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

Safia Omer,Samuel R Greenberg,Wei-Lih Lee

doi:10.7554/elife.36745

Safia Omer, Samuel R Greenberg + Show 1 more

Open Access

https://doi.org/10.7554/elife.36745

Copy DOI

Journal: eLife	Publication Date: Aug 7, 2018
Citations: 32	License type: CC BY 4.0

Affiliation: University of Massachusetts Amherst, Dartmouth College

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.

Highlights

Proper positioning of the mitotic spindle is essential for successful cell division and is crucial for a wide range of processes including creation of cellular diversity during development, maintenance of adult tissue homeostasis, and balancing self-renewal and differentiation in progenitor stem cells (Galli and van den Heuvel, 2008; Gomez-Lopez et al, 2014; Morin and Bellaıche, 2011; Siller and Doe, 2009)
We show that the population of Num1 at the bud tip appears to be independent of mitochondria and is strikingly sufficient for dynein function in nuclear migration
We found that cells lacking both cortical endoplasmic reticulum (ER) tethers Scs2 and Scs22 exhibited a dramatic loss of dim Num1 patches (Figure 1A; Video 1, bottom) and a significant reduction in the number of bright Num1 patches (Figure 1B)

Summary

Introduction

Proper positioning of the mitotic spindle is essential for successful cell division and is crucial for a wide range of processes including creation of cellular diversity during development, maintenance of adult tissue homeostasis, and balancing self-renewal and differentiation in progenitor stem cells (Galli and van den Heuvel, 2008; Gomez-Lopez et al, 2014; Morin and Bellaıche, 2011; Siller and Doe, 2009). Spindle positioning involves attachment of the minus end-directed MT motor cytoplasmic dynein to the cell cortex, where it exerts pulling force on astral MTs that emanate from the spindle poles (di Pietro et al, 2016; Kotak and Gonczy, 2013; McNally, 2013). While proteins involved in anchoring dynein have been identified (Ananthanarayanan, 2016; Couwenbergs et al, 2007; Du and Macara, 2004; Heil-Chapdelaine et al, 2000; Kotak et al, 2012; Nguyen-Ngoc et al, 2007; Saito et al, 2006; Thankachan et al, 2017) and the mechanism whereby dynein steps along the MT is becoming elucidated (DeSantis et al, 2017; DeWitt et al, 2015; Grotjahn et al, 2018; Nicholas et al, 2015; Urnavicius et al, 2018), how pulling forces are precisely regulated to achieve the appropriate spindle displacement remains incompletely understood. Dynein is recruited from the dynamic plus ends of astral MTs to cortical foci containing the attachment molecule Num; once anchored, dynein uses its minus end-directed motor activity to walk along the MT lattice, generating

Methods

Results

Conclusion