Journal of cell science最新文献

Hair cells in the utricle and saccule form two groups with oppositely oriented stereociliary bundles that enable detection of broad ranges of motion. These groups are aligned along a common polarity axis established by core planar cell polarity (PCP) proteins, which individual cells interpret differently to generate opposing bundle orientations. EMX2, GPR156 and STK32A determine how these groups integrate PCP signaling during this process. We tested functional interactions between these factors using genetic epistasis experiments and evaluating hair cells in mice with combined mutations in Gpr156 and Stk32a or Emx2 and Stk32a. We show in the utricle that: (1) GPR156 functions to reverse stereociliary bundles relative to the PCP axis but can be blocked by STK32A; and (2) EMX2 establishes the boundary between the two groups by repressing Stk32a transcription. We further demonstrate that these factors have similar functional relationships in the cochlea, despite the absence of polarity reversal in that tissue. Together, these phenotypes support a mechanism whereby EMX2 regulates Stk32a transcription, thereby allowing GPR156 to reverse the orientation of stereociliary bundles in one group of hair cells.

Individual epithelial cells that express oncogenes are often extruded from monolayers of wild-type cells, but the extrusion mechanism is not fully understood. We examined extrusion of mammary epithelial cells caused by induction of oncogenic Ras(Q61L). Ras-dependent extrusion requires phosphorylation of ERK (herein referring to ERK1 and ERK2, also known as MAPK3 and MAPK1, respectively) but not activation of AKT kinases. Unexpectedly, however, extrusion was suppressed by erlotinib, an inhibitor of epidermal growth factor receptor (EGFR), and by deletion of EGFR. In pancreatic and lung cancers, EGFR is required for full activation of Ras. However, EGFR inhibition or deletion had no impact on Ras(Q61L)-GTP levels or ERK phosphorylation. EGFR expression was not required in surrounding wild-type cells but was needed by the Ras(Q61L) cells for extrusion, yet deletion of the Ras guanine-nucleotide-exchange factors SOS1 and SOS2 (SOS1/2) did not block extrusion. Moreover, expression of a constitutively active MEK instead of Ras was sufficient to drive extrusion, and EGFR inhibition in these cells reduced extrusion. Notably, expression of Ras triggered internalization of E-cadherin (CDH1), which was partially blocked by EGFR inhibition. Together, these data demonstrate an unanticipated requirement for non-canonical EGFR signaling in cancer cell extrusion, which might act in part by promoting E-cadherin endocytosis.

Lysosome-related organelles (LROs) are a heterogeneous family of organelles found in many cell types, whose similarities to lysosomes include acidification by vacuolar-type proton ATPases (V-ATPases). However, some organelles with hallmarks of LROs are nonetheless non-acidic. Here, we investigate this phenomenon using the ciliate Tetrahymena thermophila, which has secretory LROs called mucocysts. Using three approaches, we show that mature mucocysts, poised for exocytosis, are non-acidic. However, mucocysts forming in the cytoplasm are acidic, and a specific V-ATPase a-subunit is present and indispensable for mucocyst biogenesis. In the absence of this subunit, cells show defects in at least two features of mucocyst formation, namely heterotypic vesicle fusion of mucocyst precursors and proprotein processing. The stage specificity of acidification can be explained by our finding that several other canonical V-ATPase subunits are present in the forming mucocysts but not in mature mucocysts. Based on our data, we argue that a specific V-ATPase complex is targeted to newly forming, immature mucocysts and subsequently disassembles at a later stage in the maturation pathway.

Hepatocytes, the parenchymal cells of the liver, exhibit a unique epithelial polarity phenotype in which their bile canaliculi-forming luminal domains and underlying F-actin-linked cell-cell adhesion belt organize not parallel but perpendicular to their basal extracellular matrix (ECM)-contacting domains. Hepatocytes also differ from other epithelia in that they form two basal domains on opposite sites, face only a sparse ECM and express mesenchymal rather than epithelial-typical integrins. What role these hepatocyte-specific cell-ECM interactions play in the establishment and maintenance of the unique hepatocyte polarity phenotype is unknown. We report that in primary rat hepatocyte cultures, development and maintenance of a bile canaliculi network requires the repression of contractile substrate-parallel cell-cell adhesions near matrix-contacting sites. This occurs only when cells contact ECM at two sites; it requires the integrin α1β1, and on rigid matrix, additionally an αV-integrin. We furthermore found that low matrix rigidity, as characteristic of the healthy liver, favors bile canaliculi formation, which becomes independent of p120 catenin-dependent adherens junctions. Our findings thus link the unique hepatocyte polarity phenotype to adherens junction formation downstream of their unique ECM and integrin makeup.

Increased aerobic glycolysis and increased cell motility are hallmarks of metastatic cancer. Migrating cancer cells are highly polarized, suggesting that glycolytic enzymes could be spatially regulated. Here, we investigated the role of the liver isoform of the 'gatekeeper' glycolytic enzyme phosphofructokinase-1 (PFKL) in breast cancer cell migration. Depletion of PFKL significantly decreased migration velocity and directional sensing. We have observed the localization of PFKL to lamellipodia of migrating breast cancer cells, where it colocalized with hexokinase-2 and pyruvate kinase M2. We then investigated the functional requirements of PFKL for directional migration. First, we found that expression of catalytically inactive PFKL or indirect pharmacological inhibition of PFKL activity significantly decreased directional migration. Second, we discovered that disrupting PFKL filament formation by expression of a filament-incompetent mutant decreased PFKL recruitment to lamellipodia and directional sensing, without altering migration velocity. These findings indicate that both catalytic activity and subcellular localization are required for directional migration in breast cancer cells. These results suggest a novel function of PFKL filaments in cells and provide insight into the function of compartmentalized glycolysis in the cytoplasm.

