Abstract
Microtubules are cytoskeletal polymers that perform diverse cellular functions. The plus ends of microtubules promote polymer assembly and disassembly and connect the microtubule tips to other cellular structures. The dynamics and functions of microtubule plus ends are governed by microtubule plus end-tracking proteins (+TIPs). Here we report that the Arabidopsis thaliana SPIRAL1 (SPR1) protein, which regulates directional cell expansion, is an autonomous +TIP. Using in vitro reconstitution experiments and total internal reflection fluorescence microscopy, we demonstrate that the conserved N-terminal region of SPR1 and its GGG motif are necessary for +TIP activity whereas the conserved C-terminal region and its PGGG motif are not. We further show that the N- and C-terminal regions, either separated or when fused in tandem (NC), are sufficient for +TIP activity and do not significantly perturb microtubule plus-end dynamics compared with full-length SPR1. We also found that exogenously expressed SPR1-GFP and NC-GFP label microtubule plus ends in plant and animal cells. These results establish SPR1 as a new type of intrinsic +TIP and reveal the utility of NC-GFP as a versatile microtubule plus-end marker.
Highlights
Microtubules are cytoskeletal polymers that perform diverse cellular functions
Using in vitro reconstitution experiments and total internal reflection fluorescence microscopy, we demonstrate that the conserved N-terminal region of SPR1 and its GGG motif are necessary for ؉TIP activity whereas the conserved C-terminal region and its PGGG motif are not
We further show that the N- and C-terminal regions, either separated or when fused in tandem (NC), are sufficient for ؉TIP activity and do not significantly perturb microtubule plus-end dynamics compared with full-length SPR1
Summary
To study the mechanism of SPR1’s ϩTIP activity, we took an in vitro reconstitution approach with purified recombinant SPR1-GFP protein (Fig. 1A) and dynamic microtubules using porcine tubulin. SPR1-GFP did not significantly affect the shortening rate of microtubule plus ends and only modestly increased their rescue frequency at the highest SPR1-GFP concentration tested (Fig. 1, H and J) These observations (summarized in Table S1) are largely consistent with the reported changes in microtubule plus-end dynamics in spr loss-of-function mutants compared with the WT [19]. Deletion of the C-terminal 33 amino acids (SPR1⌬87–119) diminished the signal at growing microtubule plus ends but did not eliminate it (Fig. 2D), indicating that the C-terminal region enhances the ϩTIP activity of SPR1 but is not necessary for it Consistent with these findings and with previous in vivo work [11], the chimeric N-GFP-C protein clearly localized to growing microtubule plus ends in vitro (Fig. 2D and Movie S2). N-GFP-C showed an extensive cytoplasmic signal in HEK293 cells, we were unable to detect unambiguous microtubule plus-end labeling
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