Evidence for opposing effects of calmodulin on cortical microtubules

Microtubule integrity within the cortical array was visualized in detergent-lysed carrot (Daucus carota L.) protoplasts that were exposed to various exogenous levels of Ca²⁺ and calmodulin (CAM). CaM appears to help stabilize cortical microtubules against the destabilizing action of Ca²⁺/CaM complexes at low Ca²⁺ concentrations, but not at higher Ca²⁺ concentrations. The hypothesis that CaM interacts with microtubules at two different sites, determined by the concentration of Ca²⁺, is supported by the effects of the CaM antagonists N-(6-aminohexyl)-1-naphthalene- sulfonamide and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfanamide (20 μM) and by affinity chromatography. Two classes of proteins were identified that interact with tubulin and bind to CaM. One class required Ca²⁺ for CaM binding, whereas the second class bound only when Ca²⁺ concentrations were low (<320 nM). Thus, CaM's ability to have two opposing effects upon microtubules may be regulated by the concentration of intracellular Ca²⁺ and its differential interactions with microtubule-associated proteins. Experimental manipulation of intracellular Ca²⁺ concentrations, as monitored by Indo-1, revealed that the effect of Ca²⁺ is specific to the cortical microtubules and does not affect actin microfilaments in these cells.