Molecular Biology of the Cell最新文献

Successful cutaneous wound healing requires reepithelialization by keratinocytes using a coordinated migratory process called keratinocyte collective cell migration (KCCM). Environmental stresses such as wounding induce the integrated stress response (ISR) initiated by protein kinases that phosphorylate the α subunit of eIF2 and mitigate translational control to alleviate stress damage. We previously reported that the ISR protein kinase GCN2 (EIF2AK4) facilitates KCCM via sustained phosphorylation of eIF2α and coordinated production of reactive oxygen species and amino acid transport. In this study, we show that a second ISR protein kinase, PERK (EIF2AK3), also contributes to KCCM. PERK promotes KCCM by protein-protein interactions requiring the cytoplasmic portion of PERK but independent of its catalytic functions. To discern these PERK interactions, we used BioID proximity labeling, immunoprecipitation analyses, and immunofluorescence microscopy to show that PERK interacts with multiple cell adhesion and cytoskeletal complexes important for KCCM. PERK engages with the hemidesmosome proteins ITGA6, ITGB4, COLXVII, and the desmosome proteins JUP, DSG2, and DSG3. Loss of PERK disrupts expression and localization of these cell adhesion proteins, which alters keratinocyte morphology and increases cell-substrate and intercellular adhesions. Our results define an underappreciated scaffolding function for PERK involving cell adhesions that are critical for KCCM.

Meiosis is a source of genetic variation in eukaryotes. Meiosis in the eukaryotic fission yeast Schizosaccharomyces pombe leads to the formation of spores that are particularly resistant to environmental stresses. In addition to external factors, internal processes may nevertheless contribute to cellular stress and impact the genome. This study investigates the role of Pnu1 as the major meiotic nuclease in S. pombe. Transcription and cellular expression of Pnu1 are regulated upon specific phases of meiosis, while its mitochondrial localization is also altered during this process. As a result, Pnu1 induces fragmentation of both genomic and mitochondrial DNA in the postmeiotic phase. This sugar-nonspecific endonuclease generates random double-strand breaks across the genome, an activity that appears to be mediated by direct interaction with chromatin. Given the high spore viability (∼95%) and the widespread occurrence of this phenomenon, this fragmentation appears to be physiological rather than apoptotic as observed in mammals. EndoG is the mammalian homologue of Pnu1 and is a caspase-independent apoptotic endonuclease that can allow cell survival. This study further describes the dynamics of Pnu1 action and supports the conclusion that Pnu1 is a major meiotic endonuclease of S. pombe responsible for a transient postmeiotic fragmentation of cellular DNA, potentially contributing to genetic variability.

Neuronal trafficking pathways must operate with high fidelity and speed, adapting to the dynamic demands of synaptic activity to maintain stable functionality. The biogenesis of lysosome-related organelles complex 1 (BLOC-1) is an attractive candidate to stabilize synaptic function during such challenges. BLOC-1 is an evolutionarily conserved protein complex composed of eight subunits involved in vesicle trafficking. In the nervous system, the BLOC-1 is associated with neurodevelopmental diseases and synaptic plasticity. However, the functions of each BLOC-1 component remain enigmatic. Here, we use CRISPR to mutate each Drosophila BLOC-1 gene to investigate roles in synaptic growth, function, and homeostatic plasticity. First, we show that BLOC-1 mutations are viable, with no defects in synaptic growth, morphology, or baseline function. We then demonstrate distinct synaptic localization patterns of BLOC-1 components. Finally, we show that only two of the eight BLOC-1 components, dysbindin and snapin, are necessary for presynaptic homeostatic potentiation. These results indicate separable functions and distinct synaptic localization patterns of BLOC-1 subunits, and a need to reconsider predictions made from biochemical models of BLOC-1.

To enable effective cell migration, local cell protrusion has to be coordinated with local cell attachment. Here, we investigate spatiotemporal activity patterns of key regulators of cell protrusion and adhesion, the small GTPases Rac and Rap, in migrating cells. These analyses show that Rac activity correlates very tightly with instantaneous cell protrusion events, while the Rap activity stays elevated for prolonged time periods after protrusion and is also detectable before cell protrusion. Direct analysis of activity cross-talk in living cells via light-based perturbation methods revealed that Rap can efficiently activate Rac; however, reciprocal cross-talk from Rac to Rap was not detectable. These findings suggest that Rap plays an instructive role in the generation of cell protrusions by its ability to activate Rac. Furthermore, prolonged Rap activity suggests that this molecule also plays a role in maintenance or stabilization of cell protrusions. Indeed, analysis of Rap1-depleted A431 cells revealed a significant reduction of cell attachment, suggesting that Rap-stimulated cell adhesion can stabilize newly formed protrusions. Taken together, our study suggests a mechanism, by which cell protrusion is coupled to cell adhesion via unidirectional cross-talk that connects the activity of the small GTPases Rap and Rac.

Growth is the essential vital process that drives life forward and always occurs within cells. Cell growth fuels the cell divisions that drive proliferation of single-celled organisms and growth of multicellular organisms. Mechanisms that control the extent and location of growth within cells generate the extraordinary diversity of cell sizes and shapes seen across the tree of life and within the human body, and nearly all cancers show profound defects in control of cell growth that lead to severe aberrations in cell size and shape. Yet we know little about how cell growth occurs or how it is controlled. For decades we have known how basic building blocks such as amino acids and lipids are built, but an enormous gap has always remained in our understanding of how these building blocks are used to build out cells of highly diverse sizes and shapes under varying environmental conditions and in diverse developmental contexts. Given the fundamental importance of growth in biology and cancer, our minimal understanding of cell growth is a growing problem. Here, a few of the intriguing and important questions about cell growth are considered.

