Activated ezrin controls MISP levels to ensure correct NuMA polarization and spindle orientation

Abstract

Correct spindle orientation is achieved through signaling pathways that provide a molecular link between the cell cortex and spindle microtubules in an F-actin-dependent manner. A conserved cortical protein complex, composed of LGN (also known as GPSM2), NuMA (also known as NUMA1) and dynein-dynactin, plays a key role in establishing proper spindle orientation. It has also been shown that the actin-binding protein MISP and the ERM family, which are activated by lymphocyte-oriented kinase (LOK, also known as STK10) and Ste20-like kinase (SLK) (hereafter, SLK/LOK) in mitosis, regulate spindle orientation. Here, we report that MISP functions downstream of the ERM family member ezrin and upstream of NuMA to allow optimal spindle positioning. We show that MISP directly interacts with ezrin and that SLK/LOK-activated ezrin ensures appropriate cortical MISP levels in mitosis by competing with MISP for actin-binding sites at the cell cortex. Furthermore, we found that regulation of the correct cortical MISP levels, by preventing its excessive accumulation, is essential for crescent-like polarized NuMA localization at the cortex and, as a consequence, leads to highly dynamic astral microtubules. Our results uncover how appropriate MISP levels at the cortex are required for proper NuMA polarization and, therefore, an optimal placement of the mitotic spindle within the cell.This article has an associated First Person interview with the first author of the paper.

Phosphorylation of Merlin by Aurora A kinase appears necessary for mitotic progression

“…Thus, Merlin may influence mitotic progression by modulating Ezrin functions. Interestingly, it was also reported that a correct polarized localization of NuMa is necessary for proper spindle orientation in mitosis and requires the local activation of Ezrin by the Slk kinase (39). The expression of a constitutively active form of Ezrin leads to an unpolarized recruitment of NuMa and a defect in spindle orientation in mouse apical progenitor, resulting in the same spindle orientation defect that we observed in Hela cells expressing the Merlin AA mutant.…”

Ccdc61 controls centrosomal localization of Cep170 and is required for spindle assembly and symmetry

“…For indirect IF HeLa, U2OS, HEK293T, and RPE1 cells were treated as described previously (Cizmecioglu et al , 2010; Kschonsak and Hoffmann, 2018). Coverslips were mounted onto glass slides with ProLong Gold (Molecular Probes) or Mowiol (Calbiochem).…”

ERM Proteins at the Crossroad of Leukocyte Polarization, Migration and Intercellular Adhesion

“…The PKC isoenzymes PKC-θ and PKC-α phosphorylate moesin and ezrin in vitro and associate with them in human T lymphocytes and breast carcinoma cells, respectively [44,45]. Moreover, recent attention has been given to germinal center kinases (GCK), a subfamily of the mammalian sterile 20-like kinases (Mst) including lymphocyte-oriented kinase (LOK), Mst4, SLK and Nck interacting kinase (NIK), as the main kinases that phosphorylate the regulatory Thr of ERMs during cell motility and division [46][47][48][49][50][51]. To this list of kinases, we must now add two sterile 20-like kinases identified in Drosophila, misshapen (an orthologue of NIK) and Slik/SLK [52][53][54].…”