Molecular Biology of the Cell最新文献

The maintenance of lysosome membrane integrity is vital for cell homeostasis and viability, but the underlying mechanisms are not well understood. In this study, we identified a novel role of SPHK-1, the sole Caenorhabditis elegans sphingosine kinase, in protecting lysosome membrane integrity. Loss of SPHK-1 affects lysosomal integrity and degradative function, causing cargo accumulation and lysosome membrane rupture. sphk-1(lf) mutants show severe defects in embryonic and larval development and have significantly shortened lifespan. We found that sphk-1(lf) mutants accumulate high levels of sphingosine, predominantly in lysosomes. Accordingly, sphingosine supplementation leads to the appearance of damaged lysosomes in wild-type worms. We identified sptl-1 and sptl-3 mutations that fully suppress the lysosomal integrity defects in sphk-1(lf) mutants. sptl-1 and sptl-3 encode serine palmitoyltransferases that catalyze the first and rate-limiting step of de novo sphingolipid synthesis. Loss of sptl-1 alleviates sphingosine accumulation, reverses lysosomal integrity and degradation defects, and restores normal development and longevity in sphk-1(lf) mutants. Our study indicates that sphingolipid metabolism via sphingosine kinase is important for maintaining lysosome membrane integrity and function, and is essential for animal development and longevity.

αβ-tubulin is an essential protein that is found in all eukaryotic cells. αβ-tubulins assemble into microtubule polymers that form intracellular transport networks, mitotic and meiotic spindles, and protrusive structures, including cilia and axons. Building these specialized structures creates a demand for αβ-tubulin that can vary across cell type, developmental timing, and cell cycle stage. In this review, we discuss how αβ-tubulins likely emerged from monomeric ancestors into gene families with multiple isotypes and regulatory mechanisms that meet cellular demands for αβ-tubulin. This emergence is accompanied by pathways that regulate the biogenesis and recycling of αβ-tubulin to build networks rapidly and maintain them across long timescales. We propose that the layers of regulation from αβ-tubulin gene copy number, gene sequence elements, mRNA degradation, and protein biogenesis/recycling pathways comprise an integrated program for nimble and robust response to cellular demand for αβ-tubulin. Exploring the cellular signals that control this program and program innovations across species are important next steps for the field.

Cells sense mechanical changes in their cytoskeletal network via force-sensing actin-binding proteins. Recently, a novel force-sensing mechanism was described whereby Lin11, Isl- 1, and Mec-3 (LIM) domains from diverse protein families bind directly to strained actin filaments. It remains unclear, however, how the interaction of these domains with actin is regulated in the context of full-length proteins. Here, we show that the LIM domain-containing region (LCR) of the planar cell polarity protein Prickle2 (Pk2) is associated with strained actin filaments in Xenopus mesoderm alongside known strain-sensitive LIM domains. By contrast, the full-length Pk2 did not exhibit similar recruitment along actin filaments. Structure function analysis revealed that both the structured Prickle, Espinas, Testin (PET) domain and unstructured C-terminal region of Pk2 suppress recruitment of Pk2's LCR to strained actin and promote recruitment to Pk2-rich nodes. Notably, fusion of Pk2's PET domain with the LIM domains of the cytoskeletal proteins Testin and Zyxin revealed context-dependence of this inhibitory effect. Finally, we show that two human patient-derived variants associated with epilepsy result in a loss of Pk2-LCR recruitment to actin filaments. These data provide new insights into the regulation of strain-sensitive LIM domains and may inform our understanding of planar cell polarity.

Myosins exert directed mechanical force along actin filaments. However, little is known about how myosins select particular cellular actin assemblies for their diverse physiological functions. The mammalian class IX myosins, Myo9a and Myo9b, share homologous motor and RhoGAP domains, but it remains unclear whether they target the same actin filament assemblies and thereby serve redundant functions in cells. We showed previously that Myo9b localizes to dynamic actin filament networks in extending lamellipodia and that its motor activity is both necessary and sufficient for this localization. We now show that both motor activity and additionally a predicted four-helix bundle motif in the tail region are required for the accumulation of Myo9b at the tips of filopodia. Interestingly, the class IX loop 2 insertion in the motor region is dispensable. In contrast, Myo9a does not localize to either lamellipodia or filopodia tips. However, the head domain of Myo9a alone targets actin stress fibers, while constructs that also include the neck and tail domains exhibit reduced or negligible targeting. This suggests that the head domain is sterically hindered by a folded conformation. In conclusion, Myo9a and Myo9b target different subcellular sites and actin filament assemblies, implying that they perform different physiological functions.

The Senescence-Associated Secretory Phenotype (SASP), characterized by the up-regulation of inflammatory cytokines, is triggered during senescence by antiproliferation stresses, including replicative exhaustion, γ-irradiation, Ras oncogene induction, and centrosome amplification. The elucidation of common signalling pathway(s) activated in SASP, induced by different anti-proliferation stresses, remains an important question. Indeed, micronuclei activation of the cGAS/STING pathway, which has been thought to drive SASP, remains controversial. In this report, analyses of various cell lines induced to undergo senescence by diverse stressors revealed that HIF-1α is specifically induced in senescence but not in quiescence. Consistent with our previous findings, we have further demonstrated how centrosome amplification induces a noncanonical SASP dominated by HIF-1α activation rather than the classical NFκB signaling. Finally, we revealed that during SASP, centrosome amplification-generated micronuclei do not activate the cGAS/STING-mediated interferon response. Our conclusion is consistent with recent reports, with a more rigorous focus on the analysis of individual cells, indicating that micronuclei from chromosome missegregation fail to activate cGAS/STING-mediated innate immune response. Together, our findings demonstrate that HIF-1α-activation in SASP is a defining feature of the SASP induced by diverse stressors, acting independently of micronuclei generation and cGAS/STING activation.

