Journal of cell science最新文献

Retromer mediates retrograde transport of protein cargoes 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 PS1 (also known as PSEN1), the catalytic subunit of γ-secretase. These treatments inhibited endosome-to-TGN trafficking of retromer-dependent retrograde cellular cargoes, divalent metal transporter 1 isoform II, cation-independent mannose-6-phosphate receptor and shiga toxin, whereas trafficking of retromer-independent cargoes, cholera toxin and a mutant CIMPR unable to bind retromer was not affected. Moreover, we found that γ-secretase associates with retromer cargoes even in the absence of retromer. XXI treatment and PS1 knockout did not inhibit the ability of retromer or γ-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.

Properly timed gene expression is essential for all aspects of organismal physiology. Despite significant progress, our understanding of the complex mechanisms governing the dynamics of gene regulation in response to internal and external signals remains incomplete. Over the past decade, advances in technologies like light and cryo-electron microscopy (Cryo-EM), cryo-electron tomography (Cryo-ET) and high-throughput sequencing have spurred new insights into traditional paradigms of gene expression. In this Review, we delve into recent concepts addressing 'where' and 'when' gene transcription and RNA splicing occur within cells, emphasizing the dynamic spatial and temporal organization of the cell nucleus.

The neuronal cytoskeleton comprises microtubules, actin filaments and neurofilaments, and plays a crucial role in axon outgrowth and transport. Microtubules and actin filaments have attracted considerable attention in axon regeneration studies. We have previously shown that TC10 (also known as RhoQ), a Rho family GTPase that promotes axon outgrowth through membrane addition, is required for efficient axon regeneration. This study demonstrates that TC10 on recycling endosomes, but not on the plasma membrane, balances microtubule stability and dynamics in the axons, thereby counteracting axon retraction. TC10 ablation reduced the phosphorylation of SCG10 (also known as STMN2) and MAP1B, which are neuronal microtubule-binding proteins and JNK substrates. Consistent with this, JNK phosphorylation was decreased in TC10-knockout neurons compared to in wild-type neurons. Furthermore, TC10 deletion significantly reduced PAK2 autophosphorylation. PAK2 was found on Rab11-positive endosomes in cell bodies and axons, and its localization to endosomes was reduced by TC10 loss. PAK inhibition reduced tubulin acetylation and JNK phosphorylation in axons. Furthermore, MKK4 and MKK7 (also known as MAP2K4 and MAP2K7, respectively) were found to mediate signaling from TC10-activated PAK to JNK on JIP1-positive endosomes. Overall, TC10 transmits a microtubule-regulatory signal from PAK2 to SCG10 and MAP1B via JNK on axonal endosomes.

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, 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 crucial role in maintaining the outer segment and the normal process of outer segment renewal depends on the activity of this enzyme.

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 bound to GTP (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 the interaction of talin with the plasma membrane. Previous studies identified talin as the Rap1-GTP effector for β3 integrin activation. Surprisingly, optogenetic recruitment of talin-1 (TLN1; herein referred to as 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 cytoplasm exhibits viscoelastic properties, displaying both solid and liquid-like behaviour, and can actively regulate its mechanical attributes. The cytoskeleton is a major regulator among the numerous factors influencing cytoplasmic mechanics. We explore the interdependence of various cytoskeletal filaments and the impact of their density on cytoplasmic viscoelasticity. The heterogeneous distribution of these filaments gives rise to polarised mechanical properties of the cytoplasm along the dorsoventral axis. Actin filament disassembly softens the ventral cytoplasm while stiffening the mid cytoplasm, due to increased vimentin filament assembly. Disruption of microtubules or depletion of vimentin softens both the ventral and mid cytoplasm. Cytochalasin D (Cyto D) treatment results in a localised increase of vimentin assembly in the mid cytoplasm, which is dependent on the cytolinker plectin. Nocodazole treatment has a negligible effect on F-actin distribution but significantly alters the spatial arrangement of vimentin. We demonstrate that Cyto D treatment upregulates vimentin expression via reactive oxygen species-mediated activation of NF-κΒ. This article investigates how different cytoskeletal filaments influence the rheological characteristics of various cytoplasmic regions.

Characterization of protein-protein interactions (PPIs) is a fundamental goal in the post-genomic era. Here, we document a generally applicable approach to identify cellular protein interactomes using a combination of nanobody-based affinity purification (AP) coupled with liquid chromatography and tandem mass spectrometry (LC-MS/MS). The Hippo signaling regulator TAZ (also known as WWTR1) functions as a transcriptional co-repressor or activator depending on its PPI network; we therefore undertook an unbiased proteomic screen to identify TAZ PPIs in striated muscle cells. A GFP nanotrap-based AP approach coupled with protein identification through LC-MS/MS was used to document a comprehensive list of known and novel TAZ interactome components. Informatic analysis of the interactome documented known components of the Hippo signaling pathway and multiple epigenetic regulators such as the NuRD, FACT and SWI/SNF complexes and the pro-myogenic CARM1 methyltransferase. Hippo pathway reporter gene (HOP/HIP) analysis indicated that CARM1 represses TAZ transcriptional co-activator function, promoting TAZ Ser89 phosphorylation and TAZ cytoplasmic sequestration. MS analysis revealed that CARM1 dimethylates TAZ at Arg77 in a PGPR*LAGG consensus peptide, resulting in enhanced TAZ Ser89 phosphorylation. These studies underline the utility of a nanobody-based AP approach for interactome analysis.

Cells are continuously subjected to physical and chemical cues from the extracellular environment, and sense and respond to mechanical cues via mechanosensation and mechanotransduction. Although the role of the cytoskeleton in these processes is well known, the contribution of intracellular membranes has been long neglected. Recently, it has become evident that various organelles play active roles in both mechanosensing and mechanotransduction. In this Review, we focus on mechanosensitive roles of the endoplasmic reticulum (ER), the functions of which are crucial for maintaining cell homeostasis. We discuss the effects of mechanical stimuli on interactions between the ER, the cytoskeleton and other organelles; the role of the ER in intracellular Ca2+ signalling via mechanosensitive channels; and how the unfolded protein response and lipid homeostasis contribute to mechanosensing. The expansive structure of the ER positions it as a key intracellular communication hub, and we additionally explore how this may be leveraged to transduce mechanical signals around the cell. By synthesising current knowledge, we aim to shed light on the emerging roles of the ER in cellular mechanosensing and mechanotransduction.

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 integrating 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 in maintaining a regular skeletal structure, suggesting that the membrane is not just supported by the skeleton, but actively contributes towards 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.