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Tts1, the fission yeast homologue of the TMEM33 family, functions in NE remodeling during mitosis.

Zhang D, Oliferenko S - Mol. Biol. Cell (2014)

Bottom Line: An amphipathic helix located at the C-terminus of Tts1 is important for ER shaping and modulating the mitotic NPC distribution.Of interest, the evolutionarily conserved residues at the luminal interface of the third transmembrane region function specifically in promoting SPB-NE insertion.Our data illuminate cellular requirements for remodeling the NE during "closed" nuclear division and provide insight into the structure and functions of the eukaryotic TMEM33 family.

View Article: PubMed Central - PubMed

Affiliation: Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604 zhangdan@tll.org.sg snezhana.oliferenko@kcl.ac.uk.

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Tts1 functions with Cut11 in bipolar spindle formation. (A) Morphology of microtubule arrays including the mitotic spindles (marked by GFP-Atb2) and the SPBs (marked by Pcp1-mCherry) in WT and cut11-6 cells at the restrictive temperature of 36°C. Nuclear integrity is monitored by localization of the artificial nucleoplasmic marker GST-NLS-mCherry. Cells were shifted from 24 to 36°C at 2 h before imaging. Note that nuclear integrity is lost in cut11-6 cells during mitosis (denoted by an asterisk). Shown are the maximum projections of z-stacks obtained from spinning disk confocal microscopy. (B) Extra copy of tts1 enables cut11-6 cells to grow at 36°C. (C) cut11-6 tts1Δ cells fail to form colonies at the intermediate temperature of 30°C when a single cut11-6 mutant grows normally. (D) Maximum z-projection images of spinning disk confocal stacks of cells with indicated genetic backgrounds coexpressing GFP-Atb2, Pcp1-mCherry and GST-NLS-mCherry at 24°C. (E) Quantification of spindle-related phenotypes was performed in fixed cells of the indicated genetic backgrounds at 24 and 36°C (n = 300). Cells were shifted from 24 to 36°C at 3 h before fixation. Three categories of spindle morphologies are defined in magnified views of cells in D. Scale bars, 5 μm.
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Figure 4: Tts1 functions with Cut11 in bipolar spindle formation. (A) Morphology of microtubule arrays including the mitotic spindles (marked by GFP-Atb2) and the SPBs (marked by Pcp1-mCherry) in WT and cut11-6 cells at the restrictive temperature of 36°C. Nuclear integrity is monitored by localization of the artificial nucleoplasmic marker GST-NLS-mCherry. Cells were shifted from 24 to 36°C at 2 h before imaging. Note that nuclear integrity is lost in cut11-6 cells during mitosis (denoted by an asterisk). Shown are the maximum projections of z-stacks obtained from spinning disk confocal microscopy. (B) Extra copy of tts1 enables cut11-6 cells to grow at 36°C. (C) cut11-6 tts1Δ cells fail to form colonies at the intermediate temperature of 30°C when a single cut11-6 mutant grows normally. (D) Maximum z-projection images of spinning disk confocal stacks of cells with indicated genetic backgrounds coexpressing GFP-Atb2, Pcp1-mCherry and GST-NLS-mCherry at 24°C. (E) Quantification of spindle-related phenotypes was performed in fixed cells of the indicated genetic backgrounds at 24 and 36°C (n = 300). Cells were shifted from 24 to 36°C at 3 h before fixation. Three categories of spindle morphologies are defined in magnified views of cells in D. Scale bars, 5 μm.

Mentions: Cut11 is an essential nucleoporin that is enriched at the mitotic SPBs when they are embedded within the NE (West et al., 1998). At the restrictive temperature of 36ºC, cells carrying the temperature sensitive (ts) alleles of cut11 do not properly insert the mitotic SPBs in the NE and fail to form bipolar spindles and complete mitosis (West et al., 1998; Figure 4A). Curiously, we initially identified Tts1 as a high-copy suppressor of a tight temperature-sensitive allele of cut11, cut11-6, which carries a missense mutation causing Thr-to-Ile substitution at position 498 (Figure 4B and Supplemental Figure S3A). Of interest, among the other five previously isolated cut11 ts mutants, that is, cut11-1 to cut11-5 (West et al., 1998; Supplemental Figure S3A), increase in Tts1 levels could also suppress cut11-2/3/4/5 but not cut11-1 at 36°C (Supplemental Figure S3B). At the same time, tts1Δ displayed negative genetic interactions with all six cut11 ts alleles, including cut11-1, albeit in a milder manner as compared with the rest (Figure 4C and Supplemental Figure S3C).


