Optogenetic EB1 inactivation shortens metaphase spindles by disrupting cortical force-producing interactions with astral microtubules
Alessandro Dema, Jeffrey van Haren, Torsten Wittmann
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.1Pavin N. Tolić I.M. Self-Organization and Forces in the Mitotic Spindle.Annu. Rev. Biophys. 2016; 45: 279-298Crossref PubMed Scopus (45) Google Scholar,2Elting M.W. Suresh P. Dumont S. The Spindle: Integrating Architecture and Mechanics across Scales.Trends Cell Biol. 2018; 28: 896-910Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar In mammalian cells, end-binding (EB) proteins associate with all growing microtubule plus ends throughout the cell cycle and serve as adaptors for diverse +TIPs that control microtubule dynamics and interactions with other intracellular structures.3van Haren J. Wittmann T. Microtubule Plus End Dynamics - Do We Know How Microtubules Grow?: Cells boost microtubule growth by promoting distinct structural transitions at growing microtubule ends.BioEssays. 2019; 41: e1800194Crossref PubMed Scopus (14) Google Scholar Because binding of many +TIPs to EB1 and thus microtubule-end association is switched off by mitotic phosphorylation,4van der Vaart B. Manatschal C. Grigoriev I. Olieric V. Gouveia S.M. Bjelić S. Demmers J. Vorobjev I. Hoogenraad C.C. Steinmetz M.O. Akhmanova A. SLAIN2 links microtubule plus end-tracking proteins and controls microtubule growth in interphase.J. Cell Biol. 2011; 193: 1083-1099Crossref PubMed Scopus (88) Google Scholar, 5Singh D. Schmidt N. Müller F. Bange T. Bird A.W. Destabilization of Long Astral Microtubules via Cdk1-Dependent Removal of GTSE1 from Their Plus Ends Facilitates Prometaphase Spindle Orientation.Curr. Biol. 2021; 31: 766-781.e8Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar, 6Pemble H. Kumar P. van Haren J. Wittmann T. GSK3-mediated CLASP2 phosphorylation modulates kinetochore dynamics.J. Cell Sci. 2017; 130: 1404-1412Crossref PubMed Scopus (16) Google Scholar 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, π-EB1, that allows local, acute, and reversible inactivation of +TIP association with growing microtubule ends in live cells.7van Haren J. Charafeddine R.A. Ettinger A. Wang H. Hahn K.M. Wittmann T. Local control of intracellular microtubule dynamics by EB1 photodissociation.Nat. Cell Biol. 2018; 20: 252-261Crossref PubMed Scopus (37) Google Scholar We find that acute π-EB1 photoinactivation results in rapid and reversible metaphase spindle shortening and transient relaxation of tension across the central spindle. However, in contrast to interphase, π-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 π-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.