Journal of cell science最新文献

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.

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.

In humans, inositol polyphosphate-5-phosphatase e (INPP5E) mutations cause retinal degeneration as part of Joubert and MORM syndromes and can also cause non-syndromic blindness. In mice, mutations cause a spectrum of brain, kidney, and other anomalies and prevent the formation of photoreceptor outer segments. To further explore the function of Inpp5e in photoreceptors, we generated conditional and inducible knockouts of mouse Inpp5e where the gene was deleted either during outer segment formation or after outer segments were fully formed. In both cases, the loss of Inpp5e led to severe defects in photoreceptor outer segment morphology and ultimately photoreceptor cell loss. The primary morphological defect consisted of outer segment shortening and reduction in the number of newly forming discs at the outer segment base. This was accompanied by structural abnormalities of the Golgi apparatus, mislocalized rhodopsin, and an accumulation of extracellular vesicles. In addition, knockout cells showed disruption of the actin network. Together, these data demonstrate that Inpp5e plays a critical role in maintaining the outer segment and the normal process of outer segment renewal depends on the activity of this enzyme.

Here, we apply SuperResNET network analysis of dSTORM single-molecule localization microscopy (SMLM) to determine how the clathrin endocytosis inhibitors pitstop 2, dynasore and Latrunculin A alter the morphology of clathrin-coated pits. SuperResNET analysis of HeLa and Cos7 cells identifies: 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 induce distinct homogeneous populations of Class II structures in HeLa cells suggesting that they arrest endocytosis at different stages. Inhibition is not via actin depolymerization, as the actin-depolymerizing agent latrunculin A (LatA) induces large, heterogeneous clathrin structures. Ternary analysis of SuperResNET shape features presents a distinct more planar profile for pitstop 2 blobs that align with clathrin pits identified with high-resolution MINFLUX, while control structures resemble MINFLUX clathrin vesicles. SuperResNET analysis therefore shows 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 data sets.

Cytoplasmic dynein is essential in motoneurons for retrograde cargo transport that sustains neuronal connectivity. Little, however, is known about dynein's function on the postsynaptic side of the circuit. Here we report distinct postsynaptic roles for dynein at neuromuscular junctions (NMJs). Intriguingly, we show that dynein punctae accumulate postsynaptically at glutamatergic synaptic terminals. Moreover, Skittles, a phosphatidylinositol 4-phosphate 5-kinase that produces PI(4,5)P2 to organize the spectrin cytoskeleton, also localizes specifically to glutamatergic synaptic terminals. Depletion of postsynaptic dynein disrupts the accumulation of Skittles, PI(4,5)P2 phospholipid, and organization of the spectrin cytoskeleton at the postsynaptic membrane. Coincidental with dynein depletion, we observe an increase in the size of ionotropic glutamate receptor (iGluRs) fields, and an increase in the amplitude and frequency of mEJPs. PI(4,5)P2 levels do not affect iGluR clustering, nor does dynein affect the levels of iGluR subunits at the NMJ. Our observations suggest a separate, transport independent function for dynein in iGluR cluster organization. Based on the close apposition of dynein punctae to the iGluR fields, we speculate that dynein at the postsynaptic membrane contributes to the organization of the receptor fields, hence ensuring proper synaptic transmission.

Retromer mediates retrograde transport of protein cargos from endosomes to the trans-Golgi network (TGN). γ-secretase is a protease that cleaves the transmembrane domain of its target proteins. Although retromer can form a stable complex with γ-secretase, the functional consequences of this interaction are not known. Here, we report that retromer-mediated retrograde protein trafficking in cultured human epithelial cells is impaired by the γ-secretase inhibitor XXI or by knockout of the catalytic subunit of γ-secretase. These treatments inhibited endosome to TGN trafficking of retromer-dependent retrograde cellular cargos, divalent metal transporter 1 isoform II, cation-independent mannose-6-phosphate receptor, and shiga toxin, whereas trafficking of retromer-independent cargos cholera toxin and a mutant CIMPR unable to bind retromer was not affected. Moreover, we found that g-secretase associates with retromer cargos even in the absence of retromer. XXI treatment and PS1 knockout did not inhibit the ability of retromer or g-secretase to associate with cargo and did not affect the expression of retromer subunits or Rab7-GTP, which regulates retromer-cargo interaction. These results imply that the γ-secretase-retromer interaction facilitates retromer-mediated retrograde trafficking of cellular transmembrane proteins.