Tts1, the fission yeast homologue of the TMEM33 family, functions in NE remodeling during mitosis.

Zhang D, Oliferenko S - Mol. Biol. Cell (2014)

Tts1 functions with Cut11 in bipolar spindle formation. (A) Morphology of microtubule arrays including the mitotic spindles (marked by GFP-Atb2) and the SPBs (marked by Pcp1-mCherry) in WT and cut11-6 cells at the restrictive temperature of 36°C. Nuclear integrity is monitored by localization of the artificial nucleoplasmic marker GST-NLS-mCherry. Cells were shifted from 24 to 36°C at 2 h before imaging. Note that nuclear integrity is lost in cut11-6 cells during mitosis (denoted by an asterisk). Shown are the maximum projections of z-stacks obtained from spinning disk confocal microscopy. (B) Extra copy of tts1 enables cut11-6 cells to grow at 36°C. (C) cut11-6 tts1Δ cells fail to form colonies at the intermediate temperature of 30°C when a single cut11-6 mutant grows normally. (D) Maximum z-projection images of spinning disk confocal stacks of cells with indicated genetic backgrounds coexpressing GFP-Atb2, Pcp1-mCherry and GST-NLS-mCherry at 24°C. (E) Quantification of spindle-related phenotypes was performed in fixed cells of the indicated genetic backgrounds at 24 and 36°C (n = 300). Cells were shifted from 24 to 36°C at 3 h before fixation. Three categories of spindle morphologies are defined in magnified views of cells in D. Scale bars, 5 μm.
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Figure 4: Tts1 functions with Cut11 in bipolar spindle formation. (A) Morphology of microtubule arrays including the mitotic spindles (marked by GFP-Atb2) and the SPBs (marked by Pcp1-mCherry) in WT and cut11-6 cells at the restrictive temperature of 36°C. Nuclear integrity is monitored by localization of the artificial nucleoplasmic marker GST-NLS-mCherry. Cells were shifted from 24 to 36°C at 2 h before imaging. Note that nuclear integrity is lost in cut11-6 cells during mitosis (denoted by an asterisk). Shown are the maximum projections of z-stacks obtained from spinning disk confocal microscopy. (B) Extra copy of tts1 enables cut11-6 cells to grow at 36°C. (C) cut11-6 tts1Δ cells fail to form colonies at the intermediate temperature of 30°C when a single cut11-6 mutant grows normally. (D) Maximum z-projection images of spinning disk confocal stacks of cells with indicated genetic backgrounds coexpressing GFP-Atb2, Pcp1-mCherry and GST-NLS-mCherry at 24°C. (E) Quantification of spindle-related phenotypes was performed in fixed cells of the indicated genetic backgrounds at 24 and 36°C (n = 300). Cells were shifted from 24 to 36°C at 3 h before fixation. Three categories of spindle morphologies are defined in magnified views of cells in D. Scale bars, 5 μm.
Mentions: Cut11 is an essential nucleoporin that is enriched at the mitotic SPBs when they are embedded within the NE (West et al., 1998). At the restrictive temperature of 36ºC, cells carrying the temperature sensitive (ts) alleles of cut11 do not properly insert the mitotic SPBs in the NE and fail to form bipolar spindles and complete mitosis (West et al., 1998; Figure 4A). Curiously, we initially identified Tts1 as a high-copy suppressor of a tight temperature-sensitive allele of cut11, cut11-6, which carries a missense mutation causing Thr-to-Ile substitution at position 498 (Figure 4B and Supplemental Figure S3A). Of interest, among the other five previously isolated cut11 ts mutants, that is, cut11-1 to cut11-5 (West et al., 1998; Supplemental Figure S3A), increase in Tts1 levels could also suppress cut11-2/3/4/5 but not cut11-1 at 36°C (Supplemental Figure S3B). At the same time, tts1Δ displayed negative genetic interactions with all six cut11 ts alleles, including cut11-1, albeit in a milder manner as compared with the rest (Figure 4C and Supplemental Figure S3C).

Bottom Line: An amphipathic helix located at the C-terminus of Tts1 is important for ER shaping and modulating the mitotic NPC distribution.Of interest, the evolutionarily conserved residues at the luminal interface of the third transmembrane region function specifically in promoting SPB-NE insertion.Our data illuminate cellular requirements for remodeling the NE during "closed" nuclear division and provide insight into the structure and functions of the eukaryotic TMEM33 family.

View Article: PubMed Central - PubMed

Affiliation: Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604 zhangdan@tll.org.sg snezhana.oliferenko@kcl.ac.uk.

Show MeSH