RESUMEN
Aneuploidy, which refers to unbalanced chromosome numbers, represents a class of genetic variation that is associated with cancer, birth defects and eukaryotic micro-organisms1-4. Whereas it is known that each aneuploid chromosome stoichiometry can give rise to a distinct pattern of gene expression and phenotypic profile4,5, it remains a fundamental question as to whether there are common cellular defects that are associated with aneuploidy. Here we show the existence in budding yeast of a common aneuploidy gene-expression signature that is suggestive of hypo-osmotic stress, using a strategy that enables the observation of common transcriptome changes of aneuploidy by averaging out karyotype-specific dosage effects in aneuploid yeast-cell populations with random and diverse chromosome stoichiometry. Consistently, aneuploid yeast exhibited increased plasma-membrane stress that led to impaired endocytosis, and this defect was also observed in aneuploid human cells. Thermodynamic modelling showed that hypo-osmotic-like stress is a general outcome of the proteome imbalance that is caused by aneuploidy, and also predicted a relationship between ploidy and cell size that was observed in yeast and aneuploid cancer cells. A genome-wide screen uncovered a general dependency of aneuploid cells on a pathway of ubiquitin-mediated endocytic recycling of nutrient transporters. Loss of this pathway, coupled with the endocytic defect inherent to aneuploidy, leads to a marked alteration of intracellular nutrient homeostasis.
Asunto(s)
Aneuploidia , Presión Osmótica , Proteoma/genética , Proteoma/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Estrés Fisiológico , Membrana Celular/metabolismo , Membrana Celular/patología , Proteínas de Unión al ADN/metabolismo , Endocitosis , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Homeostasis , Humanos , Cariotipo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Termodinámica , Factores de Transcripción/metabolismo , Transcriptoma/genética , Ubiquitina/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
Active fluid transport across epithelial monolayers is emerging as a major driving force of tissue morphogenesis in a variety of healthy and diseased systems, as well as during embryonic development. Cells use directional transport of ions and osmotic gradients to drive fluid flow across the cell surface, in the process also building up fluid pressure. The basic physics of this process is described by the osmotic engine model, which also underlies actin-independent cell migration. Recently, the trans-epithelial fluid flux and the hydraulic pressure gradient have been explicitly measured for a variety of cellular and tissue model systems across various species. For the kidney, it was shown that tubular epithelial cells behave as active mechanical fluid pumps: the trans-epithelial fluid flux depends on the hydraulic pressure difference across the epithelial layer. When a stall pressure is reached, the fluid flux vanishes. Hydraulic forces generated from active fluid pumping are important in tissue morphogenesis and homeostasis, and could also underlie multiple morphogenic events seen in other developmental contexts. In this review, we highlight findings that examined the role of trans-epithelial fluid flux and hydraulic pressure gradient in driving tissue-scale morphogenesis. We also review organ pathophysiology due to impaired fluid pumping and the loss of hydraulic pressure sensing at the cellular scale. Finally, we draw an analogy between cellular fluidic pumps and a connected network of water pumps in a city. The dynamics of fluid transport in an active and adaptive network is determined globally at the systemic level, and transport in such a network is best when each pump is operating at its optimal efficiency.
Asunto(s)
Actinas , Actinas/metabolismo , Transporte Biológico , Morfogénesis , ÓsmosisRESUMEN
Cells migrate in vivo through channel-like tracks. While polydimethylsiloxane devices emulate such tracks in vitro, their channel walls are impermeable and have supraphysiological stiffness. Existing hydrogel-based platforms address these issues but cannot provide high-throughput analysis of cell motility in independently controllable stiffness and confinement. We herein develop polyacrylamide (PA)-based microchannels of physiological stiffness and prescribed dimensions for high-throughput analysis of cell migration and identify a biphasic dependence of speed upon confinement and stiffness. By utilizing novel four-walled microchannels with heterogeneous stiffness, we reveal the distinct contributions of apicolateral versus basal microchannel wall stiffness to confined versus unconfined migration. While the basal wall stiffness dictates unconfined migration, apicolateral stiffness controls confined migration. By tracking nanobeads embedded within channel walls, we innovate three-dimensional traction force measurements around spatially confining cells at subcellular resolution. Our unique and highly customizable device fabrication strategy provides a physiologically relevant in vitro platform to study confined cells.
Asunto(s)
Fenómenos Mecánicos , Tracción , Movimiento Celular , Dimetilpolisiloxanos , HidrogelesRESUMEN
Tissue stem cell niches are regulated by their mechanical environment, notably the extracellular matrix (ECM). Skeletal muscles consist of bundled myofibers for force transmission. Within this macroscopic architecture, quiescent Pax7-expressing (Pax7+) muscle stem cells (MuSCs) are compressed between ECM basally and myofiber apically. Muscle injury causes MuSCs to lose apical compression from the myofiber and re-enter the cell cycle for regeneration. While ECM elasticities have been shown to affect MuSC's renewal, the significance of apical compression remains unknown. To investigate the role of apical compression, we simulate the MuSCs' in vivo mechanical environment by applying physical compression to MuSCs' apical surface. We demonstrate that compression drives activated MuSCs back to a quiescent stem cell state, regardless of basal elasticities and chemistries. By mathematical modeling and cell tension manipulation, we conclude that low overall tension combined with high axial tension generated by compression leads to MuSCs' stemness and quiescence. Unexpectedly, we discovered that apical compression results in up-regulation of Notch downstream genes, accompanied by the increased levels of nuclear Notch1&3 in a Delta ligand (Dll) and ADAM10/17 independent manner. Our results fill a knowledge gap on the role of apical compression for MuSC fate and have implications to stem cells in other tissues.