Talin regulates the adhesion and migration of cells in part by promoting the affinity of integrins for extracellular matrix proteins, a process that in cells such as endothelial cells and platelets requires the direct interaction of talin with both the small GTPase, Rap1-GTP, and the integrin β3 cytoplasmic tail. To study this process in more detail, we employed an optogenetic approach in living, immortalized endothelial cells to be able to regulate talin interaction with the plasma membrane. Previous studies identified talin as the Rap1-GTP effector for β3 integrin activation. Surprisingly, optogenetic recruitment of talin to the plasma membrane also led to the localized activation of Rap1 itself, apparently by talin competing for Rap1-GTP with SHANK3, a protein known to sequester Rap1-GTP and to block integrin activation. Rap1 activation by talin was localized to the cell periphery in suspension cells and within lamellipodia and pseudopodia in cells adherent to fibronectin. Thus, membrane-associated talin can play a dual role in regulating integrin function in endothelial cells: first by releasing Rap1-GTP from its sequestration by SHANK3 and second by serving as the relevant Rap1 effector for integrin activation.

The plasma membrane and the underlying skeleton form a protective barrier for eukaryotic cells. The molecular players forming this complex composite material constantly rearrange under mechanical stress. One of those molecules, spectrin, is ubiquitous in the membrane skeleton and linked by short actin filaments. In this work, we developed a generalized network model for the membrane skeleton integrated with myosin contractility and membrane mechanics to investigate the response of the spectrin meshwork to mechanical loading. We observed that the force generated by membrane bending is important to maintain a regular skeletal structure suggesting that the membrane is not just supported by the skeleton, but has an active contribution to the stability of the cell structure. We found that spectrin and myosin turnover are necessary for the transition between stress and rest states in the skeleton. Simulations of a fully connected network representing a whole cell show that the surface area constraint of the plasma membrane and volume restriction of the cytoplasm enhance the stability of the membrane skeleton. Furthermore, we showed that cell attachment through adhesions promotes cell shape stabilization.

This study investigated possible mechanisms underlying differences between heterophilic and homophilic cadherin adhesions that influence intercellular mechanics and multicellular organization. Results suggest that homophilic cadherin ligation selectively activates force-transduction, such that resulting signaling and mechano-transduction amplitudes are independent of cadherin binding affinities. Epithelial (E-) and neural (N-) cadherin cooperate with distinct growth factors to mechanically activate force-transduction cascades. Prior results demonstrated that E-cadherin and epidermal growth factor receptor form force-sensitive complexes at intercellular junctions. Here, results show that the reconstitution of N-cadherin force transduction required the co-expression of N-cadherin and fibroblast growth factor receptor. Mechanical measurements further demonstrated that homophilic ligation initiates receptor tyrosine kinase-dependent force transduction cascades, but heterophilic cadherin ligands fail to activate signaling or generate stereotypical mechano-transduction signatures. The all-or-nothing contrast between mechano-transduction by heterophilic versus homophilic cadherin adhesions supersedes differences in cadherin adhesion strength. This mechano-selectivity impacts cell spreading and traction generation on cadherin substrates. Homophilic ligation appears to be a key that selectively unlocks cadherin mechano-transduction. These findings may reconcile the roles of cadherin recognition and cell mechanics in the organization of multicellular assemblies.

Glycosaminoglycans (GAGs), as animal polysaccharides, are linked to proteins to form various types of proteoglycans. Bacterial GAG lyases are not only essential enzymes that spoilage bacteria use for the degradation of GAGs, but also valuable tools for investigating the biological function and potential therapeutic applications of GAGs. The ongoing discovery and characterization of novel GAG lyases has identified an increasing number of lyases suitable for functional studies and other applications involving GAGs, which include oligosaccharide sequencing, detection and removal of specific glycan chains, clinical drug development and the design of novel biomaterials and sensors, some of which have not yet been comprehensively summarized. GAG lyases can be classified into hyaluronate lyases, chondroitinases and heparinases based on their substrate spectra, and their functional applications are mainly determined by their substrates, with different lyases exhibiting differing substrate selectivity and preferences. It is thus necessary to understand the properties of the available enzymes to determine strategies for their functional application. Building on previous studies and reviews, this Review highlights small yet crucial differences among or within the various GAG lyases to aid in optimizing their use in future studies. To clarify ideas and strategies for further research, we also discuss several traditional and novel applications of GAG lyases.