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

Abstract

Current model for spindle positioning requires attachment of the microtubule (MT) motor cytoplasmic dynein to the cell cortex, where it generates pulling force on astral MTs to effect spindle displacement. How dynein is anchored by cortical attachment machinery to generate large spindle-pulling forces remains unclear. Here, we show that cortical clustering of Num1, the yeast dynein attachment molecule, is limited by its assembly factor Mdm36. Overexpression of Mdm36 results in an overall enhancement of Num1 clustering but reveals a population of dim Num1 clusters that mediate dynein-anchoring at the cell cortex. Direct imaging shows that bud-localized, dim Num1 clusters containing only ∼6 copies of Num1 molecules mediate dynein-dependent spindle pulling via lateral MT sliding mechanism. Mutations affecting Num1 clustering interfere with mitochondrial tethering but not dynein-based spindle-pulling function of Num1. We propose that formation of small ensembles of attachment molecules is sufficient for dynein anchorage and cortical generation of large spindle-pulling force.

Hierarchical integration of mitochondrial and nuclear positioning pathways by the Num1 EF hand

“…6C). The Num1 assemblies present at the sites of cortical dynein anchoring were smaller and more diffuse than mitochondria-assembled Num1 clusters, consistent with previous descriptions of dynein anchoring by Num1 in the absence of mitochondria (Omer et al 2018;Omer et al 2020).…”

“…Thus, mitochondria do not directly activate dynein activity or processivity per se, but instead help to arrange Num1 in clusters at the cell cortex that are competent to anchor dynein, and subsequently dynein is activated. As shown here in this study with the Num1 EFLM mutants and as is suggested by other studies that alter Num1 assembly (Omer et al, 2018(Omer et al, , 2020, it is possible to bypass the role of mitochondria in facilitating dynein anchoring in certain mutant conditions. However, in WT conditions, it is clear that the presence of mitochondria in the bud positively impacts dynein function in nuclear inheritance and this is dependent on a functional EFLM.…”

“…6A and B). These data indicate that in certain mutant conditions, such as disruption of the Num1 EFLM (as shown here), overexpression of Mdm36 (Omer et al 2020), or deletion of Scs2 (Omer et al 2018), the non-mitochondria associated pool of the Num1 can anchor dynein and support dynein function. However, in a WT system, it is clear that dynein anchoring by Num1 is biased by the presence of mitochondria and this bias is dependent on the ability of the EFLM of Num1 to bind Ca 2+ .…”

“…We find that dynein is preferentially anchored to clusters that are mitochondria-associated, further highlighting a role for mitochondria in dynein anchoring and function (Schmit et al, 2018). Contrasting studies suggest that the Num1 clusters that anchor mitochondria are distinct from those that anchor dynein (Omer et al, 2018(Omer et al, , 2020. In these studies, Num1 clustering is either enhanced by overexpression of Mdm36, or diminished by the deletion of Scs2, an integral ER protein thought to interact with Num1 (Hammermeister et al, 2010;Lackner et al, 2013;Chao et al, 2014;Ping et al, 2016;Omer et al, 2018).…”

“…1A; Kraft and Lackner, 2017). The only other known protein component of MECA, Mdm36, functions as an accessory component that bridges Num1-Num1 interaction to enhance Num1 clustering (Hammermeister et al, 2010;Lackner et al, 2013;Omer et al, 2020).…”