Antagonistic forces generated by myosin II and cytoplasmic dynein regulate microtubule turnover, movement, and organization in interphase cells

Abstract

Photoactivation of caged fluorescent tubulin was used mark the microtubule (MT) lattice and monitor MT behavior in interphase cells. A broadening of the photoactivated region occurred as MTs moved bidirectionally. MT movement was not inhibited when MT assembly was suppressed with nocodazole or Taxol; MT movement was suppressed by inhibition of myosin light chain kinase with ML7 or by a peptide inhibitor. Conversely, MT movement was increased after inhibition of cytoplasmic dynein with the antibody 70.1. In addition, the half-time for MT turnover was decreased in cells treated with ML7. These results demonstrate that myosin II and cytoplasmic dynein contribute to a balance of forces that regulates MT organization, movement, and turnover in interphase cells. microtubule motion T he capacity for microtubules (MTs) to undergo dynamic rearrangement is critical to the various vital cellular activities to which they contribute, including the assembly and function of the mitotic spindle, intracellular transport of organelles and vesicles, and the establishment and maintenance of cell shape and polarity. MTs are nucleated at the centrosome and maintain a generally radial organization, with plus-ends at the cell periphery. Direct observations show that peripheral plus-ends undergo dynamic instability behavior, characterized by the stochastic switching between phases of elongation and rapid shortening (1). Dynamic instability is tempered, such that turnover is more rapid in the peripheral region of the cell (2). MT turnover in the more central cytoplasmic domain is thought to occur by subunit turnover at both plus-and minus-ends (3, 4).MTs modulate the behavior of the actin cytoskeleton in a temporally and spatially regulated manner. For example, MTs are required to establish the site of contractile ring formation in cytokinesis and to restrict ruffling behavior to the leading edge of motile fibroblasts (5). The nature of the cross-talk between the MT and actin cytoskeletons is not well understood, but recent work indicates that a feedback mechanism involving Rho family members may contribute to these regulatory interactions (6). In addition, molecular motors may integrate the MT and actin systems. Actomyosin moves MTs rearward at the cell periphery (3), and myosin-generated forces are responsible for MT transport in motile cells and for axon retraction in cultured neurons (7,8). Cytoplasmic dynein contributes to MT organization in interphase cells, to the assembly and function of the mitotic spindle, and to MT transport in neurons (9, 10). These observations led us to test the hypothesis that molecular motors modulate the organization, rearrangement, and turnover of MTs in interphase cells. Materials and MethodsMaterials. All materials for cell culture were obtained from Life Technologies (Gaithersburg, MD), with the exception of FCS, which was obtained from Atlanta Biologicals (Norcross, GA). Unless otherwise noted, all other chemicals were obtained from Sigma.Cell Culture, Cell Staining, and Microinjection. Cell culture...

Myosin II regulates the shape of three-dimensional intestinal epithelial cysts

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Centrosome Reorientation in Wound-Edge Cells Is Cell Type Specific

“…The distribution was determined, by one-way analysis of variance, to be random, with 31% in the front, 29% on the side, 22% in the back, and 18% in the center, demonstrating that cytoplasmic dynein/dynactin does indeed play a role in MTOC reorientation in CHO cells at the wound edge (Table 2). Cells injected with 70.1 antibodies were characterized by less-well-organized microtubule arrays (Yvon et al, 2001) and, although lamellae did form at the leading edge, locomotion was somewhat reduced compared with control cells.…”

“…Preparation of labeled tubulins, photoactivation, and image collection were performed exactly as described previously (Yvon et al, 2001).…”