Persistence: mechanisms for triggering and enhancing phenotypic variability.

When microorganisms are exposed to lethal agents, the initial exponential decay in survival is typically followed by a slower decrease. This tailing of the survival curve is due to persister cells that have differentiated into phenotypes with reduced sensitivity to the lethal agent. We review the environmental factors that have been shown to trigger such differentiation processes, as well as the network motifs that enable the co-existence of persistent and nonpersistent cells within genetically uniform populations. Threshold amplification of noise and bi-stability from positive feedback emerge as key motifs underlying persistence.

Calculation of forces at focal adhesions from elastic substrate data: the effect of localized force and the need for regularization.

Forces exerted by stationary cells have been investigated on the level of single focal adhesions by combining elastic substrates, fluorescence labeling of focal adhesions, and the assumption of localized force when solving the inverse problem of linear elasticity theory. Data simulation confirms that the inverse problem is ill-posed in the presence of noise and shows that in general a regularization scheme is needed to arrive at a reliable force estimate. Spatial and force resolution are restricted by the smoothing action of the elastic kernel, depend on the details of the force and displacement patterns, and are estimated by data simulation. Corrections arising from the spatial distribution of force and from finite substrate size are treated in the framework of a force multipolar expansion. Our method is computationally cheap and could be used to study mechanical activity of cells in real time.

Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism.

The transition of cell-matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II-driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein-tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136-143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.

Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates.

Mechanical forces play a major role in the regulation of cell adhesion and cytoskeletal organization. In order to explore the molecular mechanism underlying this regulation, we have investigated the relationship between local force applied by the cell to the substrate and the assembly of focal adhesions. A novel approach was developed for real-time, high-resolution measurements of forces applied by cells at single adhesion sites. This method combines micropatterning of elastomer substrates and fluorescence imaging of focal adhesions in live cells expressing GFP-tagged vinculin. Local forces are correlated with the orientation, total fluorescence intensity and area of the focal adhesions, indicating a constant stress of 5.5 +/- 2 nNmicrom(-2). The dynamics of the force-dependent modulation of focal adhesions were characterized by blocking actomyosin contractility and were found to be on a time scale of seconds. The results put clear constraints on the possible molecular mechanisms for the mechanosensory response of focal adhesions to applied force.