Roles of TACC related protein, mia1p in MTOCs and microtubule dynamics in schizosaccharomyces pombe 4

Quantification the number of microtubule bundles per cell showed that 73% of wild type cells had 4 bundles, whereas majority of mia1Δ cells ~62% exhibited 2 bundles see graph in Figure

Chapter III Results

3.1 Mia1p in assembly and maintenance of persistent MTOCs at the nuclear

envelope

3.1.1 Lack of microtubule binding protein Mia1p results in fewer interphase

microtubules

3.1.1.1 Mia1p is a microtubule binding protein

During interphase, Mia1p-GFP expressed from its native promoter was localized along microtubules in a dotted pattern and was enriched at the regions where minus ends

of microtubules overlap, consistent with previously reported results (see Figure3.1.1.1 A) When interphase cells were treated with methyl benzimidazol-2-yl-carbamate (MBC), microtubules were depolymerized with short stubs remaining around the NE Mia1p-GFP was found as several distinct dots around the NE, co-localizing with short microtubule stubs and γ-tubulin (see Figure3.1.1.1 B), suggesting that Mia1p is one of components of MTOCs Far western analysis using prepolymerized taxol-stabilized microtubules suggested that the recombinant Mia1p-GST could physically associate with microtubules (carried out by Liangmeng Wee, see Supplementary Figure3.1.1.1C) Thus,

I concluded that Mia1p binds microtubules and is a component of S pombe MTOCs

Figure 3.1.1.1: Microtubule-associated protein Mia1p binds microtubules and is a component of S pombe MTOCs (A) Shown is the maximum projection of the z-stack

obtained by epifluorescence imaging of interphase cells expressing Mia1p-GFP (B) On

MBC treatment, Mia1p-GFP localized to distinct dots around the NE coinciding with short microtubule stubs (top panel) and predominantly colocalizing with γ-tubulin-rich structures (bottom panel), as seen from anti-α-tubulin, anti-γ-tubulin, and anti-GFP

immunofluorescence images of Mia1p-GFP cells (C) Mia1p binds directly to the

microtubule in vitro Filter-immobilized recombinant Mia1p-GST bound taxol-stabilized

microtubules in the Far Western assay Binding was not observed with GST alone (for details, see material and methods)

3.1.1.2 Mia1Δ cells exhibit disordered and heterogeneous microtubule bundles

Normally 4-5 anti-parallel microtubule bundles run along the axis of interphase fission yeast cells One of them is nucleated by the SPB and the rest are nucleated by the iMTOCs (interphase microtubule organizing centers) (for review, see Sawin and Tran, 2006) Thus minus ends of microtubules are anchored to the NE while plus ends face

towards cell tips (see Figure 3.1.1.2 A) In contrast, mia1Δ cells exhibited two

microtubule bundles curving around cell tips (see Figure 3.1.1.2 A) Quantification the number of microtubule bundles per cell showed that 73% of wild type cells had 4

bundles, whereas majority of mia1Δ cells (~62%) exhibited 2 bundles (see graph in

Figure 3.1.1.2 B), indicating that Mia1p was involved in organizing microtubule arrays

To examine microtubule polarity within the abnormally curved bundles

assembled in mia1Δ cells, I measured the fluorescence intensity along the length of

microtubule bundles in α-tubulin-GFP expressing wild type and mia1Δ cells Distinct medial zones of overlap (see Figure 3.1.1.2 C) were found in wild type cells, similar to

previously published data (Loiodice et al., 2005; Sawin and Tran, 2006) These high

intensity regions usually localized near the nucleus and overlapped with the iMTOCs I

found no well defined or properly positioned zones of microtubule overlap in mia1Δ cells

using similar techniques (see Figure 3.1.1.2D), suggesting that the microtubule

architecture of mia1Δ cells was disordered and heterogeneous

Figure3.1.1.2: Cells lacking Mia1p exhibit disordered and heterogeneous

microtubule bundles (A) Compared to the wild-type case, mia1Δ cells contain fewer