In the colon, the single-layered epithelium forms crypts that extend into the mucosa and are surrounded by a fibroblast network essential for extracellular matrix (ECM) production, remodeling and epithelial support. Fibroblasts are heterogeneous, but inconsistent nomenclature and lack of markers have hindered their classification. Using single-cell RNA sequencing (scRNA-seq), we identified six distinct fibroblast subpopulations in mouse colonic mucosa, each with unique molecular profiles and specialized functions. Some fibroblasts focus on ECM production and remodeling, whereas others show high contractility. Certain subsets of the fibroblasts secrete cytokines promoting epithelial differentiation or stem cell niche maintenance. Spatial mapping revealed their organization within the mucosa, and trajectory analysis suggested distinct differentiation pathways. Cell cycle scoring confirmed that fibroblasts remain largely non-proliferative under homeostasis. By integrating our dataset with published ones, we identify conserved fibroblast populations and propose a standardized nomenclature for intestinal fibroblasts. This framework enhances communication and understanding of fibroblast diversity and their roles in gut homeostasis and disease.

Mahogunin ring finger 1 (MGRN1) is a membrane-tethered E3 ligase that fine-tunes signaling sensitivity by targeting surface receptors for ubiquitylation and degradation. Although MGRN1 is known to regulate the Hedgehog signaling effector Smoothened (SMO) via the transmembrane adapter multiple epidermal growth factor-like 8 (MEGF8), the broader scope of its regulatory network has been speculative. Here, we identify attractin (ATRN) and attractin-like 1 (ATRNL1) as additional transmembrane adapters that recruit MGRN1 and regulate cell surface receptor turnover. Through co-immunoprecipitation, we show that ATRN interacts with the RING domain of MGRN1. Functional assays suggest that ATRN and ATRNL1 work with MGRN1 to promote the ubiquitylation and degradation of the melanocortin receptors MC1R and MC4R, in a process analogous to its regulation of SMO. Loss of MGRN1 or ATRN leads to increased surface and ciliary localization of MC4R in fibroblasts and elevated MC1R levels in melanocytes, resulting in enhanced eumelanin production. These findings expand the known repertoire of MGRN1-regulated receptors and provide new insight into a shared mechanism by which membrane-tethered E3 ligases utilize transmembrane adapters to facilitate substrate receptor specificity.

The endoplasmic reticulum (ER) and mitochondria are known to affect myriad cellular mechanisms processes. More recently, dynamic association between them has been identified in different eukaryotes; these interactions vary in their composition and involvement in regulation of intracellular machineries. FAM134B (also known as RETREG1), originally identified as an oncogene, regulates ER membrane shape and curvature. It is a key ER-phagy or reticulophagy receptor, which promotes autophagy of not only the ER but also simultaneous dual autophagy of ER and mitochondria. Although it is known that FAM134B can potentiate contact with mitochondria, its direct involvement in affecting mitochondrial dynamics remains unexplored. Here, we show that FAM134B can interact with the canonical fission-promoting protein DRP1 (also known as DNM1L). Functional depletion of FAM134B leads to local actin rearrangement and reduced DRP1 recruitment onto mitochondria, resulting in hyperfusion. A decrease in FAM134B levels is observed with aging in rat brains, cell and mouse models of Parkinson's disease and samples derived from individuals with disease. Our study establishes FAM134B as the ER partner that helps in maintaining mitochondrial morphology and dynamics.

CYRI proteins promote lamellipodial dynamics by opposing Rac1-mediated activation of the Scar/WAVE complex. This activity also supports resolution of macropinocytic cups, promoting internalisation of surface proteins, including integrins. Here, we show that CYRI-B also promotes focal adhesion maturation and dynamics. Focal adhesions in CYRI-B-depleted cells show accelerated maturation and become excessively large. We probed the composition of these enlarged focal adhesions, using a Bio-ID screen, with paxillin as bait. Our screen revealed changes in adhesion proteins proximal to paxillin suggesting early activation of stress fibre contraction and depletion of the integrin internalisation mediator ERC1. Lack of CYRI-B leads to more stable lamellipodia and accumulation of polymerised actin in stress fibres. This actin acts as a barrier to microtubule targeting for adhesion turnover. Thus, our studies reveal an important connection between lamellipodia dynamics controlled by CYRI-B and microtubule targeting of ERC1 to modulate adhesion maturation and turnover.

Clathrin-mediated endocytosis (CME) is an essential, highly conserved process in eukaryotic cells that facilitates the internalization of plasma membrane components, transmembrane proteins and extracellular nutrients. This complex pathway involves the concerted assembly and disassembly of many different proteins at the plasma membrane. Budding yeast has served as a powerful model for dissecting CME through combined genetic, biochemical, quantitative imaging and mathematical approaches. In this Cell Science at a Glance article, we integrate decades of quantitative data to generate a three-dimensional molecular animation depicting the full progression of CME in budding yeast (Movie 1). The animation and accompanying poster capture the spatial and temporal dynamics of key protein players. In addition, we highlight recent advances in understanding of the condensation of endocytic proteins into distinct sites and the organization of actin networks that generate the forces necessary to deform and internalize the membrane against the high internal turgor pressure of the budding yeast cell.