Journal of Cell Biology最新文献

Exosomes are multivesicular body-derived extracellular vesicles that are secreted by metazoan cells. Exosomes have utility as disease biomarkers, and exosome-mediated miRNA secretion has been proposed to facilitate tumor growth and metastasis. Previously, we demonstrated that the Lupus La protein (La) mediates the selective incorporation of miR-122 into metastatic breast cancer-derived exosomes; however, the mechanism by which La itself is sorted into exosomes remains unknown. Using unbiased proximity labeling proteomics, biochemical fractionation, superresolution microscopy, and genetic tools, we establish that the selective autophagy receptor p62 sorts La and miR-122 into exosomes. We then performed small RNA sequencing and found that p62 depletion reduces the exosomal secretion of tumor suppressor miRNAs and results in their accumulation within cells. Our data indicate that p62 is a quality control factor that modulates the miRNA composition of exosomes. Cancer cells may exploit p62-dependent exosome cargo sorting to eliminate tumor suppressor miRNAs and thus to promote cell proliferation.

The type I intermediate filament proteins keratin 14 (K14) and keratin 15 (K15) are common to all complex epithelia. K14 is highly expressed by progenitor keratinocytes, in which it provides essential mechanical integrity and gates keratinocyte entry into differentiation by sequestering YAP1, a transcriptional effector of Hippo signaling, to the cytoplasm. K15 has long been used as a marker of hair bulge stem cells, though its specific role in skin epithelia is unknown. Here, we show that the lack of two biochemical determinants, a cysteine residue within the stutter motif of the central rod domain and a 14-3-3 binding site in the N-terminal head domain, renders K15 unable to effectively sequester YAP1 in the cytoplasm like K14 does. We combine insight obtained from cell culture and transgenic mouse models with computational analyses of transcriptomics data and propose a model in which a higher K15:K14 ratio promotes a progenitor state and antagonizes differentiation in keratinocytes of the epidermis.

Dendritic cell (DC) migration via afferent lymphatics to draining LNs (dLNs) occurs in distinct steps that require the chemokine C-C motif ligand 21 (CCL21). In addition to full-length CCL21, which forms an immobilized perilymphatic gradient, a truncated soluble variant with enhanced gradient-forming capacity (CCL21-ΔC) was recently identified in tissues. We show that in skin, plasmin is continuously activated in a urokinase plasminogen activator (uPA)-dependent manner on lymphatic endothelial cells (LECs) and cleaves full-length CCL21, generating CCL21-ΔC. Inflammatory conditions, while promoting overall DC migration, markedly enhance this process, reducing immobilized perilymphatic CCL21 and increasing dermal CCL21-ΔC levels. Inhibition of uPA-mediated CCL21 cleavage causes full-length CCL21 to accumulate around dermal lymphatics, while CCL21-ΔC levels decline in the skin and dLN subcapsular sinus. Consequently, DC entry into afferent lymphatics is diminished, whereas DC egress from the subcapsular sinus into the LN parenchyma is enhanced. These findings reveal uPA/plasmin-dependent regulation of lymphatic CCL21 gradients and identify CCL21-ΔC as critical for DC migration.

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.

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.

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.

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.