Abstract
Purified actin and microtubule proteins polymerized together form a gel, while mixtures of actin with tubulin polymers lacking microtubule-associated proteins (MAPs) have low viscosities close to the sum of the viscosities of the constituents. Mixtures of actin and MAPs also have high viscosities. Our interpretation of these observations was that there is interaction of actin filaments and microtubules which is mediated by MAPs (Griffith, L. M., and Pollard, T. D. (1978) J. Cell Biol. 78, 958-965). We report here further evidence for this interaction. 1) Actin filaments and microtubules can form gels at physiological ionic strength providing the anion is glutamate rather than chloride. Both glutamate and chloride inhibit actin-MAPs interaction, but this is compensated for in glutamate where the microtubules are longer than in chloride. 2) The low shear viscosity of mixtures of isolated MAPs and actin filaments is enhanced by acidic pH and inhibited by high ionic strength. 3) MAPs can be fractionated to yield four different fractions with actin cross-linking activity: a subset of high molecular weight MAPs, purified "MAP-2" and two different fractions of tau polypeptides. 4) We have reconstituted a gel from actin, purified tubulin, and whole MAPs, but have not yet been successful with actin, purified tubulin, and any single purified MAP.
Highlights
(MAPs) have low viscosities close to the sum of the between the polymers
2) The low shear viscosity of emphasized experiments with actin and MAPs because a mixtures of isolated M A P S and actin filaments is en- direct analysis of the microtubule-actin interaction would hancedbyacidic pHand inhibited by high ionic have been restricted to those limited conditionswhich support strength
4) We mally by the factors which we tested as modifiers of the actinhave reconstituted a gel from actin, purified tubulin, MAPs interactions (Griffith and Pollard, 1982)
Summary
Purification of Proteins-Conventional rabbit skeletal muscle actin was purified by a modification of the method of Spudich and Watt (1971)as described in Grfith and Pollard (1978). The clear heat-stable supernatant of600mgof twice-cycled hog brain microtubules contained 55 mg of protein This was concentrated to a volume of 3 ml by dialysis uersus dry sucrose, dialyzed against column buffer, clarified by centrifugation at 55,000 X g for 10 min, and fractionated by gel filtration on a column (2.5 X 50 cm) of Bio-Gel A-1.5m run a t 50 cm of pressure. Stained specimens of actin filaments treated with unfractionated or purified MAPs were made as follows: 1mg/ml of actin in 0.1 M PIPES, pH 6.62,1mM EGTA, 0.1 m~ MgCL was polymerized for 30 min a t 37 "C, diluted to 0.06 or 0.12 mg/ml with warm buffer, and applied to hydrophilic carbon-coated grids for 30 s. The average length was calculated as the (total length/number of ends) X (2 ends/microtubule)
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