Molecular Biology of the Cell最新文献

Lysosome exocytosis is one of the critical functions of lysosomes in maintaining cellular homeostasis and plasma membrane (PM) repair. At the basal level, the SNAREs (soluble-N-ethylmaleimide-sensitive-factor accessory-protein receptors) regulating the lysosome fusion with the cell surface have been poorly defined. Here, we identified a Qa-SNARE STX1A, localized majorly to lysosomes and a cohort to the PM in HeLa cells. Overexpression of GFP-STX1A in HeLa cells causes decreased lysosome number and their peripheral dispersion. However, STX1A knockdown in HeLa cells displayed an accumulation of lysosomes beneath the cell surface with reduced lysosome exocytosis. Consistently, TIRF imaging microscopy demonstrated an enhanced enrichment of LAMP1-positive vesicles at the cell surface in STX1A-depleted compared with control cells. Moreover, STX1A depletion reduces proteolytic activity without affecting the lysosome content or acidity. Additionally, these cells showed enhanced lysosome dispersion and autolysosome accumulation. Functionally, GFP-STX1A also localizes to LLOMe-induced GAL3-positive damaged lysosomes and reduces their number by enhancing exocytosis. Biochemically, STX1A forms a SNARE complex with SNAP23 or SNAP25 (Qbc) and VAMP2 (R), and their knockdown in HeLa cells mimics the STX1A-depletion phenotypes. Overall, these studies demonstrate a unique function of STX1A in regulating lysosomal exocytosis by localizing to these degradative organelles.

Yeast cell fusion is equivalent to fertilization. Cell fusion requires the removal of the intervening cell wall, regulated by Fus2 and other cell fusion proteins. Fus2 is an amphiphysin-like protein that forms a complex with the BAR protein Rvs161 and the highly conserved Rho-like GTPase Cdc42 at the zone of cell fusion (ZCF); however, the function of these proteins in cell fusion has been unclear. Here, we show that the Fus2-Rvs161-Cdc42 complex regulates a mating-specific secretion event to mediate cell fusion via cell wall removal. Use of fluorogen-activated protein fusions demonstrated that the secretion of cell wall remodeling enzymes Scw4 and Gas1 is dependent on Fus2, whereas the secretion of Scw10 is independent of Fus2. We found that Cdc42 is not required for secretion per se, but instead functions to focus Fus2 at the ZCF, thereby allowing concentrated release of cell wall remodeling enzymes. Localized secretion of cell wall remodeling enzymes would overcome cell wall repair pathways. Additionally, Prm1, required for efficient membrane fusion, colocalizes with Fus2 at the ZCF. Localization of Prm1 at the ZCF is dependent on Fus2 and Rvs161. We propose that the Fus2-regulated vesicle population includes membrane fusion proteins as well as cell wall remodeling factors.

Motor-driven transport on microtubules is critical for distributing organelles throughout the cell. Most commonly, organelle movement is mediated by cargo adaptors, proteins on the surface of an organelle that directly recruit microtubule-based motors. An alternative mechanism called hitchhiking was recently discovered: some organelles move, not by recruiting the motors directly, but instead by using membrane contact sites (MCS) to attach to motor-driven vesicles and hitchhike along microtubules. Organelle hitchhiking is observed across fungi and animals. In filamentous fungi, nearly all peroxisomes move by hitchhiking on early endosomes (EE). In the fungus Aspergillus nidulans, EE-associated linker proteins PxdA and DipA are critical for establishing EE-peroxisome MCS required for peroxisome movement. Whether peroxisome-membrane proteins exist that regulate peroxisome hitchhiking on EEs is not known. Through a forward mutagenesis screen, we discovered an acyl-CoA binding (ACB) domain-containing protein AcbdA/AN1062 that localizes to peroxisomes via its tail-anchored transmembrane domain (TMD). Deleting the AcbdA gene or only its N-terminal ACB domain perturbs the movement and distribution of peroxisomes. Importantly, AcbdA is not required for the movement of EEs or for the recruitment of PxdA and DipA on EEs. Fatty acid (FA)-induced increases in peroxisome movement require AcbdA, suggesting that peroxisome hitchhiking on EEs is coupled to FA metabolism. Mutating a conserved FFAT motif, predicted to interact with the endoplasmic reticulum (ER), has no effect on peroxisome movement. Taken together, our data indicate that AcbdA is a peroxisome-membrane protein required for peroxisome hitchhiking on EEs. AcbdA's involvement in peroxisome hitchhiking represents a divergence from known functions of Acbd4/5 proteins and adds layers to our understanding of the functionality of the Acbd4/5 family of proteins.

During embryonic development, neural crest-derived melanoblasts, which are precursors of pigment-producing melanocytes, disperse throughout the skin by long-range cell migration that requires adhesion to the ECM. Members of the integrin family of cell-ECM adhesion receptors are thought to contribute to melanocyte migration in vitro. However, due to the functional redundancy between different integrin heterodimers, the precise role of integrins in melanoblast migration, as well as the mechanisms that regulate them in this process, especially in in vivo contexts, remain poorly understood. To address this, we utilize the existing transcriptomic databases to identify different integrin subunits that are specifically expressed in melanoblasts, melanocytes, and melanoma cancer cell lines. We then use mouse embryonic skin explants combined with drug and small-molecule-based perturbations to target different integrins as well as specific mechanisms that modulate integrin activity. Individual melanoblasts from live imaging movies are tracked using high-resolution, quantitative, automated analysis, and cell morphology, cell migration, and actin-based protrusions are analyzed. Overall, we uncover the nonredundant roles of different integrin heterodimers and elucidate the function of outside-in integrin activation in melanoblasts. Finally, we describe the function played, in vivo, by integrin-mediated adhesion to specific ECM ligands during melanoblast migration.