microtubule bundles in mia1Δ cells Shown is the maximum intensity projection obtained

by epifluorescence microscopy image of wild type and mia1Δ cells expressing

α-tubulin-GFP (B) Quantification of microtubule bundles number per cell in wild-type and mia1Δ

cells (n=100) (C) Fluorescence intensity scans of individual microtubule bundles in eight

wild-type cells expressing α-tubulin-GFP (D) Fluorescence intensity scans of individual

microtubule bundles in eight mia1Δ cells expressing α-tubulin-GFP Note that although there are distinct medial regions of microtubule overlap in wild-type cells (A) mia1Δ

cells (B) show heterogeneous patterns of microtubule arrangement

3.1.2 mia1Δ cells are defective in attachment of microtubules to the nucleation sites

3.1.2.1 Microtubules detached from the nucleation sites in mia1 Δ cells

To further investigate role of Mia1p in microtubule organization, I performed time-lapse analyses of microtubule and SPB dynamics in α-tubulin-GFP expressing

mia1 Δ cells that also contained the SPB marker, Sid2p-GFP In mia1Δ cells, I found that

microtubules continued to grow when they touched cell tips, producing longer and curved microtubules (see Figure 3.1.2.1 A) One of microtubules was undergoing catastrophes and shrinking from one cell end to the other (see Figure 3.1.2.1 A), indicating that microtubule minus ends were dissociated from the NE I also detected instances of the SPB detachment from the associated microtubule bundles (see Figure 3.1.2.1 A)

To ascertain the phenotypes of mia1Δ cells described above, I tracked the

movement of the SPBs and compared SPB behavior and microtubule dynamics in wild

type and mia1Δ cells expressing Sid2-GFP and α-tubulin-GFP In wild type, the SPB

always associated with underlying microtubule bundles throughout the movie, and exhibited short range oscillations along the long axis around the geometric cell center as indicated in the graph (100%, n=20 cells, observed for total of 3.5h; see Figure 3.1.2.1 B1) This behavior is due to the balanced pushing forces exerted by plus ends of

microtubules arranged in antiparallel bundles anchored at the NE (Tran et al., 2001)

When microtubules were depolymerized by MBC, the SPB stopped moving due to lack

of pushing forces (see Figure 3.1.2.1 B3) In mia1Δ cells, the SPB was either oscillating

Figure 3.1.2.1: Mia1p is required for attachment of microtubules to the SPBs and

the NE (A) Time-lapse sequence of mia1Δ cells expressing α-tubulin-GFP-and GFP show presence of free microtubules undergoing catastrophe (indicated by an arrow) and instances of microtubule bundles detaching from the SPB (indicated by a star) The SPB in the sequence is indicated by an arrowhead Shown is the maximum intensity projection of Z-stacks obtained by epifluorescence microscopy imaging Numbers refer

Sid2p-to the time, in minutes and seconds Bar, 1μm

Figure 3.1.2.1B: Mia1p is required for attachment of microtubules to the SPBs and the NE (1) Plot of SPB position over time in wild type cell expressing α-tubulin-GFP and Sid2p-GFP Some extreme positions of the SPB (indicated as 1-4) are shown on the

right of the graph (2) Plot of SPB position in mia1Δ cell expressing α-tubulin-GFP and

Sid2p-GFP Note that the SPB detached in the course of an experiment (compare

positions 2 and 3 with 1 and 4) (3) In MBC-treated wild-type cell, the SPB exhibited

only slight Brawnian motion Shown are single maximum intensity projections of stacks obtained by epifluorescence imaging

z-3.1.2.2 Microtubules remained attached to the SPB in alp14Δ cells

Interestingly, I found that in the absence of Mia1p-interacting protein of the MAP215 family, Alp14p, microtubules remained attached to the SPBs in the time-lapse sequence analysis of α-tubulin-GFP Sid2p-GFP expressing alp14Δ cell (100%, n=20 cells, observed for total 3h; see Figure 3.1.2.2 A), suggesting that Mia1p could function

in microtubule attachment to the nucleation site independently of Alp14p However, the SPB remained stationary in the course of the movie, suggesting that microtubules didn’t exert sufficient pushing force (see Figure 3.1.2.2 B)

