Differential expression of tropomyosin forms in the microfilaments isolated from normal and transformed rat cultured cells.

F Matsumura,J J Lin,S Yamashiro-Matsumura,G P Thomas,W C Topp

doi:10.1016/s0021-9258(17)44009-9

F Matsumura, J J Lin + Show 3 more

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https://doi.org/10.1016/s0021-9258(17)44009-9

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Abstract

Using a newly developed method for microfilament isolation (Matsumura, F., Yamashiro-Matsumura, S. and Lin, J. J.-C. (1983) J. Biol. Chem. 258, 6636-6644), we have analyzed protein composition of microfilaments in "normal" and transformed rat tissue culture cells. They include REF-52 (an established rat embryo cell line) cells, REF-52 transformed by DNA viruses (SV40 or adenovirus type 5), normal rat kidney cells, and normal rat kidney cells transformed by RNA viruses (Kirsten or Rous sarcoma virus). Microfilaments from normal rat culture cells contain three major tropomyosins (apparent Mr = 40,000, 36,500, and 32,400) and two relatively minor tropomyosins (apparent Mr = 35,000 and 32,000). In transformed cells the levels of one or two of the major tropomyosins (Mr = 40,000 and 36,500) are decreased and the levels of one or both of the minor tropomyosins (Mr = 35,000 and 32,000) are increased. These changes in tropomyosin patterns were also observed in temperature shift experiments with rat-1 cells transformed with a Rous sarcoma virus mutant, temperature-sensitive for transformation. Cell-free translation of whole cell mRNA generated similar tropomyosin patterns on two-dimensional gels, suggesting that changes in the pattern of tropomyosin expression were largely effected at the level of RNA rather than by post-translational modification. Such changes in the tropomyosin composition of microfilaments were consistently found to accompany the various morphological alterations associated with transformation. We suggest that alterations in the pattern of tropomyosin expression are involved in, or cause, rearrangement of stress fibers and that this may be responsible (in part) for morphological transformation.

Highlights

6644), we have analyzed protceoimn position of micro- 1977; Ericksonet al., 1980; Tooze1, 981)
We suggest that alterationsin the pattern of tropomyosinexpression are involved in, or cause, rearrangment of stress fibers and that thismay be responsible formorphological transformation
Thechangeintheorganization of stress fibersupon cell transformation, but few experiments have been done in this direction, probably because microfilaments of cultured cells are difficult to isolate

Summary

RESULTS

Washing with PBS containing 5 mM MgClz and 0.2 mM EGTA, the Triton/glycerol-insoluble residues were homogenizedin 20 mM phos-. Using our newmethod (Matsumura et al, 1983)for microfilament isolation, we have analyzetdhe protein compositions of microfilaments in normal rat culture cells and in cells phosphate buffer (pH 7.0) containing 100 mM NaCl, 5 mM MgClz, transformed by varioustypes of virus. When monolayer cells were exposed for 30 min to 10 ,ug/ml of cytochalasin B, the cells arborized Because these cells were still acrylamide gel electrophoretic analysisof microfilaments isolated from normal REF-52and four independent SV40-transformed REF-52 (REF-WT2A, -WT4A,-WTGA, and WTlOA) cell lines. We isolated amount of actin recovered inthe microfilament fraction Because these cells were detached from dishes, the procedure for Triton/glycerol extraction was modified as described below. The cells were extracted for 2 min heavy and light chains (indicated baysterisks i n Fig. 1) of t h e

Changes in TropomyEoxsipnresAsicocnompanTyirnagnsformation

NDd ND

Quantitative data on the changes in tropomyosin patterns

DISCUSSION

Findings

It is tempting to speculate that the substitutionof one set