Forced expression of chimeric human fibroblast tropomyosin mutants affects cytokinesis.

Abstract

Abstract. Human fibroblasts generate at least eight tropomyosin (TM) isoforms (hTM1, hTM2, hTM3, hTM4, hTM5, hTM5a, hTM5b, and hTMsmo0 from four distinct genes, and we have previously demonstrated that bacterially produced chimera hTM5/3 exhibits an unusually high affinity for actin filaments and a loss of the salt dependence typical for TM-actin binding (Novy, R.E., J. R. Sellers, L.-E Liu, and J. J.-C. Lin, 1993. Cell Motil. & Cytoskeleton. 26: 248-261). TO examine the functional consequences of expressing this mutant TM isoform in vivo, we have transfected CHO cells with the full-length eDNA for hTM5/3 and compared them to cells transfected with hTM3 and hTM5. Immunofiuorescence microscopy reveals that stably transfected CHO cells incorporate force-expressed hTM3 and hTM5 into stress fibers with no significant effect on general cell morphology, microfilament organization or cytokinesis. In stable lines expressing hTM5/3, however, cell division is slow and sometimes incomplete. The doubling time and the incidence of multinucleate cells in the stable hTM5/3 lines roughly parallel expression levels. A closely related chimeric isoform hTM5/2, which differs only in the internal, alternatively spliced exon also produces defects in cytokinesis, suggesting that normal TM function may involve coordination between the amino and carboxy terminal regions. This coordination may be prevented in the chimeric mutants. As bacterially produced hTM5/3 and hTM5/2 can displace hTM3 and hTM5 from actin filaments in vitro, it is likely that CHO-expressed hTM5/3 and hTM5/2 can displace endogenous TMs to act dominantly in vivo. These results support a role for nonmuscle TM isoforms in the fine tuning of microfilament organization during cytokinesis.Additionally, we find that overexpression of TM does not stabilize endogenous microfilaments, rather, the hTM-expressing cells are actually more sensitive to cytochalasin B. This suggests that regulation of microfilament integrity in vivo requires stabilizing factors other than, or in addition to, TM.

Cytoskeletal organization in tropomyosin-mediated reversion of ras-transformation: Evidence for Rho kinase pathway

“…This results in weakening of TM-actin association, exposing actin ®laments to the action of gel-severing proteins (Pittenger et al, 1995). Furthermore, increased expression of caldesmon stabilizes micro®laments (Warren et al, 1995(Warren et al, , 1996, and caldesmon levels are decreased in many transformed cells, as shown in Figure 3, and by others (Button et al, 1995;Novy et al, 1991). More recent studies implicate caldesmon in the regulation of actomyosin contractility and adhesion-dependent signaling in ®broblasts (Helfman et al, 1999).…”

“…These and other interactions may not be completely restored in the revertants of DT, notwithstanding the reemergence of micro®laments. Because of these reasons, we investigated whether TM1-induced cytoskeleton is as stable as the one in NIH3T3 cells: one established technique involves treatment of the cells with fungal toxins such as cytochalasins (Warren et al, 1995). NIH3T3, DT/TM1 and DT/TM1-TM2 cells were incubated with cytochalasin D for di erent time periods and stained for TMs with an antibody (green) and actin (red).…”

Cytoskeletal Tension Regulates Both Expression and Degradation of h2-Calponin in Lung Alveolar Cells

“…SDS-PAGE and Western blot analysis using RAH2 antibody (A) and densitometry quantification against the levels of actin or total cellular protein (B) detected decreases in h2-calponin after the reduction of cellular tension. In contrast, the levels of tropomyosin did change as shown by the Western blot quantification using mAb LC24 (54). The data are summarized as SEM from six sets of samples, *P<0.05.…”

Caldesmon and the Regulation of Cytoskeletal Functions

“…By suppressing the expression of CaD in cells and monitoring the adhesion and migration properties, one may be able to assess its overall functions. Interestingly, hampered cytokinesis was also observed upon over-expression of a HMW-and-LMW chimeric nonmuscle Tm that exhibited a much higher affinity toward actin than the wild-type Tm, 196 indicating that disassembly of microfilament organization is an essential step during cell division. Evidently, both CaD and Tm are part of stabilizing factors for the actin cytoskeleton.…”