Electron microscopy is an important technique for the study of synaptic morphology and its relation to synaptic function. The data analysis for this task requires the segmentation of the relevant synaptic structures, such as synaptic vesicles (SV), active zones, mitochondria, presynaptic densities, synaptic ribbons, and synaptic compartments. Previous studies were predominantly based on manual segmentation, which is very time-consuming and prevented the systematic analysis of large datasets. Here, we introduce SynapseNet, a tool for the automatic segmentation and analysis of synapses in electron micrographs. It can reliably segment SVs and other synaptic structures in a wide range of electron microscopy approaches, thanks to a large annotated dataset, which we assembled, and domain adaptation functionality we developed. We demonstrated its capability for (semi-)automatic biological analysis in two applications and made it available as an easy-to-use tool to enable novel data-driven insights into synapse organization and function.

Mitochondrial membrane phospholipids impact mitochondrial structure and function by influencing the assembly and activity of membrane proteins. Although the specific roles of the three most abundant mitochondrial phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL), have been extensively studied, the precise function of less abundant phosphatidylserine (PS) is not yet determined. Here, we used genetic and nutritional manipulation to engineer a set of yeast mutants, including a mutant completely devoid of PS, to assess its role in mitochondrial bioenergetics and lipid homeostasis. To circumvent the confounding effect of downstream PS products, PE and PC, we exogenously supplied ethanolamine that allows their biosynthesis via an alternate pathway. Using this system, we demonstrate that PS does not impact the abundance or the assembly of mitochondrial respiratory chain complexes; however, mitochondrial respiration is impaired. PS-lacking mitochondria cannot maintain mitochondrial membrane potential and exhibit leaky membranes. A mass spectrometry-based analysis of the cellular and mitochondrial lipidomes revealed an unexpected increase in odd-chain fatty acid-containing lipids in PS-lacking cells that may impact mitochondrial bioenergetics. Our study uncovers novel roles of PS in mitochondrial membrane biogenesis and bioenergetics and provides a viable eukaryotic system to unravel the cellular functions of PS.

Histone deacetylase 6 (HDAC6) helps cells manage misfolded proteins by transporting ubiquitin (UB)-associated structures toward the microtubule organizing center, where they can be sequestered and degraded by lysosomes. Here, we show that when cells are subjected to acute protein-folding stress in the endoplasmic reticulum (ER), HDAC6 depletion results in the appearance of enlarged endosomes that are highly decorated with UB and colocalize with both early and late endosome markers. The C-terminal UB-binding domain and adjacent disordered regions of HDAC6 are necessary and sufficient to rescue this endosomal phenotype in cells lacking endogenous HDAC6. HDAC6 deficiency does not appear to prevent the recruitment of endosomal sorting complexes required for transport (ESCRT), which coordinate endosome maturation. However, overexpression of HDAC6 can reverse endosome phenotypes associated with the depletion of the early ESCRT factor HRS. We speculate that HDAC6 facilitates the packaging and processing of endosomal cargo when the endomembrane system is under stress.

Hereditary spastic paraplegia type 21 (SPG21) is an inherited neurological disorder caused by biallelic mutations in the SPG21 gene, which encodes a protein named SPG21 or maspardin. Herein, we report that the SPG21 protein localizes to endolysosomes through interaction with the GTP-bound form of RAB7A. Disease-associated SPG21 variants reduce expression of SPG21 and disrupt its endolysosomal localization in both nonneuronal cells and neurons. Consistent with this localization, functional dependency analysis links SPG21 to endolysosomal and mTORC1 signaling pathways. Biochemical studies reveal that SPG21 depletion does not affect phosphorylation of canonical mTORC1 substrates such as ULK1, S6K1, 4E-BP1, but reduces phosphorylation of the noncanonical mTORC1 substrate TFEB. This enhances nuclear localization of TFEB and expression of a subset of TFEB-target genes. We conclude that SPG21 acts as a RAB7A effector that promotes noncanonical mTORC1-catalyzed phosphorylation of TFEB, thereby suppressing its nuclear localization and transcriptional activity. These findings link SPG21 dysfunction to altered endolysosomal signaling, offering new insights into SPG21 pathogenesis.

Neutrophils exert tumor-promoting roles in breast cancer and are particularly prominent in aggressive breast tumors. The proinflammatory signals TGF-β1 and TNF-α are upregulated in breast tumors and induce epithelial-to-mesenchymal transitions (EMT), a process linked to cancer cell aggressiveness. Here, we investigated the roles of TGF-β1 and TNF-α in the recruitment of neutrophils by breast cancer cells. Dual-treatment with TGF-β1 and TNF-α induces EMT signatures in premalignant M2 cells, which are part of the MCF10A breast cancer progression model. Conditioned media (CM) harvested from M2 cells treated with TGF-β1/TNF-α gives rise to amplified neutrophil chemotaxis compared with CM from vehicle-treated M2 cells. This response correlates with higher levels of the neutrophil chemokines CXCL1 and CXCL8, in a p38MAPK-dependent manner, and is attenuated by CXCL8-neutralizing antibodies. We combined gene editing, immunological, and biochemical assays to show that neutrophil recruitment and EMT are uncoupled in treated M2 cells. Finally, analysis of transcriptomic databases of cancer cell lines revealed a significant correlation between CXCL8 and TGF-β1/TNF-α-regulated or effector genes in breast cancer. These findings establish a novel role for the TGF-β1/TNF-α/p38 MAPK signaling axis in regulating neutrophil recruitment in breast cancer, independent of their profound impact on EMT.