Supplemental Material

General notes
Time-lapse images were generated on a Zeiss Axiovert 200M microscope equipped with UltraView RS-3 confocal system: CSU21 confocal Optical Scanner, 12 bit digital cooled Hammamatsu Orca-ER camera (OPELCO Co.) and Krypton-Argon Triple line laser illumination source. For imaging of microtubule dynamics, S. pombe cells expressing α-tubulin-GFP were grown in appropriate selective medium and placed in sealed growth chambers containing 2% agarose media. For 3D time lapse imaging, each image stack consisted of 13 sections of 0.5μm spacing and 15 sec intervals between stacks. For single section time lapse analyses, images were collected at 5 sec intervals. Experiments were carried out at room temperature. Stacks were assembled and processed using the Metamorph software package (Universal Imaging Co.).

This article contains the following supporting material:

• Table 1
• Figure 1 - Microtubules remain attached to the SPB in the absence of Alp14p.
  A, Time-lapse sequence of α-tubulin-GFP Sid2p-GFP expressing alph14Δ cell. Shown are single maximum intensity reconstructions of z-stacks. Numbers refer to the time, in minutes and seconds. B, Plot of the SPB position over time in α-tubulin-GFP and Sid2p-GFP expressing alph14Δ cell. Note that the SPB remained stationary in the course of the movie.

• Figure 2 - Interphase mia1Δ cells are capable of nucleating microtubules from around the nuclear envelope but cannot sustain the microtubule-nuclear envelope attachment.
  Time lapse sequences of cold-treated α-tubulin-GFP Uch2p-GFP expressing wild type (A) and mia1Δ (B) cells after temperature shift-up to 24°C. Note that in both cases microtubules polymerize from the nuclear surface. Numbers refer to the time, in minutes and seconds, in the time lapse sequences.

• Figure 3 - Localization of Mto1p-GFP in cells lacking the microtubule stabilizing proteins, Alp14p and Mal3p.
  Mto1p-GFP exhibits strong nuclear envelope staining, in addition to its SPB localization, in both steady state and MBC-treated alph14Δ cells (upper panel). Mto1p-GFP is found in several aggregates around the NE in non-treated and MBC-treated mal3Δ cells, similar to control. Shown are single maximum intensity reconstructions of z-stacks.

• Figure 4 - When MBC was added after the onset of the eMTOC disassembly, the nascent iMTOCs around the NE were readily detected.
  Left panel, live GFP image of a Mto1p-GFP expressing cell where microtubules were depolymerized just after the onset of the eMTOC disassembly. Right panel, live GFP image of the Mto1p-GFP expressing cell where microtubules were depolymerized after the completion of septation. Shown are single maximum intensity reconstructions of z-stacks.

• Movie 1 - Microtubule and the SPB dynamics in the interphase α-tubulin-GFP Sid2p-GFP expressing mia1Δ cell. There is a free microtubule present that undergoes a catastrophe. One can also observe the SPB detachment from the microtubule bundle. Movie is assembled from the single maximum intensity reconstructions of z-stacks Images were collected every 15 seconds.

• Movie 2 - SPB oscillations in the interphase α-tubulin-GFP Sid2p-GFP expressing wild type cell. Movie is assembled from the single maximum intensity reconstructions of z-stacks Images were collected every 15 seconds.

• Movie 3 - SPB movements in the interphase α-tubulin-GFP Sid2p-GFP expressing mia1Δ cell. Note larger amplitude of oscillations and eventual detachment of the SPB from the microtubule bundle. Movie is assembled from the single maximum intensity reconstructions of z-stacks Images were collected every 15 seconds.

• Movie 4 - SPB and microtubule dynamics in the interphase α-tubulin-GFP Sid2p-GFP expressing alp14Δ cell. Note that short microtubules remain attached to the SPB but the SPB is not oscillating. Movie is assembled from the single maximum intensity reconstructions of z-stacks Images were collected every 15 seconds.

• Movie 5 - Microtubule dynamics in the interphase α-tubulin-GFP expressing mia1Δ cell. Note microtubule nucleation event followed by ejection of newborn microtubule from the nuclear envelope. Movie is assembled from the single maximum intensity reconstructions of z-stacks Images were collected every 15 seconds.

• Movie 6 - Microtubule dynamics in the interphase α-tubulin-GFP expressing wild type cell. Shown is a single focal plane. Images were collected at 5 seconds intervals. Cell used for quantification in Fig. 4A is indicated by asterisk.

• Movie 7 - Microtubule dynamics in the interphase α-tubulin-GFP expressing mia1Δ cell. Shown is a single focal plane. Images were collected at 5 seconds intervals. Cell used for quantification in Fig. 4B is indicated by asterisk.

• Movie 8 - Microtubule dynamics in the interphase α-tubulin-GFP Sid2p-GFP expressing wild type cell upon microtubule depolymerization and recovery. Wild type cells were mounted in the flow chamber allowing the time lapse analysis of microtubule dynamics during MBC treatment (50 μg/ml) and subsequent washout. Microtubules rapidly depolymerized after MBC treatment leaving stable “stubs” at the NE. Upon washout, microtubules rapidly repolymerized from these “stubs” towards cell periphery. MBC addition and wash-out are indicated in a time-lapse sequence. Movie is assembled from the single maximum intensity reconstructions of z-stacks. Images were collected every 15 seconds.

• Movie 9 - Microtubule dynamics in the interphase α-tubulin-GFP Sid2p-GFP expressing mia1Δ cell upon microtubule depolymerization and recovery. Cells were mounted in the flow chamber allowing the time lapse analysis of microtubule dynamics during MBC treatment (50 μg/ml) and subsequent washout. Microtubules depolymerized after MBC treatment without leaving remnants in cell center. Position of the nucleus is indicated by Sid2p-GFP-positive spindle pole body. Upon washout, microtubules rapidly repolymerized from around the NE. Note that at least one of the nucleated microtubules eventually detached and shrank across the cell length upon undergoing catastrophe. MBC addition and wash-out are indicated in a time-lapse sequence. Movie is assembled from the single maximum intensity reconstructions of z-stacks. Images were collected every 15 seconds.

• Movie 10 - Microtubule re-growth from the nuclear surface following temperature shift-up of ice-treated Uch2p-GFP α-tubulin-GFP expressing wild type cell. Note that microtubules remain attached to the NE in the course of the movie. Movie is assembled from single plane time lapse images. Images were collected every 10 seconds.

• Movie 11 - Microtubule re-growth from the nuclear surface following temperature shift-up of ice-treated Uch2p-GFP α-tubulin-GFP expressing mia1Δ cell. Note that microtubules eventually detach from the nuclear envelope. Movie is assembled from single plane time lapse images. Images were collected every 10 seconds.

• Movie 12 - The PAA dynamics in the interphase α-tubulin-GFP expressing wild type cell. Note that once established, the microtubule foci are maintained and eventually form a constricting ring-like structure. Movie is assembled from the single maximum intensity reconstructions of z-stacks. Images were collected every 15 seconds.

• Movie 13 - The PAA dynamics in the interphase α-tubulin-GFP expressing mia1Δ cell. Note the absence of persistent nucleation sites in the medial region of the cell. Movie is assembled from the single maximum intensity reconstructions of z-stacks. Images were collected every 15 seconds.