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Mzt1/Tam4, a fission yeast MOZART1 homologue, is an essential component of the γ-tubulin complex and directly interacts with GCP3(Alp6).

Dhani DK, Goult BT, George GM, Rogerson DT, Bitton DA, Miller CJ, Schwabe JW, Tanaka K - Mol. Biol. Cell (2013)

Bottom Line: Mzt1/Tam4 depletion also causes cytokinesis defects, suggesting a role of the γ-tubulin complex in the regulation of cytokinesis.Yeast two-hybrid analysis shows that Mzt1/Tam4 forms a complex with Alp6, a fission yeast homologue of γ-tubulin complex protein 3 (GCP3).Together our results suggest that Mzt1/Tam4 contributes to the MTOC function through regulation of GCP3(Alp6).

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Leicester, Leicester LE1 9HN, United Kingdom Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom.

ABSTRACT
In humans, MOZART1 plays an essential role in mitotic spindle formation as a component of the γ-tubulin ring complex. We report that the fission yeast homologue of MOZART1, Mzt1/Tam4, is located at microtubule-organizing centers (MTOCs) and coimmunoprecipitates with γ-tubulin Gtb1 from cell extracts. We show that mzt1/tam4 is an essential gene in fission yeast, encoding a 64-amino acid peptide, depletion of which leads to aberrant microtubule structure, including malformed mitotic spindles and impaired interphase microtubule array. Mzt1/Tam4 depletion also causes cytokinesis defects, suggesting a role of the γ-tubulin complex in the regulation of cytokinesis. Yeast two-hybrid analysis shows that Mzt1/Tam4 forms a complex with Alp6, a fission yeast homologue of γ-tubulin complex protein 3 (GCP3). Biophysical methods demonstrate that there is a direct interaction between recombinant Mzt1/Tam4 and the N-terminal region of GCP3(Alp6). Together our results suggest that Mzt1/Tam4 contributes to the MTOC function through regulation of GCP3(Alp6).

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Mzt1 is essential, and its depletion causes abnormal MT organization and cytokinesis defect. (A) Tetrad analysis of KT3449 (h90/h90 mzt1+/mzt1::ClonNAT). Only two viable colonies out of four tetrad progenies emerged. These viable colonies are ClonNAT sensitive. (B) Those that did not form viable colonies in the tetrad analysis formed microcolonies of ∼10–30 cells that show bent-cell morphology. Scale bar, 10 μm. (C) A Mzt1 shut-off strain KT3711 that also harbors mCherry-atb2 was first cultured in the MM media in the absence of thiamine to allow expression of Mzt1-GFP. The culture was then supplemented with thiamine (2 μM), and Mzt1-GFP expression was repressed for 22 and 25 h. Whole-cell extracts were prepared, and the level of Mzt1-GFP was examined by Western blotting with anti-GFP antibody. (D, E) Cells from time 0 and 25 h after Mzt1 depletion were mixed, and mCherry-Atb2 signals were observed. Cells with Mzt1-GFP signals (time-zero sample) did not show MT defects, whereas cells without Mzt1-GFP signal (indicated by yellow and orange arrowheads) showed mitotic spindle formation defects (D) or interphase MT defect (E). In D, an anaphase spindle in a nondepleted cell (green arrowhead) elongated without a delay, whereas a monopolar spindle (yellow arrowhead) did not proceed to anaphase. Cells carrying a mitotic MTOC with very low MTOC activity (orange arrowhead) were also observed. In E, interphase cells devoid of Mzt1-GFP (yellow arrowheads) have fewer MTs, indicating that the iMTOC activity was low in these cells. Scale bar, 10 μm. (F) At 25 h after depletion of Mzt1, cells with aberrant septum started to accumulate. Scale bar, 10 μm. (G) Summary of the Mzt1 depletion phenotypes. Cells depleted of Mzt1 for 0, 22, and 25 h were filmed for 20 min at 1.5-min intervals (representative images in Supplemental Figure S1) and classified into categories as graphically illustrated in Supplemental Figure S2. Longer depletion increased the population of cells with cytokinesis defect.
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Figure 3: Mzt1 is essential, and its depletion causes abnormal MT organization and cytokinesis defect. (A) Tetrad analysis of KT3449 (h90/h90 mzt1+/mzt1::ClonNAT). Only two viable colonies out of four tetrad progenies emerged. These viable colonies are ClonNAT sensitive. (B) Those that did not form viable colonies in the tetrad analysis formed microcolonies of ∼10–30 cells that show bent-cell morphology. Scale bar, 10 μm. (C) A Mzt1 shut-off strain KT3711 that also harbors mCherry-atb2 was first cultured in the MM media in the absence of thiamine to allow expression of Mzt1-GFP. The culture was then supplemented with thiamine (2 μM), and Mzt1-GFP expression was repressed for 22 and 25 h. Whole-cell extracts were prepared, and the level of Mzt1-GFP was examined by Western blotting with anti-GFP antibody. (D, E) Cells from time 0 and 25 h after Mzt1 depletion were mixed, and mCherry-Atb2 signals were observed. Cells with Mzt1-GFP signals (time-zero sample) did not show MT defects, whereas cells without Mzt1-GFP signal (indicated by yellow and orange arrowheads) showed mitotic spindle formation defects (D) or interphase MT defect (E). In D, an anaphase spindle in a nondepleted cell (green arrowhead) elongated without a delay, whereas a monopolar spindle (yellow arrowhead) did not proceed to anaphase. Cells carrying a mitotic MTOC with very low MTOC activity (orange arrowhead) were also observed. In E, interphase cells devoid of Mzt1-GFP (yellow arrowheads) have fewer MTs, indicating that the iMTOC activity was low in these cells. Scale bar, 10 μm. (F) At 25 h after depletion of Mzt1, cells with aberrant septum started to accumulate. Scale bar, 10 μm. (G) Summary of the Mzt1 depletion phenotypes. Cells depleted of Mzt1 for 0, 22, and 25 h were filmed for 20 min at 1.5-min intervals (representative images in Supplemental Figure S1) and classified into categories as graphically illustrated in Supplemental Figure S2. Longer depletion increased the population of cells with cytokinesis defect.

