Supplemental Material

This article contains the following supporting material:

• M1_1_BipFlx_C.mov (4.54 MB) - CSF monopole labeled with X-rhodamine tubulin at speckle levels, before computational alignment.
  
  
• M1_2_BipFlx_c.mov (4.03 MB) - As M1_1 after computational alignment of sequential frames. Note lack of coherent movement of speckles towards the pole. See figure 1C.
  
  
• M1_3_BipFlx_c.mov (2.24 MB) - Second example of an aligned CSF monopoles. Note lack of coherent movement of speckles towards the pole. See figure 1E.
  
  
• M2_1_BipFlx_c.mov (4.09 MB) - CSF monopole labeled with X-rhodamine tubulin at speckle levels undergoing spontaneous bipolarization. The whole sequence is ~1350 seconds. See figure 2A.
  
  
• M2_2_BipFlx_c.mov (6.15 MB) - Second example of a CSF monopole labeled with X-rhodamine tubulin at speckle levels undergoing spontaneous bipolarization. The whole sequence is ~670 seconds. In this sequence the monopole has already started to assemble a second pole (to the right) at the beginning of the sequence. Early in the sequence there is no evidence for cohenrent speckle movement, while later in the sequence robust poleward flux can be observed, concomitant with bipolarization. See figure 2B.
  
  
• M3_1_BipFlx_c.mov (7.10 MB) - Control spindle labeled with Alexa488-IgG to TPX2 (C terminal peptide) (upper panel) and X-rhodamine tubulin at speckle levels (lower panel). Note the concerted poleward movement, at the same rate, of both tubulin speckles and TPX2 aggregates. The sequence is ~210 sec long. See figure 3A. A kymograph of this spindle is shown in figure 3C.
  
  
• M3_2_BipFlx_c.mov (8.47 MB) - Spindle treated with 2mM AMPPNP using the same labels as M3_1. Note that poleward movement of both tubulin and TPX2 are blocked. The sequence is ~210 sec long. A kymograph of this spindle is shown in figure 3C.
  
  
• M3_3_BipFlx_c.mov (8.41 MB) - Spindle treated with 0.9 mg/ml p50 dynamitin using the same labels as M3_1. Note that both tubulin and TPX2 still move poleward. The sequence is ~210 sec long. A kymograph of this spindle is shown in figure 3D.
  
  
• M4_1_BipFlx_c2.mov (5.39 MB) - Example of bipolarization by new pole assembly near chromatin. Left panel is x-rhodamine tubulin at speckle levels, right panel is Alexa488-IgG to TPX2 (C terminal peptide). See figure 4A.
  
  
• M4_2_BipFlx_c.mov (9.21 MB) - Example of bipolarization by pole splitting. Probes as M4_1. See figure 4B.
  
  
• M4_3_BipFlx_c2.mov (6.66 MB) - Example of bipolarization by both new pole assembly near chromatin and pole splitting. Probes as M4_1. Note the unidirectional flow of tubulin speckles and TPX2 aggregates from the chromtin towards the splitting poles. We believe this type of flow accounts for the mistaken conclusions that CSF monopoles can exhibit flux in Sawin and Mitchison (1994). See figure 4C.
  
  
• M5_1_BipFlx_c.mov (5.11 MB) - Spindle treated with importin-alpha (1mg/ml). Probes are x-rhodamine tubulin at speckle levels (shown green) and a mixture of Alexa488-IgG to NUMA and CenpA (both C terminal peptide). Note that the microtubule density is greatly reduced compared to control spindles, and that the remaining microtubules appear to mainly stretch between kinetochores and poles. The tubulin speckles are dim, but there is no evidence for poleward speckle movement. Note that sister kinetochores are closer together than control spindles. The sequence is 130 sec long. See figure 5A.
  
  
• M5_2_BipFlx_c.mov (2.91 MB) - Second example of a spindle treated with importin-alpha (1mg/ml). Probes as M5_1. In this sequence the spindle partly collapses. Tubulin speckles are well visualized, and a complete lack of poleward movement of speckles is clear. The sequence is 130 sec long. See figure 5B, and kymograph in figure 5C.
  
  
• M5_3_BipFlx_c.mov (4.17 MB) - Control spindle using probes as M5_1. Sister kinetochore pairs are maximally separated, and robust, coherent, poleward movement of speckles is evident The sequence is 130 sec long. See figure 5D, and kymograph in figure 5E.
  

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