Journal of Cell Biology最新文献

Importins could inhibit the condensation of RNA-binding proteins, while it remains unknown whether exportins elicit a similar function. Here, we identified that exportin CRM1 binds to the nuclear protein NPAT, which initiates and maintains the formation of the histone locus body (HLB), a membraneless nuclear body regulating histone transcription. CRM1 drives the nuclear export of NPAT by targeting a nuclear export signal (NES) within the LisH domain. The LisH domain contributes to NPAT condensation by mediating its self-association. Mechanistically, CRM1 competitively occupies the self-association sites in the NES motif, thereby suppressing NPAT condensation. In contrast, the two recurrent CRM1 E571K and E571G mutants could not regulate NPAT condensation and HLB remodeling due to their impaired binding to the NES of NPAT. Based on the "competitive occupation" model, we designed a LisH domain-derived short peptide that competes with homotypic intermolecular interactions of NPAT to perturb HLB formation. Our findings reveal that exportin regulates nuclear protein condensation via a competitive occupation strategy.

A pathological hallmark in >97% of amyotrophic lateral sclerosis (ALS) cases is the cytoplasmic mislocalization and aggregation of TDP-43, a nuclear RNA-binding protein, in motor neurons. Driving clearance of cytoplasmic TDP-43 reduces toxicity in ALS models, though how TDP-43 clearance is regulated remains controversial. We conducted an unbiased yeast screen using high-throughput dot blotting to identify genes that affect TDP-43 levels. We identified ESCRT complex genes, which induce membrane invagination (particularly at multivesicular bodies; MVBs) and genes linked to K63 ubiquitination (particularly cofactors of the E3 ubiquitin ligase Rsp5; NEDD4 in humans), as drivers of TDP-43 endolysosomal clearance. TDP-43 colocalized and bound Rsp5/NEDD4 and ESCRT proteins, and perturbations to either increased TDP-43 aggregation, stability, and toxicity. NEDD4 also ubiquitinates TDP-43. Lastly, TDP-43 accumulation induces giant MVB-like vesicles, within which TDP-43 accumulates in a NEDD4-dependent manner. Our studies shed light on endolysosomal-mediated cytoplasmic protein clearance, a poorly understood proteostasis mechanism, which may help identify novel ALS therapeutic strategies.

Insect cuticles with nano-level structures exhibit functional surface properties such as the photonic nanocrystal of the butterfly wing scale with structural color and the corneal nipple arrays of superhydrophobic compound eye lens. Despite the enormous influence the cuticle has had on biomimetic industrial applications, cellular mechanisms of cuticular nanopatterning remain poorly understood. Drosophila gore-tex/Osiris23 (gox) controls the formation of nanopores, with a molecular filtering function, on the olfactory organs. Here we used 3D electron microscopy imaging of entire hair structures to show that nanopore is formed through a novel process of bidirectional interaction of the ER and the plasma membrane trafficking. ER-resident protein Gox stimulates ER-phagy through regulation of Ref(2)P, the fly counterpart of the autophagy protein p62/SQSTM1, and initiates endocytosis. Dynamin on the plasma membrane completes endocytosis and sustains ER-phagy. The repurposing of ER-phagy for plasma membrane remodeling and the fabrication of nanoscale ECM structures sheds light on the nanopatterning mechanism of insect cuticles and their genetic control.

Constitutive integrin endocytosis and recycling control cell movement and morphology. In contrast, the role of newly synthesized integrins delivered via the biosynthetic pathway has been largely overlooked. We used the retention using selective hooks system to monitor the localization of new integrins exiting the endoplasmic reticulum in space and time. We discovered that new integrin delivery to the plasma membrane is polarized and enhances cell protrusion and focal adhesion growth in an extracellular matrix-ligand-dependent manner. Motor-clutch modeling explained the increased adhesion as higher integrin availability driving recruitment of additional receptors. Unexpectedly, live-cell imaging revealed a small subset of fast-emerging integrin vesicles rapidly transported to the cell surface to facilitate localized spreading. This unconventional secretion depended on cell adhesion and correlated with increased surface levels of immature, high-mannose glycosylated integrin, indicating bypass of the canonical Golgi-dependent secretory pathway. Thus, spatial plasma membrane-targeting of new integrins rapidly alters adhesion receptor availability, providing cells with added plasticity to respond to their environment.

Maintaining peroxisome homeostasis is crucial for cellular function and its disruption links to metabolic and neurodegenerative disorders. We developed PO-TRG mice ubiquitously expressing RFP-GFP-SKL to enable in vivo pexophagy monitoring. The probe was validated through cellular assays, immunostaining, autophagy perturbation, and age-dependent stability assessments. The model revealed tissue-specific basal pexophagy and dynamic changes during development. High-fat diet-induced obesity significantly reduced hepatic pexophagy, demonstrating metabolic sensitivity. Comparative analysis with mitophagy reporters showed both coordinated and distinct spatiotemporal patterns. We also created an inducible model (CA-PO-TRG) that eliminated cardiac artifacts and enabled neuronal analysis. These models provide robust tools for investigating pexophagy in physiological and pathological contexts.