Mentions: To gain further insight into Mzt1 function, we generated a strain from which the mzt1 ORF was deleted. A diploid strain harboring one copy of mzt1+ and one copy of a mzt1 deletion allele marked with ClonNAT was analyzed by tetrad dissection (Figure 3A). Only two of the four spores in each tetrad set were viable, and each of these was sensitive to ClonNAT, indicating that mzt1+ is an essential gene. Cells in the microcolonies that did not grow showed bent morphology (Figure 3B) reminiscent of the alp mutants that affect microtubule regulation (Radcliffe et al., 1998).


Mzt1/Tam4, a fission yeast MOZART1 homologue, is an essential component of the γ-tubulin complex and directly interacts with GCP3(Alp6).

Dhani DK, Goult BT, George GM, Rogerson DT, Bitton DA, Miller CJ, Schwabe JW, Tanaka K - Mol. Biol. Cell (2013)

Mzt1 is essential, and its depletion causes abnormal MT organization and cytokinesis defect. (A) Tetrad analysis of KT3449 (h90/h90 mzt1+/mzt1::ClonNAT). Only two viable colonies out of four tetrad progenies emerged. These viable colonies are ClonNAT sensitive. (B) Those that did not form viable colonies in the tetrad analysis formed microcolonies of ∼10–30 cells that show bent-cell morphology. Scale bar, 10 μm. (C) A Mzt1 shut-off strain KT3711 that also harbors mCherry-atb2 was first cultured in the MM media in the absence of thiamine to allow expression of Mzt1-GFP. The culture was then supplemented with thiamine (2 μM), and Mzt1-GFP expression was repressed for 22 and 25 h. Whole-cell extracts were prepared, and the level of Mzt1-GFP was examined by Western blotting with anti-GFP antibody. (D, E) Cells from time 0 and 25 h after Mzt1 depletion were mixed, and mCherry-Atb2 signals were observed. Cells with Mzt1-GFP signals (time-zero sample) did not show MT defects, whereas cells without Mzt1-GFP signal (indicated by yellow and orange arrowheads) showed mitotic spindle formation defects (D) or interphase MT defect (E). In D, an anaphase spindle in a nondepleted cell (green arrowhead) elongated without a delay, whereas a monopolar spindle (yellow arrowhead) did not proceed to anaphase. Cells carrying a mitotic MTOC with very low MTOC activity (orange arrowhead) were also observed. In E, interphase cells devoid of Mzt1-GFP (yellow arrowheads) have fewer MTs, indicating that the iMTOC activity was low in these cells. Scale bar, 10 μm. (F) At 25 h after depletion of Mzt1, cells with aberrant septum started to accumulate. Scale bar, 10 μm. (G) Summary of the Mzt1 depletion phenotypes. Cells depleted of Mzt1 for 0, 22, and 25 h were filmed for 20 min at 1.5-min intervals (representative images in Supplemental Figure S1) and classified into categories as graphically illustrated in Supplemental Figure S2. Longer depletion increased the population of cells with cytokinesis defect.
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Related In: Results  -  Collection

