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bioRxiv · 10.1101/2021.01.01.424687

Overexpression of the microtubule-binding protein CLIP-170 induces a +TIP network superstructure consistent with a biomolecular condensate

Wu, Y.-F. O.·Bryant, A. T.·Nelson, N. T.·Madey, A. G.·Fernandes, G. F.·Goodson, H. V.
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Abstract

Proper regulation of microtubule (MT) dynamics is critical for cellular processes including cell division and intracellular transport. Plus-end tracking proteins (+TIPs) dynamically track growing MTs and play a key role in MT regulation. +TIPs participate in a complex web of intra- and inter-molecular interactions known as the +TIP network. Hypotheses addressing the purpose of +TIP:+TIP interactions include relieving +TIP autoinhibition and localizing MT regulators to growing MT ends. In addition, we have proposed that the web of +TIP:+TIP interactions has a physical purpose, creating a superstructure that constrains the structural fluctuations of the fragile MT tip and thus acts as a polymerization chaperone. Many animal +TIP network proteins are multivalent and have intrinsically disordered regions, features commonly found in biomolecular condensates. This observation suggests that the +TIP network might under some conditions form a biomolecular condensate. Previous studies have shown that overexpression of the +TIP CLIP-170 induces large "patch" structures containing CLIP-170 and other +TIPs. To test the hypothesis that these patches might be biomolecular condensates, we used video microscopy, immunofluorescence staining, and Fluorescence Recovery After Photobleaching (FRAP). Our data show that the CLIP-170-induced patches have hallmarks indicative of a biomolecular condensate, one that contains +TIP proteins and excludes other known condensate markers. Moreover, bioinformatic studies demonstrate that the presence of intrinsically disordered regions is conserved in key +TIPs, implying that these regions are functionally significant. Together, these results indicate that the CLIP-170 induced patches in cells are phase-separated liquid condensates and raise the possibility that the endogenous +TIP network might form a liquid droplet at MT ends or other +TIP locations.

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Wu, Y.-F. O., Bryant, A. T., Nelson, N. T., Madey, A. G., Fernandes, G. F., Goodson, H. V.. 2021-01-01. Overexpression of the microtubule-binding protein CLIP-170 induces a +TIP network superstructure consistent with a biomolecular condensate. https://doi.org/10.1101/2021.01.01.424687

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