Lipid scramblases allow passive flip-flop of phospholipids between bilayer leaflets, thereby promoting membrane symmetry. At the endoplasmic reticulum (ER), where phospholipid synthesis is restricted to one leaflet, scramblase activity should be essential for equilibrated membrane growth. The yeast protein Ist2 contains an ER domain and a cytosolic tail that binds the plasma membrane and participates in the transfer of phosphatidylserine. We show both in vitro and in silico that the ER domain of Ist2, which bears homology to the TMEM16 proteins, possesses a lipid scramblase activity that is not regulated by Ca2+. In cells, overexpression or deletion of the ER domain of Ist2 affects ER-related processes including COPII-mediated vesicular transport, lipid droplet homeostasis, and general phospholipid transport, with a specific contribution of residues implicated in lipid scrambling. The weak phenotypes can be augmented by the deletion of another putative scramblase, the protein insertase Get1, suggesting that the combined action of different proteins supports lipid scrambling at the ER.

Diverse cell-cell fusions involve Ca2+ signaling, exposure of phosphatidylserine (PS) at the cell surface and binding of extracellular annexin A5 (Anx A5). Here we report that in the fusion stage of osteoclast formation, each of these shared hallmarks of cell fusion represents a step in a novel signaling pathway. A rise in intracellular Ca2+ activates a lipid scramblase that translocates PS from the inner to the outer leaflet of the plasma membrane. This redistribution is enhanced by binding of extracellular Anx A5 to PS. Depletion of PS in the inner leaflet weakens actin cortex-plasma membrane attachment, as evidenced by the preferential localization of the cortex detachment areas within PS-enriched regions at the cell surface. Weakening of the cortex attachment promotes osteoclast fusion. Based on these findings and theoretical analysis, we propose that PS exposure-to-cortex detachment pathway facilitates pre-fusion membrane contacts and fusion pore expansion in osteoclast fusion and other cell-cell fusions by promoting outward membrane deformations with locally elevated tension.

In response to increased intracellular calcium, the formin INF2 polymerizes 20-30% of the total cellular actin pool within 30 s, suggesting robust regulation. INF2 regulation requires an autoinhibitory interaction between the N-terminal diaphanous inhibitory domain (DID) and the C-terminal diaphanous autoregulatory domain (DAD). DID mutations are dominantly linked to two human diseases and constitutively activate INF2. However, DAD binding to actin monomers competes with DID binding, disrupting regulation. Here, we use a novel cell-free assay for the detailed investigation of INF2 regulation. Contrary to our previous findings, INF2 inhibition does not require CAP proteins but does require actin "buffering" by monomer-binding proteins such as profilin or thymosin. INF2 is activated by calcium-bound calmodulin (CALM) through CALM binding to the N terminus. In addition, the N terminus plays an important role in INF2 regulation beyond CALM binding. These findings support a role of actin monomer-binding proteins not only in regulating overall actin dynamics but also in specific regulation of an actin polymerization factor.

Synaptic specificity is governed by precise combinations of cell adhesion proteins that stabilize pre- and postsynaptic sites and appropriate neurotransmitter receptors. The postsynaptic neuroligins NL1/3 and NL2/3/4 localize to excitatory and inhibitory synapses, respectively, and regulate the corresponding neurotransmitter receptors. However, the exact molecular mechanisms that determine synaptic specificity via defined combinations of neuroligins and neurotransmitter receptors remain unclear. We found that all neuroligin isoforms form a tripartite complex with GABAA receptors and GARLH4 protein, with isoform-specific preferences, and that NL1, previously thought to be restricted to excitatory synapses, is also present at inhibitory synapses. In the absence of inhibitory synapse-specific NL2/4, NL1/3 increasingly assembles with GARLH4/GABAA receptors and relocates to inhibitory synapses. Moreover, forced interaction between NL1 and GARLH4 redirects their localization to inhibitory synapses. These findings demonstrate that GARLHs regulate the synaptic specificity of neuroligins, providing the key link between neuroligins and inhibitory GABAA receptors.

Molecular biology has benefited enormously from repurposed tools-many enzymes and antibodies evolved for other functions but are now essential for interrogating biological function by manipulating proteins or nucleic acids. In contrast, lipids have remained technically difficult to visualize or manipulate in cells. This review introduces tools that bring lipid biology into reach for molecular cell biologists, using familiar experimental approaches. We first describe adaptations of immunofluorescence and live-cell imaging of fluorescent molecules to track lipids. Then, we discuss tools for manipulating lipid levels, including pharmacologic inhibitors, synthetic biology platforms for inducible lipid generation or degradation, and optogenetic systems for precise temporal control. While some methods remain technically demanding, most tools are now broadly accessible. Our goal is to offer a practical framework for integrating lipid biology into mainstream cell biology experiments.