Chapter1: Introduction       Recent work in the field of mechanobiology have suggested that cells respond to physical signals like shear stress, substrate rigidity, stretching and even geometrical cues available in their microenvironment (1). The relevance of physical signals has been implicated in cell differentiation (2), tissue morphogenesis (3) and development (4). The transmission of these signals from plasma membrane to the nucleus involves cytoskeletal and nuclear envelope proteins (5, 6). The nuclear morphology and the nucleoskeletal organization changes upon receiving physical signals, which also correlates with alterations in chromatin dynamics leading to changes in gene expression (7-9). The induction of modular gene expression programs eventually correlates with the non-random spatial organization of chromosomes. In the introduction chapter, I shall first summarize the current understanding of (i) the transmission of physical signals from focal adhesion to the nucleus and the critical molecules involved in this process (ii) how these signals regulate gene expression. In the end, I shall state the aim of my thesis. Briefly, I study the regulation of nuclear morphology by geometrical cues and how it correlates with chromatin compaction and gene expression. The critical molecular intermediates involved in the geometry regulated chromatin compaction and gene expression are further explored. Lastly, using stem cell differentiation, I have tried to establish a link between changes in global gene expression, total chromosome activity and chromosomal organization.       1.1 Sensing and transmission of physical signals: Cells respond to the alterations in their microenvironment. This has been shown using various approaches including changes in matrix elasticity (10), cell geometry (11, 12), matrix topography (13), application of shear stress (14, 15) and substrate stretching (16). Physical sensing of extra cellular environment is initiated by focal adhesions (17-20) complex proteins and they have been identified as mechano-sensors (21, 22). Some of the well-studied mechanosensitive proteins of focal adhesion complex includes talin (23), paxillin (24), zyxin (25, 26) and p130Cas (27), which undergo post-translational modification like phosphorylation upon stretching. Application of force ~20pN, using Atomic force microscopy (AFM), resulted in extension of talin rod domain by 100nm. Again using AFM, less contractile response by zyxin null cells has been shown, which is largely due to acto-myosin complex. The stretching and extension events of the focal adhesion proteins has been proposed to be required for force sensing and presumably to further transmit them to downstream cytoskeletal proteins majorly actin. Although the connections between focal adhesions and cytoskeletal networks are very dynamic, seconds to minutes turn over-time scale, but it can efficiently propagate physical signals (28-31). Mechanical forces transduced from focal adhesions to the actin cytoskeleton (32) results in actin reorganization, which has been studied using fluid shear stress (33) and micropatterned islands (34). Actin cytoskeleton orientation changes based on the direction of fluid shear stress and the shape of the underlying pattern. By applying nanonewton force on cells using AFM, actin reorganization has been shown to be a two-step process involving short term local deformation and long term remodelling (35). Physical signals       not only alters actin organization (36) but also results in actin filament regeneration which is mediated by formins (37, 38) which also regulate its activity by modulating free pool of monomeric actin. Actin interacts with myosin to form acto-myosin complex, which are contractile in nature (39, 40) and has been described as “tension sensor” (41). Acto-myosin complex also provides a path for propagation of physical signals (42, 43) within the cell and has been implicated as the major regulator of force mediated alterations in developmental programs. Actin in the cells, with respect to nucleus, has been shown to be present on top of the nucleus and at the bottom of the nucleus known as apical actin (44) and basal actin respectively. Thick apical actin fibers directly associates with focal adhesion, are contractile in nature (45) and are highly mechano-sensitive. Within minutes of application of low shear stress apical actin get reformed and reorganized contrary to basal actin which requires 50-fold higher levels of shear stress (46). Recently, various isoforms of myosin have also been shown to display mechanosensitive behaviour (47-51). In a recent work, using optical tweezers, the strain sensing ability of myosin I have been characterized (52). Micro-pipette experiments have also revealed mechano-sensitivity of myosin II, whereby it has been elucidated that myosin II gets accumulated at the point of mechanical perturbation (53). Contractile nature of acto-myosin complex has been shown to applies tensile load on the nucleus (54) largely via apical actin. Decrease in nuclear area and increase in height has been shown by using low dose of Lantrunculin-A, which only depolyemerize apical actin (44). Contrary to actin, microtubules exert compressive load on the nucleus (54) whereas role of vimentin, an intermediate filaments, in nuclear dynamics are still largely unknown.       