Figure 3.1.2.2: Although SPBs remain stationary in alp14Δ cells, microtubules are

attached to the SPBs (A) Time-lapse sequence of alp14Δ cell expressing GFP and Sid2p-GFP Shown are the maximum intensity projections of z-stacks obtained

α-tubulin-by epifluorescence imaging Numbers refer to the time, in minutes and seconds (B) Plot

of the SPB position over time in alp14Δ cell expressing α-tubulin-GFP and Sid2p-GFP

Note that the SPB remained stationary in the course of the experiment

3.1.3 Mia1p is not required for microtubule nucleation

3.1.3.1 Microtubules could be nucleated on the preexisting microtubules in mia1Δ

cells

Since mia1Δ cells exhibited reduced numbers of microtubule bundles, I was interested in finding out whether mia1Δ cells are defective in nucleating microtubules

However, time-lapse analysis of microtubule dynamics in α-tubulin-GFP expressing

mia1Δ cell showed that microtubules could be nucleated from the NE but did not maintain their attachment to it: a growing microtubule was ejected from its nucleating site and continued to grow at its plus end, pushing the stable minus end toward the cell center (see Figure 3.1.3.1 A) Subsequently, I observed a second nucleation event, as judging by a sudden increase in fluorescence intensity in the vicinity of the minus end in kymograph (as marked by a cross in Figure 3.1.3.1 A & B) The newly born microtubule quickly formed antiparallel bundle with mother microtubule I measured microtubule growth rate and found that the resulting bundle grew at both plus ends Transition from monopolar growth to bipolar growth is shown in Figure 3.1.3.1 C

My data suggested that nucleation and bundling could occur in cells lacking Mia1p However, these cells exhibited a compromised anchoring of minus ends of microtubules to the nucleation sites

Figure 3.1.3.1: Microtubules could be nucleated on the preexisting microtubules in

mia1Δ cells (A) Time-lapse sequence of mia1Δ cell expressing α-tubulin-GFP A

microtubule nucleation event (indicated by an arrowhead) is followed by microtubule ejection from the NE (indicated by a star) A second nucleation event on the preexisting microtubule is marked by a cross Shown are single maximum intensity reconstructions

of z-stacks obtained by epifluorescence microscopy imaging Numbers refer to the time,

in minutes and seconds (B) Kymograph of the growing microtubule ejection from the

time-lapse sequence presented in Figure 3.1.3.1A Time 0 corresponds to the time of microtubule ejection from the NE (marked by a star in the Figure 3.1.3.1A) Note that microtubule initially grew at the plus end facing the cell tip, and consequently pushing its minus end toward the center of the cell A second nucleation event was followed by formation of the antiparallel bundle (marked by a cross; also see corresponding image in A) The resulting bipolar microtubule bundle continued to grow on both sides Eventually

the older bundle underwent catastrophe (C) Graph of microtubule growth rate obtained

from the kymograph

3.1.3.2 Microtubules could be nucleated from several sites around the NE in

GFP Sid2p-GFP-expressing wild type and mia1Δ cells in a perfusion chamber mounted at

the microscope stage In wild type cells, microtubules rapidly depolymerized upon addition of 50 μg/ml MBC (within 45 sec), leaving microtubule remnants that appeared

as several dots around the NE When the drug was washed out with fresh medium, repolymerization of microtubules was initiated from these dots in a bidirectional manner

