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. 44 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). 45 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 bundles, whereas majority of mia1Δ cells (~62%) exhibited 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. 46 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-tubulinGFP. (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. 47 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 mia1Δ 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 mia1Δ 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 on a much broader scale around the cell center or abruptly stopped for periods of time (see graph in Figure 3.1.2.1 B2). During this period, microtubule bundle disassociated from the SPB (26%, n=19 cells, observed for a total of 9.5h; see Figure 3.1.2.1 B2). 48 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 Sid2pGFP 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 to the time, in minutes and seconds. Bar, 1μm. 49 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 and with and 4). (3) In MBC-treated wild-type cell, the SPB exhibited only slight Brawnian motion. Shown are single maximum intensity projections of zstacks obtained by epifluorescence imaging. 50 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). 51 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 α-tubulinGFP and Sid2p-GFP. Shown are the maximum intensity projections of z-stacks obtained 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. 52 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. 53 3.2.4 The SPBs ensure the NE division by preventing abnormal deformation of the NE by the mitotic spindle Since the NE was deformed by the intranuclear spindles and the tips of protrusion always lacked SPBs in Mia1p-overexpressing cells, I hypothesized that it was the SPBs that provided mechanical resistance to allow the spindle poles to push against the NE without deforming it, thus to ensure its successful division. This hypothesis was confirmed in collaboration with the laboratory of Dr. A. Khodjakov using laser microsurgery. When both SPBs were cut off, spindles continued elongation due to sliding forces produced in the spindle midzone. These spindle remnants readily formed panhandle-shaped protrusion at opposite sides of the nucleus (see Figure 3.2.4A), evocative of the phenotype observed in Mia1p-overexpressing cells (see Figure3.2.2F), and similar to what has been previously noted (Khodjakov et al., 2004). When the SPB was severed from one spindle pole, spindles deformed the NE at this location, whereas no NE deformation was observed at the opposite spindle pole still tethered to the other SPB (see Figure3.2.4B). Based on these experiments, I concluded that the SPBs ensured NE division by preventing NE deformation by the spindle. 89 Figure3.2.4: NE division fails when the SPBs are not positioned at poles of elongated mitotic spindle. (A) Time-lapse sequence of a Cut11p-GFP α-tubulin-GFP expressing wild type cell when both SPBs were severed from the elongating anaphase spindle by laser microsurgery. (B) Time-lapse sequence of a Cut11p-GFP α-tubulin-GFP expressing wild type cell when one SPB was cut off the spindle. Note that the NE is forming the panhandle shaped protrusions at the sites of contact with the elongating spindle remnants. DIC images at the end of the sequence show normally proceeding septation. Shown are maximum projection images of z-stacks obtained by time lapse spinning disk confocal imaging. Numbers refer to the time, in minutes and seconds. 90 3.2.5 Failure of chromosome segregation was not due to defects in kinetochore attachment In Mia1p-overexpressing cells, DNA masses could not properly segregate. I wondered whether failure in chromosome segregation could be due to the defects in attachment of microtubules to kinetochores. So I overexpressed Mia1p in cells carrying the mitotic checkpoint protein, Mad2p-GFP, and found that the majority of cells exhibited NE localization of Mad2-GFP, indicating that most Mia1p-overexpressing cells overcame the spindle assembly checkpoint (see Figure3.2.5) (even though approximately 12% of cells exhibited Mad2p-GFP on kinetochores, indicative of some aspect of spindle malfunction). 