Acto-myosin complex directly interacts with proteins on nuclear envelope and hence establish a physical connection between plasma membrane and nucleus. Physical signals transmitted via acto-myosin contractility impinges on nucleus through connections between actin cytoskeleton and nuclear envelope (55), which are highly dynamic in nature. There are various structural elements in the nuclear envelope (56, 57) major being inner and outer nuclear membrane (INM and ONM respectively). INM and ONM is separated by a gap size of ~50nm and harbour LINC (Linker of nucleus and cytoskeleton) complex, group of proteins bridging cytoskeleton and nucleus (58, 59). Transmission of physical signals between the cytoskeleton and the nucleus is largely mediated by LINC complex since impaired propagation of intracellular forces upon LINC complex perturbation has been shown (60, 61). Using RNAi studies, the role of nesprin, a LINC complex protein on ONM, in nuclear deformation upon force application has been studied (62). Actin and other cytoskeletal proteins including microtubules and intermediate filaments directly interacts with LINC complex proteins (63). 1.2 Response upon receiving physical signals: Physical signals transmitted via cytoskeleton and nuclear envelope proteins impinge on nuclear architecture and dynamics (8, 9, 64, 65) including nuclear rotation, movement and positioning (46, 66-72). Imposing differential geometrical constraints on the cells resulted in changes in nuclear volume, height, shape and positionins (64, 65, 73). Similarly, changes in substrate rigidity from 0.4KPa to 300KPa resulted in significant reduction in nuclear height (74) further showing effect of physical signals on nuclear architecture. Externally applied forces also changes nuclear stiffness, which ranges from 0.1KPa to 10KPa depending on the cell type (75-78) and has been quantified by micropipette aspiration of cells. Physical signals regulated       acto-myosin contractility dependant changes in morphology, stiffness and spatiotemporal dynamics of nucleus has been suggested to correlate with chromatin compaction and gene expression (81) and are hallmark of stem cell differentiation (79) (80). Several studies have shown that stretching of human osteoblasts (82, 83), fibroblasts (84) and other cell types (85, 86), application of fluid shear stress on human mesenchymal stem cells (87) and culturing stem cells on varying geometrical patterns (43) affects gene expression profile. Application of geometrical cues on smooth muscle cells, directly impinges on nuclear volume, DNA synthesis and gene expression (88). Culturing stem cells on micro and nanoscale patterns modulates their self-renewal and differentiation potential (2, 89, 90). By applying varying geometric cues on mesenchymal stem cells (MSCs), lineage specification was found to get altered which was largely sensitive to the curvature of the underlying patterns (43). The contribution of various physical signals to human embryonic stem cell lineage commitment further strengthens the role of physical signals in modulating genetic profiles (89). However, the transmission of physical signals to the nucleus are not sufficient for inducing changes in gene expression. This requires interaction of chromatin with specific transcription factors and co-factors as described later. 1.3 Chemical intermediates involved in physical signals transduction: Changes in gene expression largely requires specific activity of transcription factors (TFs) and cofactors (91). NF-κB (92), FosB (93), JNK-AP1 (94), Egr1 (95), BMP2 (96) are some of the TFs which has been suggested to be mechano-sensitive. These TFs are compartmentalized in the nucleus and the cytoplasm. Depending upon the physical signal their shuttling dynamics between two compartments alters resulting in modular changes       in gene expression. One of the important factor which translate varied physical signals into differential gene expression is YAP\TAZ (97). Nuclear to cytoplasmic redistribution of YAP\TAZ occurs upon culturing cells on patterns of varying sizes or using substrates and pillars of different stiffness and rigidity. As the cell size and the substrate stiffness decreases YAP\TAZ translocates to the nuclues which inturn influences the differentiation potential of MSCs and the survival of endothelial cells (97, 98). Another important mechano-sensitive transcription co-factor is MRTF-A (99). MRTF-A is known to form complex with Serum response factor (SRF) and regulates a large number