Coordinating Role of IQGAP1 in the Regulation of Multivesicular Endosomal Compartment Dynamics

Abstract

binding to the fibers obtained only when multiple holoenzyme subunits are engaged (Khan et al. 2016. Biophys. J.111,395-408). We have now measured the binding of GFP-CaMKII molecules to rhodamine-phalloidin labeled, single F-actin filaments and characterized the architectural transitions of the formed filament networks to mechano-osmotic stress. CaMKII induced formation of mechanically-resilient networks with aster-like nodes. Cryo-electron tomography showed CaMKII localized in small clusters (n < 5) at the nodes. Although nodes disappeared rapidly upon addition of calcium calmodulin, the network structure took several minutes to dissipate. Both C. elegans and human CaMKIIs formed networks at stoichiometric concentrations, revealing an evolutionary-conserved interaction. At the micromolar concentrations investigated, CaMKII-actin bundles were sparsely distributed in the networks. Macromolecular crowding by addition of polyethylene glycol or force generated by addition of myosin motors plus ATP bundled the filaments; but CaMKII retarded, rather than assisted, this transition. CaMKII also protected against filament fragmentation by motor forces. We propose that stimulationtriggered disruption of spine CaMKII-actin interactions has two distinct effects, formation of a compliant F-actin network primarily due to loss of nodal contacts and polymerization of the released G-actin. We are simulating these processes in a 3-dimensional ''virtual'' spine to determine CaMKII, G and F-actin concentrations and affinities that best mimic the observed spine dynamics upon stimulation.