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Cytomatrix synthesis in MDCK epithelial cells
Author(s) -
Mitchell John J.,
Low Robert B.,
WoodcockMitchell Janet L.
Publication year - 1990
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041430315
Subject(s) - cytoskeleton , vimentin , keratin , tubulin , biology , microbiology and biotechnology , leucine , intermediate filament , biochemistry , cell culture , protein biosynthesis , polyacrylamide gel electrophoresis , cell , microtubule , amino acid , genetics , enzyme , immunology , immunohistochemistry
Detailed information regarding the synthesis rates of individual protein components is important in understanding the assembly and dynamics of the cytoskeletal matrix of eukaryotic cells. As an approach to this topic, the dual isotope technique of Clark and Zak J. Biol. Chem. , 256 : 4863–4870, 1981), was employed to measure fractional synthesis rates (FSRs) in growing and quiescent cultures of MDCK epithelial cells. Cell protein was labeled to equilibrium with [ 14 C] leucine over several days and then pulse‐labeled for 4 hours with [ 3 H] leucine. FSRs (as percent per hour) were calculated from the 3 H/ 14 C ratio of cell extracts or individual proteins separated by two‐dimensional polyacrylamide gel electrophoresis and the 3 H/ 14 C ratio of free leucine in the medium. Synthesis of total cell protein rose from approximately 1.4%/hour in quiescent cells to 3.5%/hour in the growing cultures. The latter rate was sufficient to account for the rate of protein accumulation and a low level of turnover in the growing cultures. The FSR of the buffered‐Triton soluble extract was higher and the cytoskeletal FSR significantly lower than that for total protein in quiescent monolayers. This difference, however, was not observed in growing cultures. A distinct pattern of differences was seen in the FSRs of individual cytoskeletal proteins in the quiescent cultures. Vimentin synthesis was significantly lower than that of the keratins and the keratin FSRs were not obviously matched in pairwise fashion. Unexpectedly, the FSRs of α‐ and β‐tubulin diverged in quiescent cells with α‐tubulin turnover exceeding β‐tubulin. Likewise, components of the microfilament lattice showed unequal fractional synthesis rates, myosin and α‐actinin being faster than actin. In addition, the FSR for globular actin exceeded that of the cytoskeletal associated form. The results suggest that metabolic coupling between individual cellular filament systems is not strict. The data are, however, consistent with models that predict that assembly of a subcellular structure influences the turnover of its component proteins.