Mutations affecting Num1 clustering interfere with mitochondrial tethering but do not interfere with the dynein-based spindle-pulling function of Num1. and cortical generation of large spindle-pulling forces. This short article has an connected First Person EGF Chromafenozide interview with the first author of the paper. hallmark of dynein-mediated spindle pulling (Adames and Cooper, 2000; Yeh et al., 2000), hence the size of Num1 clusters required for this classic dynein-dependent microtubuleCcortex connection remains unfamiliar. Additionally, recent studies have begun to reveal that organelles such as mitochondria and the endoplasmic reticulum (ER) are involved in regulating Num1 cluster formation, having a subset of clusters requiring mitochondria for his or her assembly in the bud (Kraft and Lackner, 2017), and another subset requiring the ER tethering proteins Scs2 and Scs22 for his or her formation throughout the cell cortex (Chao et al., 2014; Omer et al., 2018). Chromafenozide The Num1 patches put together by mitochondria can in turn bind and tether the organelle to the cell cortex (Kraft and Lackner, 2017; Ping et al., 2016). Intriguingly, like Num1, the fission candida homologue Mcp5 also forms cortical foci for dynein anchoring and employs a similar CC website for connection with both dynein and mitochondria (Chacko et al., 2019; Kraft and Lackner, 2019; Saito et al., 2006; Thankachan et al., 2017; Yamashita and Yamamoto, 2006), suggesting the mechanism regulating cortical cluster formation might be conserved. The general hypothesis growing from these studies is that unique populations of Num1 clusters might exist in the cell periphery, but whether different swimming pools of Num1 could be carrying out different Num1 functions C namely, dynein anchoring and mitochondrial tethering C remains unclear. Here, using Mdm36 as a tool to cluster Num1, we found that, in contrast to the prevailing notion for dynein-anchoring proteins, enhancing Num1 clustering unexpectedly reduces dynein recruitment to the cell cortex, but without influencing dynein function in spindle placing. We report direct observation of MT sliding that occurs when an astral MT plus end encounters a cortical Num1 cluster comprising only a small number of Num1 molecules. The observed sliding events do not appear to require Mdm36 and mitochondria. Furthermore, mutations that interfere with Num1 clustering disrupt mitochondrial-tethering activity but not dynein-based spindle-pulling activity of Num1, highlighting a more essential part for clustering in mitochondrial-anchoring rather than dynein-anchoring function. RESULTS Overexpression of Mdm36 dramatically enhances Num1 clustering We 1st investigated how Num1 distribution is definitely affected by Mdm36 in the cell cortex. As previously shown, deletion of Mdm36 resulted in smaller and dimmer Num1 patches (Lackner et al., 2013) (Fig.?1A), consistent with a clustering part for Mdm36. To determine whether Mdm36 level is definitely limiting for Num1 clustering, we examined the effects of overexpressing Mdm36. We integrated the inducible promoter in the 5 end of the endogenous chromosomal locus of the gene and assayed for Num1CGFP intensity and distribution. Chromafenozide In Mdm36-overexpressing cells, hereafter referred to as Mdm36OX cells, we observed a striking enhancement in the intensity of cortical Num1CGFP patches (Fig.?1A; Fig.?S1A). The level of cytoplasmic Num1CGFP fluorescence in Mdm36OX cells was dramatically reduced compared to the levels in promoter) resulted in an enhancement in the intensity of Num1 patches (Fig.?S1A). However, cortical patches in Num1-overexpressing cells were markedly more dispersed compared to those in Mdm36OX cells (Fig.?S1B), indicating that Mdm36 is a clustering element for Num1 in the cell membrane. Open in a separate windowpane Fig. 1. Mdm36 enhances Num1 clustering. (A) Representative unadjusted images of Num1CGFP in WT, Mdm36OX and or deletions require the dynein pathway for normal growth (Geiser et al., 1997; Gerson-Gurwitz et al., 2009; Miller and Rose, 1998). Tetrad dissection analysis showed that is overexpressed. In agreement with the observed reduction in dynein offloading, the mean intensity of the cortical Dyn1C3GFP foci in Mdm36OX cells was significantly decreased (Fig.?3B). The.
Mutations affecting Num1 clustering interfere with mitochondrial tethering but do not interfere with the dynein-based spindle-pulling function of Num1