Mitotic Spindle Organization by the Preprophase Band Ambrose J. Christian , Cyr Richard   Molecular Plant  Volume 1, Issue 6, Pages 950-960 (November 2008) DOI: 10.1093/mp/ssn054 Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 1 Prophase Cells Lacking PPBs Have No Bridge MTs and Fail to Exhibit Spindle Bipolarity. A BY-2 cell, stably expressing the GFP:MBD MT reporter, is shown with its cell outline indicated by dotted lines at t = 0:00. NEB occurs at 9:00. The cell has reached metaphase by 67:30. Times are min:s. Scale bar = 10 μm. Molecular Plant 2008 1, 950-960DOI: (10.1093/mp/ssn054) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 2 Prophase Cells with PPBs Have Bridge Microtubules and Bipolar Spindles. A BY-2 cell, stably expressing the GFP:MBD MT reporter, is shown at a midplane optical section. (A) Images taken from a timelapse series (see Supplemental Video 1). Arrowheads indicate bridge MTs. (B) Kymograph generated from a transverse slice (indicated by dotted line) along the spindle equator. Black vertical lines indicate the equatorial boundaries of the nucleus/prophase spindle. Asterisks indicate bulge corresponding to lateral stretching of the equatorial spindle boundaries toward the PPB at late prophase. Arrowheads indicate constriction corresponding to lateral collapse at NEB/early prometaphase. Times are in minutes. Scale bar = 10 μm. Molecular Plant 2008 1, 950-960DOI: (10.1093/mp/ssn054) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 3 Prophase Cells with Non-Uniform Distribution of Bridge MTs Have Off-Centered Nuclei. A BY-2 cell, stably expressing the GFP:MBD MT reporter, is shown at a midplane optical section. (A) Images taken from a time lapse series (see Supplemental Video 2). Arrows indicate the initial position of the nucleus/prophase spindle at 0:00. Arrowheads indicate bridge MTs. (B) Kymograph generated from a transverse slice (indicated by dotted lines) along the spindle equator of the same cell. Black vertical lines indicate the initial equatorial boundaries of the nucleus/prophase spindle. Arrowhead indicates width decrease on the right side of the spindle during prometaphase. Open arrowhead indicates width decrease on the left side of the spindle during prometaphase. Times are min:s. Scale bar = 10 μm. Molecular Plant 2008 1, 950-960DOI: (10.1093/mp/ssn054) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 4 Taxol Induces Non-Uniform Bridge MT Distribution and Spindle Eccentricity in Prophase Cells. A BY-2 cell, treated with 5 μm taxol and stably expressing the GFP:MBD MT reporter, is shown at a midplane optical section. (A) Images taken from time series (see Supplemental Video 3). Taxol was added at roughly 30 s after the first frame. (B) Kymograph generated from a transverse slice (indicated by dotted lines) along the spindle equator of the same cell. Black vertical lines indicate the initial equatorial boundaries of the nucleus/prophase spindle. Scale bar = 10 μm. Molecular Plant 2008 1, 950-960DOI: (10.1093/mp/ssn054) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 5 Taxol-Induced Prophase Spindle Migration Does Not Require F-actin. A BY-2 cell, treated with 5 mM taxol and 20 mM Latrunculin B and stably expressing the GFP:MBD MT reporter, is shown at a midplane optical section. (A) Images taken from time series (see Supplemental Video 4). Taxol and Latrunculin B were added at roughly 30 s after the first frame. (B) Kymograph generated from a transverse slice (indicated by dotted lines) along the spindle equator of the same cell. Black vertical lines indicate the initial equatorial boundaries of the nucleus/prophase spindle. Scale bar = 10 μm. Molecular Plant 2008 1, 950-960DOI: (10.1093/mp/ssn054) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 6 Off-Centered Prophase/Prometaphase Spindles of atk5-1-Null Mutants Bend with their Midzones toward the Nearest Region of the PPB. An Arabidopsis root cell in an atk5-1-null mutant plant, stably expressing the GFP:TUB6 MT reporter, is shown at optical midplane during late prophase/prometaphase. Dotted line indicates cell boundary. Times are in minutes. Scale bar = 5 μm. Molecular Plant 2008 1, 950-960DOI: (10.1093/mp/ssn054) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 7 Closing of Polar Annulus Coincides Temporally with Prophase Spindle Migration, Equatorial Clearing, and Lateral Deformation. Various optical planes and image reconstruction from a BY-2 cell, stably expressing the GFP:MBD MT reporter, are shown. (A) Four-dimensional imaging of a cell with non-uniform bridge MT distribution undergoing the prophase-to-prometaphase transition. Frames represent 3-D reconstructions of Z-series rotated 90° to produce a pole view of the cell over time. (B) Closing of polar annulus is coincident with lateral deformation and equatorial clearing of the prophase spindle. Two time points are shown, corresponding to early and late prophase. For each time point, on the left is shown a median section from the Z-series, and on the right are 3-D reconstructions taken from the top pole (indicated by box) tilted from 0° to 90°. Times are in minutes. Scale bars = 10 μm. Molecular Plant 2008 1, 950-960DOI: (10.1093/mp/ssn054) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 8 Model for Bridge MT-Dependent Organization of Prophase Spindle. The column on the left illustrates uniform bridge MT distribution and the resultant symmetrical bipolarity and lack of migration. The column on the right illustrates non-uniform bridge MT distribution and the resultant nuclear migration and asymmetric bipolarity. Note annular spindle poles during late prophase. Horizontal axis is exaggerated for clarity. Arrow indicates time vector. Molecular Plant 2008 1, 950-960DOI: (10.1093/mp/ssn054) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions