Journal of cell science最新文献

The actin-based motor myosin-19 (Myo19) exerts force on mitochondrial membrane receptors Miro1/2, influencing endoplasmic reticulum (ER)-mitochondria contact sites and mitochondrial cristae structure. The mitochondrial intermembrane bridging (MIB) complex connects the outer and inner mitochondrial membranes at the cristae junction through the mitochondrial contact site and cristae organization system (MICOS). However, the interaction between Myo19, Miro1 and Miro2 (hereafter Miro1/2), and the MIB-MICOS complex in cristae regulation remains unclear. This study investigates the roles of Miro1/2 and metaxin 3 (Mtx3), a MIB complex component, in linking Myo19 to MIB-MICOS. We show that Miro1/2 interact with Myo19 and the MIB complex but not with Mtx3. Their mitochondrial membrane anchors are not essential for MIB interaction or cristae structure. However, Mtx3 is crucial for the connection between MIB-MICOS and the Myo19 and Miro1/2 proteins. Deleting Miro1/2 mimics the effects of Myo19 deficiency on ER-mitochondria contacts and cristae structure, whereas Mtx3 deletion does not. Notably, the loss of Myo19 and Miro1/2 alters mitochondrial lipid composition, reducing cardiolipin and its precursors, suggesting Myo19 and Miro1/2 influence cristae indirectly via lipid transfer at ER-mitochondria contact sites.

To rapidly adapt to harmful changes to their environment, cells activate the integrated stress response (ISR). This results in an adaptive transcriptional and translational rewiring, and the formation of biomolecular condensates named stress granules (SGs), to resolve stress. In addition to this first line of defence, the mitochondrial unfolded protein response (UPRmt) activates a specific transcriptional programme to maintain mitochondrial homeostasis. We present evidence that the SG formation and UPRmt pathways are intertwined and communicate. UPRmt induction results in eIF2α phosphorylation and the initial and transient formation of SGs, which subsequently disassemble. The induction of GADD34 (also known as PPP1R15A) during late UPRmt protects cells from prolonged stress by impairing further assembly of SGs. Furthermore, mitochondrial functions and cellular survival are enhanced during UPRmt activation when SGs are absent, suggesting that UPRmt-induced SGs have an adverse effect on mitochondrial homeostasis. These findings point to a novel crosstalk between SGs and the UPRmt that might contribute to restoring mitochondrial functions under stressful conditions.

Cells form multiple, molecularly distinct membrane contact sites (MCSs) between organelles. Despite knowing the molecular identity of several of these complexes, little is known about how MCSs are coordinately regulated in space and time to promote organelle function. Here, we examined two well-characterized mitochondria-endoplasmic reticulum (ER) MCSs - the ER-mitochondria encounter structure (ERMES) and the mitochondria-ER-cortex anchor (MECA) in Saccharomyces cerevisiae. We report that loss of MECA results in a substantial reduction in the number of ERMES contacts. Rather than reducing ERMES protein levels, loss of MECA results in an increase in the size of ERMES contacts. Using live-cell microscopy, we demonstrate that ERMES contacts display several dynamic behaviors, such as de novo formation, fusion and fission, that are altered in the absence of MECA or by changes in growth conditions. Unexpectedly, we find that the mitochondria-plasma membrane (PM) tethering, and not the mitochondria-ER tethering, function of MECA regulates ERMES contacts. Remarkably, synthetic tethering of mitochondria to the PM in the absence of MECA is sufficient to rescue the distribution of ERMES foci. Overall, our work reveals how one MCS can influence the regulation and function of another.

