RESUMEN
Live-cell super-resolution microscopy enables the imaging of biological structure dynamics below the diffraction limit. Here we present enhanced super-resolution radial fluctuations (eSRRF), substantially improving image fidelity and resolution compared to the original SRRF method. eSRRF incorporates automated parameter optimization based on the data itself, giving insight into the trade-off between resolution and fidelity. We demonstrate eSRRF across a range of imaging modalities and biological systems. Notably, we extend eSRRF to three dimensions by combining it with multifocus microscopy. This realizes live-cell volumetric super-resolution imaging with an acquisition speed of ~1 volume per second. eSRRF provides an accessible super-resolution approach, maximizing information extraction across varied experimental conditions while minimizing artifacts. Its optimal parameter prediction strategy is generalizable, moving toward unbiased and optimized analyses in super-resolution microscopy.
Asunto(s)
Artefactos , Microscopía Fluorescente/métodosRESUMEN
Cellular mechanics play a crucial role in tissue homeostasis and are often misregulated in disease. Traction force microscopy is one of the key methods that has enabled researchers to study fundamental aspects of mechanobiology; however, traction force microscopy is limited by poor resolution. Here, we propose a simplified protocol and imaging strategy that enhances the output of traction force microscopy by increasing i) achievable bead density and ii) the accuracy of bead tracking. Our approach relies on super-resolution microscopy, enabled by fluorescence fluctuation analysis. Our pipeline can be used on spinning-disk confocal or widefield microscopes and is compatible with available analysis software. In addition, we demonstrate that our workflow can be used to gain biologically relevant information and is suitable for fast long-term live measurement of traction forces even in light-sensitive cells. Finally, using fluctuation-based traction force microscopy, we observe that filopodia align to the force field generated by focal adhesions.
Asunto(s)
Microscopía de Fuerza Atómica/métodos , Fenómenos Biomecánicos , Línea Celular Tumoral , Adhesiones Focales/ultraestructura , Humanos , Microscopía de Fuerza Atómica/instrumentación , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Imagen Óptica/instrumentación , Imagen Óptica/métodos , Seudópodos/ultraestructuraRESUMEN
Cell fate transitions are frequently accompanied by changes in cell shape and mechanics. However, how cellular mechanics affects the instructive signaling pathways controlling cell fate is poorly understood. To probe the interplay between shape, mechanics, and fate, we use mouse embryonic stem cells (ESCs), which change shape as they undergo early differentiation. We find that shape change is regulated by a ß-catenin-mediated decrease in RhoA activity and subsequent decrease in the plasma membrane tension. Strikingly, preventing a decrease in membrane tension results in early differentiation defects in ESCs and gastruloids. Decreased membrane tension facilitates the endocytosis of FGF signaling components, which activate ERK signaling and direct the exit from the ESC state. Increasing Rab5a-facilitated endocytosis rescues defective early differentiation. Thus, we show that a mechanically triggered increase in endocytosis regulates early differentiation. Our findings are of fundamental importance for understanding how cell mechanics regulates biochemical signaling and therefore cell fate.
Asunto(s)
Células Madre Embrionarias , Células Madre Embrionarias de Ratones , Animales , Diferenciación Celular , Endocitosis , Ratones , Transducción de SeñalRESUMEN
Filopodia are adhesive cellular protrusions specialized in the detection of extracellular matrix (ECM)-derived cues. Although ECM engagement at focal adhesions is known to trigger the recruitment of hundreds of proteins ("adhesome") to fine-tune cellular behavior, the components of the filopodia adhesions remain undefined. Here, we performed a structured-illumination-microscopy-based screen to map the localization of 80 target proteins, linked to cell adhesion and migration, within myosin-X-induced filopodia. We demonstrate preferential enrichment of several adhesion proteins to either filopodia tips, filopodia shafts, or shaft subdomains, suggesting divergent, spatially restricted functions for these proteins. Moreover, proteins with phosphoinositide (PI) binding sites are particularly enriched in filopodia. This, together with the strong localization of PI(3,4)P2 in filopodia tips, predicts critical roles for PIs in regulating filopodia ultra-structure and function. Our mapping further reveals that filopodia adhesions consist of a unique set of proteins, the filopodome, that are distinct from classical nascent adhesions, focal adhesions, and fibrillar adhesions. Using live imaging, we observe that filopodia adhesions can give rise to nascent adhesions, which, in turn, form focal adhesions. We demonstrate that p130Cas (BCAR1) is recruited to filopodia tips via its C-terminal Cas family homology domain (CCHD) and acts as a mechanosensitive regulator of filopodia stability. Finally, we demonstrate that our map based on myosin-X-induced filopodia can be translated to endogenous filopodia and fascin- and IRSp53-mediated filopodia.
Asunto(s)
Adhesión Celular/fisiología , Proteína Sustrato Asociada a CrK/fisiología , Adhesiones Focales/metabolismo , Seudópodos/metabolismo , Animales , Línea Celular Tumoral , Humanos , RatasRESUMEN
While it is clear that key transcriptional programmes are important for maintaining pluripotency, the requirement for cell adhesion to the extracellular matrix remains poorly defined. Human pluripotent stem cells (hPSCs) form colonies encircled by an actin ring and large stable cornerstone focal adhesions (FA). Using superresolution two-colour interferometric photo-activated localisation microscopy, we examine the three-dimensional architecture of cornerstone adhesions and report vertical lamination of FA proteins with three main structural features distinct from previously studied focal adhesions: 1) integrin ß5 and talin are present at high density, at the edges of cornerstone FA, adjacent to a vertical kank-rich protein wall, 2) vinculin localises higher than previously reported, displaying a head-above-tail orientation, and 3) surprisingly, actin and α-actinin are present in two discrete z-layers. Finally, we report that depletion of kanks diminishes FA patterning, and actin organisation within the colony, indicating a role for kanks in hPSC colony architecture.
