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. Author manuscript; available in PMC: 2011 Jun 1.
Published in final edited form as: Nat Neurosci. 2010 Oct 31;13(12):1463–1471. doi: 10.1038/nn.2665

Figure 2. Microtubule organization in radial glial progenitors throughout the cell cycle.

Figure 2

(a) Microtubules of radial glial progenitors expressing GFP-Tubulin (green) in E18 rat brain slices. Left panel: Tubulin is distributed throughout interphase cells, with bundles of microtubules parallel to the long axis of the cell seen in favorable, dilated cell regions (arrows). Right panels: Dividing cell showing mitotic spindle. Basal process remains visible during division (top, arrows), as detected using soluble RFP, whereas tubulin-GFP cannot be found in this region (bottom). (b) Time-lapse images of plus-ends of growing microtubules labeled with GFP-EB3 (green) in an interphase radial glial progenitor cell at E18. (Centrosome is indicated in magenta (arrowhead) by DsRed-centrin co-expression.) Fluorescence images were taken every second. Time series of EB3 images in three regions correspondent to the basal process (box 1), soma (box 2) and apical process (box 3) are aligned to form a kymograph. “Comet tail”-like EB3 streaks are readily observed to move predominantly in the basal direction (arrowheads). (c), (d) GFP-EB3 behavior and microtubules organization in radial glial cells at different cell-cycle stages. (c) Superimposed negative contrast images of EB3 streaks (arrowheads) and centrosomes (double arrowheads, red) from 30 sec - 2 min time-lapse movies. (d) Tracings of the EB3 streaks using multiple colors to distinguish among individual microtubules. When soma is in the upper portion of the VZ (G2), EB3 streaks are mostly found to originate from the centrosomal region within the endfeet, curve around the nucleus, and enter the basal process (arrows; Movie S5, S6). During mitosis (M), EB3 streaks radiate from the two spindle poles to form the mitotic spindle. No detectable EB3 streaks enter the basal process (Movie S7). During cytokinesis (Cyto), the microtubules still radiate from the centrosomes in each daughter cell, with many microtubules aimed toward the midbody. EB3 streaks remain absent from the basal processes at this stage (Movie S8). (Non-radial glial cells are seen in uppar portion of image.) Paired cells following probable symmetric RGPC division (Symm) as evidenced by persistence of centrosomes at the endfeet of both daughter cells (Movie S9). EB3-tipped microtubules are oriented upward in both cells and now re-enter the basal fibers (arrows). Paired cells following probable asymmetric (Asym) RGPC division. The centrosome of daughter cell at right is shifted away with EB3 streaks emerging radially to now form a bidirectional microtubule array (Movie S10). Bars = 5 μm. (e) Velocity distribution shows EB3 movements to be comparable to migrating neurons and other cells. KIF1 A RNAi does not significantly affect the rate of EB3 movements in these cells. (f) Direction distribution of the EB3 movements during interphase indicates that 93% are oriented toward the basal directions. KIF1A RNAi has no obvious effects on the overall orientation of EB3 movements.