Dynamic Localization of CLIP-170 to Microtubule Plus Ends Is Coupled to Microtubule Assembly

Abstract

CLIP-170 is a cytoplasmic linker protein that localizes to plus ends of microtubules in vivo. In this study, we have characterized the microtubule-binding properties of CLIP-170, to understand the mechanism of its plus end targeting. We show that the NH2-terminal microtubule-interacting domain of CLIP-170 alone localizes to microtubule plus ends when transfected into cells. Association of CLIP-170 with newly-formed microtubules was observed in cells microinjected with biotinylated tubulin, used as a tracer for growing microtubules. Using in vitro assays, association of CLIP-170 with recently polymerized tubulin is also seen. Cross-linking and sedimentation velocity experiments suggest association of CLIP-170 with nonpolymerized tubulin. We conclude from these experiments that the microtubule end targeting of CLIP-170 is closely linked to tubulin polymerization.

Microtubule binding proteins CLIP‐170, EB1, and p150Glued form distinct plus‐end complexes

“…Here we have reconstituted microtubule plus-end localization of CLIP-170 and EB1 in vitro. CLIP-170 binds to tubulin and co-polymerizes into growing microtubules, as previously proposed [15], resulting in preferential localization to the faster-growing plus end. In contrast, we have shown that in vitro, EB1 alone does not preferentially localize to microtubule plus ends.…”

“…As CLIP-170 binds directly to tubulin, co-polymerization may explain the robust plus-end specificity observed in vivo [15]. To test this, CLIP-170-H2 (3.0 lM) was pre-incubated with GTP-tubulin (15 lM) and then mixed with microtubule seeds and shifted to 37°C to allow a brief pulse of new polymerization.…”

T Lymphocyte-triggering Factor of African Trypanosomes Is Associated with the Flagellar Fraction of the Cytoskeleton and Represents a New Family of Proteins That Are Present in Several Divergent Eukaryotes

“…6I). The punctate distribution of GAS11-(115-258)-GFP along a subset of microtubules is strikingly similar to that seen for CLIP-170 (24), a "cytoplasmic linker protein" that links subcellular organelles to microtubules and has been implicated in regulating the assembly of microtubules at their plus-end (42)(43)(44). DISCUSSION The trypanosome cytoskeleton is distinguished from that of most eukaryotic cells by the fact that it does not contain any detectable trans-cellular filaments and is composed almost entirely of microtubules and microtubule-associated proteins (4).…”

Neutrophil microtubules suppress polarity and enhance directional migration

“…Transient expression of a GFP-tagged version of the Nterminal domain of Clip170 (24), which binds to growing ends of microtubules (25), revealed the dynamic behavior of microtubules in living cells: comet-like streaks of GFP-N-Clip170 orig- inated at a common origin (presumably the MTOC) and moved at Ϸ3 m͞min toward the cell's periphery ( Fig. 2 A Right; Movies 3 and 4, which are published as supporting information on the PNAS web site), comets directed toward the back of the cell reached all of the way to the cell periphery and often traveled an additional distance (Ϸ1-3 m) in a direction parallel to the membrane, and comets directed toward the front of the cell stopped before entering the pseudopod (see Movie 4).…”