Cortical dynein pulling mechanism is regulated by differentially targeted attachment molecule Num1

Abstract

Cortical dynein generates pulling forces via microtubule (MT) end capture-shrinkage and lateral MT sliding mechanisms. In Saccharomyces cerevisiae, the dynein attachment molecule Num1 interacts with endoplasmic reticulum (ER) and mitochondria to facilitate spindle positioning across the mother-bud neck, but direct evidence for how these cortical contacts regulate dynein-dependent pulling forces is lacking. We show that loss of Scs2/Scs22, ER tethering proteins, resulted in defective Num1 distribution and loss of dynein-dependent MT sliding, the hallmark of dynein function. Cells lacking Scs2/Scs22 performed spindle positioning via MT end capture-shrinkage mechanism, requiring dynein anchorage to an ER- and mitochondria-independent population of Num1, dynein motor activity, and CAP-Gly domain of dynactin Nip100/p150Glued subunit. Additionally, a CAAX-targeted Num1 rescued loss of lateral patches and MT sliding in the absence of Scs2/Scs22. These results reveal distinct populations of Num1 and underline the importance of their spatial distribution as a critical factor for regulating dynein pulling force.

Overexpression of Mdm36 reveals Num1 foci that mediate dynein-dependent microtubule sliding in budding yeast

“…Similar to Num1 patches in WT cells (Omer et al, 2018), Num1-GFP patches in Mdm36 OX cells redistributed to the polarized ends of the cell (the distal bud tip and the mother cell apex) upon deletion of the ER tethering proteins, Scs2 and Scs22 (Fig. S1C).…”

“…In WT cells, cortical dynein pulls the spindle into the bud cell compartment via lateral MT sliding mechanism (Adames and Cooper, 2000). More recently, however, several studies showed that cortical dynein can also pull on the astral MT via end-on MT capture-shrinkage mechanism (Laan et al, 2012; Omer et al, 2018). To assess the mechanism in Mdm36 OX cells, we monitored dynein-dependent astral MT interaction with the bud cortex using a spindle correction assay, scoring for MT behavior during anaphase spindle re-alignment from a misoriented position.…”

“…The CC domain also binds Mdm36, a 65 kDa protein implicated in promoting Num1 cluster formation and mitochondrial division (Hammermeister et al, 2010; Lackner et al, 2013). Although it is well-accepted that dynein exerts spindle pulling force at cortical Num1 sites, the abundance and heterogeneity of Num1 patches along the cell cortex (Heil-Chapdelaine et al, 2000; Omer et al, 2018; Schmit et al, 2018) has made it impossible to follow the effects of astral MT plus end interaction with individual cortical Num1 sites, a prerequisite for understanding how clustering might impact dynein force amplification. To our knowledge, contacts between astral MT plus end and individual Num1 foci have not been observed for MT sliding, the in vivo 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 microtubule-cortex interaction remains unknown.…”

“…To our knowledge, contacts between astral MT plus end and individual Num1 foci have not been observed for MT sliding, the in vivo 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 microtubule-cortex interaction remains unknown. Additionally, recent work shows that organelles such as mitochondria and endoplasmic reticulum (ER) are involved in regulating Num1 cluster formation: a subset of cortical Num1 clusters appears to require mitochondria for their assembly (Kraft and Lackner, 2017), whereas another population requires the ER tethering proteins Scs2/Scs22 for their distribution throughout the cell cortex (Chao et al, 2014; Omer et al, 2018). The general hypothesis emerging from these studies is that distinct populations of Num1 clusters might exist at the cell periphery, but whether different pools of Num1 could be performing different Num1 functions – namely, dynein anchoring and mitochondrial tethering – remains a total mystery.…”

Insights into LIS1 function in cargo-adapter-mediated dynein activation in vivo

“…In filamentous fungi and budding yeast, dynein, dynactin and LIS1-NudE all accumulate at the microtubule (MT) plus ends (Han et al, 2001;Efimov, 2003;Lee et al, 2003;Sheeman et al, 2003;Zhang et al, 2003;Li et al, 2005;Lenz et al, 2006;Moore et al, 2008;Callejas-Negrete et al, 2015). The MT plus-end accumulation of dynein is important for spindle-orientation/nuclear migration and for early endosome transport Sheeman et al, 2003;Lenz et al, 2006;Omer et al, 2018;Xiang, 2018). In A. nidulans and Ustilago maydis, the plus-end dynein accumulation depends on dynactin and kinesin-1 but not NudF/LIS1 (Zhang et al, 2003;Lenz et al, 2006;Zhang et al, 2010;Egan et al, 2012;Yao et al, 2012).…”