Sequential activities of Dynein, Mud and Asp in centrosome–spindle coupling maintain centrosome number upon mitosis

Abstract

Centrosomes nucleate microtubules and are tightly coupled to the bipolar spindle to ensure genome integrity, cell division orientation and centrosome segregation. While the mechanisms of centrosome-dependent microtubule nucleation and bipolar spindle assembly have been the focus of numerous works, less is known about the mechanisms ensuring the centrosome-spindle coupling. The conserved NuMA protein (Mud in ) is best known for its role in spindle orientation. Here, we analyzed the role of Mud and two of its interactors, Asp and Dynein, in the regulation of centrosome numbers in epithelial cells. We found that Dynein and Mud mainly initiate centrosome-spindle coupling prior to nuclear envelope breakdown (NEB) by promoting correct centrosome positioning or separation, while Asp acts largely independently of Dynein and Mud to maintain centrosome-spindle coupling. Failure in the centrosome-spindle coupling leads to mis-segregation of the two centrosomes into one daughter cell, resulting in cells with supernumerary centrosomes during subsequent divisions. Altogether, we propose that Dynein, Mud and Asp operate sequentially during the cell cycle to ensure efficient centrosome-spindle coupling in mitosis, thereby preventing centrosome mis-segregation to maintain centrosome number.

The role of Patronin in Drosophila mitosis

“…5a,c). Although recent work has shown that epithelial cells of asp mutants frequently exhibit supernumerary centrosomes [53], Asp depletion in S2 cells did not lead to an increase in the frequency of spindles associated with more than two centrosomes (Table 1). The latter finding is consistent with observations on brain cells from asp mutants [16], and suggests that the centrosomes of different cell types respond differently to Asp depletion.…”

Control of Intestinal Cell Fate by Dynamic Mitotic Spindle Repositioning Influences Epithelial Homeostasis and Longevity

“…Depleting either Pins or Mud through RNAi increased planar spindles and relative D1+ cell numbers, suggesting that both proteins are required to orient oblique spindles ( Figures 4E, 5D, and S6G). Because Mud plays additional roles at the spindle pole during spindle assembly (Bosveld et al, 2017;Du et al, 2002;Izumi et al, 2006;Silk et al, 2009), we expressed GFPtagged Mud (using a Bac-based transgene in which GFP-tagged Mud is integrated at landing site 50E1, 62E1, and 65B2) (Bosveld et al, 2016) to visualize its localization and differentiate between the two functions of Mud. After inducing a regenerative response by Ecc15 treatment, we found that 55% of ISCs had Mud suggest that the activity of JNK promotes release of Mud from the cell cortex and thus prevents oblique spindles.…”

The LINC complex inhibits excessive chromatin repression

“…The following stocks were used: koi 84 /Cyo-dfd-eYfp (FBst0025105) have been described previously (Kracklauer et al, 2007), koi-RNAi (FBst0040924), delta KASH (MSP-300 deltaKASH ;klar mCD4 /TM6B from J.A Fischer, University of Texas, Austin, TX), tubP-GAL80 ts /TM2 (FBst0007017), GAL4-Mef2.R (FBst0027390), , UAS-Spastin (obtained from V. Brodu, Institute Jacques Monod, Paris), ubi-H2B-mRFP/CyO; Dr/TM6B (Bosveld et al, 2017), UAS-klar-GFP (Elhanany-Tamir et al, 2012), Tub-Gal80ts; Mef2-Gal4 (obtained from F. Schnorrer IBDM, Marseille), UAS-2E12LI-EGFP(III)/TM6B (live H3K27me3-GFP mintbody obtained from H. Kimura, Tokyo Institute of Technology), His2Av-GFP (FBst0005941). Fly lines for Targeted Dam-ID (obtained from A.…”