In response to external stress, mitochondrial dynamics is often disrupted, but the associated physiologic changes are often uncharacterized. In many cancers, including glioblastoma (GBM), mitochondrial dysfunction has been observed. Understanding how mitochondrial dynamics and physiology contribute to treatment resistance will lead to more targeted and effective therapeutics. This study aims to uncover how mitochondria in GBM cells adapt to and resist ionizing radiation (IR), a component of the standard of care for GBM. Using several approaches, we investigated how mitochondrial dynamics and physiology adapt to radiation stress, and we uncover a novel role for Fis1, a pro-fission protein, in regulating the stress response through mitochondrial DNA (mtDNA) maintenance and altered mitochondrial bioenergetics. Importantly, our data demonstrate that increased fission in response to IR leads to removal of mtDNA damage and more efficient oxygen consumption through altered electron transport chain (ETC) activities in intact mitochondria. These findings demonstrate a key role for Fis1 in targeting damaged mtDNA for degradation and regulating mitochondrial bioenergetics through altered dynamics.

Macrophages play a pivotal role in clearing debris and microbes from the microenvironment via phagocytosis and orchestrating local inflammation. While mostly understood to be through the synthesis and secretion of soluble mediators such as cytokines and eicosanoids, it has been recently proposed that macrophages can release previously phagocytosed and processed PAMPs and DAMPs into the local microenvironment via a process termed eructophagy, and that these, in turn, can activate recently vicinal leukocytes. Additionally, it has been commonly observed that local macrophages physically interact with other leukocytes, such as neutrophils and T cells, recruited to sites of inflammation. This study demonstrated that eructophagy in macrophages is significantly induced during physical interaction with neutrophils and T cells, which is mediated by ICAM1 on macrophages and LFA1 on neutrophils/T cells. Notably, ICAM1 activation alone is sufficient to trigger eructophagy in macrophages and is dependent on Lyn kinase. Through this mechanism, it is proposed that neutrophils and lymphocytes can influence their own activation by interacting with local macrophages containing PAMP-containing phagolysosomes, which subsequently triggers PAMP release into the local microenvironment through eructophagy.

Pancreatic stellate cells (PSCs) are primarily responsible for producing the stiff tumor tissue in pancreatic ductal adenocarcinoma (PDAC). Thereby, PSCs generate a stiffness gradient between the healthy pancreas and the tumor. This gradient induces durotaxis, a form of directional cell migration driven by differential stiffness. However, the molecular sensors behind durotaxis are still unclear. To investigate the role of mechanosensitive ion channels in PSC durotaxis, we established a two-dimensional stiffness gradient mimicking PDAC. Using pharmacological and genetic methods, we investigated the contribution of the ion channels Piezo1, TRPC1, and TRPV4 in PSC durotaxis. We found that PSC migration towards a stiffer substrate is diminished by altering Piezo1 activity. Moreover, disrupting TRPC1 along with TRPV4 abolishes PSC durotaxis even when Piezo1 is functional. Our results demonstrate that optimal PSC durotaxis requires an intermediary level of ion channel activity, which we simulated via a numerically discretized mathematical model. These findings suggest that mechanosensitive Piezo1 channels detect the differential stiffness microenvironment. The resulting intracellular signals are amplified by TRPV4 and TRPC1 channels to guide efficient PSC durotaxis.

When starved of nitrogen, fission yeast Schizosaccharomyces pombe cells enter a quiescent "G0" state with smaller nuclei and transcriptional repression. The genomics of S. pombe G0 cells has been well studied, but much of its nuclear cell biology remains unknown. Here we use confocal microscopy, immunoblots, and electron cryotomography to investigate the cytological, biochemical, and ultrastructural differences between S. pombe proliferating, G1-arrested, and G0 cell nuclei, with an emphasis on the histone acetylation, RNA polymerase II fates, and macromolecular complex packing. Compared to proliferating cells, G0 cells have lower levels of histone acetylation, nuclear RNA polymerase II, and active transcription. The G0 nucleus has similar macromolecular crowding yet fewer chromatin-associated multi-megadalton globular complexes. Induced histone hyperacetylation during nitrogen starvation results in cells that have larger nuclei and therefore less compact chromatin. However, these histone-hyperacetylated cells remain transcriptionally repressed with similar nuclear crowding. Canonical nucleosomes - those that resemble the crystal structure - are rare in proliferating, G1-arrested, and G0 cells. Our study therefore shows that extreme changes in nucleus physiology are possible without extreme reorganisation at the macromolecular level.

