Critical regulation of microtubule density by CLASP during interphase Depleting microtubules: How low can we go?

Abstract

Microtubules are indispensable components of the cellular cytoskeleton, responsible for maintaining cellular integrity. They are polar structures that form through the polymerization of αβ-tubulin heterodimers. On its own, spontaneous nucleation of microtubules is energetically unfavorable. Despite this, molecular factors like the gamma-tubulin ring complex (γ-TuRC) can promote nucleation. Following their nucleation, microtubules are inherently unstable, constantly fluctuating between phases of growth and shrinkage, termed dynamic instability. An extensive group of regulatory proteins modulates this dynamic instability and collectively form the microtubule-associated proteins (MAPs). This regulation is required for an array of microtubule-based processes, such as, microtubule-based transport, cell division, and the organization of intracellular machinery throughout the cytosol. One such regulatory MAP, Cytoplasmic-linker-associated proteins (CLASPs) inhibit catastrophe and stabilize microtubules at their growing ends, tether and stabilize free microtubule ends at the Golgi, promote microtubule nucleation at the Golgi, and repair damaged microtubules by incorporating new tubulin heterodimers at damaged-sites. Here, we present CLASP as a more robust regulator of the microtubule network in cells than a microtubule polymerase like chTOG. We show that retinal pigment epithelial cells (RPE1) thoroughly depleted of CLASPs have severe reductions in microtubule density and organization. Microtubules in these cells grow slightly faster and have decreased catastrophe frequencies, likely owing to a higher pool of free tubulin and a stable remaining population of microtubules. Furthermore, we report that microtubule nucleation from the Golgi and cytosol are perturbed almost wholly, whereas centrosomal-based microtubule nucleation is reduced in the absence of CLASPs. Moreover, we show a substantial reduction in γ-TuRC, NEDD1, and Ninein, but not Pericentrin, CEP152, CEP192, and CDK5RAP2 localization to the centrosome, yet this does not translate to the robust decrease in microtubule density we observe in the absence of CLASPs. We also present a p53 knock-out based strategy that intriguingly improves the viability of microtubule scarce cells. Removing the centrosome in cells lacking CLASPs reduced the microtubule density even further, which to our knowledge has not been observed before, revealing a critical threshold of microtubule density required for the proper organization and distribution of organelles throughout the cell.