1. Partitioning and Remodeling of the Schizosaccharomyces japonicus Mitotic Nucleus Require Chromosome Tethers 
Candice Yam, Ying Gu, Snezhana Oliferenko 
Current Biology 
Volume 23, Issue 22, Pages (November 2013)
DOI: /j.cub
Copyright © 2013 Elsevier Ltd Terms and Conditions

2. Figure 1
Nuclear Pores Are Inherited by the Daughter Nuclei and Are Found in Close Association with Chromatin in Mitotic S. japonicus Cells
(A) A time-lapse sequence of the wild-type cell undergoing mitosis. The nuclear pore complexes (NPCs) are marked by Nup85-mCherry, and the chromatin is labeled by the histone H3-GFP. Late-segregating chromatin strands at time point 4 min 30 s are indicated by arrows.
(B) A sequence of scanning confocal images indicating the dynamics of the Dendra-tagged nucleoporin Nup189 during mitosis before and after photoconversion. Note that the photoconverted Dendra-marked NPCs (area of photoconversion is shown within a dotted outline) that initially localize to the nuclear equator move toward the poles of the elongating mother nucleus and are inherited by the daughter nuclei.
(C) A time-lapse sequence of a mitotic wild-type cell, with telomeres labeled by Taz1-mCherry and NPCs labeled by Nup85-GFP. A cluster of the NPCs remaining in the proximity of the lagging telomeres is indicated by arrows.
(D) The late-segregating chromatin strand is the short arm of chromosome III that carries the rDNA repeats, as shown by this time-lapse sequence of a wild-type cell expressing the nucleolar marker Erb1-mCherry and the rDNA marker Nhp6-GFP and carrying a LacO array integrated at the site immediately proximal to the rDNA. The LacO integration site is visualized by GFP-LacI. Note the bright LacO-GFP-LacI dots at the edge of the Nhp6-GFP-enriched chromatin strands.
(E) A time-lapse sequence of the mitotic mad2Δ cell treated with low doses of the microtubule-depolymerizing drug TBZ and expressing H3-GFP to mark chromosomes and Nup85-mCherry to mark the NPCs (upper panel). Magnified images of the lost chromosome surrounded by the Nup85-mCherry-labeled NPCs at time point 6 min 25 s are shown in the lower panel. Scale bars for close-up images represent 2.5 μm. For all panels, the maximum z projection images of spinning-disk confocal stacks are shown. Elapsed time is shown in minutes and seconds.
For all images except the lower panel in (E), scale bars represent 5 μm.
Current Biology  , DOI: ( /j.cub )
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3. Figure 2
S. japonicus Cells Lacking the LEM-Domain Protein Man1 Function Exhibit Defects in Chromatin Association with the Nuclear Periphery and Abnormal Nuclear Pore Dynamics during Anaphase of Mitosis
(A) GFP-Man1 colocalizes with Nup85-mCherry-labeled nuclear pores during mitosis. Note that both markers clear the nuclear equator as anaphase progresses, as shown in this time-lapse sequence.
(B) The NPCs visualized by Nup85-GFP do not clear the nuclear equator and remain evenly distributed around the NE in cells lacking Man1. Approximately 18% of nuclei (n = 49 cells) divide into two parts (bottom panel).
(C) A sequence of scanning confocal images indicating the dynamics of the Dendra-tagged nucleoporin Nup189 during mitosis in man1Δ cells, before and after photoconversion. Note that a large fraction of photoconverted Dendra-marked NPCs (area of photoconversion is shown within the dotted outline) remain in between the daughter nuclei.
(D) Longitudinal medial sections of the nuclei of wild-type and man1Δ cells expressing H3-GFP and Nup85-mCherry, obtained by scanning confocal sectioning and image deconvolution. Included are two examples for man1Δ genetic background. Scale bar represents 5 μm.
(E) Time-lapse sequence of a wild-type cell throughout mitosis. Telomeres are labeled by Taz1-mCherry, and the NPCs are marked by Nup189-GFP. Microtubules marked by GFP-tagged α-tubulin Atb2 indicate the mitotic progression.
(F) Time-lapse sequence of Taz1-mCherry Nup189-GFP GFP-Atb2-expressing man1Δ cell undergoing mitosis. Note that the defect in association of telomeres with the nuclear periphery in man1Δ cells is specific to anaphase B.
(G) Time-lapse sequence of a mitotic cell expressing GFP-tagged Man1ΔHEH mutant as the sole source of Man1 protein. The NPCs are marked by Nup85-mCherry. Note that similarly to man1Δ cells, the NPCs are not cleared from the nuclear equator.
(H) A time-lapse sequence of a mitotic cell expressing GFP-tagged Man1(1–488) mutant as a sole source of Man1 protein. The NPCs are marked by Nup85-mCherry. Note that GFP-Man1(1–488) is enriched at the chromatin and that, similarly to man1Δ cells, NPCs are not redistributed toward the poles of the dividing nucleus in this genetic background. Shown are the maximum z projection images of spinning-disk confocal stacks. Elapsed time is in minutes and seconds. Scale bars represent 5 μm.