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Figure 3: Mzt1 is essential, and its depletion causes abnormal MT organization and cytokinesis defect. (A) Tetrad analysis of KT3449 (h90/h90 mzt1+/mzt1::ClonNAT). Only two viable colonies out of four tetrad progenies emerged. These viable colonies are ClonNAT sensitive. (B) Those that did not form viable colonies in the tetrad analysis formed microcolonies of ∼10–30 cells that show bent-cell morphology. Scale bar, 10 μm. (C) A Mzt1 shut-off strain KT3711 that also harbors mCherry-atb2 was first cultured in the MM media in the absence of thiamine to allow expression of Mzt1-GFP. The culture was then supplemented with thiamine (2 μM), and Mzt1-GFP expression was repressed for 22 and 25 h. Whole-cell extracts were prepared, and the level of Mzt1-GFP was examined by Western blotting with anti-GFP antibody. (D, E) Cells from time 0 and 25 h after Mzt1 depletion were mixed, and mCherry-Atb2 signals were observed. Cells with Mzt1-GFP signals (time-zero sample) did not show MT defects, whereas cells without Mzt1-GFP signal (indicated by yellow and orange arrowheads) showed mitotic spindle formation defects (D) or interphase MT defect (E). In D, an anaphase spindle in a nondepleted cell (green arrowhead) elongated without a delay, whereas a monopolar spindle (yellow arrowhead) did not proceed to anaphase. Cells carrying a mitotic MTOC with very low MTOC activity (orange arrowhead) were also observed. In E, interphase cells devoid of Mzt1-GFP (yellow arrowheads) have fewer MTs, indicating that the iMTOC activity was low in these cells. Scale bar, 10 μm. (F) At 25 h after depletion of Mzt1, cells with aberrant septum started to accumulate. Scale bar, 10 μm. (G) Summary of the Mzt1 depletion phenotypes. Cells depleted of Mzt1 for 0, 22, and 25 h were filmed for 20 min at 1.5-min intervals (representative images in Supplemental Figure S1) and classified into categories as graphically illustrated in Supplemental Figure S2. Longer depletion increased the population of cells with cytokinesis defect.
Mentions: To gain further insight into Mzt1 function, we generated a strain from which the mzt1 ORF was deleted. A diploid strain harboring one copy of mzt1+ and one copy of a mzt1 deletion allele marked with ClonNAT was analyzed by tetrad dissection (Figure 3A). Only two of the four spores in each tetrad set were viable, and each of these was sensitive to ClonNAT, indicating that mzt1+ is an essential gene. Cells in the microcolonies that did not grow showed bent morphology (Figure 3B) reminiscent of the alp mutants that affect microtubule regulation (Radcliffe et al., 1998).

Bottom Line: Mzt1/Tam4 depletion also causes cytokinesis defects, suggesting a role of the γ-tubulin complex in the regulation of cytokinesis.Yeast two-hybrid analysis shows that Mzt1/Tam4 forms a complex with Alp6, a fission yeast homologue of γ-tubulin complex protein 3 (GCP3).Together our results suggest that Mzt1/Tam4 contributes to the MTOC function through regulation of GCP3(Alp6).

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Leicester, Leicester LE1 9HN, United Kingdom Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom.

ABSTRACT
In humans, MOZART1 plays an essential role in mitotic spindle formation as a component of the γ-tubulin ring complex. We report that the fission yeast homologue of MOZART1, Mzt1/Tam4, is located at microtubule-organizing centers (MTOCs) and coimmunoprecipitates with γ-tubulin Gtb1 from cell extracts. We show that mzt1/tam4 is an essential gene in fission yeast, encoding a 64-amino acid peptide, depletion of which leads to aberrant microtubule structure, including malformed mitotic spindles and impaired interphase microtubule array. Mzt1/Tam4 depletion also causes cytokinesis defects, suggesting a role of the γ-tubulin complex in the regulation of cytokinesis. Yeast two-hybrid analysis shows that Mzt1/Tam4 forms a complex with Alp6, a fission yeast homologue of γ-tubulin complex protein 3 (GCP3). Biophysical methods demonstrate that there is a direct interaction between recombinant Mzt1/Tam4 and the N-terminal region of GCP3(Alp6). Together our results suggest that Mzt1/Tam4 contributes to the MTOC function through regulation of GCP3(Alp6).

Show MeSH
Related in: MedlinePlus