Asunto(s)
Adhesión Celular , Matriz Extracelular/metabolismo , Adhesiones Focales/metabolismo , Microscopía de Interferencia/métodos , Células Madre Pluripotentes/metabolismo , Actinina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular , Proteínas del Citoesqueleto/metabolismo , Humanos , Cadenas beta de Integrinas/metabolismo , Microscopía Confocal , Células Madre Pluripotentes/citología , Unión Proteica , Talina/metabolismo , Vinculina/metabolismoRESUMEN
Cell-type-specific functions and identity are tightly regulated by interactions between the cell cytoskeleton and the extracellular matrix (ECM). Human pluripotent stem cells (hPSCs) have ultimate differentiation capacity and exceptionally low-strength ECM contact, yet the organization and function of adhesion sites and associated actin cytoskeleton remain poorly defined. We imaged hPSCs at the cell-ECM interface with total internal reflection fluorescence microscopy and discovered that adhesions at the colony edge were exceptionally large and connected by thick ventral stress fibers. The actin fence encircling the colony was found to exert extensive Rho-ROCK-myosin-dependent mechanical stress to enforce colony morphology, compaction, and pluripotency and to define mitotic spindle orientation. Remarkably, differentiation altered adhesion organization and signaling characterized by a switch from ventral to dorsal stress fibers, reduced mechanical stress, and increased integrin activity and cell-ECM adhesion strength. Thus, pluripotency appears to be linked to unique colony organization and adhesion structure.
Asunto(s)
Actinas/metabolismo , Adhesiones Focales/metabolismo , Células Madre Pluripotentes/citología , Actinas/ultraestructura , Fenómenos Biomecánicos , Adhesión Celular , Diferenciación Celular , División Celular , Línea Celular , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Adhesiones Focales/ultraestructura , Humanos , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/ultraestructura , Transducción de Señal , Fibras de Estrés/metabolismo , Fibras de Estrés/ultraestructuraRESUMEN
MicroRNAs (miRNA) are central regulators of diverse biological processes and are important in the regulation of stem cell self-renewal. One of the widely studied miRNA-protein regulators is the Lin28-Let-7 pair. In this study, we demonstrate that contrary to the well-established models of mouse ES cells (mESC) and transformed human cancer cells, the pluripotent state of human ES cells (hESC) involves expression of mature Let-7 family miRNAs with concurrent expression of all LIN28 proteins. We show that mature Let-7 miRNAs are regulated during hESC differentiation and have opposite expression profile with LIN28B. Moreover, mature Let-7 miRNAs fine tune the expression levels of LIN28B protein in pluripotent hESCs, whereas silencing of LIN28 proteins have no effect on mature Let-7 levels. These results bring novel information to the highly complex network of human pluripotency and suggest that maintenance of hESC pluripotency differs greatly from the mESCs in regard to LIN28-Let-7 regulation.
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Células Madre Embrionarias Humanas/metabolismo , MicroARNs/metabolismo , Células Madre Pluripotentes/metabolismo , Proteínas de Unión al ARN/biosíntesis , Diferenciación Celular/fisiología , Línea Celular Tumoral , Regulación hacia Abajo , Humanos , MicroARNs/biosíntesis , MicroARNs/genética , Células Madre Pluripotentes/citología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , TransfecciónRESUMEN
Epigenomic regulation is likely to be important in the maintenance of genomic integrity of human pluripotent stem cells, however, the mechanisms are unknown. We explored the epigenomes and transcriptomes of human pluripotent stem cells before and after spontaneous transformation to abnormal karyotypes and in correlation to cancer cells. Our results reveal epigenetic silencing of Catalase, a key regulator of oxidative stress and DNA damage control in abnormal cells. Our findings provide novel insight into the mechanisms associated with spontaneous transformation of human pluripotent stem cells towards malignant fate. The same mechanisms may control the genomic stability of cells in somatic tissues.
Asunto(s)
Cariotipo Anormal , Catalasa/genética , Silenciador del Gen , Células Madre Pluripotentes/metabolismo , Neoplasias Testiculares/genética , Estudios de Casos y Controles , Catalasa/metabolismo , Línea Celular , Humanos , Masculino , Estrés Oxidativo , Células Madre Pluripotentes/enzimología , Neoplasias Testiculares/metabolismo , TranscriptomaRESUMEN
The RNA-binding protein L1TD1 is one of the most specific and abundant proteins in pluripotent stem cells and is essential for the maintenance of pluripotency in human cells. Here, we identify the protein interaction network of L1TD1 in human embryonic stem cells (hESCs) and provide insights into the interactome network constructed in human pluripotent cells. Our data reveal that L1TD1 has an important role in RNA splicing, translation, protein traffic, and degradation. L1TD1 interacts with multiple stem-cell-specific proteins, many of which are still uncharacterized in the context of development. Further, we show that L1TD1 is a part of the pluripotency interactome network of OCT4, SOX2, and NANOG, bridging nuclear and cytoplasmic regulation and highlighting the importance of RNA biology in pluripotency.