CELL GEOMETRIC CONSTRAINTS INDUCE ACTOMYOSIN CONTRACTILITY DEPENDENT CHANGES IN GENE EXPRESSION AND CHROMOSOME POSITIONING

Abstract

The field of biology is making a transition from understanding the role of chemical signals in tissue development and cellular functioning towards exploring the role of mechanical forces. Tissue patterning and remodelling, cell differentiation and other cellular processes respond to the available physical and mechanical cues in the extracellular environment. To study the transduction and the functional implications of these physical cues in the cells, micro-patterns were used to modulate the cell geometry. Cells were cultured on fibronectin coated patterns of varying sizes, shapes and aspect ratios to understand the role of geometric cues in modulating nuclear architecture, cytoskeletal organization and gene expression patterns. Changes in cell geometry resulted in large scale changes in nuclear architecture and acto-myosin contractility. Geometric cues also impinges on the compaction state of chromatin as characterized by the changes in histone acetylation level which was correlated with changes in nuclear volume. Further, the molecular mechanism of geometry modulated chromatin compaction was elucidated by characterizing the mechano-sensitivity of histone deacetylase (HDACs). HDAC regulated alterations in the chromatin compaction depends on their cytoplasmic to nuclear shuttling which in turn was found to be governed by the acto-myosin contractility. Geometry mediated changes in the actin polymerization also provide specificity to the gene expression by modulating the activity and chromatin binding of MRTF-A, an actin dependant transcription co-factor. Lastly, a geometry sensitive antagonistic relation between MRTF-A and NF-?B was established. Further, combined changes in nuclear architecture, acto-myosin contractility and gene expression were probed in the context of spatial redistribution of chromosomes. Early onset of stem cell differentiation was marked with the emergence of actin stress fibers, alterations in nuclear volume and marked changes in the global transcriptional levels. Using 3D Fluorescence in-situ hybridization (FISH) technique, large scale repositioning of chromosomes was observed during early onset of differentiation. Interestingly, the reorganization was found to be dependent on the transcriptional state of the chromosomes. Chromosomes with higher mean activity tend to reposition towards nuclear periphery whereas the low active chromosomes remain randomly distributed even after 24 hours of differentiation. Another level of chromosome organization was characterized depending on the Interchromosome activity difference (IAD). Chromosomes with low IAD tends to remain in close proximity whereas the chromosomes with higher IAD value tends to move apart significantly as the differentiation proceeds. Hence, compelling evidences towards non-random organization of chromosomes, depending on their transcriptional state, were provided. Altogether, in this thesis, I have explored how mechano-chemical signaling alters acto-myosin contractility which than impinge on nuclear architecture and chromatin compaction by regulating the shuttling dynamics of transcription regulators and how in turn these processes couple with spatial reorganization of chromosomes.