Actin-regulating activities in cultured BHK cells.

Area of Science:

• Cell Biology
• Biochemistry
• Cytoskeletal Dynamics

Background:

• Actin polymerization is crucial for cellular processes.
• Regulation of actin dynamics involves various accessory proteins.
• Understanding these regulators is key to cell function.

Purpose of the Study:

• To investigate the role of cellular fractions from baby hamster kidney (BHK) cells in actin polymerization.
• To identify and characterize actin-binding proteins, including capping and cross-linking proteins.
• To explore the influence of calcium and membrane components on actin dynamics.

Main Methods:

• High-speed centrifugation of BHK cell lysates.
• Deoxyribonuclease (DNase) assay for G-actin quantification.
• Fluorescence assay using pyrene-conjugated actin for polymerization.
• Assay for actin cross-linking activity via low-speed centrifugation.
• Treatment with calcium and cytochalasin E.

Main Results:

• BHK cell lysates contain a high proportion of monomeric G-actin.
• Cellular fractions exhibit actin nucleation and capping activities, reducing net polymerization.
• A 'pointed'-end capping protein is identified in the cytoplasm.
• Plasma membrane fractions show nucleation activity, and supernatant fractions enhance actin cross-linking, especially when membrane-derived.

Conclusions:

• BHK cell cytoplasm contains factors that inhibit actin polymerization, including barbed-end and pointed-end capping proteins.
• Plasma membranes contribute to actin nucleation, and membrane-associated proteins are involved in actin cross-linking.
• Calcium ions accelerate nucleation but further inhibit polymerization by increasing the critical monomer concentration.
• Actin dynamics are complexly regulated by interactions between cytoplasmic and membrane-associated proteins.