Actin assembly induced by polylysine beads or purified phagosomes: quantitation by a new flow cytometry assay.
BACKGROUND
Actin assembly on biological membranes is a poorly understood process. We have previously shown that phagosomal membranes could induce actin assembly in the presence of thymosin beta4 (an actin sequestering protein that inhibits nonspecific nucleation), via the barbed ends of actin filaments.


METHODS
Here, we have developed an in vitro system based on fluorescein-labeled G (monomeric) actin and flow cytometry analysis, which allowed us to quantify de novo actin assembly on the cytoplasmic side of purified phagosomes. To standardize the system, we also used latex beads covalently coupled with polylysine, which efficiently promote actin nucleation.


RESULTS
Flow cytometry analysis showed that the percentage of polylysine beads positive for F-actin filaments increased in a time- and G-actin concentration-dependent manner. Incubation of phagosomes with reagents affecting actin dynamics allowed us to extend our previous data showing that the phagosomal membranes assemble actin filaments de novo. Finally, our results pin-point a potential role for gelsolin as a positive regulator of actin assembly on the phagosomal membrane.


CONCLUSIONS
We propose that our system could facilitate the development of other in vitro assays for the analysis of actin assembly and its links to signaling in cells.
Defacque
H
H
European Molecular Biology Laboratory, Heidelberg, Germany.
Egeberg
M
M
Antzberger
A
A
Ansorge
W
W
Way
M
M
Griffiths
G
G
D000199:Actins / Q000378:metabolism* / Q000493:pharmacokinetics; D000818:Animals; D002478:Cells, Cultured; D015638:Cytochalasin D / Q000494:pharmacology; D003850:Deoxyribonuclease I; D005434:Flow Cytometry / Q000379:methods*; D018260:Gelsolin / Q000494:pharmacology; D007840:Latex; D008264:Macrophages / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008863:Microspheres; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010588:Phagosomes / Q000378:metabolism*; D011107:Polylysine / Q000494:pharmacology*; D013947:Thymosin / Q000378:metabolism; D000099201:Thymosin beta(4)
D000199:Actins; D015638:Cytochalasin D; D003850:Deoxyribonuclease I; D018260:Gelsolin; D007840:Latex; D019384:Nucleic Acid Synthesis Inhibitors; D011107:Polylysine; D013947:Thymosin; D000099201:Thymosin beta(4)