Abstract
We have previously shown that microtubule-associated protein 2 (MAP2) and Tau, two major microtubule-associated proteins, interact with actin differently as measured by low-shear viscosity and that their activities are modified by phosphorylation (Nishida, E., Kotani, S., Kuwaki, T., and Sakai, H. (1982 in Biological Functions of Microtubules and Related Structures (Sakai, H., Mohri, H., and Borisy, G. G., eds) pp. 297-309, Academic Press, Japan). In the present study we further examined their interaction using turbidimetry, electron microscopy, low- and high-shear viscometry. MAP2 increased the low-shear viscosity of actin filament but had weaker effect on high-shear viscosity and turbidity of actin filaments. In contrast, Tau reduced high-shear viscosity of actin filaments and enhanced the turbidity which were due to formation of actin filament bundles as shown by electron microscopy. We conclude that MAP2 is a gelation factor, while Tau is a bundling factor. A well-known Ca2+-dependent regulatory protein, calmodulin, inhibited both MAP2-actin and Tau-actin interaction in a Ca2+-dependent manner. The calmodulin-dependent inhibition was canceled by higher concentrations of MAP2 or Tau, and calmodulin had no effect on the viscosity of actin filament alone, indicating that this inhibition is based on the stoichiometric interaction of calmodulin with MAP2 or Tau.
Highlights
From the Departmento.f~Biophysics and Biochemistry, Faculty of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan
Interaction between actinfilamentsandmicrotubules in uitro was first demonstrated by Griffith and Pollard [1]and has been further characterized by Pollard and his co-workers [1,2,3,4,5]. They revealed that microtubules and actin filaments are cross-linked by microtubule-associated proteins (MAPs’) and that MAPs alone cross-link actin filaments
We investigated if calmodulin modulates MAPS-actininteraction, and show that calmodulin inhibits both MAP2-actin and Tauactin interaction in a Ca2+-dependent manner
Summary
Protein Preparation-Microtubule protein was isolatefdrom freshly slaughtered porcine brains by the method of Shelanski et al [14] withsome modifications [15]. Purified Tawu as composed of five polypeptides, molecular weights of which were in a range between 55,000 and 62,000 on a polyacrylamide gel as recently observed by Lindwall and Cole [17], and was free from other polypeptides (Fig. l b ). Rabbit skeletal muscle actin was prepared by t,he method of Spudich and Watt [18],and further purified by gel filtration on Sephadex G-100. The actin concentrationwas determined by UV absorption measurement using Ai$ = 6.5. Actin polymerization in theabsence of shear stresswas monitored by measuring the change in absorbance at 237 nm using a Gilford 260 spectrophotometer. Chemicals-Reagent grade materials were obtained from the following sources; MES and EGTA from Sigma; DE52 from Whatman; Bio-Gel A-15m from Bio-Rad; ATP and GTP from Yamasa Shoyu Co. (Choshi, Japan); 2-mercaptoethanol from Nakarai Chemicals Ltd. (Kyoto, Japan)
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