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Actin associated with membranes from 3T3 mouse fibroblast and HeLa cells
Abstract:
A protein component of membranes isolated from 3T3 mouse fibroblasts and HeLa cells has been identified as actin by peptide mapping. Extensive but apparently not total coincidence was found between the peptide maps of these two nonmuscle membrane-associated actins compared to chick skeletal muscle actin. Between 2 and 4 percent of the total membrane protein appears in the actin band on sodium dodecyl sulfate polyacrylamide gels of 3T3 membranes while about 4 percent of the membrane protein appears as the actin band from HeLa membranes. These values represent approximately the same proportion of actin to total protein found in the cell homogenates. Treatment of intact cells with levels of cytochalasin B sufficient to cause pronounced morphological changes did not change the amount of actin associated with the membrane in either 3T3 or HeLa cells. However, incubation of isolated membranes under conditions favoring conversion of actin from filamentous to monomeric form resulted in dissociation of approximately 80 and 60 percent of the actin from 3T3 and HeLa membranes, respectively. Thus, approximately 20 percent of 3T3 membrane actin and 40 percent of HeLa membrane actin remained associated with the membrane even under actin depolymerizing conditions.
Insights
Researchers identified actin as a key protein in 3T3 fibroblast and HeLa cell membranes. While most actin dissociated under depolymerizing conditions, a significant portion remained membrane-associated, suggesting a stable interaction.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Actin is a crucial cytoskeletal protein involved in various cellular processes.
- The presence and role of actin in nonmuscle cell membranes are not fully understood.
- Previous studies have focused on muscle actin, leaving gaps in knowledge regarding membrane-associated actin in other cell types.
Purpose of the Study:
- To identify and characterize actin as a membrane protein in 3T3 mouse fibroblasts and HeLa cells.
- To investigate the association of membrane-associated actin with cellular conditions and its stability.
- To compare membrane-associated actin from nonmuscle cells with that of skeletal muscle actin.
Main Methods:
- Peptide mapping was employed to identify actin in isolated cell membranes.
- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) quantified actin levels in membrane protein.
- Cytochalasin B treatment and actin depolymerization conditions were used to assess actin-membrane association stability.
Main Results:
- Actin was identified as a significant membrane protein component in both 3T3 (2-4%) and HeLa (4%) cells.
- Peptide maps showed extensive similarity between nonmuscle membrane actins and chick skeletal muscle actin.
- While cytochalasin B did not affect membrane actin levels, actin depolymerization conditions led to significant dissociation (80% from 3T3, 60% from HeLa), with a notable portion (20% and 40%, respectively) remaining membrane-bound.
Conclusions:
- Actin is a substantial component of 3T3 and HeLa cell membranes, present in proportions similar to total cell homogenates.
- A significant fraction of membrane-associated actin exhibits stable binding, persisting even under conditions that favor actin depolymerization.
- These findings highlight a potentially important, stable role for actin in nonmuscle cell membrane structure and function.