Supplemental Material

This article contains the following supporting material:

• movie1.mov - Post-anaphase array (PAA) formation and interphase microtubule dynamics in wild-type cells expressing nmt81:GFP-atb2. Projection of deconvolved image stacks (0.6 um spacing) comprising entire cell volume; 30 second frame interval (time compression 300X). In mitotic cells, note the stability of the equatorial microtubule-organizing center (eMTOC) at the end of mitosis, and the continuous microtubule nucleation from the eMTOC through the completion of septation. Also note the rapid dynamics of cytoplasmic astral microtubules during mitosis. In interphase cells, note the rapid microtubule dynamics and nucleation from many independent sites within the cell.
  
  
• movie2.mov - PAA formation and interphase microtubule dynamics in mto2Δ cells expressing nmt81:GFP-atb2. Projection of deconvolved image stacks (0.6 um spacing) comprising entire cell volume; 30 second frame interval (time compression 300X). In mitotic cells note that in spite of the absence of the stable eMTOC structure seen in wild-type cells, microtubules are nevertheless nucleated from an eMTOC, although at lower frequency. Note also that cytoplasmic astral microtubules can grow longer than in wild-type cells, as seen in the left-hand mitotic cell. In interphase cells, note that microtubules can curve around cell tips and sometimes undergo bend-breakage as a result (top cell), and that, in contrast to wild-type cells, new microtubule nucleation (as opposed to new microtubules generated by bend-breakage) appears restricted to a single site on the nuclear surface.
  
  
• movie3.mov - PAA formation and interphase microtubule dynamics in mto2Δ cells expressing nmt81:GFP-atb2, at higher time-resolution. Projection of deconvolved image stacks (0.6 um spacing) comprising entire cell volume; 15 second frame interval (time compression 150X). In the mitotic cell, note that at this higher time resolution, microtubule growth from the eMTOC is unambiguously due to new microtubule nucleation as opposed to growth from discarded fragments of the mitotic spindle. Note also that microtubules nucleated by the eMTOC can persist as bundles for relatively long periods. In interphase cells, microtubule nucleation and dynamics, and bend-breakage are similar to that seen in movie02.mov.
  
  
• movie4.mov - PAA formation and interphase microtubule dynamics in mto2Δ cells expressing nmt81:GFP-atb2, at higher time-resolution. Projection of deconvolved image stacks (0.6 um spacing) comprising entire cell volume; 15 second frame interval (time compression 150X). In the mitotic cell, note that eMTOC activity is slightly different from that seen in movie03.mov; here, eMTOC microtubule nucleation is initially seen even before mitotic spindle disassambly, and then later occurs sporadically. In interphase cells, microtubule nucleation and dynamics, and bend-breakage are similar to that seen in movie02.mov and movie03.mov.
  
  
• movie5.mov - PAA formation and interphase microtubule dynamics in mto2Δ cells expressing nmt41:GFP-atb2 (higher levels of GFP-atb2 than in movies 01-04). Projection of deconvolved image stacks (0.6 um spacing) comprising entire cell volume; 30 second frame interval (time compression 300X). In mitotic cells, note that the eMTOC is still capable of nucleating microtubules, as are the spindle pole bodies (SPBs). In interphase cells, note that new microtubule nucleation appears restricted to a single site on the nuclear surface, as seen before in cells expressing nmt81:GFP-atb2 (i.e., in movies 02-04). However, at this higher level of GFP-atb2 expression, the number of microtubule bundles at steady-state appears reduced compared to cells expressing nmt81:GFP-atb2, and the microtubule ends are in general less dynamic (compare especially with movie02, which has same time compression).
  
  
• movie6.mov - Through-focus sequence showing localization of mto2-GFP. Single time point, 0.6 um interval between frames, non-deconvolved images. Note that mto2-GFP can be seen along microtubules, at SPBs, and at eMTOCs, but not along spindle microtubules (i.e., between mitotic SPBs). Also note that some faint spots of mto2-GFP are visible on microtubules towards the cell tips, although these are not always obvious when the entire image stack is viewed as a single projection (Fig. 4).
  
  
• movie7.mov (1.24 MB) - Dynamics of mto2-GFP. Projection of three non-deconvolved sections (0.6 um spacing) comprising approximately one-third of the cell thickness; 5 second frame interval (time compression 50X). Note rapid apparent back-and-forth movement of mto2-GFP satellite particles on interphase microtubules, as well as on PAA microtubules in the dividing cell.
  
  
• movie8.mov - Through-focus sequence showing localization of mto1-YFP in wild-type background. Single time point, 0.6 um interval between frames, non-deconvolved images. Note localization of mto1-YFP to SPBs and eMTOC and to satellite particles on interphase microtubules.
  
  
• movie9.mov - Through-focus sequence showing localization of mto1-YFP in mto2Δ cells. Single time point, 0.6 um interval between frames, non-deconvolved images. Imaging conditions identical to movie08; the reasons for increased photobleaching in movie09 are not understood. Note localization of mto1-YFP to SPBs and eMTOC and to satellite particles on interphase microtubules, although because of the relatively faint signal these are not always obvious when the entire image stack is viewed as a single projection (Fig. 6). Note also that not every interphase cell shows similar numbers of satellite particles.
  
  
• movie10.mov (1.05 MB) - Dynamics of mto1-YFP in wild-type background. Projection of three non-deconvolved sections (0.6 um spacing) comprising approximately one-third of the cell thickness; 5 second frame interval (time compression 50X). Note rapid back-and-forth movement of mto1-YFP satellite particles on interphase microtubules.
  
  
• movie11.mov - Dynamics of mto1-YFP in mto2Δ cells. Projection of three non-deconvolved sections (0.6 um spacing) comprising approximately one-third of the cell thickness; 5 second frame interval (time compression 50X). Imaging conditions identical to movie08; the reasons for increased photobleaching in movie10 are not understood. Note that mto1-YFP interphase satellites appear to exhibit back-and-forth movements similar to those in wild-type cells, although these may not stand out as much as movement of the brightest (SPB) spot (see especially cells in upper-right quadrant, and dividing cell in lower-left quadrant).
  
  

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