition, Vph1NT-GFP rapidly relocalizes to a region adjacent to the vacuole in a PI(3,5)P₂-dependent manner. The intensity and duration of this response depend on salt concentration, but the response is diminished by 30-45 min, even if salt is readded. Vph1NT-GFP returns to the same location upon repeated salt challenge, suggesting that PI(3,5)P₂ synthesis occurs at a localized domain/contact site that may be endosomal. When the high osmolarity glycerol pathway, which coordinates long-term transcriptional changes, is disrupted, Vph1NT-GFP recruitment is significantly extended. This underscores the integration of lipid signaling and transcriptional regulation in osmoadaptation. These findings suggest activation of endolysosomal targets by PI(3,5)P₂ synthesis provides immediate protection that primes cells for longer-term survival strategies.

The regulation of the actin cytoskeleton is key for controlling cell shape and structure. While the Rho GTPase RhoA is well known to regulate the actomyosin cytoskeleton, its function in controlling the septin cytoskeleton remains unclear. As RhoA interactions can vary in both time and space, they can be challenging to discern from traditional bulk biochemical assays. Here we use multiple optogenetic tools to spatially and temporally increase myosin localization, stimulate contractile force, and activate RhoA, to investigate how RhoA and its downstream effector myosin impact the septin cytoskeleton. We find that neither local accumulation of myosin nor increased activity of myosin is sufficient to alter septin architecture. Local activation of RhoA, however, results in a local increase in septin accumulation. Importantly, this septin increase is independent of the scaffolding protein anillin, which can directly bind both septin and RhoA. Together these data expand the potential role of septins in mediating RhoA signaling by stimulating the remodeling of the septin cytoskeleton. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

Endoplasmic reticulum-plasma membrane (ER-PM) contact sites play important roles in maintaining lipid homeostasis at plasma membrane (PM), cellular calcium homeostasis and cell signaling. Here, we show that MCTP1 and MCTP2 are at ER subdomains that form membrane contact sites (MCS) with multiple organelles using proximity labelling assay. MCTPs are three C2 domain-containing transmembrane proteins. We show that upon overexpression, MCTPs promote ER-PM contact sites in C2 domain dependent manner. MCTP C2 domains bind to PI(4)P and PI(4,5)P₂, phosphoinositides that are enriched in the PM. Furthermore, we show that deletion of MCTP1 or MCTP2 increases PI(4)P levels in the PM and promote cell migration. Thus, our study identifies MCTPs as multiple ER-organelle contact site proteins and establishes its role at ER-PM contact sites in regulating lipid homeostasis and cell migration.

Keratin 17 (K17) is a stress-responsive intermediate filament protein that is upregulated in chronic skin diseases and in several carcinomas. We previously showed that K17 is induced in epidermal keratinocytes following exposure to DNA damaging agents, promoting keratinocyte survival and chemically-induced papilloma formation in mouse skin. Molecularly, K17 is recruited to the nucleus where it impacts nuclear architecture, gene expression, and the DNA damage response (DDR). Here, we report on efforts to delineate K17-dependent processes during DDR by focusing on its interacting partners. Using mass spectrometry, we identified a network of K17-interacting Rho GTPase signaling proteins, including Rac1 and its activator Dock7. Biochemically, we confirmed that Rac1 and K17 interact directly in vitro and in A431 tumor keratinocytes, both at baseline and after ionizing radiation. We show that KRT17 deletion leads to decreased levels of Rac1 protein, its DNA damage-related effector TOP2A, and Rac1-dependent decrease in cellular proliferation following ionizing radiation. Remarkably, key K17-dependent readouts are rescued by expression of constitutively-active, but not dominant-negative, Rac mutants in KRT17 null A431 keratinocytes. These findings uncover a K17-Rac1-TOP2A signaling axis that promotes DNA damage response and associated proliferation, with implications for cancer and chronic skin diseases.

Migratory cells can adopt membrane protrusions like blebbing or lamellipodia for efficient migration. The underlying mechanisms of how switching contributes to cell migration are not clearly understood. Here we found that nonmuscle myosin II (NM II) mediated blebbing to lamellipodia conversion (BLC) increased the speed of migration whereas lamellipodia to blebbing conversion (LBC) decreased it in various cells like cancerous cells, mesenchymal stem cells, and T-lymphocytes. Cells with lamellipodia had larger and greater number of focal adhesions compared with blebbing cells, suggesting a link between adhesion strength with membrane protrusions and migration. Migratory cells seeded on collagen I, but not on poly-L-lysine, exhibited a faster BLC and greater migration speed compared with cells seeded on an uncoated surface. Knockdown of integrinβ1 reduced cell migration but these cells were able to undergo conversion of membrane protrusions, albeit with a substantial delay. These findings suggest that cells can fine tune the migration strategy by controlling NM II-mediated protrusion switching and modulating integrin dependent adhesion strength. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