of genes majorly related to actin cytoskeleton and focal adhesion. By providing varying geometrical cues to keratinocytes, MRTF-A localization, activity and hence differential effect on differentiation was determined (100). Application of a static tensile load of 0.65pN/µm2 on cells using collagen coated beads also resulted in nuclear translocation of MRTF-A further showing mechano-senstive behavior of MRTF-A. Nuclear to cytoplasmic redistribution of majority of these factors and co-factors is largely attributed to acto-myosin contractility and cytoskeletal tension. Maintenance of nuclear YAP/TAZ in MSCs is reported to be Rho GTPase and stress fibers dependent whereby treatment with Latrunculin-A resulted in lower nuclear levels of YAP\TAZ. Tensile load mediated nuclear translocation of MRTF-A also depends on activation of RhoA phosphorylation which results in actin polymerization (101, 102). Higher levels of polymerized actin results in MRTF-A nuclear translocation since monomeric actin (G-actin) sequesters MRTF-A in the cytoplasm. Hence, these factors have been characterized as the chemical link between the physical signals and changes in gene expression which are regulated by altered cytoskeleton dynamics and tension.       Once these mechano-sensitive TFs translocates to the nucleus, they need to converge on their downstream target genes residing on various chromosomes. This points towards the argument that there must be a well defined spatial organization of chromosomes, which is physiologically and energetically more favourable than random positioning, so that TFs can access their targets efficiently. In the next section, a detailed description of chromosome organization and gene clustering is discussed. 1.4 Non random organization of chromosomes territories: Packaging of DNA inside the nucleus is highly ordered with different levels of organization. DNA packaging starts with its interaction with an octamer of core histone proteins (two copies each of H2A, H2B, H3 and H4) called the 10nm chromatin structure. Negatively charged DNA interacts with positively charged histone with around 147 bps wrapped around each histone octamer (103, 104) collectively known as nucleosome. By interacting with linker histones these structures undergo condensation to form 30nm chromatin fiber and further interaction with non histone proteins condenses chromatin into coiled coil 300nm higher order structures (105-109). These structures further get remodelled to form compact heterochromatin (transcriptionally silent) and loosely wound euchromatin (transcriptionally active) regions which finally results in the formation of “chromosome territories” (110-113). Large scale reorganization also results in highly compacted form of chromatin which is inaccessible for transcription regulators. Various chromatin modifications including acetylation, phosphorylation, sumolyation, methylation and ubiquitylation, maintain a dynamic balance between condensed and open chromatin (114). Currently the mechanism by which TFs find their gene targets in this highly complex chromatin arrangement is understood to be sliding and hopping mechanism along the DNA. This non specific binding with DNA has been postulated to help in searching for the correct binding sequence (115).       Such a hit and trial method along with the complexity in chromatin organization argues alternative or specific approaches taken by the cell to organize genes and chromosome so as to provide specificity to gene expression and easy accessibility for TFs. Using fluorescence in-situ hybridization (FISH) (116), the spatial arrangement of chromosomes in various cell types has been characterized. Chromosome size has been elucidated as the first level of chromosome organization. By separately labelling micro and macro chromosomes in chicken nuclei, it was observed that macro chromosomes largely position themselves at the periphery compared to micro chromosomes which were positioned more centrally (117). Even in human cells, size based positioning of chromosomes has been observed (118). Along with the size, the radial positioning of chromosomes also depends on gene density as observed in chicken (117), human (110, 119) and primates cells (120). Recently, correlation between chromosomal transcriptional activity and spatial organization has been modelled and experimentally shown (121). Using stem cells, it has been demonstrated that positions of chromosomes are developmentally regulated. Large scale spatial repositioning of chromosomes was observed during adipocyte differentiation (122). Further, tissue specific chromosomal organization has been documented (123, 124). Various structural proteins also contribute to chromosome repositioning. Nuclear lamina which includes lamin and lamin binding proteins mediates chromosomal positioning (125, 126). Along with chromosome positioning, gene clustering has also been considered to be an alternative approach for efficient and energetically favourable transcription regulation. 