Molecular Biology of the Cell最新文献

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. Prior 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 prior to 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.

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.

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.

Rab GTPases are key regulators of endosomal trafficking in eukaryotes. In mammalian cells, Rab4 and Rab7 were shown to localize to distinct compartments, with Rab4 on early endosomes for fast recycling and Rab7 on late endosomes for degradation. Here, we show that in Drosophila, endogenous Rab4 and Rab7 extensively colocalize across tissues and developmental stages. Recruited to the same compartments through mechanisms that do not require the activity of the other, they have opposing effects on endolysosomal size: Rab4 overexpression or Rab7 impairment leads to enlarged endolysosomes, whereas Rab4 loss or constitutively active Rab7 reduces their sizes. Rab4 deficiency suppresses the swelling induced by Rab7 impairment, and conversely, Rab7 activation mitigates the swelling induced by Rab4 overexpression. Genetically, Rab4 loss selectively compromises the viability of rab7-deficient flies but not rab5 or rab11 mutants, supporting a functional overlap between Rab4 and Rab7. Moreover, the levels of endogenous βPS-Integrin, a cargo recycled by Rab4 and degraded via Rab7, are elevated in rab4 mutants and reduced with Rab4 overexpression. Lastly, Rab4 and Rab7 show notable colocalization in mammalian cells and mouse brains, and live imaging reveals dynamic β1-integrin trafficking between Rab4- and Rab7-positive endosomes. Together, these data support that in addition to recycling, Rab4 plays a role in degradation by directing its cargos such as β1-integrin into Rab7-mediated late endolysosomal pathway. [Media: see text].

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.