Dr. Feroz H Musthafa's Talk

Aug 28, 2014 - 17:00

Aug 28, 2014 - 18:00

Room 118 Chemical Engg.

Seminars

Dr. Feroz H Musthafa Microfluidics and Microfabrication Facility Centre for Cellular and Molecular Platforms.

Abstract: 1) Eukaryotic cells modulate their gene expression profile and differentiation in response to the rigidity of their micro-environment. The exact mechanism by which cells sense the rigidity of the substrate is not understood. To better understand the mechanism involved we varied the center-to-center spacing of the 500 nm micropillars from 1 to 3 micrometers and compared two different cell lines. We observed that the displacements of the pillars were constant over an order of magnitude variation in stiffness of pillars and a five-fold variation in edge-to-edge spacing between pillars. Mouse embryonic fibroblast (RPTPα+/+) and human foreskin fibroblast (HFF) cells produced consistent maximal pillar displacements of ~60 nm (with a minor peak at ~120nm observed in the mouse cells). In all cases displacements were due to localized contractions in ~2-3 micrometer regions at the cell edge. Perturbation of the actomyosin machinery by the myosin inhibitor blebbistatin or over-expression of myosin ATPase inhibitory protein caldesmon caused a decrease in the frequency of the pulling events without changing the average displacements. Inhibition of Src family kinases using PP2 caused the cells to have a wider range of displacements than was observed in untreated cells. Our results indicate that the contractile machinery is powered by a localized (up to 3 micrometers) actomyosin cassette. Delineating the components of the localized contraction units is crucial to understand the sensing of the substrate stiffness by cells and has implications for cell differentiation and metastasis. 2) In the body soft tissues often undergo cycles of stretch and relaxation but it is not known if those affect cell behavior. We find that soft pillar arrays will not support cell spreading and growth. Since the rigidity of the pillars will not change dramatically when the substrate is stretched we asked if the application of force by stretching a matrix of nearly constant rigidity would affect behavior. Surprisingly we found that although 5% static stretch of the pillars did not alter cell spreading or growth 5% cyclic stretch over a frequency range of 0.01 to 10 Hz caused significant increases in spreading and stress fiber formation with the optimum at 0.1 Hz. Similarly there was an increase in the rate of cell growth as measured by BrdU incorporation into DNA over this frequency range. At 0.01 Hz we found that cyclic stretching for 10 s (relaxation for 90s) did not cause significant spreading whereas cyclic stretching for 50 or 90 s (50 and 10 s of relaxation) did. We conclude that the application of force to cells by a soft matrix can substitute for a stiff matrix in stimulating (this can explain why some cells may be stimulated to grow upon physical activity—i.e. use it or lose it).