Journal of cell science最新文献

Vesicle-associated membrane protein-associated protein A (VAPA) is a protein of the endoplasmic reticulum (ER) and a component of several membrane contact sites (MCSs). We show here that VAPA also localizes to the inner nuclear membrane (INM), in close proximity to nuclear lamins, INM proteins and nucleoporins. Using our proteomics approach 'rapamycin- and APEX-dependent identification of proteins by SILAC' (RAPIDS), we identified several nuclear proximity partners of VAPA, including emerin, different LAP2 isoforms, lamin A/C and Nup153. Depletion of VAPA in various cellular systems resulted in reduced nuclear lamin levels and aberrant nuclear morphology, including the formation of membrane invaginations and tunnels. Furthermore, histone acetylation levels were altered. Our data suggest that VAPA has distinct nuclear functions, in addition to its established role as an ER organizer.

Microtubule organization plays a central role in cell differentiation, orchestrating essential processes such as cell polarization, mechanotransduction, organelle positioning and intracellular transport. A hallmark of many differentiated cells is the transition from a centrosomal to a non-centrosomal microtubule-organizing center (MTOC). Here, we demonstrate that both centrosomal and nuclear envelope (NE)-associated MTOCs coexist in osteoclasts. We show that the key players for NE-MTOC formation, the AKAP6 and nesprin-1 (SYNE1) isoforms AKAP6β and nesprin-1α, previously considered muscle specific, are upregulated during osteoclast differentiation, suggesting a conserved role in NE-MTOC assembly across cell types. Targeted depletion of AKAP6 in RAW264.7-derived osteoclasts led to the displacement of the Golgi and MTOC-associated proteins PCM1, pericentrin and CDK5RAP2 from the NE, while their centrosomal localization remained intact. This selectively impaired microtubule nucleation from the NE without disrupting centrosomal microtubule activity, enabling a functional dissection of the two MTOCs. Loss of NE-MTOC activity, through AKAP6 depletion, impaired podosome formation and significantly reduced bone resorption capacity, highlighting the distinct and essential role of NE-derived microtubules in osteoclast function.

Regulation of lamin A/C levels and distribution is crucial for nuclear integrity and mechanotransduction via the linker of nucleoskeleton and cytoskeleton (LINC) complex. Dysregulation of lamin A/C correlates with poor cancer prognosis, and its levels determine sensitivity to the microtubule-stabilising drug paclitaxel. Paclitaxel is well-known for disrupting mitosis, yet it also reduces tumour size in slow-dividing tumours, indicating an additional, poorly characterised interphase mechanism. Here, we reveal that paclitaxel induces nuclear aberrations in interphase through SUN2-dependent lamin A/C disruption. Using advanced optical imaging and electron cryo-tomography, we show the formation of aberrant microtubule-vimentin bundles during paclitaxel treatment, which coincides with nuclear deformation and altered lamin A/C protein levels and organisation at the nuclear envelope. SUN2 is required for lamin A/C reduction upon paclitaxel treatment and is in turn regulated by polyubiquitylation. Furthermore, lamin A/C expression levels determine not only cell survival during treatment but also recovery after drug removal. Our findings support a model in which paclitaxel acts through both defective mitosis and interphase nuclear-cytoskeletal disruption, providing additional mechanistic insights into a widely used anticancer drug.

Within the nucleus of each human cell, ∼2 m of linear DNA is compacted and organized. The structures and principles of genome organization are developmentally regulated and broadly evolutionarily conserved. However, conclusive links between genome structure and function have been difficult to find. In this Review, we provide an overview of mammalian genome organization, highlight recent studies demonstrating how it interacts with evolutionary diversity, and explore its contributions to development. We propose an innovative perspective - that variability in genome organization supports plastic cell fates in multicellular organisms - and draw analogies to show how evolutionary variation can inform study of the function of genome organization.

Sensing the mechanical environment enables cells to rapidly respond to physical tissue parameters, influencing decisions regarding shape, function, and fate. Despite the well-established effects of physical cues on cellular morphological characteristics, the effect of these cues, including surface topography and confinement, on organelle architecture has not yet been thoroughly investigated. This study aims to identify the Golgi-specific transcriptional program involved in cellular adaptation to pillar-induced confinement using bone marrow-derived mesenchymal stem cells as a model system. We make use of seven polystyrene micro-pillar arrangements with unique designs that induce a high diversity in structural Golgi organization. Transcriptional analysis of functionally relevant Golgi genes shows a unique fingerprint at the gene expression level for each pillar design, and variation in the amount of affected genes can partly be explained by the level of confinement and disruption of Golgi organization respectively. We outline a method using pillar-enhanced substrates as a model system to reveal cellular adaptation to extracellular mechanical cues such as confinement, encouraging further exploration of the effects of physical cues on organelle structural reorganization and functional adaptation.

