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

Yueh-Fu O. Wu,Nora T. Nelson,Annamarie T. Bryant,Holly V. Goodson,Gail F. Fernandes,Alexander G. Madey,Bipul R. Acharya

doi:10.1371/journal.pone.0260401

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 dynamic scaffold that constrains the structural fluctuations of the fragile MT tip and thus acts as a polymerization chaperone. Here we examine the possibility that this proposed scaffold is a biomolecular condensate (i.e., liquid droplet). Many animal +TIP network proteins are multivalent and have intrinsically disordered regions, features commonly found in biomolecular condensates. Moreover, previous studies have shown that overexpression of the +TIP CLIP-170 induces large “patch” structures containing CLIP-170 and other +TIPs; we hypothesized that these structures might be biomolecular condensates. To test this hypothesis, 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.

Highlights

Microtubules (MTs) compose one of the three major filament networks of the eukaryotic cytoskeleton, and they are required for basic cellular functions such as cell polarity, cell division, and intracellular transport
We focused our efforts on three intrinsically disordered regions (IDRs) predictors, which were used with default parameters unless otherwise indicated: 1) “ESpritz version 1.3” [50], used with prediction type as X-Ray and decision threshold as 5% false positive rate (5% FPR; chosen because the default setting [“Best Sw”–a weighted score rewarding prediction of IDRs] tends to overestimate IDRs); 2) “Interpro” (EMBL, https://www.ebi.ac.uk/interpro/), which provides as part of its standard analysis disorder predictions from the MobiDB-lite database [51]); and 3) “IUPred2”, used with “short disordered” setting, as obtained from the IUPred2A server [52,53]
Proteins included in droplets generally have the following structural characteristics that are important for droplet formation: they have multiple sites for binding other droplet members, and they contain intrinsically disordered regions (IDRs)

Summary

Introduction

Microtubules (MTs) compose one of the three major filament networks of the eukaryotic cytoskeleton, and they are required for basic cellular functions such as cell polarity, cell division, and intracellular transport. Microtubules display a surprising behavior known as dynamic instability, which describes the approximately random alteration between phases of slow growth (polymerization) and rapid shrinkage (depolymerization). This behavior is regulated by MT binding proteins and is central to MT function because it enables MTs to explore space to respond rapidly to internal and external signals and find organelles to be transported (reviewed by [2]). While many +TIPs and their MT regulatory roles have been identified, it is not yet fully understood why so many +TIPs bind to other +TIPs. One favored explanation is that the interactions of the +TIP network create regulatory pathways by relieving the autoinhibition feature present in many +TIP proteins (Fig 1A). Another explanation is that +TIP:+TIP interactions serve to localize and deliver proteins in a spatiotemporal manner (e.g. localizing +TIPs to the MT ends, and facilitating the surfing of proteins to cell edge) (reviewed in [3])

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