(see Figure 3.1.3.2 A) In contrast in mia1Δ cells, although the process of microtubule

depolymerization was somewhat slower (~90sec) than in wild type cells, most microtubules eventually disassembled, leaving no microtubule remnant but just a single fluorescent structure representing the SPB (see Figure 3.1.3.2 B) Surprisingly, microtubules could be nucleated from several sites around the NE upon MBC wash-out (see Figure 3.1.3.2 B) However, these microtubules eventually detached In the case shown in the Figure 3.1.3.2 B, one of microtubules was undergoing catastrophe and shrinking across the cell body exhibiting a plus-end depolymerization rate of ~12μm/min (see Figure 3.1.3.2 B 11’30”-12’15”), suggesting that Mia1p was not required for

expressing the NE marker Uch2p-GFP and α-tubulin-GFP to the room temperature and performed time-lapse analyses to observe microtubule dynamics I found that

microtubule nucleation could be initiated from the NE in both wild type and mia1Δ cells

(see Figure 3.1.3.2 C&D) However, compared to wild type cells in which microtubules remained attached to the NE and nucleus was deformed by the polymerizing

microtubules when they touched cell ends (see Figure 3.1.3.2 C), in mia1Δ cells

microtubules were released from the nucleation sites and were eventually rearranged as one long microtubule bundle curving around cell tips (see Figure 3.1.3.2 D)

Figure 3.1.3.2: Microtubules could be nucleated from several sites around the NE in

mia1Δ cells (A) Wild- type cells expressing α-tubulin-GFP Sid2p-GFP were mounted in

the flow chamber allowing the time-lapse analysis of microtubule dynamics during MBC treatment (50μg/ml) and subsequent washout MBC treatment caused microtubule depolymerization, leaving stable microtubule “stubs” at the NE On washout, microtubules rapidly repolymerized from these “stubs” toward cell periphery Shown are the maximum projection image of the z-stack obtained by epifluorescence imaging

Figure 3.1.3.2: Microtubules could be nucleated from several sites around the NE in

mia1Δ cells (B) mia1Δ cells expressing α-tubulin-GFP Sid2p-GFP were mounted in the

flow chamber allowing the time-lapse analysis of microtubule dynamics during MBC treatment (50μg/ml) and subsequent washout Microtubules depolymerized after MBC treatment leaving one bright dot presented the SPB at the NE On washout with fresh medium, I detected efficient nucleation of microtubules from around the NE At least one microtubule eventually detached and underwent catastrophe (as indicated by an arrow) Shown are maximum projection images of z-stacks obtained by epifluorescence imaging Numbers refer to the time, in minutes and seconds Bar, 1μm

Figure 3.1.3.2: Microtubules could be nucleated from several sites around the NE in

mia1Δ cells (C) Time lapse sequences of cold-treated α-tubulin-GFP Uch2p-GFP

expressing wild type after temperature shift-up to 24oC Microtubules depolymerized after incubated on ice, leaving stable “stubs” at the NE On washout, microtubules rapidly

repolymerized from these “stubs” toward cell periphery (D), Time lapse sequences of

cold-treated mia1Δ cells expressing α-tubulin-GFP and Uch2p-GFP after temperature

shift-up to 24oC Note that microtubules polymerize from the nuclear surface in mia1Δ cells However, mia1Δ cells could not sustain the microtubule-nuclear envelope

attachment Shown is the maximum projection image of the z-stack obtained by epifluorescence imaging

3.1.4 Mia1p is required for maintenance of microtubule bundles

My results from microtubule depolymerization and recovery experiments

suggested that microtubule bundles were unstable in mia1Δ cells To confirm this

phenotype, I performed fast (every 5 sec) time-lapse analyses of single focal planes in

wild type and mia1Δ cells expressing α-tubulin-GFP I found that microtubule bundles

were relatively stable in wild type cells Rare events of disappearance of preexisting bundles were observed (1.2 events per bundle per hour, n=8, total time=3.5) (see Figure 3.1.4 A), and microtubule nucleation was mainly maintained to preexisting MTOCs ( I observed only 4.7 de novo nucleation events per cell per hour, n=6, total time=2.5h) In

contrast, in mia1Δ cells the lifetime of microtubule bundles was significantly reduced

