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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 promotes formation of the SPB fenestrae. (A) Time-lapse maximum z-projection images of spinning disk confocal stacks of cut11-6 or cut11-6 tts1Δ cells coexpressing indicated marker proteins at 36°C. The temperature was shifted from 24 to 36°C at 3 h before imaging. Loss of the NE integrity is indicated by leakage of the artificial nucleoplasmic marker GST-NLS-mCherry into cytoplasm in cut11-6 cells (denoted by an asterisk). The elapsed time is shown in minutes. (B) Scanning confocal micrographs of cut11-6 tts1Δ cells coexpressing Pcp1-mCherry and GFP-ADEL. The temperature was shifted from 24 to 36°C at 3 h before imaging. Shown are four serial planes from a z-stack with a step size of 0.3 μm. White arrows denote NE projection adjacent to the two mitotic SPBs. Scale bars, 5 μm. (C) Micrographs representing two sections of a cut11-6 tts1Δ cell where the mitotic SPBs are positioned within a NE bleb. Membrane boundaries are indicated by yellow dashed lines. NE, nuclear envelope; SPB, spindle pole body. Scale bars, 200 nm.
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Figure 6: Tts1 promotes formation of the SPB fenestrae. (A) Time-lapse maximum z-projection images of spinning disk confocal stacks of cut11-6 or cut11-6 tts1Δ cells coexpressing indicated marker proteins at 36°C. The temperature was shifted from 24 to 36°C at 3 h before imaging. Loss of the NE integrity is indicated by leakage of the artificial nucleoplasmic marker GST-NLS-mCherry into cytoplasm in cut11-6 cells (denoted by an asterisk). The elapsed time is shown in minutes. (B) Scanning confocal micrographs of cut11-6 tts1Δ cells coexpressing Pcp1-mCherry and GFP-ADEL. The temperature was shifted from 24 to 36°C at 3 h before imaging. Shown are four serial planes from a z-stack with a step size of 0.3 μm. White arrows denote NE projection adjacent to the two mitotic SPBs. Scale bars, 5 μm. (C) Micrographs representing two sections of a cut11-6 tts1Δ cell where the mitotic SPBs are positioned within a NE bleb. Membrane boundaries are indicated by yellow dashed lines. NE, nuclear envelope; SPB, spindle pole body. Scale bars, 200 nm.

Mentions: In closed mitosis, the nuclear membrane is rapidly remodeled to fenestrate the NE and enable integration of the duplicated SPBs in the NE without breaching the nucleocytoplasmic barrier (Gonzalez et al., 2009; Tallada et al., 2009). Cells deficient in Cut11 function lose nuclear integrity and exhibit large gaps in the NE adjacent to SPBs upon unproductive SPB insertion (Tallada et al., 2009; Figure 4A; a cell in which the nuclear integrity is lost is indicated with an asterisk). In line with this, we observed leakage of the artificial nuclear marker GST-NLS-mCherry into the cytoplasm in mitotic cut11-6 cells at the restrictive temperature of 36°C (Figure 6A; 10 of 10 cells). Of interest, mitotic cut11-6 tts1Δ cells frequently maintained NE integrity (Figure 6A; four of eight cells). In these cells, spindle microtubules were not nucleated from the SPBs throughout the course of imaging, consistent with our immunofluorescence data (Figure 4E).


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

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

Tts1 promotes formation of the SPB fenestrae. (A) Time-lapse maximum z-projection images of spinning disk confocal stacks of cut11-6 or cut11-6 tts1Δ cells coexpressing indicated marker proteins at 36°C. The temperature was shifted from 24 to 36°C at 3 h before imaging. Loss of the NE integrity is indicated by leakage of the artificial nucleoplasmic marker GST-NLS-mCherry into cytoplasm in cut11-6 cells (denoted by an asterisk). The elapsed time is shown in minutes. (B) Scanning confocal micrographs of cut11-6 tts1Δ cells coexpressing Pcp1-mCherry and GFP-ADEL. The temperature was shifted from 24 to 36°C at 3 h before imaging. Shown are four serial planes from a z-stack with a step size of 0.3 μm. White arrows denote NE projection adjacent to the two mitotic SPBs. Scale bars, 5 μm. (C) Micrographs representing two sections of a cut11-6 tts1Δ cell where the mitotic SPBs are positioned within a NE bleb. Membrane boundaries are indicated by yellow dashed lines. NE, nuclear envelope; SPB, spindle pole body. Scale bars, 200 nm.
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Figure 6: Tts1 promotes formation of the SPB fenestrae. (A) Time-lapse maximum z-projection images of spinning disk confocal stacks of cut11-6 or cut11-6 tts1Δ cells coexpressing indicated marker proteins at 36°C. The temperature was shifted from 24 to 36°C at 3 h before imaging. Loss of the NE integrity is indicated by leakage of the artificial nucleoplasmic marker GST-NLS-mCherry into cytoplasm in cut11-6 cells (denoted by an asterisk). The elapsed time is shown in minutes. (B) Scanning confocal micrographs of cut11-6 tts1Δ cells coexpressing Pcp1-mCherry and GFP-ADEL. The temperature was shifted from 24 to 36°C at 3 h before imaging. Shown are four serial planes from a z-stack with a step size of 0.3 μm. White arrows denote NE projection adjacent to the two mitotic SPBs. Scale bars, 5 μm. (C) Micrographs representing two sections of a cut11-6 tts1Δ cell where the mitotic SPBs are positioned within a NE bleb. Membrane boundaries are indicated by yellow dashed lines. NE, nuclear envelope; SPB, spindle pole body. Scale bars, 200 nm.
Mentions: In closed mitosis, the nuclear membrane is rapidly remodeled to fenestrate the NE and enable integration of the duplicated SPBs in the NE without breaching the nucleocytoplasmic barrier (Gonzalez et al., 2009; Tallada et al., 2009). Cells deficient in Cut11 function lose nuclear integrity and exhibit large gaps in the NE adjacent to SPBs upon unproductive SPB insertion (Tallada et al., 2009; Figure 4A; a cell in which the nuclear integrity is lost is indicated with an asterisk). In line with this, we observed leakage of the artificial nuclear marker GST-NLS-mCherry into the cytoplasm in mitotic cut11-6 cells at the restrictive temperature of 36°C (Figure 6A; 10 of 10 cells). Of interest, mitotic cut11-6 tts1Δ cells frequently maintained NE integrity (Figure 6A; four of eight cells). In these cells, spindle microtubules were not nucleated from the SPBs throughout the course of imaging, consistent with our immunofluorescence data (Figure 4E).

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
Related in: MedlinePlus