Focal adhesion mediated regulation of pluripotency : The role of focal adhesions and contractile forces in human pluripotent stem cells

Human pluripotent stem cells (hPSCs) hold great promise for future medicine. They can differentiate to all adult cell types and self-replicate virtually endlessly. These features would make hPSCs a valuable tool for applications of regenerative medicine. Pluripotency is controlled by extracellular cues which establish the correct culture conditions. Appropriate growth factors and extracellular matrix (ECM) composition must be provided for maintenance of viability, pluripotency and self-renewal in vitro. However, the stem cell field has overlooked many of the basic cell biological phenomena that could affect the regulation of pluripotency. Focal adhesions (FAs) connect the ECM via integrin receptors to the cellular cytoskeleton. They are dynamic protein complexes responsible for broadcasting the information of composition and mechanical properties of the ECM to biochemical intracellular signalling cascades. Furthermore, they provide the physical anchoring points needed for cell adherence and movement. FAs have not been studied in the context of human pluripotency before. In this thesis, I utilise high-resolution microscopy to describe for the first time the characteristics of FAs in hPSCs. We show that hPSCs have large cornerstone FAs connected via contractile actin stress fibres at colony periphery. We provide evidence that structures at the colony edge create traction forces needed for compaction of the cells. Also, we show that FAs function as signalling platforms. We employ 3D superresolution microscopy and unveil unique ultrastructural features of large FAs and show that perturbation of the structure accelerates hPSC differentiation. Finally, we introduce a versatile, easy access method for implementation of fluctuation based super-resolution microscopy to measure cellular forces in nanoscale. In summary, this thesis provides in detail characterisation of cornerstone FAs in hPSCs and highlights their role for morphological features of the colonies and pluripotency. In addition, it provides a new method for studying cellular forces exerted by the FAs

A Strong Contractile Actin Fence and Large Adhesions Direct Human Pluripotent Colony Morphology and Adhesion

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.Peer reviewe

Filopodome Mapping Identifies p130Cas as a Mechanosensitive Regulator of Filopodia Stability

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

Fluctuation-Based Super-Resolution Traction Force Microscopy

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

The L1TD1 Protein Interactome Reveals the Importance of Post-transcriptional Regulation in Human Pluripotency

Peer reviewe

A Strong Contractile Actin Fence and Large Adhesions Direct Human Pluripotent Colony Morphology and Adhesion

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.</p

Superresolution architecture of cornerstone focal adhesions in human pluripotent stem cells

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 β5 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.</p

Functional role of novel RNA binding protein L1TD 1 in human Embryonic stem cells

Siirretty Doriast

Membrane Tension Gates ERK-Mediated Regulation of Pluripotent Cell Fate.

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 β-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.This work was supported b\ the European Union¶s Hori]on 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 641639 (ITN Biopol, HdB and EKP), the Medical Research Council UK (MRC programme award MC_UU_12018/5, HdB and EKP), the Human Frontier Science Program (Young InvestigatorGrant RGY 66/2013 to EKP), the Leverhulme Trust (Prize in Biological Sciences to EKP), an ERC Consolidator Grant (CellFateTech, 772798, KC), a core support grant from the Wellcome Trust and Medical Research Council to the Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute (KJC). KJC is a Royal Society University Research Fellow

Mature Let-7 miRNAs fine tune expression of LIN28B in pluripotent human embryonic stem cells

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.Peer reviewe