Anti-actin antibodies generated against profilin : actin distinguish between non-filamentous and filamentous actin, and label cultured cells in a dotted pattern

• Actin polymerization is a prominent feature of migrating cells, where it powers the protrusion of the leading edge. Many studies have characterized the well-ordered and dynamic arrangement of filamentous actin in this submembraneous space. However, less is known about the organization of unpolymerized actin. Previously, we reported on the use of covalently coupled profilin:actin to study actin dynamics and presented evidence that profilin-bound actin is a major source of actin for filament growth. To locate profilin:actin in the cell we have now used this non-dissociable complex for antibody generation, and obtained monospecific anti-actin and antiprofilin antibodies from two separate immunizations. Fluorescence microscopy revealed drastic differences in the staining pattern generated by the anti-actin antibody preparations. With one, distinct puncta appeared at the actin-rich leading edge and sometimes aligned with microtubules in the interior of the lamella, while the other displayed typical actin filament staining. Labelling experiments in vitro demonstrated failure of the first antibody to recognize filamentous actin and none of the two bound microtubules. The two anti-profilin antibodies purified in parallel generated a punctated pattern similar to that seen with the first anti-actin antibody. All antibody preparations labelled the nuclei.

Nyckelord

profilin-actin; antibodies; fluorescence microscopy; immunohistochemistry; microfilament dynamics

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