Optogenetic reconstitution reveals that Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble
Preprint posted on March 06, 2018 https://www.biorxiv.org/content/early/2018/03/06/277202
Mitotic cortical force machinery assembles on the plasma membrane and provides pulling forces on astral microtubules (MTs) that are important for the correct positioning of the spindle. Correct spindle positioning is important for both symmetric and asymmetric divisions, determining both cell size and fate following segregation of genetic material. In human cells, this machinery consists of the cortically anchored complex of Nuclear Mitotic Apparatus protein (NuMA), LGN and G alpha i (NuMA-LGN-Gαi), with cytoplasmic dynein and dynactin. The complex generates pulling forces on astral MTs through dynein’s minus-end directed motility and/or the control of MT dynamics. The authors use a light-induced membrane tethering system (iLID) to assess the mechanism by which NuMA contributes to cortical force generation (Figure 1A).
Using the iLID system, the authors show that spindles displace towards light-activated regions of the cortex upon NuMA, Dynein Heavy Chain (DHC) and p150 recruitment, and are able to show that repositioning of the light-induced cortical NuMA allows rotational reorientation of spindles. Although dynein-dynactin is required for cortical pulling, it is not sufficient for spindle displacement in the absence of NuMA. It contains a 200nm long central coiled-coil domain that is necessary for spindle pulling, and two additional microtubule-binding domains in the C-terminus. Together with dynein-dynactin, these regions allow NuMA clusters to efficiently capture and maintain associations with the plus-tips of astral microtubules in order to generate cortical pulling forces that are required for spindle positioning.
Why we chose this preprint:
We were drawn to this preprint thanks to the authors’ elegant use of a light induced system to reconstitute spindle pulling forces at the cortex. This preprint further highlights the power of using optogenetic tools to investigate protein function with precise spatial and temporal control, with the potential for much wider applications within other areas of cell biology. Here, by targeting the dynein-dynactin-NuMA (DDN) complex to the cortex, they further elucidate the underlying mechanisms controlling spindle positioning which is a topic of great interest to our lab.
This paper makes a significant step in dissecting the mechanism of cortical pulling by highlighting the requirement for both active dynein and additional microtubule-binding domains in NuMA to generate cortical forces. The authors note that NuMA tends to form distinct punctae on the membrane and hypothesize that it may be forming a higher-order complex in order to generate force. What might this structure look like? Finally, the authors show that slow spindle displacement can even be seen when microtubules are taxol-stabilized, removing microtubule depolymerisation at the cortex. In this case, what role are microtubule dynamics and pushing on the cortex playing during the process of spindle centering?
Posted on: 10th May 2018Read preprint
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