Cancer deaths are largely attributed to the dissemination of cancer cells from a primary tumor to a secondary metastatic site. The metastatic cascade is initiated by cancer cell invasion that is facilitated by cytoskeletal remodeling to produce ventral cell protrusions, termed invadopodia, that degrade the extracellular matrix to promote motility. Conventional invadopodia studies rely on techniques with embedded cells in 3D matrices to observe and determine protein behavior, which often utilize immunolabeling strategies and struggle to visualize individual invadopodia, thereby limiting investigations of protein and invadopodia dynamics. Here, the design and utilization of an axial invasion chamber is described for live-cell imaging of elongating invadopodia in 3D. Results identify that cytoskeletal and microtubule-associated proteins within invadopodia exist in an organized framework, and determine the functional contribution by which noncentrosomal microtubules promote cancer cell invasion and migration.

The attachment of cells to the extracellular matrix (ECM) is essential for morphogenesis. The activity of Integrins, the main mediators of cell-ECM adhesion in animals, is required for morphogenesis and must be precisely regulated to ensure proper development. However, the mechanisms that ensure precise integrin activity during animal development are poorly understood. The best characterized mechanism for integrin regulation is conformational change driven by either extracellular signals ("outside-in activation") or by intracellular signals ("inside-out activation"). The cytoplasmic protein talin is a key regulator of inside-out activation. We used mutations in talin to demonstrate, for the first time, that modulation of integrin activation is essential for early mammalian development. We find that integrin activation mutants die by E8.5-E9.5 and show developmental delay and abnormal growth. Intriguingly, disrupting integrin regulation does not impinge on embryonic patterning and ECM distribution. Analysis of embryonic stem cells isolated from integrin activation mutants revealed a reduction in the strength of cell-ECM attachment but only mild defects in focal adhesion number and maturation. Notably, activation mutants at E7.5 showed increased cell death and reduced cell-proliferation Overall, we find that inside-out integrin activation strengthens cell-ECM attachment in early mouse development that is essential for cell survival and proliferation.

The nucleolus is a nonmembrane-bound compartment that forms around tandem arrays of ribosomal RNA genes and provides the cell with ribosomes. Multiple nucleoli within the same nucleus coalesce, and fusion is thought to result mainly from intrinsic properties of nucleoli. However, ribosomal DNA (rDNA) arrays are mostly in chromosomal context, and chromosomes are not randomly organized. How the spatial arrangement of chromosomes affects nucleolar fusion is largely unknown. Using fluorescence microscopy, we investigated nucleolar fusion in diploid budding yeast. Nucleoli forming around homologous rDNA arrays efficiently fused during interphase but often individualized during late anaphase. Although nucleoli were far from the spindle pole body (SPB) in interphase, they came close during mitosis, suggesting that SPB-dependent positioning may affect nucleolar fusion. Indeed, disruption of microtubule-dependent centromere anchorage to the SPB by nocodazole promoted individualization of nucleoli. In contrast, impairment of rDNA tethering to the nuclear envelope had little or no effect. Hence, chromosome positioning by non-rDNA sequences facilitates nucleolar fusion.

Cardiac sarcomere assembly is a highly orchestrated process requiring integration between intracellular contractile machinery and extracellular adhesions. While α-actinin-2 (ACTN2) is well known for its structural role at the cardiac Z-disc, the sarcomere border, the function of the "non-muscle" paralog α-actinin-1 (ACTN1) in cardiac myocytes remains unclear. Using human induced pluripotent stem cell-derived cardiac myocytes (hiCMs), we demonstrate that siRNA-mediated depletion of ACTN1 disrupts sarcomere assembly, and that exogenous re-introduction of ACTN1 but not ACTN2 restores assembly, revealing non-redundant functions. Unlike ACTN2, ACTN1 localized predominantly to cardiac myocyte focal adhesions, and was required for adhesion enlargement during sarcomere assembly, suggesting ACTN1 but not ACTN2 is required for adhesion maturation. Live-cell imaging of vinculin dynamics showed decreased stability of adhesion-associated vinculin in ACTN1-deficient cells, whereas paxillin dynamics were unaffected. These results suggest that ACTN1 stabilizes focal adhesions to promote effective force transmission during sarcomere assembly.

Autophagy is critical for the homeostasis and function of neurons, as misregulation of autophagy has been implicated in age-related neurodegenerative diseases, and neuron-specific knockdown of early autophagy genes results in early neurodegeneration in mice. We previously found that autophagosome formation decreases with age in murine neurons. Sex differences have been intensely studied in neurodegenerative diseases, but whether sex differences influence autophagy at the neuronal level has not been investigated. We compared protein expression of 22 autophagy components between neural tissues of female and male mice across development and aging. We found minimal sex-related differences in autophagy protein expression throughout the murine lifespan. Additionally, we assayed the recruitment of autophagy complexes and autophagosome biogenesis; we found no sex-dependent differences in multiple stages of autophagosome formation in neurons, independent of age. Our data suggest that biological sex does not influence autophagosome formation in neurons across development and aging.