(bundles disappeared with the rate of 13.6 events per bundle per hour, n=10, total time=3.5h) (see Figure 3.1.4 B), and microtubules were frequently nucleated from different sites around the NE (23 events per cell per hour, n=11, total time=3.3h) I concluded that Mia1p was not required for microtubule nucleation but was required for maintenance of microtubule bundles

Figure3.1.4: Mia1p is required for maintenance of microtubule bundles (A) Graph

and representative still images (1 to 3) following number of microtubule bundles present

in one focal plane of the wild type cell over a period of time Note that microtubule

nucleation was mainly maintained to preexisting MTOCs in wild type cells (B) Graph

and representative still images (1 to 5) following number of microtubule bundles present

in one focal plane of the mia1Δ cell over a period of time Note that microtubules were

frequently nucleated from different sites around the NE Shown is one focal plane time

lapse image with 5 seconds temporal resolution of wild type and mia1Δ cells expressing

α-tubulin-GFP

3.1.5 Mia1p is required for sustaining proper post-anaphase array dynamics

3.1.5.1 PAA dynamics is abnormal in mia1Δ cells

At the end of mitosis, the eMTOC localizes at the cell center to nucleate a specialized microtubule network called the PAA (post anaphase array), producing symmetrical radial arrays of microtubules at both sides of the eMTOC with their plus ends facing to mother cell tips (for review see Hagan 1998) I found that Mia1p-GFP localized to the equatorial γ-tublin ring (eMTOC), as judged by live GFP immunofluorescence (see Figure 3.1.5.1 A) and immunoEM analyses (see Supplementary Figure 3.1.5.1 B) Since Mia1p has been found to be involved in maintaining stable microtubule bundles during interphase, I decided to check whether any aspects of the

eMTOC/PAA dynamics could be affected in mia1Δ cells I performed time-lapse

analyses in α-tubulin-GFP wild type and mia1Δ cells In wild type cells, microtubule nucleating-structures started as two foci at the cell center in late anaphase (see Figure 3.1.5.1C, marked with stars in 1’30” time point) These foci persisted and eventually fused together to form a single ring-like structure that constricted with the actomyosin ring (see Figure 3.1.5.1C) I tracked paths of these focal structures over the course of cell division and combined them on a single superimposed image (see Figure 3.1.5.1C) My analysis confirmed that once assembled, these microtubule-nucleating structures remained fairly stable In contrast, I observed that nucleation of microtubules did occur at

the medial region of mia1Δ cells, consistent with the results obtained from interphase

cells, but that the nucleating structures were unstable (see Figure 3.1.5.1D) Tracking analysis indicated that the lifetime of each structure was short and that cells initiated multiple nucleation events from different sites at the eMTOC (see Figure 3.1.5.1D) This

behavior resulted in a poorly organized PAA and a loss of microtubule from the medial region (see Figure 3.1.5.1D)

Figure 3.1.5.1: Mia1p is required for PAA organization (A) Mia1p-GFP localizes to

both the SPBs and the eMTOC at the division site during cell division Shown is the

maximum projection image of the z-stack obtained by epifluorescence imaging (B)

Mia1p localizes to the eMTOC at the division site Immunoelectron micrography of cell expressing Mia1p-GFP stained with gold paricle-coupled anti-GFP antibodies Mia1p-GFP is found to localize on both sides of the division site (indicated by stars) Bar,

300nm (for details, see material and methods) (C)Time-lapse sequence of the dividing

wild type cells expressing α-tubulin-GFP Once established, the microtubule-organizing structures are maintained as judged by tracking from their appearance to ring constriction (as shown in C’) Stars mark the medial microtubule foci Different colors in C’ denote

independent nucleation sites (D) Time-lapse sequence of the dividing mia1Δ cell

expressing α-tubulin-GFP suggests that although microtubule can be nucleated from the eMTOC There are no persistent nucleation sites Different colors in D’ represent independent nucleation sites Ejection event is marked by the arrow at time point 1’30” Shown is the maximum projection image of the z-stack obtained by epifluorescence imaging Numbers refer to the time, in minutes and seconds Bar, 1μm

