Cell edges accumulate gamma tubulin complex components and nucleate microtubules following cytokinesis in Arabidopsis thaliana

Abstract

Microtubules emanate from distinct organizing centers in fungal and animal cells. In plant cells, by contrast, microtubules initiate from dispersed sites in the cell cortex, where they then self-organize into parallel arrays. Previous ultrastructural evidence suggested that cell edges participate in microtubule nucleation but so far there has been no direct evidence for this. Here we use live imaging to show that components of the gamma tubulin nucleation complex (GCP2 and GCP3) localize at distinct sites along the outer periclinal edge of newly formed crosswalls, and that microtubules grow predominantly away from these edges. These data confirm a role for cell edges in microtubule nucleation, and suggest that an asymmetric distribution of microtubule nucleation factors contributes to cortical microtubule organization in plants, in a manner more similar to other kingdoms than previously thought.

Figure 1. Localization of GCP2-GFP in roots.

A Post-cytokinetic epidermal root division zone cells. Sequential images from confocal stack, starting at outer periclinal face, ending at median optical plane. Right panel shows a maximum Z projection of the series, a Y-axis orthogonal view, and a fluorescence intensity plot corresponding to the white line. B Root epidermal cells from early elongation zone. Sequential images from confocal stack, starting at outer periclinal face, ending at median optical plane. Right panel shows the outermost optical slice of the series, X- and Y-axis orthogonal views, and a fluorescence intensity plot corresponding to the dotted line. C Root epidermal cells from the late elongation zone. Sequential images from confocal stack, starting at outer periclinal face, ending at median optical plane. Arrowheads indicate edge enrichment. Arrows indicate perinuclear and cortical punctae. n = nucleus. Confocal planes correspond to 0.5 µm slice intervals. Scale Bars = 5 µm.

Figure 2. Localization of GCP2-GFP in cotyledons and hypocotyls.

A Cotyledon post-cytokinetic epidermal cells. Sequential images from confocal stack, starting at outer periclinal face, ending near the inner periclinal face. Right panel shows a maximum Z projection of the series and X-axis orthogonal view corresponding to the dotted line. Brackets indicate edge enrichment in post-cytokinetic cells. Arrows indicate perinuclear punctae. B Post cytokinetic and early expanding cotyledon epidermal cells. Cells at multiple stages are present. Sequential images from confocal stack, starting at outer periclinal face, ending near the median optical plane. Inset in B shows high contrast image corresponding to boxed region. n = nucleus. Phrag = phragmoplast. C Expanded hypocotyl epidermal cells. Sequential images from confocal stack, starting at outer periclinal face, ending near the median optical plane. Confocal planes correspond to 1 µm slice intervals. Scale Bars = 5 µm.

Figure 3. MT growth directions in post-cytokinetic cells.

(A–C) EB1b-GFP in post-cytokinetic cotyledon epidermal cells. For A and B, the top images are single time points, the middle panels are time projections of the same cells, and the bottom panels are kymographs corresponding to the dotted black lines in the top and middle panels. A Recently divided pavement epidermal cell. B A second example of recently divided cells. MTs are difficult to visualize due to high autofluorescence from vacuolar anthocyanins present in young cells. These cells are from the guard cell lineage. C Top (red) panel shows time projection of RFP-TUB6 corresponding to the time projection of EB1b-GFP in the middle panel. D Quantification of growth polarities with respect to newly formed and old cell edges in cotyledon pavement epidermal cells. E Post-cytokinetic root epidermal division zone cells. Two examples are shown, both show nucleation primarily from the bottom edge, and both contain multiple polarities with respect to these edges. Dotted lines indicate cell outlines. F Time projection and corresponding X and Y kymographs of EB1b-GFP showing nuclear initiation. Projections are taken from XYTZ series with 5 second intervals, and are 5 µm thick projections. Scale Bars = 5 µm.