Asunto(s)
Células Satélite del Músculo Esquelético , Nicho de Células Madre , Músculo Esquelético/metabolismo , Células Madre , Células Satélite del Músculo Esquelético/metabolismoRESUMEN
Although metastasis is the leading cause of cancer deaths, it is quite rare at the cellular level. Only a rare subset of cancer cells (~ 1 in 1.5 billion) can complete the entire metastatic cascade: invasion, intravasation, survival in the circulation, extravasation, and colonization (i.e. are metastasis competent). We propose that cells engaging a Polyaneuploid Cancer Cell (PACC) phenotype are metastasis competent. Cells in the PACC state are enlarged, endocycling (i.e. non-dividing) cells with increased genomic content that form in response to stress. Single-cell tracking using time lapse microscopy reveals that PACC state cells have increased motility. Additionally, cells in the PACC state exhibit increased capacity for environment-sensing and directional migration in chemotactic environments, predicting successful invasion. Magnetic Twisting Cytometry and Atomic Force Microscopy reveal that cells in the PACC state display hyper-elastic properties like increased peripheral deformability and maintained peri-nuclear cortical integrity that predict successful intravasation and extravasation. Furthermore, four orthogonal methods reveal that cells in the PACC state have increased expression of vimentin, a hyper-elastic biomolecule known to modulate biomechanical properties and induce mesenchymal-like motility. Taken together, these data indicate that cells in the PACC state have increased metastatic potential and are worthy of further in vivo analysis.
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Neoplasias , Línea Celular TumoralRESUMEN
Aging is a major risk factor in microvascular dysfunction and disease development, but the underlying mechanism remains largely unknown. As a result, age-mediated changes in the mechanical properties of tissue collagen have gained interest as drivers of endothelial cell (EC) dysfunction. 3D culture models that mimic age-mediated changes in the microvasculature can facilitate mechanistic understanding. A fibrillar hydrogel capable of changing its stiffness after forming microvascular networks is established. This hydrogel model is used to form vascular networks from induced pluripotent stem cells under soft conditions that mimic young tissue mechanics. Then matrix stiffness is gradually increased, thus exposing the vascular networks to the aging-mimicry process in vitro. It is found that upon dynamic matrix stiffening, EC contractility is increased, resulting in the activation of focal adhesion kinase and subsequent dissociation of ß-catenin from VE-Cadherin mediated adherens junctions, leading to the abruption of the vascular networks. Inhibiting cell contractility impedes the dissociation of ß-catenin, thereby preventing the deconstruction of adherens junctions, thus partially rescuing the age-mediated vascular phenotype. The findings provide the first direct evidence of matrix's dynamic mechano-changes in compromising microvasculature with aging and highlight the importance of hydrogel systems to study tissue-level changes with aging in basic and translational studies.
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Uniones Adherentes , beta Catenina , Uniones Adherentes/metabolismo , Células Endoteliales/metabolismo , Hidrogeles , Fenotipo , beta Catenina/metabolismoRESUMEN
The role of mechanical forces driving kidney epithelial fluid transport and morphogenesis in kidney diseases is unclear. Here, using a microfluidic platform to recapitulate fluid transport activity of kidney cells, we report that renal epithelial cells can actively generate hydraulic pressure gradients across the epithelium. The fluidic flux declines with increasing hydraulic pressure until a stall pressure, in a manner similar to mechanical fluid pumps. For normal human kidney cells, the fluidic flux is from apical to basal, and the pressure is higher on the basal side. For human Autosomal Dominant Polycystic Kidney Disease cells, the fluidic flux is reversed from basal to apical. Molecular and proteomic studies reveal that renal epithelial cells are sensitive to hydraulic pressure gradients, changing gene expression profiles and spatial arrangements of ion exchangers and the cytoskeleton in different pressure conditions. These results implicate mechanical force and hydraulic pressure as important variables during kidney function and morphological change, and provide insights into pathophysiological mechanisms underlying the development and transduction of hydraulic pressure gradients.
Asunto(s)
Proteínas de Transporte de Membrana , Riñón Poliquístico Autosómico Dominante , Células Epiteliales/metabolismo , Femenino , Humanos , Riñón , Masculino , Proteínas de Transporte de Membrana/metabolismo , Riñón Poliquístico Autosómico Dominante/metabolismo , ProteómicaRESUMEN
A method of microscale pressure measurement based on immiscible fluid/fluid interface is proposed. This method utilizes observed curvature changes in a fluid/fluid interface, and can accurately report hydraulic pressure in fluids at length scales of 10 microns. The method is especially suited for measuring fluid pressure in micro-scale biological samples. Using this method, we probe fluid pressure build up in epithelial domes, murine mammary gland organoids embedded in hydrogel, and lumen pressure in the developing mouse embryo. Results reveal that the pressure developed across epithelial barriers is on the order of 100~300 Pa, and is modulated by ion channel activity.