Nelson, N. T. | Search bioRxiv

bioRxiv2021

Characterizing interactions between the microtubule-binding protein CLIP-170 and F-actin

The cooperation between the actin and microtubule (MT) cytoskeletons is important for cellular processes such as cell migration and muscle cell development. Full understanding of how this cooperation occurs has yet to be sufficiently developed. The MT plus-end tracking protein (+TIP) CLIP-170 has been implicated in this actin-MT coordination by associating with the actin-binding signaling protein IQGAP1, and by promoting actin polymerization through binding with formins. Thus far, CLIP-170s interactions with actin were assumed to be indirect. Here, we demonstrate that CLIP-170 can bind to filamentous actin (F-actin) directly. The affinity is relatively weak, but is strong enough to be significant in the actin-rich cortex, where actin concentrations can be extremely high. Using CLIP-170 fragments and mutants, we show that the direct CLIP-170:actin interaction is independent of the FEED domain, the region that mediates formin-dependent actin polymerization, and that the CLIP-170 F-actin-binding region overlaps with the MT-binding region. Consistent with these observations, in vitro competition assays indicate that CLIP-170:F-actin and CLIP-170:MT interactions are mutually exclusive. Taken together, these observations lead us to speculate that direct CLIP-170:F-actin interactions may function to reduce the stability of MTs in actin-rich regions of the cell, as previously proposed for EB1.

biochemistry↗

bioRxiv2021

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

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.

cell biology↗