Search

To initiate directed movement, cells must become polarized, establishing a protrusive leading edge and a contractile trailing edge. This symmetry-breaking process involves reorganization of cytoskeleton and asymmetric distribution of regulatory molecules. However, what triggers and maintains this asymmetry during cell migration remains largely elusive. Here, we established a micropatterning-based 1D motility assay to investigate the molecular basis of symmetry breaking required for directed cell migration. We show that microtubule (MT) detyrosination drives cell polarization by directing kinesin-1-based transport of the adenomatous polyposis coli (APC) protein to cortical sites. This is essential for the formation of cell's leading edge during 1D and 3D cell migration. These data, combined with biophysical modeling, unveil a key role for MT detyrosination in the generation of a positive feedback loop linking MT dynamics and kinesin-1-based transport. Thus, symmetry breaking during cell polarization relies on a feedback loop driven by MT detyrosination that supports directed cell migration.

Protein phosphatase 2A (PP2A) is an abundant phosphoprotein phosphatase that acts as a tumor suppressor. For this reason, compounds able to activate PP2A are attractive anticancer agents. The compounds iHAP1 and DT-061 have recently been reported to selectively stabilize specific PP2A-B56 complexes to mediate cell killing. We were unable to detect direct effects of iHAP1 and DT-061 on PP2A-B56 activity in biochemical assays and composition of holoenzymes. Therefore, we undertook genome-wide CRISPR-Cas9 synthetic lethality screens to uncover biological pathways affected by these compounds. We found that knockout of mitotic regulators is synthetic lethal with iHAP1 while knockout of endoplasmic reticulum (ER) and Golgi components is synthetic lethal with DT-061. Indeed we showed that iHAP1 directly blocks microtubule assembly both in vitro and in vivo and thus acts as a microtubule poison. In contrast, DT-061 disrupts both the Golgi apparatus and the ER and lipid synthesis associated with these structures. Our work provides insight into the biological pathways perturbed by iHAP1 and DT-061 causing cellular toxicity and argues that these compounds cannot be used for dissecting PP2A-B56 biology.

The detyrosination-tyrosination cycle involves the removal and religation of the C-terminal tyrosine of α-tubulin and is implicated in cognitive, cardiac, and mitotic defects. The vasohibin–small vasohibin-binding protein (SVBP) complex underlies much, but not all, detyrosination. We used haploid genetic screens to identify an unannotated protein, microtubule associated tyrosine carboxypeptidase (MATCAP), as a remaining detyrosinating enzyme. X-ray crystallography and cryo–electron microscopy structures established MATCAP’s cleaving mechanism, substrate specificity, and microtubule recognition. Paradoxically, whereas abrogation of tyrosine religation is lethal in mice, codeletion of MATCAP and SVBP is not. Although viable, defective detyrosination caused microcephaly, associated with proliferative defects during neurogenesis, and abnormal behavior. Thus, MATCAP is a missing component of the detyrosination-tyrosination cycle, revealing the importance of this modification in brain formation.

Mitotic spindle assembly is crucial for chromosome segregation and relies on bundles of microtubules that extend from the poles and overlap in the middle. However, how these structures form remains poorly understood. Here we show that overlap bundles arise through a network-to-bundles transition driven by kinetochores and chromosomes. STED super-resolution microscopy revealed that PRC1-crosslinked microtubules initially form loose arrays, which become rearranged into bundles. Kinetochores promote microtubule bundling by lateral binding via CENP-E/kinesin-7 in an Aurora B-regulated manner. Bundle separation is driven by steric interactions of the bundle-associated chromosomes at the spindle midplane since spindles with uncondensed or uncongressed chromosomes are narrower. In agreement with experiments, theoretical modeling suggests that bundles arise through competing attractive and repulsive mechanisms. Finally, perturbation of overlap bundles led to inefficient correction of erroneous kinetochore-microtubule attachments. Thus, kinetochores and chromosomes drive coarsening of a uniform microtubule array into overlap bundles, which promote not only spindle formation but also chromosome segregation fidelity.