Lipid droplets (LD) are neutral lipid storage organelles that emerge from the endoplasmic reticulum (ER). Their assembly occurs in ER regions enriched with seipin, which, through its homooligomeric ring-like structure, facilitates neutral lipid nucleation. In yeast, Seipin (Sei1) partners with Ldb16, Ldo45 (yeast homologue of human LDAF1), and Ldo16, which regulate LD formation and consumption. How the molecular architecture of the yeast seipin complex and its interaction with regulatory proteins adapt to different metabolic conditions remains poorly understood. Here, we show that multiple Ldb16 regions contribute differently to recruiting Ldo45 and Ldo16 to the seipin complex. Using an in vivo site-specific photo-crosslinking approach, we further show that Ldo45 resides at the center of the seipin ring both in the absence and presence of neutral lipids. Interestingly, neutral lipid synthesis leads to the recruitment of Ldo45 but not Ldo16 to the complex. Our findings suggest that the seipin complex serves as a preassembled scaffold for lipid storage that can be remodeled in response to increased neutral lipid availability.

Hepatocellular carcinoma (HCC) remains a lethal malignancy with a persistently poor prognosis. While our previous studies established the anti-tumor function of Deoxyribonuclease I Like Protein 3 (DNASE1L3) in HCC, the underlying mechanisms involving the immune microenvironment are less understood. Here, we demonstrate that loss of DNASE1L3 accelerated HCC progression by impairing M1-type macrophage polarization in Dnase1l3 knockout (KO) mice, co-culture models, RNA-seq, and comprehensive molecular/cellular analyses. Mechanistically, DNASE1L3 deletion suppresses tumor-associated macrophages (TAMs) polarization toward the M1 phenotype and inhibits pyroptosis by attenuating the NLRP3 inflammasome/gasdermin D (GSDMD) pathway in vitro and in vivo, thereby reducing pyroptosis in HCC cells. This regulation involves impaired nuclear translocation of NF-κB p65. Crucially, NLRP3 agonism partially reversed DNase1L3-deletion-induced suppression of the NLRP3-GSDMD axis and restored M1 polarization. Our findings reveal DNase1L3 as a pivotal regulator of TAM phenotype via the NF-κB/NLRP3-GSDMD axis and highlight its potential for immunotherapy targeting macrophage reprogramming in HCC.

The late David Baltimore will long be remembered as a towering figure in the modern era of virology and immunology. But less well known is that early in his career, he discovered the existence of eukaryotic RNA-binding proteins, in collaboration with Alice Huang. This work was an extension of previous experiments he had done in which poliovirus RNA added to HeLa cell cytoplasmic extracts underwent an increase in its sucrose gradient sedimentation velocity. The subsequent work revealed the existence of a soluble pool of RNA-binding proteins and had two impacts. On the one hand, it was the beginning of the eukaryotic RNA-binding protein field. On the other hand, it led some investigators to challenge the reality of isolated mRNP complexes. As we know, the latter concern was settled by the introduction of in vivo UV-mediated RNA-protein crosslinking. The mRNA-protein interaction landscape now is at a very advanced and richly enabling stage, but as always in scientific epistemology, it is appropriate to recall from whence it arose.

Neurons have long, thin axons and branched dendritic processes which rely on an extensive microtubule network that functions as a cellular scaffold and substrate for cargo transport. Microtubule defects are a defining pathological feature of neurological disorders. The highly arborized, long, polarized neuronal processes pose challenges for imaging-based assays. Available methods use either dispersed cultures, which are inefficient for compartment-specific analyses, or microfluidic chambers, which allow clear separation of somatodendritic and axonal compartments but are expensive and difficult to maintain. Here, we introduce an "i³Neurosphere" culture model of induced pluripotent stem cell (iPSC)-derived human cortical i³Neurons that enables high-throughput imaging of hundreds of axons without specialized equipment. We characterize neurite outgrowth, polarization, microtubule dynamics, and motility of diverse cargo, providing a reference for future work on microtubule processes in this system. The high-throughput compartment-specific imaging we present, combined with facile genetic engineering in i³Neurons provides a powerful tool to study human neurons.

Yeast vacuolar fusion is driven by Sec17, Sec18, SNAREs of four families (R [Nyv1], Qa [Vam3], Qb [Vti1], Qc [Vam7]) and HOPS, a catalyst of SNARE assembly. Qc, the only vacuolar SNARE that is not membrane-anchored, has a unique path of assembly with other fusion catalysts. Qc is the only SNARE that binds Sec17 with high affinity. Sec18 confers a high affinity for Qc (but not Qb) on HOPS-dependent fusion, but it has been unclear how Sec18 acts. The membrane complex of Sec17 and Sec18 binds Qc to form a membrane:Sec18:Sec17:Qc complex. Sec18 ATP hydrolysis, though dispensable for fusion, provides a measure of the physical and functional interactions between Qc, Sec17, Sec18, and membranes. Each binary interface in this quaternary complex regulates Sec18 ATPase and fusion. Qc is better than other SNAREs, alone or in combination, for stimulating ATP hydrolysis. We propose a working model in which membrane-bound Qc:Sec17:Sec18 associates with the trans complex of HOPS:R:QaQb, displacing HOPS while providing both Qc for complete SNARE zippering and localized Sec17 apolar loops, the twin driving forces for fusion.