91 Figure3.2.5: The majority of Mia1p-over-expressing cells exhibit Mad2p-GFP on the NE. Graph quantifying the proportion of cells with Mad2p-GFP on kinetochores in wild type and cells over-expressing Mia1p (n=100). Inset displays typical appearance of Mad2p-GFP in Mia1p-over-expressing cells, either on kinetochores or evenly spread around the NE. Shown are the maximum projection images of the z-stacks obtained by epifluorescence microscopy imaging. 92 3.2.6 Genetic ablation of the NE allows chromosome segregation in Mia1poverexpressing cells Based on my data, I reasoned that should the undivided NE restrict the movement of chromosomes in cells overexpressing Mia1p, the removal of this obstacle could enable some degree of chromosome segregation through the action of acentrosomal spindles. Thus, I tested whether the Mia1p-induced spindles could segregate DNA at least at some frequency in an experimental situation in which cells underwent mitosis in the absence of the NE. Genetic ablation of the NE in Mia1p-overexpressing cells to allow cells undergoing “open mitosis” was carried out in cooperation with Dr S. Oliferenko. The temperature–sensitive mutant in the Ran-GEF, pim1-1, has been reported to induce NE fragmentation upon passage through mitosis (Demeter et al., 1995). Time-lapse analyses of spindle and NE dynamics in α-tubulin-GFP Uch2p-GFP pim1-1 cells at the permissive (see Supplementary Figure 3.2.6A) and restrictive (see Supplementary Figure 3.2.6B) temperatures suggested that lack of Pim1p function induced an irreversible NE fragmentation in early anaphase B. Spindles continued elongating (see Supplementary Figure 3.2.6B), and cells eventually underwent cytokinesis, arresting at this point in the cell cycle (Demeter et al., 1995) (see Supplementary Figure 3.2.6F). I induced overexpression of Mia1p in pim1-1 cells containing the integral SPB marker, Pcp1p-GFP, and the NE marker, Uch2p-GFP, at either permissive or restrictive temperature. Panhandle-shaped NE protrusions could be induced in pim1-1 cells at 24oC at a rate similar to control Uch1p-GFP cells (see Supplementary Figure 3.2.6C). Only 10% of Pcp1p-GFP Uch2p-GFP pim1-1 cells overexpressing Mia1p segregated DNA in two masses when the NE was intact (see Supplementary Figure 3.2.6D), consistent with the 93 above experiments. When the NE was disassembled upon shifting cells to the restrictive temperature, the proportion of binucleate cells increased considerably (32%) (see Supplementary Figure 3.2.6D). I found that few cells with spindles lacking the SPBs at least at one spindle pole exhibited two or more closely positioned DNA masses when the NE was intact (4% of cells, see Supplementary Figure 3.2.6E). However, when the NE was fragmented, I observed an appearance of divided cells with DNA segregated to daughter compartments. Upon the shift to the restrictive temperature, 27% of cells exhibiting duplicated, but not separated, SPBs on one side of the septum showed presence of chromosomes in both daughter cells (n=100, see Supplementary Figure 3.2.6F). I repeated this experiment using cells expressing the centromer I marker, Cen1-GFP, and the SPB marker, Sad1pDSRed. I found that 32% of cells (n=68) with acentrosomal spindles properly segregated sister chromatids when the NE was fragmented, suggesting that failure in NE division could hinder DNA segregation (see Supplementary Figure 3.2.6G). Taken together, I concluded that failure to divide the NE could result in restriction of DNA masses movement and, ultimately, in a chromosome segregation defect. 94 Sup-Figure3.2.6: DNA segregation occurs in cells overexpressing Mia1p when the NE is fragmented. (A) Time-lapse sequence of an Uch2p-GFP α-tubulin-GFP expressing pim1-1 cell undergoing anaphase at the permissive temperature of 24oC. (B) Time-lapse sequence of an Uch2p-GFP α-tubulin-GFP expressing pim1-1ts cell undergoing anaphase at the restrictive temperature of 36oC. Cells were shifted to the restrictive temperature hours prior to imaging to allow Pim1p protein inactivation. Note that the NE is fragmented shortly after anaphase B onset. Shown are maximum projection images of the z-stacks obtained by epifluorescence microscopy imaging. Numbers refer to the time, in minutes. 