3.1.5.2 Mia1p is not required for the assembly of the eMTOC

I wondered whether the defects of PAA organization could be attributed to the

absence of the properly assembled eMTOC in mia1Δ cells However, I found no evidence

of this scenario: Several eMTOC markers, including Alp4p-GFP, Mto1p-GFP and

Rsp1p-GFP, localized to the eMTOC in both wild type and mia1Δ cells (see Figure

3.1.5.2) The eMTOC constriction and disappearance of the equatorial ring material

proceeded similarly in mia1Δ and wild type cells I concluded that although Mia1p was

not required for eMTOC assembly, it was necessary to sustain the continuing use of nucleating sites through the course of cell division

Figure 3.1.5.2: Mia1p is non-essential for the assembly of the eMTOC Proteins

associated with the γ-tubulin complex, Rsp1p-GFP, Alp4p-GFP and Mto1p-GFP localize

at the cell equator in both wild type and mia1Δ cells Shown is the maximum projection

image of the z-stack obtained by epifluorescence imaging

3.1.6 There are no detectable γ-tubulin-rich iMTOC structures in mia1Δ cells

Since nucleation of microtubules could occur from around the NE in mia1Δ cells,

suggesting that Mia1p was downstream of MTOC components, I suspected that γ-TuRC components localized normally in the absence of Mia1p and the iMTOC structures could

be detected in interphase mia1Δ cells In the steady-state interphase wild type cells,

Mto1p-GFP and Alp4p-GFP localized to the SPB and to several bright dots around the nucleus and along microtubules (see Figure 3.1.6A), likely representing the MTOCs In

mia1Δ cells, I detected only the SPB signal together with very faint and even staining

along microtubules and around the NE (see Figure 3.1.6A)

When microtubules were depolymerized by addition of MBC in both wild type

cells and mia1Δ cells expressing fluorescent MTOC markers, I detected several bright

dots around the NE in wild type cells, as expected (see Figure 3.1.6B) One of them is the SPB, and the rest are the iMTOCs Surprisingly, the MTOC markers appeared as sole dot

which represented SPB, but no iMTOC structures were detected around the NE in mia1Δ

cells after microtubule depolymerization (see Figure 3.1.6B)

I concluded that although the microtubule-nucleating complexes were active at

the NE of mia1Δ cells, they were not organized into discernible iMTOC structures,

probably reflecting the lack of microtubule/NE attachment sites Thus, it seemed that

mia1Δ cells were deficient in establishing the iMTOC structures at the NE

Figure 3.1.6: Components of the γ-tubulin complex are not found as prominent

structures around the NE or on microtubules in interphase mia1Δ cells (A)

Mto1p-GFP and Alp4p-Mto1p-GFP localize to the SPB, several large aggregates around the NE and

along microtubules in interphase wild-type cells (top panel) In mia1Δ cells, Mto1p and

Alp4p-GFP mainly localize to a single dot, most likely the SPB (bottom panel) (B) Upon

MBC treatment, components of the iMTOCs are detected as several distinct dots at the

nuclear envelope in wild-type but not in mia1Δ cells Shown is the maximum projection

image of the z-stack obtained by epifluorescence imaging

3.1.7 Microtubules are required for emergence of iMTOC structures upon entry into the new cell cycle

3.1.7.1 Why iMTOC structures could be absent from mia1Δ cells

I hypothesized that lack of large MTOC structures was related to the

disassociation of microtubule minus ends from the NE in mia1Δ cells In S pombe cells,

in addition to the iMTOCs and eMTOC, the microtubule-nucleating material is also found in weakly fluorescent dots (defined as “satellites”) traveling along the interphase

microtubules in a bi-directional manner (Zimmerman et al., 2004a) It was recently

proposed that satellites were capable of nucleating new microtubules as they traveled

along the preexisting microtubules (Janson et al., 2005) These microtubules could later slide toward the cell center (Carazo-Salas et al., 2005) and eventually get anchored at the

