Molecular Biology of the Cell最新文献

Neuropeptide release by Drosophila sLNv clock neurons controls circadian behaviors and sleep. Strikingly, neuropeptide content in sLNv terminals is rhythmic with late-night accumulation occurring while the axon arbor is expanding in preparation for midmorning synaptic exocytosis of neuropeptide-containing dense-core vesicles (DCVs). Past studies showed increased synaptic neuropeptide content can be produced by delivery of more neuropeptide to terminals or activity-dependent capture of circulating DCVs. To distinguish between these mechanisms, neuropeptide-containing DCVs were imaged in the ex vivo brain explant preparation. First, post-exocytosis DCV axonal transport and presynaptic neuropeptide accumulation following retrograde transport inhibition show that sLNv DCVs circulate. Furthermore, anterograde transport to terminals is constant throughout the day demonstrating there is no increase in DCV delivery. Rather, capture of circulating DCVs produces the daily boost in terminal neuropeptide content. Remarkably, this capture occurs before the daily increase in Ca²⁺ spike activity and is independent of concurrent IP₃ signaling and axon arbor expansion. Finally, a per clock gene mutation inhibits rhythmic DCV capture. Thus, rather than responding to Ca²⁺ signaling or axonal plasticity, capture of circulating DCVs in sLNv presynapses is increased by the molecular clock in anticipation of activity-induced release hours later. [Media: see text].

Meiosis ensures formation of haploid gametes through two rounds of chromosome segregation after one round of DNA replication. How this cell cycle process is restricted to two and only two divisions is poorly understood. In budding yeast, RNA-binding protein Rim4 binds various mRNAs to prevent their translation. At the onset of meiosis II, phosphorylation and degradation of Rim4, along with the concomitant release of sequestered mRNA, has an important role in ensuring meiotic exit. Building on previous work, we developed a parsimonious mathematical model of meiotic termination that elucidates the role of Rim4-mRNA release and translation of AMA1 mRNA in the fidelity of meiotic exit. Central to our model is the accumulation of Ama1 protein, a meiosis-specific activator of APC/C. Our mathematical model predicted further outcomes, which we tested experimentally. We found that either slowing Rim4 degradation or disrupting APC/C^Ama1 activity delayed meiosis II. In some cells, this disruption prevented meiotic exit entirely, leading them to re-enter cell cycle oscillations after meiosis II. These findings demonstrate that the timely activation of this regulatory network is crucial for ensuring irreversible meiotic exit.

Epithelia maintain their barrier function through the proliferative and plastic behavior of stem cells that drive continuous tissue regeneration. However, these same properties render epithelia susceptible to tumorigenesis. The skin, the largest epithelial barrier, is the source of the most prevalent human cancers, yet the molecular mechanisms by which stem cells and their microenvironment cooperate to promote cutaneous cancer development remain incompletely defined. Previous work demonstrated that genotoxic injury in normal skin activates epithelial-dermal inflammasome signaling that drives epithelial stem cell hyperproliferation and misspecification. Here, we investigated whether this mechanism also operates in diseased skin. We found that stem cell misspecification is a broadly conserved feature across pathological skin conditions but is absent in normally proliferating tissue. Notably, inflammasome activation is detected in both epithelial and dermal compartments of cutaneous squamous cell carcinoma (cSCC), but not in other skin pathologies. Mechanistically, oncogenic KRAS expression in keratinocytes triggers inflammasome activation before tumor formation non-cell autonomously. Furthermore, IL-1 signaling is activated in fibroblasts adjacent to the cSCC tumor interface, but not in the overlying epithelium. Taken together, these findings support a model in which KRAS-driven epithelial-fibroblast inflammasome crosstalk establishes a feed-forward IL-1 signaling loop that enhances the tumor-promoting microenvironment in cSCC.

β-catenin is a critical effector of the Wnt pathway and a key component of the cadherin complex. Whether Wnt-regulated cytoplasmic β-catenin interacts with the cadherin-associated pool under physiological conditions is unclear. Using a cell line depleted of N- and E-cadherins, we demonstrate that cadherin-depleted cells exhibit lower levels of basal b-catenin that plateau to a similar level as the parental line with Wnt3a stimulation. The cadherin-depleted line exhibits significantly enhanced levels of Wnt signaling by comparison to its parental control; these effects are reversed by wild-type E-cadherin but not E-cadherin with disrupted b-catenin-binding. Enhanced Wnt signaling in the cadherin-depleted line is consistent with a previous study showing that Wnt pathway activation correlates with fold changes in β-catenin levels, rather than the absolute concentration. Our mathematical modeling suggests a mechanism in which β-catenin binding to cadherins acts as a sink to maintain elevated cytoplasmic b-catenin levels in the face of b-catenin destruction complex activity, and also limits pathway response. Our bioinformatic analysis reveals a correlation between elevated Wnt target gene expression and E-cadherin loss in a Wnt-driven model of thyroid cancer. Our results have relevance for tumorigenesis, as cadherin loss is commonly associated with poor prognosis and increased metastatic potential.

The elevated occurrence and fatality rates of colorectal cancer (CRC) can be attributed not only to the biological attributes of uncontrolled tumor cell proliferation but also to the intricate interplay between tumor cells and surrounding cells in the microenvironment. Among these, cancer-associated fibroblasts (CAFs) stand out as a predominant type of mesenchymal cell in the microenvironment, with their biological functions in tumor advancement becoming increasingly recognized. CAFs play a significant role in the progression of CRC through diverse mechanisms, such as direct interactions with tumor cells and the secretion of cytokines that modulate the recruitment and function of macrophages and immune cells. This review explored various facets of the involvement of CAFs in the progression of CRC, including their origins, subtypes, marker molecules, interactions with other cells, and the potential clinical significance of targeting CAFs or CAFs-derived molecules in the context of CRC therapy.