Current Biology  , DOI: ( /j.cub )
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4. Figure 3
S. japonicus Cells Deficient in Man1 Function Exhibit Defects in Nucleolar Disassembly during Mitosis
(A) The mother nucleolar mass often segregates into one of the daughter nuclei in the man1Δ genetic background, as shown by color-composite time-lapse dynamics of the nucleolar marker Erb1-GFP and the NPC protein Nup189-mCherry.
(B) Time-lapse sequences of wild-type (top panel) and man1Δ (bottom panel) mitotic cells coexpressing histone H3-GFP and the nucleolar marker Erb1-mCherry. The color-composite images are shown at the bottom of each panel. Note that the abnormally segregating nucleolar mass in man1Δ cells is associated with a poorly compacted chromatin strand.
(C) A color-composite time-lapse sequence of a mitotic cell expressing the nucleolar marker Erb1-mCherry and the GFP-tagged Man1ΔHEH mutant as the sole source of Man1 protein. 11 of 18 cells exhibited unequal nucleolar partitioning, and 3 of 18 cells completely failed to disassemble the mother nucleolus.
(D) A time-lapse sequence of a mitotic cell coexpressing Erb1-mCherry and the GFP-tagged Man1(1–488) mutant as a sole source of Man1 protein. 10 of 18 cells showed unequal segregation of nucleolar material, and 4 of 18 cells did not disassemble the nucleolus. Shown are maximum z projection images of spinning-disk confocal stacks. Elapsed time is in minutes and seconds. Scale bars represent 5 μm.
Current Biology  , DOI: ( /j.cub )
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5. Figure 4
Association of Chromatin with the Nuclear Periphery during Anaphase May Promote Efficient Inheritance of the NPCs, Nucleolar Remodeling, and Formation of Equally Sized Daughter Nuclei
(A) Graph summarizing the nuclear membrane surface area (x axis) and NPC inheritance (y axis) in daughter nuclei in relation to the mother nucleus in wild-type (blue dots) and man1Δ (red dots) cells. Data were obtained from 30 mother nuclei dividing into 60 daughters. Average fluorescence intensity of Nup85-GFP was used as a proxy for NPC density. Note that the wild-type data points are clustered at the intersection of the two dotted lines, indicating that most of the mother NPCs are divided equally between the daughter nuclei and that the surface area of most daughter nuclei is approximately half that of the mother. The man1Δ cells exhibit defects in NPC recovery in daughter nuclei and irregular nuclear sizes upon division. Insets: representative images of dividing nuclei labeled by Nup85-GFP in wild-type and man1Δ cells.
(B) Boxplot showing differences in NPC recovery in daughter nuclei in wild-type (blue) and man1Δ (red) cells.
(C) An empirical cumulative distribution function plot of surface area differences within pairs of daughter nuclei in wild-type (blue) and man1Δ (red) cells. Note that lack of Man1 leads to dramatically elevated incidence of daughter pairs with unequally sized nuclei.
(D) Time-lapse sequences of GFP-AHDL dynamics in mitotic wild-type (top) and man1Δ (bottom) cells show that the NE breaks in the vicinity of the equator in the wild-type but often ruptures off-center in cells lacking Man1.
(E) Color-composite time-lapse sequences of Taz1-GFP mCherry-AHDL-expressing wild-type (top) and man1Δ (bottom) cells indicate that in the wild-type, the last pair of telomeres is associated with the location of the NE break in anaphase. The two telomeres were located directly at the opposite edges of the broken NE in 7 of 12 cells, with the rest of cells exhibiting one or both telomeres positioned just to one end of the ruptured NE. This association is not detected in cells lacking Man1. Single-plane spinning-disk confocal images are shown.
(F) Tethering of chromatin to the nuclear pores by coexpressing the GFP-binding linker construct [Man1(1–488)-GFPbinding-mCherry] and Nup189-GFP in man1Δ cells is capable of rescuing poleward redistribution of the NPCs during anaphase.
(G) Graph summarizing the nuclear membrane surface area (x axis) and NPC inheritance (y axis) in daughter nuclei in relation to the mother nucleus in Nup189-GFP man1Δ (blue dots) and Nup189-GFP man1Δ cells coexpressing the GFP-binding chromatin linker construct (red dots). Data were obtained from ten mother nuclei. Insets: representative images of dividing nuclei labeled by Nup189-GFP.
(H) Time-lapse sequence of the nucleolar marker Erb1-mCherry in the Nup189-GFP man1Δ mitotic cell coexpressing the GFP-binding chromatin linker construct. For (D), (E), (F), and (H), elapsed time is in minutes and seconds. Scale bars represent 5 μm.
(I) Diagram of progression of mitosis in wild-type (upper panel) and man1Δ (lower panel) nuclei. Pictorial legend is shown in the upper left corner.
Current Biology  , DOI: ( /j.cub )
Copyright © 2013 Elsevier Ltd Terms and Conditions