NE I proposed a model where fluctuations in initial distribution and/ or activity of tubulin complexes are positively reinforced when additional material is delivered to the nucleating sites via attached microtubules Thus, the resulting large structures could serve

γ-as dominant microtubule-organizing centers This positive feedback loop would be

disrupted in mia1Δ cells due to the lack of microtubule attachment to the nucleation sites,

leading to a defect in γ-tubulin complexes coalescence into large NE-bound MTOCs (see Figure 3.1.7.1)

Figure 3.1.7.1: A model of the iMTOC formation in S pombe cells Fluctuations in

initial distribution and /or activity of γ-tubulin complexes are positively reinforced when additional complexes are delivered to the nucleating sites via attached microtubules This

positive feedback loop is disrupted in mia1Δ cells due to the lack of microtubule

attachment to the nucleating sites, leading to a defect in coalescence of γ-tubulin complexes into larger MTOC structures

3.1.7.2 Experimental confirmation of hypothesis

This model predicts that emergence of interphase MTOCs in each cell cycle

should depend on microtubules To test my hypothesis, I mimicked mia1Δ phenotype in

two ways First, I disrupted microtubule organization in the cells by introducing a sensitive β-tubulin mutation nda3-KM311 into a deletion mutant of mitotic checkpoint

cold-protein, Mad2p When grown at 18oC cells undergo septation without any microtubules Then I checked the iMTOCs establishment by following the fluorescent MTOC marker Mto1p-GFP throughout the cell cycle I found that at the restrictive temperature of 18oC,

Mto1p-GFP mad2Δ nda3-KM311 cells lost all microtubules (confirmed by α-tubulin

immunofluorescent staining) and underwent septation with only one daughter cell inheriting the undivided nucleus Mto1p-GFP localized to non-separated SPBs and in an evenly fluorescent pattern around the NE (see Figure 3.1.7.2A; 85%; n=66 cells) In contrast, I detected distinct bright fluorescent structures around the NE of both daughter

cells in control Mto1p-GFP mad2Δ cells upon division (see Figure 3.1.7.2A; 95%; n=62

cells) This experiment suggested microtubules were required for establishment of the iMTOCs around the NE in each new cell cycle

Secondly I depolymerized microtubules in wild type cells by using the microtubule depolymerizing drug MBC, and checked the emergence of iMTOCs as cells entered new cell cycle I immobilized the Mto1p-GFP-expressing wild-type cells in a perfusion chamber mounted at the fluorescence microscope stage and searched for cells

that Mto1p-GFP localized to the SPB, but no detectable MTOC structures were found around the NE in majority of MBC-treated daughter cells (75%; n=38 cells), although eMTOC disassembly and septation proceeded normally, confirming my hypothesis that emergence of the iMTOCs required microtubules In contrast, nascent iMTOCs around the NE were detected in the daughter cells when MBC was added just after the onset of the eMTOC / actomyosin ring constriction, suggesting that iMTOC establishment proceeded simultaneously with the eMTOC disassembly

To test whether microtubules were required to establish iMTOCs’ structures, I prepared Mto1p-GFP cells lacking iMTOCs as described above (Figure3.1.7.2C) and allowed microtubule polymerization by washing out MBC I found that 20min after MBC washout, 100% of cells (n=20) exhibited Mto1p-GFP –positive aggregates at the NE and spread along microtubules I then briefly depolymerized microtubules in these cells to allow a better visualization of the fluorescent material and found that Mto1p-GFP appeared as several dots around the NE, indicating that microtubules were required for establishment of the iMTOCs