Collagen remodelling by fibroblasts has a crucial role in organizing tissue structures that are essential to motility during wound repair, development and regulation of cell growth. However, the mechanism of collagen fibre movement in three-dimensional (3D) matrices is not understood. Here, we show that fibroblast lamellipodia extend along held collagen fibres, bind, and retract them in a 'hand-over-hand' cycle, involving alpha2beta1 integrin. Wild-type fibroblasts move collagen fibres three to four times farther per cycle than fibroblasts lacking myosin II-B (myosin II-B(-/-)). Similarly, myosin II-B(-/-) fibroblasts contract 3D collagen gels threefold less than controls. On two-dimensional (2D) substrates, however, rates of collagen bead and cell movement are not affected by loss of myosin II-B. Green fluorescent protein (GFP)-tagged myosin II-B, but not II-A, restores normal function in knockout cells and localizes to cell processes, whereas myosin II-A is more centrally located. Additionally, GFP-myosin II-B moves out to the periphery and back during hand-over-hand fibre movement, whereas on 2D collagen, myosin II-B is more centrally distributed. Thus, we suggest that cyclic myosin II-B assembly and contraction in lamellipodia power 3D fibre movements.

A truncated fragment of the nonmuscle myosin II-A heavy chain (NMHC II-A) lacking amino acids 1-591, ⌬N592, was used to examine the cellular functions of this protein. Green fluorescent protein (GFP) was fused to the amino terminus of full-length human NMHC II-A, NMHC II-B, and ⌬N592 and the fusion proteins were stably expressed in HeLa cells by using a conditional expression system requiring absence of doxycycline. The HeLa cell line studied normally expressed only NMHC II-A and not NMHC II-B protein. Confocal microscopy indicated that the GFP fusion proteins of full-length NMHC II-A, II-B, and ⌬N592 were localized to stress fibers. However, in vitro assays showed that baculovirus-expressed ⌬N592 did not bind to actin, suggesting that ⌬N592 was localized to actin stress fibers through incorporation into endogenous myosin filaments. There was no evidence for the formation of heterodimers between the fulllength endogenous nonmuscle myosin and truncated nonmuscle MHCs. Expression of ⌬N592, but not full-length NMHC II-A or NMHC II-B, induced cell rounding with rearrangement of actin filaments and disappearance of focal adhesions. These cells returned to their normal morphology when expression of ⌬N592 was repressed by addition of doxycycline. We also show that GFPtagged full-length NMHC II-A or II-B, but not ⌬N592, were localized to the cytokinetic ring during mitosis, indicating that, in vertebrates, the amino-terminus part of mammalian nonmuscle myosin II may be necessary for localization to the cytokinetic ring.

The effects of the discrete cellular structure on propagation of electrical excitation in cardiac muscle were studied in a one-dimensional fiber model containing a periodic intercalated disk structure. Globally, the macroscopic velocity of propagation follows the behavior associated with propagation in a continuous tissue (except for high values of disk resistance). In addition, the computed spatial extracellular potential along the fiber is a smooth biphasic waveform and does not reflect the underlying discrete cellular structure of the tissue. Other results of the simulations demonstrate the discontinuous nature of propagation and the importance of the structure in arrhythmogenesis. Vmax displays a biphasic behavior as a function of increasing intercalated disk resistance. An initial "paradoxical" increase in Vmax (with a simultaneous decrease in conduction velocity) is followed by a decrease that leads to decremental propagation and conduction block. The time constant of the foot of the action potential (tau foot) increases monotonically with increasing intercalated disk resistance. An increase in the leakage current to extracellular space brings about a significant decrease in the action potential duration and a loss of the plateau. This major effect is accompanied by a relatively smaller decrease in conduction velocity. Collision of two activation wavefronts results in a significant (100%) increase in Vmax and a very small (0.6%) decrease in tau foot.

The cleavage furrow is created by an actomyosin contractile ring that is regulated by small GTPase proteins such as Rac1 and RhoA. Guanine nucleotide exchange factors (GEFs) are positive regulators of the small GTPase proteins and have been implicated as important factors in regulating cytokinesis. However, it is still unclear how GEFs regulate the contractile ring during cytokinesis in mammalian cells. Here we report that a novel GEF, which is termed MyoGEF (myosin-interacting GEF), interacts with non-muscle myosin II and exhibits activity toward RhoA. MyoGEF and non-muscle myosin II colocalize to the cleavage furrow in early anaphase cells. Disruption of MyoGEF expression in U2OS cells by RNA interference (RNAi) results in the formation of multinucleated cells. These results suggest that MyoGEF, RhoA, and non-muscle myosin II act as a functional unit at the cleavage furrow to advance furrow ingression during cytokinesis.