95 96 Sup-Figure3.2.6: DNA segregation occurs in cells overexpressing Mia1p when the NE is fragmented. (C) Shown are maximum projection images of the z-stack obtained by epifluorescence microscopy images of the NE labeled by Uch2p-GFP in wild type and Mia1p-overexpressing pim1-1 cells at 24oC. Note that overexpression of Mia1p induces abnormal NE deformations in pim1-1 cells at the permissive temperature (top panel). Graph representing the proportion of cells forming the panhandle-shaped protrusions in both genetic backgrounds (bottom panel). (D) Quantification of binucleate Pcp1p-GFP Uch2p-GFP pim1-1 cells over-expressing Mia1p at 24oC and 36oC (n=300). (E) Shown are maximum projection images of the z-stack obtained by epifluorescence images of formaldehyde fixed Mia1p-over-expressing pim1-1 cells expressing the core SPB marker, Pcp1p-GFP, and the outer NE marker, Uch2p-GFP, at 24oC. DNA is stained with DAPI. Note that although it is rare, phenotype exhibiting two or more closely positioned DNA masses at the permissive temperature could be observed in pim1-1 cells overexpressing Mia1p with acentrosomal spindles. (F) Graph quantifying the proportion of uninucleate and binucleate Mia1p-over-expressing Pcp1p-GFP Uch2p-GFP pim1-1 cells that not separate the SPBs at 36oC (n=100). Insets, examples of the observed phenotypes. Shown are maximum projection images of the z-stacks obtained by epi-fluorescence microscopy images of fixed Mia1p-over-expressing pim1-1 cells expressing the core SPB marker, Pcp1p-GFP, and the outer NE marker, Uch2p-GFP, at 36oC upon NE fragmented. DNA is stained with DAPI. 97 3.3 Role of Mia1p in maintenance of microtubule polarity 3.3.1 Mia1p was required for symmetrical distribution of cortical tip proteins at cell tips Since mia1Δ cells had been shown to exhibit altered cell shape (Oliferenko and Balasubramanian, 2002), I was interested in establishing localizations of cortical tip proteins in mia1Δ genetic background. Immunofluorescence analyses revealed that both cell end marker, Tea1p-GFP (expressed from its native promoter) spread symmetrically at cell tips and also localized to the microtubule plus ends in wild type cells (see Figure 3.3.1A). Although it also appeared at microtubule plus ends in mia1Δ cells, they were asymmetrically localized to cell tips due to the curving interphase microtubule bundles (see Figure 3.3.1B). The asymmetrical distribution of cortical tip protein illuminated that cells lacking Mia1p were deficient in maintaining the normal axis of growth and failed to position growth sites correctly, thus forming bent cell shapes. 98 Figure3.3.1: Symmetrical cell tip localization of the cell end marker requires Mia1p. Compared to wild type cells expressing Tea1p-GFP (A), symmetrical localization of Tea1p-GFP to the cell tips is disturbed in mia1Δ cells (B). Note that Tea1p-GFP localized to the ends of curved microtubule bundles in mia1Δ cells. Shown are one plane images obtained by epifluorescence microscopy of formaldehyde fixed cells using anti-GFP antibodies (left panel) and α-tubulin antibody (middle panel). The merged image is shown in the right panel. 99 3.3.2 Polarity of microtubules is altered in mia1Δ cells To further investigate role of Mia1p in efficient distribution of cortical tip proteins, I performed time lapse analyses of plus end directed kinesin, Tea2p-GFP, in wild type and mia1Δ cells. Tea2p-GFP localized to cell tips and appeared as dots representing plus ends of microtubules, in both wild type and mia1Δ cells. I found that in wild type cells, Tea2p-GFP was delivered from cell center to cell tips. It was mainly unidirectional movement (see Figure 3.3.2A). In contrast, Tea2p-GFP moved around cell tips in mia1Δ cells. Some Tea2p-GFP was delivered back from cell tips to the cell center. It appeared to be bidirectional (see Figure 3.3.2B), indicating that either polarity of interphase microtubules was altered in the cells lacking Mia1p or Tea2p had troubles in unloading from plus end of microtubule to cell tips. The Kar3-type kinesin, Klp2p, is involved in sliding of newly nucleated microtubules towards cell center along preexisting microtubules to maintain anti-parallel configuration of interphase microtubule arrays (Carazo-Salas et al., 2005). Time lapse analyses of Klp2p dynamics revealed that Klp2p-GFP dots often moved from cell tips to the cell center in wild type cells, consistent with sliding function of Klp2p (see Figure 3.3.2C). In contrast, in mia1Δ cells I found that some Klp2p-GFP dots moved from cell center to cell tips (see Figure3.3.2D). I traced Klp2p-GFP in wild type and mia1Δ cells and found that moving rates of dots were quite similar in wild type cells (n=3, 3.9μm/min) compared to mia1Δ cells (n=10, 3.44 μm/min), indicating that function of Klp2p was not affected in mia1Δ cells. Thus I concluded that change in moving direction was likely due to disruption of antiparallel microtubule arrangement in mia1Δ cells. 