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Membrane-associated actin from the microvillar membranes of ascites tumor cells
Abstract:
A membrane fraction (MF2) has been purified from isolated microvilli of the MAT-C1 subline of the 13762 rat mammary ascites adenocarcinoma under conditions which cause F-actin depolymerization. This membrane preparation contains actin as a major component, although no filamentous structures are observed by transmission electron microscopy. Membranes were extracted with a Triton X-100-containing actin-stabilizing buffer (S buffer) or actin-destabilizing buffer (D buffer). In D buffer greater than 90% of metabolically labeled protein and glycoprotein was extracted, and 80-90% of these labeled species was extracted in S buffer. When S buffer extracts of MF2 were fractionated by either gel filtration on Sepharose 6 B or rate-zonal sucrose density gradient centrifugation, most of the actin was found to be intermediate in size between G- and F-actin. In D buffer most of the MF2 actin behaved as G-actin. Extraction and gel filtration of intact microvilli in S buffer also showed the presence of the intermediate form of actin, indicating that it did not arise during membrane preparation. When [35S]methionine-labeled G-actin from ascites cells was added to S buffer extracts of MF2 and chromatographed, all of the radioactivity chromatographed as G-actin, indicating that the intermediate form of actin did not result from an association of G-actin molecules during extraction or chromatography. The results of this study suggest that the microvillar membrane fraction is enriched in an intermediate form of actin smaller than F-actin and larger than G-actin.
Insights
This study purified a microvillar membrane fraction (MF2) from rat mammary ascites adenocarcinoma. MF2 contains an intermediate form of actin, smaller than filamentous actin (F-actin) but larger than globular actin (G-actin).
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Microvilli are crucial for cellular functions, and their structure is maintained by actin dynamics.
- The MAT-C1 subline of 13762 rat mammary ascites adenocarcinoma provides a model for studying cellular components.
- Actin exists in different forms, including globular (G-actin) and filamentous (F-actin).
Purpose of the Study:
- To characterize the form of actin present in a purified microvillar membrane fraction (MF2) from the MAT-C1 rat mammary ascites adenocarcinoma.
- To investigate the relationship between actin forms and microvillar membrane structures.
- To determine if an intermediate form of actin exists in this specific cellular context.
Main Methods:
- Purification of a membrane fraction (MF2) from isolated microvilli.
- Extraction of MF2 using actin-stabilizing (S buffer) and actin-destabilizing (D buffer) conditions.
- Fractionation of extracted proteins using gel filtration (Sepharose 6 B) and sucrose density gradient centrifugation.
- Metabolic labeling of proteins and glycoproteins for detection.
- Chromatography of labeled G-actin to assess associations.
Main Results:
- The MF2 preparation contained actin as a major component, without observable filamentous structures.
- Extraction in D buffer released over 90% of labeled protein and glycoprotein, with most actin behaving as G-actin.
- Extraction in S buffer released 80-90% of labeled species, and fractionated actin was found to be an intermediate size between G- and F-actin.
- The intermediate actin form was also present in intact microvilli, suggesting it is not an artifact of preparation.
- Experiments with added G-actin indicated the intermediate form did not arise from G-actin association during extraction or chromatography.
Conclusions:
- The microvillar membrane fraction (MF2) is enriched in a distinct form of actin.
- This form of actin is intermediate in size, larger than G-actin and smaller than F-actin.
- The findings suggest a unique actin conformation or complex within the microvillar membrane.