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Profilin is predominantly associated with monomeric actin in Acanthamoeba
D A Kaiser1, V K Vinson, D B Murphy
1Structural Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
Abstract:
We used biochemical fractionation, immunoassays and microscopy of live and fixed Acanthamoeba to determine how much profilin is bound to its known ligands: actin, membrane PIP(2), Arp2/3 complex and polyproline sequences. Virtually all profilin is soluble after gentle homogenization of cells. During gel filtration of extracts on Sephadex G75, approximately 60% of profilin chromatographs with monomeric actin, 40% is free and none voids with Arp2/3 complex or other large particles. Selective monoclonal antibodies confirm that most of the profilin is bound to actin: 65% in extract immunoadsorption assays and 74-89% by fluorescent antibody staining. Other than monomeric actin, no major profilin ligands are detected in crude extracts. Profilin-II labeled with rhodamine on cysteine at position 58 retains its affinity for actin, PIP(2) and poly-L-proline. When syringe-loaded into live cells, it distributes throughout the cytoplasm, is excluded from membrane-bounded organelles, and concentrates in lamellapodia and sites of endocytosis but not directly on the plasma membrane. Some profilin fluorescence appears punctate, but since no particulate profilin is detected biochemically, these spots may be soluble profilin between organelles that exclude profilin. The distribution of profilin in fixed human A431 cells is similar to that in amoebas. Our results show that the major pool of polymerizable actin monomers is complexed with profilin and spread throughout the cytoplasm.
Insights
Profilin, a protein essential for cell structure, primarily binds to actin monomers in Acanthamoeba. This binding ensures actin is available for polymerization throughout the cytoplasm.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Profilin is a ubiquitous eukaryotic protein known to bind actin monomers.
- Its interactions with other cellular components, like membrane phosphoinositides and polyproline sequences, are also established.
- The precise in vivo distribution and binding status of profilin within the cell remain incompletely understood.
Purpose of the Study:
- To quantify the binding of profilin to its known ligands in Acanthamoeba.
- To determine the subcellular localization and distribution of profilin within live cells.
- To elucidate the role of profilin in regulating the availability of actin monomers.
Main Methods:
- Biochemical fractionation using gel filtration (Sephadex G75).
- Immunoassays including selective monoclonal antibody-based immunoadsorption.
- Fluorescence microscopy of live and fixed Acanthamoeba and A431 cells using rhodamine-labeled profilin.
Main Results:
- Approximately 60% of cellular profilin co-elutes with monomeric actin during gel filtration, indicating strong binding.
- Immunoadsorption and fluorescent antibody staining confirm significant profilin-actin complex formation (65-89%).
- Live cell imaging shows profilin distributed in the cytoplasm, concentrated in lamellipodia and endocytic sites, but excluded from organelles.
Conclusions:
- The major pool of polymerizable actin monomers in Acanthamoeba is complexed with profilin.
- Profilin is soluble and distributed throughout the cytoplasm, with specific enrichment at sites of dynamic actin remodeling.
- These findings highlight profilin's critical role in maintaining cytoplasmic actin monomer availability for cellular processes.