Polymerization of actin by positively charged liposomes.

Abstract

Abstract. By cosedimentation, spectrofluorimetry, and electron microscopy, we have established that actin is induced to polymerize at low salt concentrations by positively charged liposomes. This polymerization occurs only at the surface of the liposomes, and thus monomers not in direct contact with the liposome remain monomeric. The integrity of the liposome membrane is necessary to maintain actin in its polymerized state since disruption of the liposome depolymerizes actin. Actin polymerized at the surface of the liposome is organized into two filamentous structures: sheets of parallel filaments in register and a netlike organization. Spectrofluorimetric analysis with the probe N-pyrenyl-iodoacetamide shows that actin is in the F conformation, at least in the environment of the probe. However, actin assembly induced by the liposome is not accompanied by full ATP hydrolysis as observed in vitro upon addition of salts.TIN is the major protein of muscle cells and has been found in the cytoplasm of all other eukaryotic cells (6,18,31). Actin exists as a monomer, G-actin and as a polymer, F-actin. Polymerization of actin may be induced by millimolar concentrations of divalent cations and/or physiological ionic strength (10,15,16,20,27,35,39,49). A molecule of ATP is bound to each actin monomer and is hydrolyzed during polymerization (25, 28-30, 36, 50). In an excess of divalent cations, such as Mg + § actin filaments associate into paracrystals (1,8,12,25,41,42,46,51).The reversible monomer-polymer transition of cytoplasmic actin in nonmuscle cells is a fundamental phenomenon which is thought to be the basis of many cellular functions such as motility, cytokinesis, phagocytosis. Thus, actin polymerization has been extensively studied in vitro (9,18,33).In a recent paper (38), we have shown that the polymer, F-actin is able to interact directly with the positively charged lipids of artificial membranes. In the present paper, we show that the monomer, G-actin may also interact with positively charged liposomes. However, in these instances, actin polymerizes at the surface of the liposomes even at low salt concentrations. Materials and Methods Preparation and Labeling of Actin Preparation of LiposomesLiposomes were prepared in G-buffer by the reverse phase technique of Szoka and Papahadjopoulos (47). Neutral liposomes were made solely from phosphatidyl choline, whereas positively charged liposomes were prepared from phosphatidyl choline and 1-20% stearylamine. Negatively charged liposomes were made from phosphatidyl choline and 10% oleic acid. All lipids were purchased from Sigma Chemical Co. (St. Louis, MO) and used without further purification. Measurements of Interaction between Actin and Liposomes by TurbidimetryG-actin and liposomes were mixed in G-buffer at various final concentrations indicated in the results section. After 10 min of incubation at room temperature, the turbidity of the solution was determined by optical density at 550 nm. Liposomes were then sedimented by centrifugation at 90,000 g x 120 min a...

Binding of actin filaments to charged lipid monolayers: Film balance experiments combined with neutron reflectivity

“…But it is clear from the experiments done in Gbuffer that actin has to be in the form of polymerised filaments to adsorb to positively charged monolayers. This is in contradiction with the results of Laliberte and Gicquaud [30] who observed a polymerisation on positively charged vesicles (phosphatidylcholine and stearylamine 90/10), although the experiments were done in depolymerising buffer. However, our results are consistent with those of Taylor and Taylor who observed that quaternary ammonium surfactants are better promoter of 2-D crystal growth than stearylamine.…”

Morphological changes in liposomes caused by polymerization of encapsulated actin and spontaneous formation of actin bundles.

“…A previous study (12) demonstrated that actin binding to the lipid-bilayer membrane could be suppressed when the membrane was made from a mixture ofneutral and negatively charged lipids.…”

“…In fact, we found that actin solutions at higher concentrations became birefringent upon actin polymerization, indicating that spontaneous alignment of actin filaments occurred without liposomes (unpublished work). However, this alignment occurred on a much larger scale than that with liposomes; the size of the birefringent area was -10-20 Aum wide and 100 Am long, whereas the size ofthe liposomes was only up to [10][11][12][13][14][15][16][17][18][19][20] Aum in width and length in most cases. In addition, we observed no tendency of the aligned filaments in solution to form circular structures.…”

Actin polymerization induces a shape change in actin-containing vesicles.

“…This mixture also avoided aggregation of vesicles (apparent with PtdCho or PtdEtn/phosphatidylserine vesicles) (17). The negatively charged surface prevented actin polymerization directly from the membrane (18 There were no detectable differences in the shapes of vesicles prepared by dilution or by using Centrex concentrators. FPR.…”