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

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.

Phase separation of +TIP-networks regulates microtubule dynamics

“…The role of different mammalian EB family members in regulating LLPS will be an interesting direction for future studies. The ability of these +TIP-networks to phase separate depends on intrinsically disordered regions (Maan et al, 2021;Song et al, 2021) and multivalent interaction modules (Figure 2; Meier et al, 2021), consistent with the observation that these features are highly evolutionarily conserved across +TIPs (Wu et al, 2021). Further studies will be necessary to investigate whether additional +TIPs contribute to the formation and regulation of +TIP-droplets.…”

A liquid +TIP-network drives microtubule dynamics through tubulin condensation

“…The role of different mammalian EB family members in regulating LLPS will be an interesting direction for future studies. The ability of these +TIP-networks to phase separate depends on intrinsically disordered regions (Maan et al, 2021;Song et al, 2021) and multivalent interaction modules (Figure 2; Meier et al, 2021), consistent with the observation that these features are highly evolutionarily conserved across +TIPs (Wu et al, 2021). Further studies will be necessary to investigate whether additional +TIPs contribute to the formation and regulation of +TIP-droplets.…”

“…Previous studies indicate that CLIP-170 overexpressed in cells has the potential to undergo LLPS (Pierre et al, 1994;Goodson et al, 2003;Wu et al, 2021). Indeed, overexpression of mCherry-CLIP-170 in CRISPR/Cas9 knock-in GFP-tubulin RPE-1 cells resulted in cytosolic CLIP-170 patch formation in addition to microtubule plus-end tracking (Figure 1A).…”

“…Early studies of CLIP-170 expression in cells revealed that in addition to its microtubule plus-end localization, it also formed cytoplasmic "patches" that co-localized with EB1 and the dynein-activating protein dynactin (Pierre et al, 1994;Goodson et al, 2003). Based on the physical properties of these patches, it has recently been suggested that they form by liquid-liquid phase separation (LLPS) (Wu et al, 2021). LLPS is the process by which molecules spontaneously condensate into droplets and de-mix from their surrounding solution, resulting in the co-existence of two unique liquid phases (reviewed in Boeynaems et al, 2018;Hyman et al, 2014;Shin and Brangwynne, 2017).…”

“…A key shared feature of these processes is the co-condensation of tubulin with LLPS-potent microtubule-associated proteins to catalyze biochemical reactions. Along these lines, condensation of a microtubule +TIP-network has been proposed based on transient multivalent interactions between the +TIPs (Akhmanova and Steinmetz, 2015;Wu et al, 2021). Despite this, LLPS at the microtubule plus-end has never been shown.…”