Mammalian female meiosis is uniquely regulated to produce a developmentally competent egg capable of supporting embryogenesis. During meiosis I, homologous chromosomes segregate, with half extruded into the first polar body. The egg then arrests at metaphase II and only resumes meiosis and extrudes the second polar body following fertilization. The MOS/MAPK signaling pathway is important for maintaining the metaphase II arrest; in mos-/- mutants, a subset of eggs undergo spontaneous parthenogenetic activation and exhibit additional abnormal cell divisions. To further understand the cell cycle mis-regulation in activated mos-/- eggs, we used time-lapse microscopy to monitor the abnormal divisions. We discovered that, following parthenogenetic activation, the first polar body can assemble a spindle, segregate chromosomes, and divide with timings similar to anaphase II onset in the egg. This behavior contrasts with wildtype polar bodies, which do not divide and typically degenerate. We demonstrate that mos-/- oocytes and polar bodies can exchange cytoplasm for a longer duration due to an extension of telophase I, likely allowing the transfer of cell cycle regulators between the two compartments. Further inspection revealed that mos-/- oocytes have defective meiotic midbody assembly with most oocytes lacking a cap structure, which is needed to separate the oocyte and the polar body prior to abscission. We report that polar bodies of mos-/- eggs can re-enter the cell cycle and undergo additional aberrant divisions. These findings identify MOS as a critical regulator of meiotic midbody formation and uncover a novel consequence of disrupted MOS/MAPK signaling: the potential for polar bodies to become mitotically active.

The strict control, yet dynamic nature of adhesive structures that form in the extracellular environment are crucial for the development and homoeostasis of multicellular organisms. A gradual increase in the strength of the myotendinous junction (MTJ) occurs as ligands accumulate in the ECM and bind to opposing integrin complexes at muscle junction interfaces. While proteomic studies of the muscle-tendon junction in mice and humans have revealed the complexity of protein classes in this extracellular environment, the functions of many ECM proteins remain elusive. To fill this gap in knowledge, we performed a sensitized genetic screen to expose MTJ-relevant genes in Drosophila melanogaster whose functions may be redundant or sensitive to mechanical strain. Aside from the expected ECM proteins that comprise the basement membrane, we uncovered functional roles for other classes of ECM-affiliated proteins. Here we follow up on the sole ortholog of Transglutaminase (Tg) encoded in the Drosophila genome. Either Tg RNAi knockdown or expression of catalytically inactive Tg causes detachment of direct muscle-cuticle attachments at different stages in development. In adults, MTJ stability is further weakened in response to increased mechanical tension. These studies together describe a previously unappreciated role for Tg crosslinking in preserving muscle attachments in response to tension.

Mechanical coupling and chemical communication between cardiomyocytes are facilitated through a specialized adhesive structure called the intercalated disc (ICD). The ICD is essential for heart organization and contraction. Yet, the network of adhesion, adaptor and signaling proteins that form the ICD remains poorly defined. Here, we combined proximity labeling and quantitative mass spectrometry to identify proteins associated with the desmosomal cadherin desmoglein 2 (DSG2), in cultured neonatal cardiomyocytes. We identified over 300 proteins in the DSG2 interactome, half of which are shared with the N-cadherin (CDH2) interactome in cardiomyocytes. Proteins unique to DSG2 include connexin 43 and the plakin family of cytolinker proteins. Comparison of the cardiomyocyte DSG2 interactome with the interactomes of desmosomal proteins from epithelia revealed few shared proteins. In cardiomyocytes, plakoglobin and plakophilin 2 (PKP2) were the most abundant shared proteins between the DSG2 and CDH2 interactomes. We show that PKP2 is a dynamic protein whose membrane recruitment in cardiomyocytes is dependent on tension. Our analysis of the DSG2 interactome provides a crucial new dimension to the proteomic atlas of the essential molecular complexes required for cardiomyocyte adhesion.

Lipid membranes form the essential barriers that compartmentalize life, separating intracellular processes from the external environment. To maintain cellular function and viability, both the plasma membrane and internal organelle membranes undergo continuous compositional and functional remodeling in response to environmental fluctuations. Traditionally, glycerophospholipids have been primarily considered structural components of these membranes. However, their dynamic synthesis plays a crucial role in modulating membrane functions and, consequently, cellular adaptability. This Review discusses how cells orchestrate complex glycerophospholipid metabolism to adapt to diverse environmental challenges. By examining membrane adaptation to various changes, including temperature shifts, pH imbalances and nutrient availability, we propose that responsive alterations in glycerophospholipid synthesis act as a central metabolic hub. This hub influences overall cellular metabolism and regulatory networks. This Review highlights an often overlooked aspect of lipid biology: the pivotal role of glycerophospholipid metabolism in modulating cellular adaptability and resilience.

Studying how actin filaments are assembled into different subcellular structures can provide insights into both physiological processes and the mechanisms of disease. However, quantifying the size, abundance, and organization of different classes of actin structure from optical microscopy data remains a challenge. To address this, we developed a deep learning based Filamentous Actin Segmentation Tool (FAST) to accurately and efficiently segment and quantify different classes of actin structure from Phalloidin stained confocal microscopy images. We evaluated the performance of this tool to segment and quantify the abundance of different classes of actin structure in different cell lines and with dynamic changes in actin organization using lifeact-GFP during drug treatments. FAST enables quantification of different classes of actin structure from actin images alone, without the need for specific antibodies against proteins in different actin structures and hence can be a useful tool for researchers studying actin related pathways involved in cell motility, cancer metastasis, and drug development.