Grant: Discovery Grants Program - Individual

The long-term goal of my NSERC Discovery research program is to address the fundamental question in the Natural Sciences of how cells receive and respond to signals from their surrounding environment and from neighbouring cells during tissue development and maintenance. As part of our efforts to fulfill this goal, we have examined molecular mechanisms involved in modulation by extracellular matrix proteins of mechanical signals. Integrins are major cell surface receptors for the extracellular matrix, and are essential for formation and maintenance of the epidermis. Through their association with kindlins and other cytoplasmic proteins, integrins modulate multiple processes in epidermal cells, including responses to mechanical signals, survival and maintenance of stem cell potential. Kindlins are scaffold proteins that bind beta integrins, mediate integrin activation and regulate cell adhesion, proliferation, and motility. Kindlin 1 is important for epidermal integrity, but its role on mechanotransduction remains virtually unexplored. To address this knowledge gap, we used CRISPR/Cas9 gene editing to inactivate the Kindlin 1-encoding FERMT1 gene in N-TERT epidermal cells. Compared with Kindlin 1-expressing cells (hereafter termed WT cells), Kindlin-1 deficient (KO) cells exhibit impaired responses to changes in substrate stiffness, alterations in the perinuclear F-actin cap, and abnormal nuclear morphology. These alterations are consistent with a link between Kindlin-1 and regulation of mechanotransduction. The focus of our short-term objectives is: Short Term Objective 1. To determine how Kindlin 1 transmits integrin-dependent mechanical tension to the nucleus. We will investigate the role that Rho and Rho kinase play downstream from Kindlin 1 and integrins in transmitting tension to the nucleus, as well as alterations in nuclear tension in response to changes in ECM stiffness in WT and KO keratinocytes using tension biosensors. We will also determine the role of Kindlin 1 in the formation of Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes at the interface between the perinuclear actin cap and the nuclear envelope. Short Term Aim 2: To determine the functional role of Kindlin 1 in LINC complex function, genomic integrity, gene expression, and stemness. The role of Kindlin in modulating mechanotransduction to the nucleus will be assessed by comparing in WT vs. KO keratinocytes LINC complex function, alterations in genomic integrity resulting from abnormal mechanotransduction in KO cells, and ability of KO cells to give rise to 3D epidermal tissues. Deciphering the pathways and networks involved in mechanosensing and responses to mechanical cues from the extrallular environment will provide key insight into fundamental processes involved in formation, maintenance and function of many essential tissues, including the epidermis.