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

Area of Science:

• Cell Biology
• Molecular Motors
• Cytoskeleton Dynamics

Background:

• Cortical dynein generates forces through microtubule (MT) end capture-shrinkage and lateral MT sliding.
• In Saccharomyces cerevisiae, Num1 links dynein to cortical organelles (endoplasmic reticulum [ER] and mitochondria) for spindle positioning.
• The precise role of cortical contacts in regulating dynein-dependent forces remains unclear.

Purpose of the Study:

• To investigate how ER tethering proteins (Scs2/Scs22) influence Num1 distribution and dynein-mediated pulling forces.
• To elucidate the mechanisms of spindle positioning in the absence of ER-dynein-cortical interactions.
• To determine the distinct roles of different Num1 populations in dynein function.

Main Methods:

• Genetic deletion of ER tethering proteins Scs2/Scs22 in Saccharomyces cerevisiae.
• Analysis of Num1 protein distribution using microscopy.
• Assessment of dynein-dependent microtubule (MT) sliding and spindle positioning.
• Functional rescue experiments using CAAX-targeted Num1.

Main Results:

• Loss of Scs2/Scs22 disrupted Num1 distribution and abolished dynein-dependent MT sliding.
• Spindle positioning in Scs2/Scs22-deficient cells relied on MT end capture-shrinkage, requiring dynein anchorage to an ER/mitochondria-independent Num1 pool.
• This alternative mechanism necessitated dynein motor activity and the CAP-Gly domain of dynactin (Nip100/p150Glued).
• Targeting Num1 to the plasma membrane (CAAX-Num1) rescued MT sliding defects.

Conclusions:

• Distinct Num1 populations exist and play specific roles in dynein-mediated force generation.
• Spatial distribution of Num1 is critical for regulating dynein pulling forces, particularly MT sliding.
• ER and mitochondria interactions are important for efficient dynein-dependent MT sliding, but alternative mechanisms exist.