Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Supplemental Figure 1: Distribution of HDEL-YFP in living and fixed cells of Arabidopsis. HDEL-YFP does not colocalise with microtubules in living cells (A) nor in fixed cells co-stained with anti-tubulin antibodies (B-J): preprophase (B-D), metaphase (E-G) and telophase (H-J). Anti-GFP (B, E, H), anti-tubulin (C, F, I), merge (D, G, J: anti-GFP = green, anti-tubulin = red). Note that only limited overlap between the signals of YFP-HDEL and AtEB1-GFP can be seen: i.e. at the spindle poles and nuclear envelopes of telophase cells. Nuclear envelope labelling is evident in cells containing a PPB. Also note (K) that caution is necessary when processing YFP-HDEL cells for localisation studies since microtubule-like patterns can be observed following extraction of living cells expressing YFP-HDEL with Triton X-100. Bar, in A-E =7 μm; in H = 6 μm.
• Supplemental Movie 1 - Movie 1: Polarity of microtubules at prophase. At prophase, plus-end comets (see arrowheads) grow out from perinuclear accumulations of AtEB1a-GFP towards the cell cortex. Bar = 10 μm.
• Supplemental Movie 2 - Movie 2: Polarity of microtubules at anaphase and telophase. AtEB1a-GFP shows comets grow out from perinuclear accumulations of AtEB1a-GFP during late anaphase/early telophase. The heavy fluorescent signal in the midline can be seen to result from the overlap of the plus ends (comets). A subset of nucleus-associated microtubules, reminiscent of astral microtubules found in animal cells, can also be observed to grow from the upper surfaces of the daughter nuclei with the comets clearly directed towards the cell cortex. Bar = 10 μm.
• Supplemental Movie 3 - Movie 3: Polarity of microtubules during cytokinesis. AtEB1a-GFP marks foci at the nuclear envelope (arrowheads: 1-5) from which plus-end comets of AtEB1a-GFP (small arrowheads) can be seen to grow towards the cell cortex or phragmoplast. Focus 1 disappears following the emergence of a plus-end comet in frame 30 sec ? microtubule growth then ceases from this spot. Foci 2 and 3 demonstrate mobility (both circumferentially and radially) at the periphery of the nucleus concomitant with microtubule nucleation events. In frame 110 sec, two plus-end comets can be seen to emerge from focus 2 and split in divergent angles. Focus 4 appears in frame 110 sec and demonstrates that microtubule growth occurs towards the midline of the phragmoplast. Focus 5 illustrates that the brightness of foci decreases following comet growth; two comets can also be observed to emerge from a focus and grow at divergent angles in frame 235 sec. T = time (sec), arrowheads = minus end foci of AtEB1a-GFP, small arrowheads = plus-end comets, * = outer edges of the phragmoplast, N = upper nucleus of daughter pair. Bar = 10 μm.
• Supplemental Movie 4 - Movie 4: cell division in the presence of the PPB. In the presence of a PPB, the orientation of spindle and phragmoplast is predictable. Note that the spindle forms with its equatorial plane perpendicular to the cell?s long axis and the phragmoplast grows along the plane predicted by the PPB. Cells were imaged at 10 min time intervals using a spinning disc confocal microscope. Bar = 10 μm.
• Supplemental Movie 5 - Movie 5: Cell division in the absence of a PPB. In the absence of a PPB, the orientations of spindle and phragmoplast are variable. Note that the spindle forms with its equatorial plane parallel to the cell?s long axis and the phragmoplast reorientates prior to selection of the division plane. Cells were imaged at 10 min time intervals on a spinning disc confocal microscope. Bar = 10 μm.