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

Abstract

The fission yeast Schizosaccharomyces pombe undergoes "closed" mitosis in which the nuclear envelope (NE) stays intact throughout chromosome segregation. Here we show that Tts1, the fission yeast TMEM33 protein that was previously implicated in organizing the peripheral endoplasmic reticulum (ER), also functions in remodeling the NE during mitosis. Tts1 promotes insertion of spindle pole bodies (SPBs) in the NE at the onset of mitosis and modulates distribution of the nuclear pore complexes (NPCs) during mitotic NE expansion. Structural features that drive partitioning of Tts1 to the high-curvature ER domains are crucial for both aspects of its function. 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.

FIGURE 1:

Evolutionarily conserved motifs in Tts1 are required for its partitioning to the tubular ER. (A) Predicted structural features and conserved motifs in the TMEM33 protein Tts1 in S. pombe. Note that the motif shown in red is conserved throughout the TMEM33 family, whereas the motif in blue contains two charged arginines that are highly conserved among its fungal orthologues. Highlighted are conserved residues that were mutated in this study. The helical wheel showing that helix 3 in Tts1 C-terminus potentially has amphipathic properties was generated using HeliQuest (http://heliquest.ipmc.cnrs.fr/). Mutated residues at the hydrophobic side are indicated by red points. (B) Localization of GFP-fused Tts1 mutants in tts1Δ cells expressing Rtn1-mCherry. The cartoons show the logic of construction of Tts1 mutants. Scanning confocal micrographs of cells expressing indicated proteins were taken from either top or middle (for Tts1-Cter) planes of z-stacks. Scale bars, 5 μm. Normalized colocalization factors shown in red indicate the significant decrease of specificity in the tubular ER localization of the corresponding Tts1 mutants (mean ± SD; 10 < n < 35). *p ≪ 10⁻⁴ in comparison to the wild type; two-tailed Student's t test.

FIGURE 2:

Genetic complementation analysis reveals domains important for Tts1 ER-shaping function. (A) Epifluorescence images of Calcofluor-stained cells with indicated genotypes. (B) Quantification of the division septa positioning phenotypes in cells of indicated genetic backgrounds (500 < n < 1500). o/c, off-center. Tts1 mutants that did not rescue the division site mispositioning in Δtry cells are indicated by red asterisks. Mutants that were partially compromised in correcting division site positioning defects in Δtry cells are denoted by blue asterisks.

FIGURE 3:

Tts1 is required for maintaining normal morphology of the dividing nuclear envelope. (A, B) Time-lapse maximum z-projection images of wild-type (WT) and tts1Δ cells coexpressing indicated marker proteins. The abnormal NE protrusions are marked by arrows. (C) Time-lapse maximum z-projection images of tts1Δ cells coexpressing the artificial luminal ER marker protein mCherry-ADEL and Cut11-GFP. Bottom, middle stacks from the framed region with 2× magnification. The clusters of Cut11-marked NPCs at the ER-NE junctions are indicated by arrows. The elapsed time is shown in minutes. Scale bars, 5 μm.

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.

FIGURE 5:

The mitotic SPBs are compromised in recruiting the γ-tubulin complex in cut11-6 tts1Δ cells. (A) Time-lapse maximum z-projection images of spinning disk confocal stacks of cells with indicated genetic backgrounds coexpressing GFP-Atb2, Pcp1-mCherry, and the nucleoplasm marker protein GST-NLS-mCherry. An aster-like short spindle in early mitotic tts1Δ cells is denoted by an arrow. (B, C) Time-lapse maximum z-projection images of spinning disk confocal stacks of cells with indicated genetic backgrounds coexpressing Pcp1-mCherry and Alp4-GFP (B) or Alp6-GFP (C). Cells were grown and imaged at 24°C, the permissive temperature for cut11-6 mutant. The elapsed time is shown in minutes. Scale bars, 5 μm.

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.

FIGURE 7:

Evolutionarily conserved TMEM33 motifs are required for Tts1 function in SPB-NE interaction and spindle assembly. (A) Quantification of spindle morphologies in cells with the indicated genetic backgrounds at 24°C (n = 500). Spindle morphologies are categorized as in Figure 4D. The red asterisks highlight Tts1 mutants that were unable to rescue spindle defects in cut11-6 tts1Δ cells. Tts1 mutants that were partially compromised in correcting spindle abnormalities in cut11-6 tts1Δ cells are indicted by blue asterisks. (B) Diagram showing structural features of Tts1 important for its functions in structuring the cortical ER and the mitotic NE and in the mitotic SPB-NE insertion. A possible topology of the third transmembrane domain (TM3) with four conserved residues is shown as well.