Giardia intestinalis is a globally prevalent cause of waterborne diarrheal disease, yet about 40% of its proteome remains functionally uncharacterized due to the lack of conserved homologous proteins and limited experimental validation of protein function. To begin addressing this gap, we created a large-scale subcellular localization resource by fluorescently tagging and imaging 608 Giardia fusion proteins (12% of the proteome) expressed in live cells from native promoters. This dataset includes 240 hypothetical proteins, 215 domain-family proteins (including ankyrin repeat and NEK kinase families), 171 diplomonad- or Giardia-specific proteins, 69 conserved eukaryotic proteins, and 77 proteins with known functions that were previously unlocalized. Imaging revealed localization to cytoskeletal and Giardia-specific organelles (eight flagella, the ventral disk, and the median body), along with novel components of the plasma membrane and endomembrane systems. Integrating localization data with domain architecture, homology, and Giardia-specific Gene Ontology terms, we produced a "localization-informed" gene annotation with a standardized, structured nomenclature. This resource provides the largest experimentally-validated functional annotation of the Giardia proteome to date, linking predicted gene models to cellular structures, creating testable hypotheses for protein function and establishing a durable framework for future studies of cell biology, pathogenesis, and eukaryotic evolution in this deeply divergent diplomonad lineage.

Stem cells sense biophysical cues within their extracellular microenvironment and respond via mechanotransduction signaling pathways that induce changes in gene expression and associated cell fate outcomes. Histone modifying enzymes are known to drive stem cell differentiation through changes in chromatin accessibility, but little is understood as to how extracellular matrix (ECM) mechanics regulate epigenomic remodeling. Here, we utilized alginate hydrogels with tunable mechanical properties to investigate the role of both matrix stiffness and stress relaxation on histone demethylase expression and activity during osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). Our results revealed that the expression of two histone demethylases, KDM4B and KDM6B, was upregulated during osteogenesis in response to stiff and fast stress-relaxing matrix conditions. Additionally, CUT&Tag profiling coupled with RNA-sequencing demonstrated that repressive histone methylation was decreased at osteogenic-specific loci in stiff, fast-relaxing matrices. Further, inhibition of mechanotransduction signaling pathways reduced expression of KDM4B and KDM6B and hindered osteogenic differentiation overall. Interestingly, phosphorylation of SMAD 1/5/8 increased in cells cultured in stiff, stress relaxing matrices, and pharmacological inhibition of SMAD 1/5/8 activation reduced expression of KDM4B and KDM6B. Together, our results establish novel impacts of stem cell mechanotransduction signaling events that promote osteogenesis through epigenetic remodeling.

It is well established that many tumor types possess defective autophagic pathways. Several studies have reported that the transmembrane, autophagic lipid scramblase ATG9B is altered in multiple cancers, suggesting that this dysregulation could contribute to oncogenesis. Therefore, the goal of this study was to define the cellular distribution of ATG9B in two different tumor cell types and to provide insights into its cellular function. Surprisingly, we found that ATG9B shows a modest association with autophagic structures and exhibits a unique and prominent localization to mitochondria, in contrast to its related form ATG9A. Upon expression of tagged ATG9B forms, this mitochondrial distribution was accompanied by aberrant changes in mitochondrial morphology as well as a reduction in the mitochondrial membrane potential and the release of mtDNA. Few indicators for ATG9B-dependent mitophagy were noted. Instead, ATG9B overexpression led to pronounced apoptotic cell death as assessed by a variety of indicators. Further, we find that the N-terminal sequence of ATG9B acts as a mitochondrial targeting domain and that expression of this peptide alone can induce apoptotic cell death. These findings provide new insights into a putative cellular localization and function for ATG9B. [Media: see text] [Media: see text] [Media: see text].

Microtubule dynamics change during cell division to enable rapid microtubule network remodeling. The switching from microtubule growth to shrinkage is attributed to the loss of a stabilizing GTP-cap structure at the growing microtubule end. The size of the GTP-cap is a result of a balance between GTP-tubulin addition to the microtubule end and subsequent GTP-hydrolysis in the microtubule lattice. Whether the cell-cycle-dependent changes in microtubule dynamics are supported by concurrent modulation of the stabilizing GTP-cap size is not known. Here, we use high spatiotemporal resolution live-cell imaging of EB1, an established marker for the GTP-cap, to directly determine the relationship between GTP-cap size and microtubule growth rate throughout the cell cycle. Our data reveal that GTP-cap size for matching growth rates is reduced during mitosis. Comparison of EB1 comets on astral versus spindle microtubules reveals that the scaling between the GTP-cap size and microtubule growth rate is not spatially regulated in mitosis. We find that these regulatory patterns are conserved across epithelial cells from two different species. Taken together, our findings reveal modulation of GTP-cap size as a cell-cycle-regulated mechanism for tuning microtubule stability. [Media: see text].