Microtubule-associated proteins promote microtubule generation in the absence of γ-tubulin in human colon cancer cells

Abstract

{gamma}-Tubulin complex acts as the predominant microtubule (MT) nucleator that initiates MT formation and is therefore an essential factor for cell proliferation. Nonetheless, cellular MTs are formed after experimental depletion of the {gamma}-tubulin complex, suggesting that cells possess other factors that drive MT nucleation. Here, by combining gene knockout, auxin-inducible degron, RNA interference, MT depolymerisation/regrowth assay, and live microscopy, we identified four microtubule-associated proteins (MAPs), ch-TOG, CLASP1, CAMSAPs, and TPX2, which are involved in {gamma}-tubulin-independent MT generation in human colon cancer cells. In the mitotic MT regrowth assay, nucleated MTs organised non-centriolar MT organising centres (ncMTOCs) in the absence of {gamma}-tubulin. Depletion of CLASP1 or TPX2 substantially delayed ncMTOC formation, suggesting that they promote MT nucleation in the absence of {gamma}-tubulin. In contrast, depletion of CAMSAPs or ch-TOG did not affect the timing of ncMTOC appearance. CLASP1 also accelerates {gamma}-tubulin-independent MT regrowth during interphase. Thus, MT generation can be promoted by MAPs without the {gamma}-tubulin template.