Molecular Biology of the Cell最新文献

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.

Cancer cell invasion relies on dynamic cell shape changes, which originate from protrusive and contractile intracellular forces. Previous studies revealed that contractile forces are controlled by positive-feedback amplification of the contraction regulator Rho by Lbc GEFs. These GEFs were previously linked to tumor progression; however, the underlying mechanisms are poorly understood. Here, we generated a mouse melanoma model in which cytosolic levels of the Lbc GEF GEF-H1 are controlled by light. Using this model, we found that increased GEF-H1 levels strongly stimulate cell contraction dynamics. Interestingly, increased contraction dynamics rapidly induced expansion of tumor spheroids via a focal adhesion kinase-dependent mechanism. Furthermore, long-term stimulation led to the escape of individual cells from spheroids. These findings reveal new insights into the oncogenic roles of Lbc GEFs and how they might promote tumor cell invasion. We propose a mechanism in which increased cell contraction dynamics result in asymmetric pulling forces at the tumor border, promoting the detachment and escape of individual cells.

Lipid droplets are increasingly recognized as necessary organelles. However, the cellular pathways that regulate lipid droplets have only been defined in select fungi, algae, plants, and animals. Our experiments expand the study of lipid droplets to an evolutionarily distinct model organism, the ciliate Tetrahymena thermophila. We identify conserved pathways that promote lipid droplet homeostasis while also uncovering features that suggest adaptation. We show that Tetrahymena accumulate lipid droplets in response to nutrient deprivation, including starvation and the stationary phase. Pulse-chase experiments with a fluorescent fatty acid analogue demonstrate lipid trafficking to lipid droplets in starved cultures. Unlike other cell types, starved Tetrahymena appear to use both peroxisomes and mitochondria (not vacuoles) for further fatty acid catabolism. We observe cooccurence of the fluorescent fatty acid analogue with markers of peroxisomes and a subpopulation of mitochondria, suggesting specialized catabolic roles for both organelles. We demonstrate a decrease in survival following starvation in the presence of inhibitors of mitochondrial fatty acid import or peroxisomal fatty acid metabolism. Together, our experiments add Tetrahymena to the expanding list of eukaryotes that increase lipid droplets in response to nutrient depletion while also uncovering important and distinct roles for mitochondrial and peroxisomal catabolism in survival pathways.

Centromeres are essential chromosomal components that ensure proper cell division by serving as assembly sites for kinetochores, which connect chromosomes to spindle microtubules. Centromeres are marked by the evolutionarily conserved centromere-specific histone H3 variant, CENP-A, which is deposited into centromere nucleosomes during G₁ in human cells. Centromeres retain cohesin, a ring-like protein complex, during mitosis, protecting sister chromatid cohesion and centromere transcription to prevent chromosome missegregation. Previous work in Drosophila has suggested that centromere transcription and centromeric RNAs are important for CENP-A deposition in chromatin. During mitosis, centromeric cohesin is critical for centromere transcription. However, it is not clear how or whether centromeric transcription and cohesin contribute to CENP-A deposition in G₁ in human cells. To address these questions, we combined a cell synchronization strategy with the Auxin Inducible Degron technology and transcription inhibition in human cells. In contrast with Drosophila cells, our results demonstrated that neither centromeric transcription nor cohesin is required for CENP-A deposition in human cells. Our data demonstrate clear differences in the CENP-A deposition mechanism between human and Drosophila cells. These findings provide deeper insights into the plasticity underlying centromere maintenance and highlight evolutionary divergence in centromere maintenance systems across species.

Phenotypic heterogeneity-distinct molecular and behavioral variations within a population-significantly influences collective invasion and tumor progression. Here, we use a molecular approach to explore how the underlying metabolic heterogeneity in non-small cell lung cancer (NSCLC) influences invasion and pack patterning. Assessment of  three-dimensional (3D) pack patterning revealed invasive heterogeneity across NSCLC cell lines and patient-derived samples. Flow cytometry identified IL13RA2 as a biomarker for invasive potential, enabling isolation of subpopulations with distinct invasive characteristics. By integrating a cell surface biomarker (IL13RA2±) with mitochondrial membrane potential (TMRM), we identified and isolated three distinct subpopulations. Two-dimensional (2D) analyses revealed differences in mitochondrial polarity and transcriptional programs associated with migration and oxygensensitivity. In 3D, these subpopulations invaded with distinct patterns, from contiguous circular packs to structured chains. Assessments under varied oxygen tension demonstrated that oxygen availability and subpopulation metabolism together influence collective invasion patterning. When recombined at ratios recapitulating the original population, both stochastic and opportunistic cooperative dynamics emerged, dependent on subpopulation composition and oxygen levels. Our molecular approach, integrating cell surface and metabolic characteristics, enables the isolation of unique subpopulations and demonstrates that phenotypic and metabolic heterogeneity, population composition, and oxygen availability collectively pattern invasion packs and drive collective invasion.

Actomyosin rings are specializations of the nonmuscle actomyosin cytoskeleton that drive cell shape changes during division, wound healing, and other events. Contractile rings throughout phylogeny and in a range of cellular contexts are built from conserved components, including nonmuscle myosin II, actin filaments, and cross-linking proteins. To explore whether diverse actomyosin rings generate contractile force and close via a common mechanism, we studied three instances of ring closure within the continuous cytoplasm of the Caenorhabditis elegans syncytial oogenic germline: mitotic cytokinesis of germline stem cells, apoptosis of meiotic compartments, and cellularization of oocytes. The three ring types exhibited distinct closure kinetics and component protein abundance dynamics. We formulated a physical model to relate measured closure speed and molecular composition dynamics to ring active stress and viscosity. We conclude that these ring intrinsic factors vary among the ring types. Our model suggests that motor and nonmotor cross-linkers' abundance and distribution along filaments are important to recapitulate observed closure dynamics. Thus, our findings suggest that across ring closure contexts, fundamental contractile mechanics are conserved, and the magnitude of contractile force is tuned via regulation of ring component abundance and distribution. These results motivate testable hypotheses about cytoskeletal regulation, architecture, and remodeling.