Abstract
SPIRAL2 (SPR2) of Arabidopsis thaliana is a microtubule-associated protein containing multiple HEAT repeats that are found only in the plant lineage. We show that SPR2 and SP2L, their closest Arabidopsis homolog, are expressed in various tissues with partially overlapping patterns, and spr2-sp2l double mutants exhibit enhanced right-handed helical growth. Fusion to green fluorescent protein (GFP) expressed under the control of the native regulatory elements showed that both SPR2 and SP2L were localized to cortical microtubules, mainly in particles of various sizes. Along the microtubule, the GFP-fused forms also distributed partly at the plus ends. In the spr2-mutant background, cortical microtubules were less dynamic, and the pause state - in which microtubules undergo neither growth nor shrinkage - increased at the plus ends. The continuous plus-end tracking of GFP-EB1 was occasionally interrupted in the mutant cells. Recombinant SPR2 protein promoted microtubule polymerization, and bound to microtubules with an N-terminal segment that contained two HEAT repeats as well as to those with a C-terminal region. In vitro analyses of microtubule dynamics revealed that SPR2 and SP2L suppressed the pause state at microtubule ends, thereby leading to enhanced microtubule growth. We propose that the SPR2-family proteins act on the pause state to facilitate a transition to microtubule growth.
Highlights
Microtubules display dynamic instability in vitro and in vivo
Many regulations of microtubule dynamics have been identified over the years; some regulator families are well conserved in yeasts, animals and plants, but others are lost or gained in the plant lineage (Gardiner and Marc, 2003)
We show that SPR2 is partially enriched at the microtubule plus end in vivo, reduces microtubule pausing and facilitates continued growth of cortical microtubules
Summary
Microtubules display dynamic instability in vitro and in vivo. Individual microtubules undergo polymerization and depolymerization with sporadic changes between the two states (Desai and Mitchison, 1997). Polymerization dynamics of microtubules have been shown to be important for many cellular functions, such as cell division, polarization, motility and intracellular transport. The end binding 1 (EB1) family shows robust plus-end-tracking activity in vivo, and accumulates selectively at the growing microtubule ends without other proteins in vitro, as demonstrated for the yeast EB1 homologue Mal (Bieling et al, 2007). The +TIP family members EB1, CLASP, and XMAP215 are evolutionally conserved in plants (Bisgrove et al, 2004). An example of plantspecific +TIPs is SPIRAL1, which accumulates at the growing microtubule end in some cell types of Arabidopsis plants (Nakajima et al, 2004; Sedbrook et al, 2004). Cellular and biochemical functions of these plant +TIPs, are poorly understood
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