Microtubule plus-end tracking by CLIP-170 requires EB1

Abstract

Microtubules are polarized polymers that exhibit dynamic instability, with alternating phases of elongation and shortening, particularly at the more dynamic plus-end. Microtubule plus-end tracking proteins (؉TIPs) localize to and track with growing microtubule plus-ends in the cell. ؉TIPs regulate microtubule dynamics and mediate interactions with other cellular components. The molecular mechanisms responsible for the ؉TIP tracking activity are not well understood, however. We reconstituted the ؉TIP tracking of mammalian proteins EB1 and CLIP-170 in vitro at single-molecule resolution using time-lapse total internal reflection fluorescence microscopy. We found that EB1 is capable of dynamically tracking growing microtubule plus-ends. Our singlemolecule studies demonstrate that EB1 exchanges rapidly at microtubule plus-ends with a dwell time of <1 s, indicating that single EB1 molecules go through multiple rounds of binding and dissociation during microtubule polymerization. CLIP-170 exhibits lattice diffusion and fails to selectively track microtubule ends in the absence of EB1; the addition of EB1 is both necessary and sufficient to mediate plus-end tracking by CLIP-170. Single-molecule analysis of the CLIP-170 -EB1 complex also indicates a short dwell time at growing plus-ends, an observation inconsistent with the copolymerization of this complex with tubulin for plus-end-specific localization. GTP hydrolysis is required for ؉TIP tracking, because end-specificity is lost when tubulin is polymerized in the presence of guanosine 5-[␣,␤-methylene]triphosphate (GMPCPP). Together, our data provide insight into the mechanisms driving plus-end tracking by mammalian ؉TIPs and suggest that EB1 specifically recognizes the distinct lattice structure at the growing microtubule end.ϩTIP ͉ single molecule ͉ total internal reflection fluorescence (TIRF) microscopy ͉ dynamic instability

The Microtubule Plus-End Tracking Proteins SPR1 and EB1b Interact to Maintain Polar Cell Elongation and Directional Organ Growth in Arabidopsis

“…EBs have been shown to autonomously bind to MTs, while many other +TIPs require EBs for proper MT binding and/or tip localization (Dixit et al, 2009;Akhmanova and Steinmetz, 2010;Slep, 2010;Li et al, 2011b). While we found SPR1 to behave like other +TIPs in that it physically interacts with EB1b, our data show that SPR1 can function, at least in part, autonomously of EB1b.…”

Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

“…The binding process of CLIP-170 is rather complex and details can be found in [207,38,37,99,96]. Here, we just notice that CLIP-170 temporarily binds to the plus-end of MTs and is released with a given rate.…”

“…The properties of the motors could be changed by the viscoelasticity of the cytoplasm (in a similar way as it has been shown that mechanical properties of MTs can be quite different in a viscoelastic medium [53]). Complementary proteins, as for example the tau protein, exist which can bind to MTs and reduce the binding affinity of molecular motors [96]. The attachment rate could also be influenced by regulatory proteins binding directly to the head region of molecular motors [244].…”

Interactions between EB1 and Microtubules

“…histidine or glutathione S-transferase), but the tags and purification strategies differ. Some studies used proteins with the tags left intact (26 -28), whereas others used tag-cleaved proteins (19,24,29,30). In some cases where the tags were cleaved off, several amino acids still remained after the main tag was removed.…”