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bioRxiv · 10.1101/2021.09.29.462463

Optogenetic EB1 inactivation shortens metaphase spindles by disrupting cortical force-producing interactions with astral microtubules

Dema, A.·van Haren, J.·Wittmann, T.
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Abstract

Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to spindle function: Kinetochore microtubules attach and transmit forces to chromosomes, antiparallel interpolar microtubules support spindle structure, and astral microtubules connect spindle poles to the cell cortex [1,2]. In mammalian cells, End Binding (EB) proteins associate with all growing microtubule plus ends throughout the cell cycle and serve as adaptors for a diverse group of +TIPs that control microtubule dynamics and interactions with other intracellular structures [3]. Because binding of many +TIPs to EB1 and thus microtubule-end association is switched off by mitotic phosphorylation [4-6] the mitotic function of EBs remains poorly understood. To analyze how EB1 and associated +TIPs on different spindle microtubule populations contribute to mitotic spindle dynamics, we use a light sensitive EB1 variant, {pi}-EB1, that allows local, acute and reversible inactivation of +TIP association with growing microtubule ends in live cells [7]. We find that acute {pi}-EB1 photoinactivation results in rapid and reversible metaphase spindle shortening and transient relaxation of tension across the central spindle. However, in contrast to interphase, {pi}-EB1 photoinactivation does not inhibit microtubule growth in metaphase, but instead increases astral microtubule length and number. Yet, in the absence of EB1 activity astral microtubules fail to engage the cortical dynein/dynactin machinery and spindle poles move away from regions of {pi}-EB1 photoinactivation. In conclusion, our optogenetic approach reveals mitotic EB1 functions that remain hidden in genetic experiments likely due to compensatory molecular systems regulating vertebrate spindle dynamics.

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Dema, A., van Haren, J., Wittmann, T.. 2021-10-01. Optogenetic EB1 inactivation shortens metaphase spindles by disrupting cortical force-producing interactions with astral microtubules. https://doi.org/10.1101/2021.09.29.462463

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