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Integrative biology : quantitative biosciences from nano to macro 4(4):422-430.     135       [...]... changes in gene expression with chromosome activity Further, I have explored how the chromosome activity changes during the early onset of differentiation and couple with emergence of chromosomal positioning   12                   Chapter 2: Cell geometric constraints induce modular gene expression pattern via redistribution of HDAC3 regulated by actomyosin contractility 13       2.1 Introduction: Cellular... alterations in cellular geometry influence the fate of mesenchymal stem cells and human embryonic stem cells (43, 137, 138) Depending on the geometry of underlying substrate, these cells differentiate to multiple lineages including mesoderm and endoderm Perliminary studies has largely attributed these changes in differentiation potential to the changes in acto-myosin contractility generated by differential cellular... avoid heterogeneity and uncertainty in gene expression arising due to multioccupancy, we standardized the cell seeding density for all the patterns of different geometries Multi-occupancy of cells on the pattern results in differential tension between cells; cells at the edges have higher acto-myosin contractility while cells in the centre have reduced acto-myosin contractility Such variability in mechanical... particular gene (Acta1 in this case) was mined for transcription 33       factor binding sites, binding score and its E-Value was obtained Scatter plot of score vs E value shows the threshold used to select the significant binding sites Each point in the plot denotes a TF The points in red are the TFs with significant binding efficiency and the rest of the TFs are shown in black (D) Distribution of binding... (AR 1:1) and rectangle (AR 1:5) of equal area (1800µm2) and triangle and circle of equal area (1800µm2) respectively are shown in Fig 2.5A, B and C Gene expression profiles between cells of varying sizes revealed actin and actin related genes to be significantly altered For example, actinin, an actin cross linker, was found to be upregulated in cells of larger area (Fig 2.6A) Simultaneously, genes directly... sensitive to geometric cues Genomic programs were found to be largely sensitive to changes in cell size Matrix related genes were found to be upregulated in cells of larger area whereas reduced cell- substrate contact resulted in up-regulation of genes involved in cellular homeostasis Changes in geometric cues also resulted in differential modulation of nuclear morphology, acto-myosin contractility and histone... regulated gene expression, chromatin compaction and nuclear dynamics needs to be characterized In continuation, how these links impinges on the spatial reorganization of chromosomes needs to be characterized to understand the non-random organization of chromosomes In this thesis, I have used approaches like application of geometrical constraints on differentiated cells and differentiation of stem cells... dishes and incubated for 45 minutes before washing them (B) The protocol used for seeding cells resulted in 80% efficiency of obtaining singlet in culture Data is given as Mean±S.E 26       Figure 2.3: Standardization of cell seeding density: (A) Cells grown on triangular pattern of area 500µm2 (B) Cells grown on circular pattern of area 500µm2 (C) Cells grown on AR 1:5 of area 1800µm2 65,000 cells... the category of cell migration, cellsubstrate adhesion and actin-cytoskeleton In contrast, comparison between small triangle with larger ones belong to regulation of gene expression, negative regulation of transcription and chromatin DNA binding (Fig 2.4C) The main GO groups for the genes up-regulated in bigger sizes falls into the category of cell motility, cell motion, regulation of cell proliferation,... 2.3.1 Cell geometry impose modular changes in gene expression: To understand the changes in gene expression imposed by cellular geometry, whole transcriptome analysis using microarray was performed on cells grown on un-patterned or on different geometries As shown in Fig 2.1A and B, NIH3T3 cells acquire a normal spreading area of around 850+30µm2 when plated on plastic substrate whereas on fibronectin . repositioning. Nuclear lamina which includes lamin and lamin binding proteins mediates chromosomal positioning (125, 126). Along with chromosome positioning, gene clustering has also been considered. 64, 65) including nuclear rotation, movement and positioning (46, 66-72). Imposing differential geometrical constraints on the cells resulted in changes in nuclear volume, height, shape and positionins. alterations in chromatin dynamics leading to changes in gene expression (7-9). The induction of modular gene expression programs eventually correlates with the non-random spatial organization of chromosomes.