100 To further support my conclusion that polarity of interphase microtubules was altered in mia1Δ cells, I carried out the fluorescence recovery after photobleaching (FRAP) experiments. Wild type and mia1Δ cells expressing α-tubulin-GFP were bleached in lateral microtubule surface. I found that in wild type cells the photobleached region could recover from cell tip to cell center, presumedly due to sliding of newly nucleated microtubules by Klp2p (see Figure 3.3.2E). Unlike in wild type cells, I found that in mia1Δ cells, bleached region moved from left to right, eventually crossing the nuclear region (see Figure 3.3.2F). I concluded that minus ends of microtubules didn’t anchor at the NE. Based on my data, I concluded that Mia1p was involved in organizing interphase microtubule arrays in fission yeast. 101 Figure3.3.2: Orientation of microtubules is altered in mia1Δ cells. (A) Shown is timelapse image obtained by epifluorescence imaging of wild type cells expressing Tea2pGFP. Note that Tea2p moved unidirectional from cell center to the cell tips. (B) Shown is time-lapse image obtained by epifluorescence imaging of mia1Δ cell expressing Tea2pGFP. Note that Tea2p movement is bi-directional, from cell center towards cell tips and from cell tips towards cell center. 102 Figure3.3.2 Orientation of microtubules is altered in mia1Δ cells. (C) In wild type cells, Klp2p-GFP moved from cell tips to the cell center. (D) Klp2p-GFP movement is bidirectional in mia1Δ cells, from cell tips to cell center and from cell center to cell tips. Shown are the maximum projection images of the z-stacks obtained by time-lapse spinning disk confocal microscopy of cells expressing Klp2p-GFP. 103 Figure3.3.2: Orientation of microtubules is altered in mia1Δ cells. Shown is time lapse images obtained by scanning confocal microscopy images of α-tubulin-GFP fluorescence recovery after photobleaching at lateral surface of microtubule bundles in wild type (E) and mia1Δ (F) cells. Note that the photobleached region moved across nuclear region in mia1Δ cells. 104 [...]... overexpressing Mia1p arrest in mitosis with fully elongated spindles and nonsegregated chromosomes 3.2.1.1 Mia1p- overexpressing cells arrest in mitosis Mia1p was found to be involved in anchoring minus ends of microtubules to the NE and for the proper organization of mitotic spindle (Oliferenko and Balasubramanian, 2002; Sato et al., 20 04) in my previous studies To further investigate functions of Mia1p, ... required for maintenance of microtubule bundles 61 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... presence of actomyosin rings in Mia1p- overexpressing cells as judged by localization of the myosin II light chain, Rlc1p-GFP (see Figure 3.2.1.1B) Quantification of proportion of cells exhibiting mitotic localization of marker proteins is shown in graph (see Figure 3.2.1.1C) I concluded that cells overexpressing Mia1p arrested in mitosis 76 Figure 3.2.1.1 Fission yeast cells over-expressing Mia1p arrest in. .. 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... 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. .. 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... structures (right panel) In B and C, shown is the maximum projection image of z-stacks obtained by epifluorescence imaging 74 3.1.7.3 Model of establishment of the iMTOCs in fission yeast I proposed a model where fluctuations in initial distribution and / or activity of γtubulin complexes are positively reinforced when additional material is delivered to the nucleating sites via attached microtubules (see... 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. .. 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... leading to a defect in γ-tubulin complexes coalescence into large NE-bound MTOCs (see Figure 3.1.7.1) 70 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 . 44 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. that Mia1p binds microtubules and is a component of S. pombe MTOCs. 45 Figure 3.1.1.1: Microtubule- associated protein Mia1p binds microtubules and is a component of S. pombe. imaging. 51 3.1.2.2 Microtubules remained attached to the SPB in alp 14 cells Interestingly, I found that in the absence of Mia1p- interacting protein of the MAP215 family, Alp14p, microtubules