Fission yeast myosin I facilitates PI(4,5)P ₂ -mediated anchoring of cytoplasmic dynein to the cortex

Abstract

Several key processes in the cell, such as vesicle transport and spindle positioning, are mediated by the motor protein cytoplasmic dynein, which produces force on the microtubule. For the functions that require movement of the centrosome and the associated nuclear material, dynein needs to have a stable attachment at the cell cortex. In fission yeast, Mcp5 is the anchor protein of dynein and is required for the oscillations of the horsetail nucleus during meiotic prophase. Although the role of Mcp5 in anchoring dynein to the cortex has been identified, it is unknown how Mcp5 associates with the membrane as well as the importance of the underlying attachment to the nuclear oscillations. Here, we set out to quantify Mcp5 organization and identify the binding partner of Mcp5 at the membrane. We used confocal and total internal reflection fluorescence microscopy to count the number of Mcp5 foci and the number of Mcp5 molecules in an individual focus. Further, we quantified the localization pattern of Mcp5 in fission yeast zygotes and show by perturbation of phosphatidylinositol 4-phosphate 5-kinase that Mcp5 binds to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P]. Remarkably, we discovered that the myosin I protein in fission yeast, Myo1, which is required for organization of sterol-rich domains in the cell membrane, facilitates the localization of Mcp5 and that of cytoplasmic dynein on the membrane. Finally, we demonstrate that Myo1-facilitated association of Mcp5 and dynein to the membrane determines the dynamics of nuclear oscillations and, in essence, dynein activity.

A conserved mechanism for mitochondria-dependent dynein anchoring

“…In meiotic cells, SpNum1 localized along the cortex as well as in clusters enriched at the poles, as previously described (Saito et al. , 2006; Yamashita and Yamamoto, 2006; Thankachan et al. , 2017).…”

“…, 1999), which is anchored to the PM by clustered SpNum1 (Saito et al. , 2006; Yamashita and Yamamoto, 2006; Thankachan et al. , 2017).…”

“…Similar to Saccharomyces cerevisiae Num1 (ScNum1), the CC domain of SpNum1 is required for its interaction with dynein, and the PH domain binds to PI 4,5 P 2 in the PM (Saito et al. , 2006; Yamashita and Yamamoto, 2006; Thankachan et al. , 2017).…”

Cortical tethering of mitochondria by the anchor protein Mcp5 enables uniparental inheritance

“…Additionally, when deleting Mcp5 to test its role in mitochondrial tethering, we not only knocked down Mcp5 but also abrogated the oscillations that occur during the meiotic prophase (Saito et al, 2006; Yamashita and Yamamoto, 2006; Thankachan et al, 2017). To delineate the specific role of the oscillations, if any, in facilitating parental mitochondrial segregation, we sought to attenuate the oscillations of the horsetail nucleus while keeping Mcp5 intact.…”

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

“…In various cell types, 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 Gönczy, 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.…”