Feroz M. Hameed

Cyclic stretching of soft substrates induces spreading and growth

In the body, soft tissues often undergo cycles of stretching and relaxation that may affect cell behaviour without changing matrix rigidity. To determine whether transient forces can substitute for a rigid matrix, we stretched soft pillar arrays. Surprisingly, 1-5% cyclic stretching over a frequency range of 0.01-10 Hz caused spreading and stress fibre formation (optimum 0.1 Hz) that persisted after 4 h of stretching. Similarly, stretching increased cell growth rates on soft pillars comparative to rigid substrates. Of possible factors linked to fibroblast growth, MRTF-A (myocardin-related transcription factor-A) moved to the nucleus in 2 h of cyclic stretching and reversed on cessation; but YAP (Yes-associated protein) moved much later. Knockdown of either MRTF-A or YAP blocked stretch-dependent growth. Thus, we suggest that the repeated pulling from a soft matrix can substitute for a stiff matrix in stimulating spreading, stress fibre formation and growth.

Dynamics of passive and active particles in the cell nucleus.

Inspite of being embedded in a dense meshwork of nuclear chromatin, gene loci and large nuclear components are highly dynamic at C. To understand this apparent unfettered movement in an overdense environment, we study the dynamics of a passive micron size bead in live cell nuclei at two different temperatures ( and C) with and without external force. In the absence of a force, the beads are caged over large time scales. On application of a threshold uniaxial force (about 10 pN), the passive beads appear to hop between cages; this large scale movement is absent upon ATP-depletion, inhibition of chromatin remodeling enzymes and RNAi of lamin B1 proteins. Our results suggest that the nucleus behaves like an active solid with a finite yield stress when probed at a micron scale. Spatial analysis of histone fluorescence anisotropy (a measure of local chromatin compaction, defined as the volume fraction of tightly bound chromatin) shows that the bead movement correlates with regions of low chromatin compaction. This suggests that the physical mechanism of the observed yielding is the active opening of free-volume in the nuclear solid via chromatin remodeling. Enriched transcription sites at C also show caging in the absence of the applied force and directed movement beyond a yield stress, in striking contrast with the large scale movement of transcription loci at C in the absence of a force. This suggests that at physiological temperatures, the loci behave as active particles which remodel the nuclear mesh and reduce the local yield stress.

Integrin adhesion drives the emergent polarization of active cytoskeletal stresses to pattern cell delamination

Tissue patterning relies on cellular reorganization through the interplay between signaling pathways and mechanical stresses. Their integration and spatiotemporal coordination remain poorly understood. Here we investigate the mechanisms driving the dynamics of cell delamination, diversely deployed to extrude dead cells or specify distinct cell fates. We show that a local mechanical stimulus (subcellular laser perturbation) releases cellular prestress and triggers cell delamination in the amnioserosa during Drosophila dorsal closure, which, like spontaneous delamination, results in the rearrangement of nearest neighbors around the delaminating cell into a rosette. We demonstrate that a sequence of “emergent cytoskeletal polarities” in the nearest neighbors (directed myosin flows, lamellipodial growth, polarized actomyosin collars, microtubule asters), triggered by the mechanical stimulus and dependent on integrin adhesion, generate active stresses that drive delamination. We interpret these patterns in the language of active gels as asters formed by active force dipoles involving surface and body stresses generated by each cell and liken delamination to mechanical yielding that ensues when these stresses exceed a threshold. We suggest that differential contributions of adhesion, cytoskeletal, and external stresses must underlie differences in spatial pattern.

Concerted modulation of paxillin dynamics at focal adhesions by Deleted in Liver Cancer-1 and focal adhesion kinase during early cell spreading.

Deleted in Liver Cancer‐1 (DLC1) is a RhoGTPase‐activating protein (GAP) and a tumor suppressor often downregulated in cancers. It is localized to the focal adhesions (FAs) and its absence leads to enhanced cell migration, invasion, and metastasis. Although DLC1 interacts with focal adhesion kinase (FAK), talin, and tensin, its role in focal adhesions dynamics remains unclear. We examined the effect of DLC1 in Human Foreskin Fibroblasts and determined its localization, dynamics and impact on paxillin by Fluorescence Recovery After Photobleaching at both nascent and mature focal adhesions. During early cell spreading, DLC1 is preferentially localized at the inner/mature adhesions whereas phosphorylated paxillin occupies the outer/nascent FAs. In addition, DLC1 downregulates paxillin turnover in a process, that does not require its GAP activity. Instead, it requires the presence of FAK. Acting in concert, both DLC1 and FAK could provide a unique spatio‐temporal mechanism to regulate paxillin function in tissue homeostasis.

Probing structural stability of chromatin assembly sorted from living cells

Post-translational modifications of the histone tails and other chromatin binding proteins affect the stability of chromatin structure. In this study, we have purified chromatin from live cell nuclei using a fluorescence activated cell sorter (FACS) and studied the structural stability of this self-assembled structure. Using total internal reflection fluorescence (TIRF) microscopy, we map the effect of covalent modifications on the interaction of histone-DNA complex, by measuring the dissociation rates of histones from the chromatin fiber in the presence of different salt concentrations. Dynamic force spectroscopy (DFS) experiments were carried out to measure the structural disintegration of large chromatin globules under force. The characteristic rupture of multiple linkages in the large chromatin globules show differences in the stiffness of the higher order structure of chromatin with altered epigenetic states. Our studies reveal a direct correlation between histone modifications and the structural stability of higher order chromatin assembly.

Micropillar displacements by cell traction forces are mechanically correlated with nuclear dynamics

Cells sense physical cues at the level of focal adhesions and transduce them to the nucleus by biochemical and mechanical pathways. While the molecular intermediates in the mechanical links have been well studied, their dynamic coupling is poorly understood. In this study, fibroblast cells were adhered to micropillar arrays to probe correlations in the physical coupling between focal adhesions and nucleus. For this, we used novel imaging setup to simultaneously visualize micropillar deflections and EGFP labeled chromatin structure at high spatial and temporal resolution. We observed that micropillar deflections, depending on their relative positions, were positively or negatively correlated to nuclear and heterochromatin movements. Our results measuring the time scales between micropillar deflections and nucleus centroid displacement are suggestive of a strong elastic coupling that mediates differential force transmission to the nucleus.