Molecular Biology of the Cell最新文献

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.

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.

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.

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.

Understanding why isogenic cancer cells respond differently to equivalent oncogenic stimuli is vital for optimizing anticancer therapies. Emerging evidence suggests that pre-existing differences in cell state may modulate signaling responses to new stimuli, but the interplay of specific cell states and signals remains unclear. We investigated whether epithelial-mesenchymal (E/M) state, a major axis of cancer cell heterogeneity, influences signaling responses to epidermal growth factor (EGF), a critical oncogenic stimulus in non-small cell lung cancer (NSCLC). We imaged >64,000 A549 NSCLC cells labeled for DNA, F-actin, and alternate signaling markers (p-AKT-S473, p-AKT-T308, p-ERK, or p-S6) after acute stimulation. Quantitative single-cell morphological and spatial profiling defined a stimulus-invariant "E/M state landscape" over which EGF signaling responses were compared. This revealed state-dependent differences in signal-activation magnitudes, dynamics, and subcellular routing. AKT responses exhibited phosphosite- and compartment-specific dynamics across states, with epithelial cells showing strong, transient membrane-localized S473 and higher internalized T308, whereas mesenchymal cells displayed weaker but sustained nuclear and ruffle-localized S473. Regression-based computational multiplexing concurrently inferred all signaling responses per cell, mapping state-dependent divergence in multimolecular signaling trajectories. E/M state thus predetermines distinctive spatiotemporal profiles of EGF-induced signaling, with implications for signaling functions and antisignaling therapy responses across E/M state-diverse tumors.

Lysosomes, as central organelles of the endolysosomal system, support cell growth by releasing nutrients derived from hydrolytic digestion of macromolecules. Additionally, they serve as storage organelles for ions and amino acids and must respond to changes in osmolarity by adjusting their membrane to maintain membrane integrity. The nutrient-sensing target of rapamycin complex 1 (TORC1) and the lipid kinase Fab1 (PIKfyve in mammals) are key regulators of these processes on yeast vacuoles. TORC1 phosphorylates Fab1, yet how their activities are functionally coupled is unknown. Here, we show that yeast TORC1 is essential for the sorting of Fab1-derived phosphatidylinositol-3,5-bisphosphate (PI(3,5)P₂) from vacuoles to signaling endosomes (SEs), whose formation depends on the CROP membrane remodeling complex. TORC1 phosphorylation activates Fab1, presumably to maintain elevated PI(3,5)P₂ levels on SEs toward cell growth. In mutants defective in endosome-vacuole fusion, PI(3,5)P₂ accumulates on endosomes adjacent to the vacuole, indicating that its hydrolysis primarily occurs on the vacuolar membrane. Our findings reveal that synthesis and spatial distribution of the vacuolar signaling lipid PI(3,5)P₂ are directly coordinated by TORC1, coupling nutrient sensing to membrane remodeling and endosomal signaling.

Endoplasmic reticulum (ER) homeostasis is maintained through tightly regulated processes that coordinate lipid metabolism and proteostasis. The ER-resident acyl-CoA diphosphatase FIT2, and its yeast homologue Scs3, are key regulators of this balance; their loss disrupts ER morphology and induces chronic ER stress, though the underlying mechanisms remain unclear. To uncover factors involved in Scs3-dependent ER maintenance, we conducted a genome-wide multicopy suppressor screen in SCS3 knockout yeast cells, which display inositol auxotrophy. This analysis identified IZH1, a zinc-related ER membrane protein homologous to the human PAQR (Progestin and AdipoQ Receptor) family, as a genetic interactor of SCS3. IZH1 overexpression enhanced INO1 expression, partially restored the growth of SCS3 knockout cells in inositol-deprived conditions, and reduced ER stress levels without correcting ER morphology defects. Moreover, IZH1 overexpression attenuated unfolded protein response signaling during acute proteotoxic stress and normalized ER-associated degradation kinetics. Together, these findings identify Izh1 as a novel regulator of ER homeostasis and provide new insight into how FIT2/Scs3 influences ER function.

Tardigrades are microscopic animals that can survive exceptional levels of ionizing radiation or desiccation-DNA-damaging conditions that would kill most animals. Irradiation or radiomimetic drug treatment of the tardigrade Hypsibius exemplaris can induce remarkably high expression levels of DNA repair genes, primarily those in the base excision repair and nonhomologous end joining pathways. How tardigrades can repair widespread DNA damage without producing frequent, large-scale chromosome structural abnormalities, like chromosome translocations and fusions, is unknown. Here, we report the results of examining chromosome and nuclear architecture throughout the cell cycle in early embryos of H. exemplaris. We found that H. exemplaris chromosomes are maintained in an individualized form throughout the cell cycle. We were surprised to also find that each chromosome is housed in a fully or partially separate lamin-lined compartment, instead of all chromosomes being housed in a single, nearly spherical nuclear lamina and envelope. Our results reveal unusual chromosomal and nuclear organization in a tardigrade. We speculate that these unexpected features might limit chromosomal rearrangements during DNA damage repair in extreme conditions.

The barriers limiting the number of women appointed, retained, and promoted in academia are well documented. Addressing these barriers is key to increasing participation in science, technology, engineering, and mathematics (STEM), which in turn is essential for maintaining a well-trained and competitive workforce and educational system. The success stories of tenacious individuals who persisted despite the many barriers placed before them serve both as potent reminders of hard-fought gains in equal access and opportunities and as inspiration to current and aspiring scientists. When concepts like diversity, equity, and inclusion are rebranded as discriminatory, how do academic institutions respond to the challenge to encourage broad participation in STEM?