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CAMSAP-driven microtubule release from γ-TuRC and its regulation by nucleation-promoting factors

ABSTRACT: γ-tubulin ring complex (γ-TuRC) is the major microtubule-nucleating factor. After nucleation, microtubules can be released from γ-TuRC and stabilized by other proteins, such as CAMSAPs, but the biochemical cross-talk between minus-end regulation pathways is poorly understood. Here, we reconstituted this process in vitro using purified components. We found that all CAMSAPs could bind to the minus-ends of γ-TuRC-attached microtubules. CAMSAP2 and CAMSAP3, which decorate and stabilize growing minus ends, but not the minus-end tracking protein CAMSAP1 induced microtubule release from γ-TuRC. CDK5RAP2, a γ-TuRC-interactor, and CLASP2, a regulator of microtubule growth, strongly stimulated γ-TuRC-dependent microtubule nucleation, but only CDK5RAP2 suppressed CAMSAP binding to γ-TuRC-anchored minus ends and their release. CDK5RAP2 also improved selectivity of γ-tubulin-containing complexes for 13- rather than 14-protofilament microtubules in microtubule-capping assays. Knockout and overexpression experiments in cells showed that CDK5RAP2 inhibits formation of CAMSAP2-bound microtubules detached from the microtubule-organizing center. We conclude that CAMSAPs can release newly nucleated microtubules from γ-TuRC, whereas nucleation-promoting factors can differentially regulate this process.

INSTRUMENT(S): Orbitrap Exploris 480, Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Early Embryonic Cell

SUBMITTER: Kelly Stecker

LAB HEAD: Kelly E. Stecker

PROVIDER: PXD048637 | Pride | 2024-01-30

REPOSITORIES: Pride

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			Action	DRS
	20191022_GCP3_GFP_SII_TuRC.raw	Raw
	20191022_GCP3_GFP_SII_TuRC.xlsx	Xlsx
	20191022_GCP3_GFP_SII_TuRC_02.raw	Raw
	20191022_GCP3_GFP_SII_TuRC_02.xlsx	Xlsx
	20191022_GCP6_GFP_SII_TuRC.raw	Raw

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