The endolysosomal pathway, with its interconnected endosomes and lysosomes, has key functions in cellular nutrient and ion uptake, metabolic adaptation, as well as protein and organelle turnover via autophagy. Rab5 GTPases are organelle identity markers on endosomes, though it remains unclear why cells have several Rab5 isoforms and guanine nucleotide exchange factors (GEFs) as their activators. Using yeast, we demonstrate that the key Rab5 GEFs Vps9 and Muk1 overlap in their Rab5 specificity in vitro but cover distinct cellular territories in vivo. Vps9 functions between the Golgi and endosomes, while Muk1 is primarily found in the early endocytic pathway. Using targeting approaches, we show that Rab5 GEFs can only partially replace each other, demonstrating that each GEF is specific for its cellular niche. Intriguingly, Muk1 functions in vivo as an oligomer through its C-terminal domain, which is sufficient to also oligomerize a chimeric Vps9. Overall, our data suggest the spatial distribution of Rab5 GEFs and their substrate Rab5s fine-tune the endolysosomal system for cellular needs and metabolic cues.

Yeast vacuolar protein sorting 13 (Vps13) is a bridge-like transporter that directs lipid flow between membranes at organelle contact sites. Vps13 targeting relies on organelle-specific adaptors containing proline-X-proline (PxP) motifs, which compete for binding to the Vps13 adaptor-binding (VAB) domain. Though a VAB-PxP interface has been identified for the mitochondrial adaptor Mcp1, whether other adaptors use identical binding mechanisms is unknown. Moreover, not every Vps13 function is connected to a known PxP adaptor, suggesting other adaptors may exist. Here, we validate the significance of the shared VAB-PxP interface by showing that mutations within this region inhibit both adaptor binding and Vps13 membrane targeting in vivo. Using predictive modeling, we demonstrate that while adaptors share a common Vps13-binding interface, slight differences between these interfaces may contribute to preferential binding and adaptor competition. Notably, we find that the VPS pathway functions independently of the PxP motif binding site. Our results indicate that Vps13 likely employs a non-PxP adaptor mechanism in this pathway, yet the structural integrity of the VAB domain remains essential for proper pathway function.

During autophagy induction in Saccharomyces cerevisiae, over 20 autophagy-related (Atg) proteins localize to the site of autophagosome formation to generate the pre-autophagosomal structure (PAS), where phase-separated condensates of the Atg1 kinase complex serve as a scaffold for recruiting other Atg proteins. The lipid transfer protein Atg2 forms a complex with the phosphatidylinositol 3-phosphate (PI3P)-binding protein Atg18 and mediates lipid influx from the endoplasmic reticulum to the PAS for membrane expansion. In this study, we discover that the Atg2-Atg18 complex interacts with the Atg1 complex. This interaction involves the C-terminal regions of Atg2 and the Atg1 complex subunit Atg29, and is enhanced by Atg1-dependent phosphorylation of Atg29. This interaction, together with Atg18 binding to PI3P, promotes PAS localization of the Atg2-Atg18 complex. These findings provide new insight into PAS organization and highlight the Atg1 complex as a central hub coordinating Atg protein assembly during autophagosome formation.

Two main pathways are responsible for protein secretion across the cytoplasmic membrane in prokaryotes. While the general secretory (Sec) pathway transports proteins across the membrane in an unfolded state, the twin-arginine translocation (Tat) pathway exports proteins primarily in their folded conformation. Although the Tat system appears dispensable in multiple model bacteria, some species require it for viability, and the reason for the distinction is nebulous. Here we show that all three subunits of the Tat complex-TatA, TatB, and TatC-are essential in the alpha-proteobacterium Caulobacter crescentus. Additionally, depletion of the Tat complex results in abnormal cell morphology. We found that localization to the cell periphery, as well as midcell localization upon osmotic upshift, of the essential peptidoglycan transpeptidase PBP2 is dependent on the Tat apparatus. In contrast, subcellular localization of the actin homolog MreB and the penicillin-binding protein PBP1a is not perturbed upon depletion of the Tat complex. As PBP2 transpeptidase activity links glycan chains at sites of cell wall remodeling and is essential for cell elongation, localization results and leader sequence analysis together suggest that PBP2 translocation is a key responsibility of the Tat system in Caulobacter and possibly other alpha-proteobacteria.

Actin filaments and microtubules are fundamental components of the cytoskeleton in eukaryotic cells, orchestrating cellular processes such as intracellular transport, migration, and division. Traditionally studied as distinct entities, growing evidence highlights their intricate crosstalk, mediated by crosslinking proteins, motor proteins, and signaling pathways. Reconstituted in vitro systems provide a powerful framework for isolating and probing these interactions under controlled conditions, enabling direct connections between molecular coupling and higher-order cytoskeletal organization. Such approaches have revealed how actin-microtubule interactions shape network architecture, coordinate force transmission, and give rise to emergent mechanical behavior that cannot be inferred from either system alone. This review synthesizes mechanistic principles of actin-microtubule crosstalk revealed by reconstituted systems, spanning molecular interactions, network-scale organization, and mechanical feedback. These insights advance understanding of cytoskeletal coordination in cells and identify key challenges toward developing predictive frameworks that link molecular interactions to emergent cellular mechanics.