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Organizational forms of actin in 13762 ascites mammary tumor cell microvilli
Abstract:
The organization of microvillus actin and its associated proteins have been investigated in sublines of mammary ascites tumors (MAT) with mobile (MAT-B1) and immobile (MAT-C1) cell surface receptors. Microvilli isolated from these sublines differ in morphology (branched for MAT-C1 versus unbranched for MAT-B1) and the presence of a 58,000-dalton polypeptide (58K). 58K is found associated with MAT-C1 microvilli, microvillar cytoskeletons obtained by nonionic detergent extractions, and microvillar membranes prepared under conditions which depolymerize actin microfilaments. By extraction with actin-stabilizing buffers (isotonic Triton-Mg-ATP) microvillar actin can be fractionated into four forms. About 40% of the actin is sedimented at low speed (7,500g, 15 min). The pellets contain microfilaments; actin and alpha-actinin are the predominant proteins. High-speed pellets from these low-speed supernates contain about 10% of the actin as a transmembrane complex with a cell surface glycoprotein (cytoskeleton-associated glycoprotein, [CAG] 75-80,000 daltons) in MAT-B1 cells or with CAG and 58K in MAT-C1 cells. Transmembrane complexes can be purified from MAT-B1 and MAT-C1 microvillar membranes in Triton-containing buffer by gel filtration or sucrose density gradient centrifugation. The presence of only CAG and actin in the MAT-B1 transmembrane complex strongly suggests the direct interaction of actin and a cell surface component. The high-speed supernates contain soluble actin. By gel filtration or rate-zonal sucrose density gradient centrifugation about 30% of the microvillar actin is found as small oligomers and about 10% as G-actin in this extraction buffer. We suggest that the actin-containing transmembrane complexes may serve as membrane-association sites for oligomeric actin segments and microfilaments and as initiation sites for actin polymerization.
Insights
Researchers studied microvillus actin organization in tumor cells, revealing differences in actin complexes and a 58K protein. These findings suggest actin-cytoskeleton-associated glycoprotein complexes may initiate actin polymerization at the cell membrane.
Area of Science:
- Cell Biology
- Biochemistry
- Cancer Research
Background:
- Microvilli are crucial for cell surface interactions and signaling.
- The organization of actin cytoskeleton within microvilli is key to their structure and function.
- Differences in cell surface receptors can influence microvillar organization and associated proteins.
Purpose of the Study:
- To investigate the organization of microvillus actin and associated proteins in mammary ascites tumor (MAT) sublines with distinct cell surface receptor mobility.
- To characterize the differences in microvillar morphology and protein composition between mobile (MAT-B1) and immobile (MAT-C1) cell lines.
- To elucidate the role of a 58,000-dalton polypeptide (58K) and transmembrane complexes in microvillar structure.
Main Methods:
- Isolation and morphological analysis of microvilli from MAT-B1 and MAT-C1 sublines.
- Fractionation of microvillar actin using actin-stabilizing buffers (isotonic Triton-Mg-ATP).
- Biochemical analysis of protein complexes using gel filtration and sucrose density gradient centrifugation.
Main Results:
- Microvilli from MAT-C1 cells exhibited branched morphology and contained a 58K polypeptide, unlike MAT-B1 cells.
- Actin was fractionated into sedimentable microfilaments, transmembrane complexes, soluble oligomers, and G-actin.
- Transmembrane complexes containing actin and cytoskeleton-associated glycoprotein (CAG) were identified, with 58K present in MAT-C1 complexes.
Conclusions:
- The 58K polypeptide is specifically associated with microvilli of the immobile MAT-C1 cell line.
- Actin-cytoskeleton-associated glycoprotein (CAG) complexes represent direct interactions between cell surface glycoproteins and microvillar actin.
- These transmembrane complexes likely serve as sites for actin association and polymerization initiation at the cell membrane.