The γ -Tubulin Complex Protein GCP4 Is Required for Organizing Functional Microtubule Arrays inArabidopsis thaliana 

Abstract

Microtubule (MT) nucleation and organization depend on the evolutionarily conserved protein g-tubulin, which forms a complex with GCP2-GCP6 (GCP for g-Tubulin Complex Protein). To date, it is still unclear how GCP4-GCP6 (the non-core GCPs) may be involved in acentrosomal MT nucleation in plant cells. We found that GCP4 was associated with g-tubulin in vivo in Arabidopsis thaliana. When GCP4 expression was repressed by an artificial microRNA, transgenic plants exhibited phenotypes of dwarfism and reduced organ size. In mitotic cells, it was observed that the g-tubulin signal associated with the mitotic spindle, and the phragmoplast was depleted when GCP4 was downregulated. Consequently, MTs failed to converge at unified spindle poles, and the bipolar phragmoplast MT array frequently had discrete bundles with extended minus ends, resulting in failed cytokinesis as reflected by cell wall stubs in leaf epidermal cells. In addition, cortical MTs in swollen guard cells and pavement cells of the leaf epidermis became hyperparallel and bundled, which was likely caused by frequent MT nucleation with shallow angles on the wall of extant MTs. Therefore, our results support the notion that GCP4 is an indispensable component for the function of g-tubulin in MT nucleation and organization in plant cells.

TheArabidopsisTRM1–TON1 Interaction Reveals a Recruitment Network Common to Plant Cortical Microtubule Arrays and Eukaryotic Centrosomes

“…Apart from proteins of the bona fide g-tubulin complex (Liu et al, 1994;Erhardt et al, 2002;Binarová et al, 2006;Pastuglia et al, 2006;Nakamura and Hashimoto, 2009;Kong et al, 2010), only a handful of plant proteins with similarity with animal centrosomal proteins have been identified and characterized (Pastuglia and Bouchez, 2007). In Arabidopsis thaliana, this includes NEDD1 (Zeng et al, 2009), Cyclin-Dependent Kinase A;1 (Weingartner et al, 2004), FASS/TONNEAU2 (TON2) (Camilleri et al, 2002), and TON1 (Azimzadeh et al, 2008).…”

Interaction of Antiparallel Microtubules in the Phragmoplast Is Mediated by the Microtubule-Associated Protein MAP65-3 inArabidopsis

“…Plant growth conditions and agrobacterium-mediated transformation are as described previously (Kong et al, 2010).…”

TONNEAU2/FASS Regulates the Geometry of Microtubule Nucleation and Cortical Array Organization in Interphase Arabidopsis Cells

“…Recent data provide indications that the conformation of g-TURC subunits may be important in determining the branching nucleation angle. A missense mutation in GCP2 and downregulation of GCP4 have been shown to change the angle of microtubule branching in Arabidopsis (Nakamura and Hashimoto, 2009;Kong et al, 2010). Of note, human g-TURC was shown to interact with subunits of the chaperonin containing T-complex protein, suggesting that conformation of the nucleation complex may be actively regulated (Teixidó -Travesa et al, 2010).…”

“…Biochemical isolations have shown that core proteins are assembled into a complex with stoichiometry similar to the g-TURCs from yeast and animals (Nakamura et al, 2010). g-TURC components g-tubulin, GCP2, GCP4, and GCP-WD have been shown to play an essential role in organization of cortical microtubules, the spindle, and phragmoplast arrays in Arabidopsis thaliana (Binarová et al, 2006;Pastuglia et al, 2006;Nakamura and Hashimoto, 2009;Zeng et al, 2009;Kong et al, 2010).…”