Nuclear-localized subtype of end-binding 1 protein regulates spindle organization in Arabidopsis

Abstract

SummaryEnd-binding 1 (EB1) proteins are evolutionarily conserved plus-end-tracking proteins that localize to growing microtubule plus ends where they regulate microtubule dynamics and interactions with intracellular targets. Animal EB1 proteins have acidic C-terminal tails that might induce an autoinhibitory conformation. Although EB1 proteins with the same structural features occur in plants (EB1a and EB1b in Arabidopsis thaliana), a variant form (EB1c) is present that lacks the characteristic tail. We show that in Arabidopsis the tail region of EB1b, but not of EB1c, inhibits microtubule assembly in vitro. EB1a and EB1b form heterodimers with each other, but not with EB1c. Furthermore, the EB1 genes are expressed in various cell types of Arabidopsis, but the expression of EB1c is particularly strong in the meristematic cells where it is targeted to the nucleus by a nuclear localization signal in the C-terminal tail. Reduced expression of EB1c compromised the alignment of spindle and phragmoplast microtubules and caused frequent lagging of separating chromosomes at anaphase. Roots of the eb1c mutant were hypersensitive to a microtubule-disrupting drug and complete rescue of the mutant phenotype required the tail region of EB1c. These results suggest that a plant-specific EB1 subtype has evolved to function preferentially on the spindle microtubules by accumulating in the prophase nucleus.

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

“…Similarly, we examined the MT binding properties of EB1b, which, like SPR1, has been shown to preferentially localize to the growing ends of MTs (Mathur et al, 2003). Consistent with previous reports that EB proteins bind directly to MTs (Berrueta et al, 1998;Tirnauer et al, 2002;Komaki et al, 2010), E. coliexpressed and -purified N-terminal 6xHis-tagged EB1b fusion protein (H-EB1b) was found to cosediment with taxol-stabilized MTs when centrifuged at 25,000g, while less H-EB1b protein sedimented in the absence of MTs ( Figure 5A). Error bars in (E) and (F) represent SD.…”

“…Alternatively, if the SPR1 GGG and PGGG motifs bind adjacent tubulin proteins, the internal linker portion of SPR1 would loop out. Note that the C termini of two EB1b proteins interact to form a dimer (Komaki et al, 2010). While our data show that SPR1 interacts physically with EB1b, it is unclear exactly where; the interaction is likely not facilitated by an Iso70-Pro71 motif used by other proteins to bind EB1.…”

“…Fluorescent protein fusions with EB1a and EB1b exquisitely label growing cortical MT plus ends (Chan et al, 2003;Mathur et al, 2003). Disruption of EB1c function affects spindle and phragmoplast alignment and confers anti-MT drug hypersensitivity (Komaki et al, 2010). Subtle cell expansion defects were found associated with mutations in all three Arabidopsis EB1 genes, with loss of EB1b function causing the most notable root directional growth phenotypes (Bisgrove et al, 2008).…”

The Microtubule-Associated Protein END BINDING1b, Auxin, and Root Responses to Mechanical Cues

“…From these studies, EB1 family members have emerged as key regulatory +TIPs because they bind directly to microtubule ends, modify microtubule dynamics, and interact with a diverse array of additional proteins [26]. Like their animal and fungal counterparts, plant EB1 proteins preferentially accumulate on growing microtubule ends where they regulate microtubule dynamics [11,[29][30][31][32]. Presumably, they also recruit other proteins to microtubules, although plant-specific proteins that interact with EB1 are largely unknown [25].…”

Microtubules During the Cell Cycle of Higher Plant Cells

“…EB1 proteins are highly conserved in animals and fungi, yet SPR1 appears to be plant specific. Among known functions of EB1 proteins are the promotion of microtubule polymerization, stabilization, regulation of spindle positioning and chromosome segregation (Komaki et al, 2010). SPR1 and EB1 may act together to regulate directional cell expansion in response to the environmental stimulation.…”