The ability to quantitatively study mRNA translation using SunTag imaging is transforming our understanding of the translation process. Here, we expand the SunTag method to study new aspects of translation regulation in Drosophila. Repression of the maternal hunchback (hb) mRNA in the posterior of the Drosophila embryo is a textbook example of translational control. Using SunTag imaging to quantitate translation of maternal SunTag-hb mRNAs, we show that repression in the posterior is leaky as ∼5% of SunTag-hb mRNAs are translated. In the anterior of the embryo, the maternal and zygotic SunTag-hb mRNAs show similar translation efficiency despite having different UTRs. We demonstrate that the SunTag-hb mRNA can be used as a reporter to study ribosome pausing at single-mRNA resolution, by exploiting the conserved xbp1 mRNA and A60 pausing sequences. Finally, we adapt the detector component of the SunTag system to visualise and quantitate translation of the short gastrulation (sog) mRNA, encoding an essential secreted extracellular BMP regulator, at the endoplasmic reticulum in fixed and live embryos. Together, these tools will facilitate the future dissection of translation regulatory mechanisms during development.

The guided-entry of tail-anchored proteins (GET) pathway is a post-translational targeting route to the endoplasmic reticulum. Upon glucose withdrawal, the soluble GET proteins re-localize to dynamic cytosolic foci, here termed GET bodies. Our data reveal that the pre-targeting complex components, Sgt2 and the Get4-Get5 heterodimer, and the Get3 ATPase play important roles in the assembly of these structures. More specifically, the TPR region of Sgt2 is required as a GET body scaffold. Systematic compositional analyses of GET bodies reveal their chaperone-rich nature and the presence of numerous proteins involved in metabolic processes. Temporal analyses of GET body assembly demonstrate the sequential recruitment of different chaperones, and we discover the requirement of Sis1 and Sti1 for maintaining the dynamic properties of these structures. In vivo, NADH derived from the oxidation of ethanol to acetaldehyde can induce GET body disassembly in a reaction depending on the alcohol dehydrogenase Adh2 and in vitro, addition of NADH resolves GET bodies. This suggests a mechanistic basis for their formation and disassembly in response to the metabolic shift caused by glucose withdrawal and re-addition.

SuperResNET is a network analysis pipeline for the analysis of point cloud data generated by single-molecule localization microscopy (SMLM). Here, we applied SuperResNET network analysis of SMLM direct stochastic optical reconstruction microscopy (dSTORM) data to determine how the clathrin endocytosis inhibitors pitstop 2, dynasore and latrunculin A (LatA) alter the morphology of clathrin-coated pits. SuperResNET analysis of HeLa and Cos7 cells identified three classes of clathrin structures: small oligomers (class I), pits and vesicles (class II), and larger clusters corresponding to fused pits or clathrin plaques (class III). Pitstop 2 and dynasore treatment induced distinct homogeneous populations of class II structures in HeLa cells, suggesting that they arrest endocytosis at different stages. Inhibition of endocytosis was not via actin depolymerization, as the actin-depolymerizing agent LatA induced large, heterogeneous clathrin structures. Ternary analysis of SuperResNET shape features presented a distinct more planar profile for blobs from pitstop 2-treated cells, which aligned with clathrin pits identified with high-resolution minimal photon fluxes (MINFLUX) microscopy, whereas control structures resembled MINFLUX clathrin vesicles. SuperResNET analysis therefore showed that pitstop 2 arrests clathrin pit maturation at early stages of pit formation, representing an approach to detect the effect of small molecules on target structures in situ in the cell from SMLM datasets.