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...
We have investigated in the present study the interaction between G-actin and various types of liposomes, zwitterionic, positively charged, and negatively charged. To investigate at the molecular level the conformation of actin in the presence of lipids, we have selectively attached a fluorinated probe, 3-bromo-1,1,1-trifluoropropanone, to the actin cysteine residues 10, 285, and 374 and used high-resolution 19F nuclear magnetic resonance spectroscopy to investigate the probe resonances. The results indicate a change in the mobility of the 19F labels when G-actin is in the presence of positively charged liposomes made of DMPC and stearylamine and in the presence of DMPG, a negatively charged lipid. No conformational change was observed in the actin molecule in the presence of neutral liposomes. Electron micrographs of these systems reveal the formation of paracrystalline arrays of actin filaments at the surface of the positively charged liposomes, while no evidence of actin polymerization or paracrystallization was observed in the presence of DMPG. The interaction between actin and the lipid polar headgroup has also been investigated using solid-state phosphorus and deuterium NMR. The results indicate no evidence of interaction between actin and zwitterionic liposomes but show an interaction between the positively charged liposomes and a negative charge on the actin molecules. Interestingly, the negatively charged liposomes interact with a positive charge, which is most likely associated with the three residues (His-Arg-Lys) preceding the cysteine 374 residue in the protein.
One of the current dogmas in cytoskeleton research holds that actin filaments are attached to the cell membrane through integral membrane actin-binding proteins. We have challenged this concept, using an in vitro system composed of pure actin and liposomes, and have found that actin may also interact with membrane lipids. Differential scanning calorimetry (DSC) shows that when the actin molecule is in contact with such lipids, it undergoes a major conformational change which results in the complete disappearance of its phase transition. Conversely, DSC scans reveal that the phase transition of the membrane lipids is only weakly affected by the presence of actin. Indeed, the lipids' main transition shows only slight shifts in Tm, from 56.6 to 57 degrees C, and delta Hcal, from 10.1 to 8.8 kcal/mol. In the lipids' pretransition, Tp is shifted from 52.7 to 53.7 degrees C, and delta Hcal is shifted from 0.75 to 0.33 kcal/mol. This interaction between purified actin and membrane lipids is inhibited by high concentrations of KCl, thus indicating that the phenomenon is primarily electrostatic in nature. The ultrastructural consequences of this change in actin conformation were investigated by electron microscopy, which revealed the formation of paracrystalline arrays of actin filaments at the surface of the liposomes. We therefore propose a model in which a limited number of lipid molecules may interact with specific sites on the actin molecule, resulting in the protein's observed conformational change.
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