Abstract
The cooperation between the actin and microtubule (MT) cytoskeletons is important for cellular processes such as cell migration and muscle cell development. However, a full understanding of how this cooperation occurs has yet to be sufficiently developed. The MT plus-end tracking protein 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, the interactions of CLIP-170 with actin were assumed to be indirect. Here, we demonstrate using high-speed cosedimentation assays that CLIP-170 can bind to filamentous actin (F-actin) directly. We found that the affinity of this binding is relatively weak but 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–F-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 MT end-binding protein 1.
Highlights
We used a combination of cosedimentation assays and microscope-based filamentous actin (F-actin)-bundling assays to show that CLIP-170 can bind directly to F-actin in vitro
By studying CLIP-170 fragments and mutants, we found that the F-actin-binding domain is independent of the formin-activating FEED domain, requires the second CLIP170 cytoskeleton-associated protein glycine-rich (CAP-GLY) motif (i.e., CAP-GLY 2 or "CG2") for efficient binding, and overlaps with the MT-binding surface of the CAP-GLY domain
These results suggested that CLIP-170 can bind to F-actin directly via its N-terminal head domain
Summary
Components of the cytoskeleton are often described as having apparently independent localizations and activities. By studying CLIP-170 fragments and mutants, we found that the F-actin-binding domain is independent of the formin-activating FEED domain, requires the second CLIP170 cytoskeleton-associated protein glycine-rich (CAP-GLY) motif (i.e., CAP-GLY 2 or "CG2") for efficient binding, and overlaps with the MT-binding surface of the CAP-GLY domain. Consistent with these observations, our competition assays further indicate that CLIP-170 cannot bind to Factin and MTs simultaneously. We speculate that binding of CLIP-170 to actin may function helping to destabilize MTs at the cell periphery
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