1. Volume 38, Issue 5, Pages 463-477 (September 2016)
A Nucleoporin Docks Protein Phosphatase 1 to Direct Meiotic Chromosome Segregation and Nuclear Assembly 
Neil Hattersley, Dhanya Cheerambathur, Mark Moyle, Marine Stefanutti, Amelia Richardson, Kian-Yong Lee, Julien Dumont, Karen Oegema, Arshad Desai 
Developmental Cell 
Volume 38, Issue 5, Pages (September 2016)
DOI: /j.devcel
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2. Developmental Cell 2016 38, 463-477DOI: (10.1016/j.devcel.2016.08.006)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1 MEL-28 Is Required for Meiosis I Chromosome Segregation
(A) Schematic highlighting why oocyte meiosis I in C. elegans provides unique access for analyzing chromosome segregation functions of Y-complex nucleoporins. See text for details. The essential nucleoporin depleted here is MEL-28; images for NPP-6/Nup160 depletion are shown in Figure S1A. Scale bars, 10 μm.
(B) Schematics of Y complex (box) and of meiotic kinetochore protein localization in C. elegans oocytes. Images below are of the spindle region of live meiosis I prometaphase oocytes expressing GFP fusions of Y-complex subunits together with mCherry::H2b. Scale bars, 5 μm. Immunofluorescence is shown on the bottom row for the outer kinetochore protein KNL-1 (red) and MEL-28 (green; detected using an antibody to GFP); DNA was visualized using Hoechst (blue). Scale bars, 2 μm. See also Figure S1A.
(C) Stills at indicated times from time-lapse sequences of oocyte meiosis I chromosome segregation for the indicated conditions. Scale bar, 5 μm. See also Figure S1C.
(D) (Top) Images from time-lapse sequences of oocyte meiosis I for control and mel-28(RNAi). Dashed line indicates anterior embryo edge and schematic above highlights imaged region. Green arrowheads denote univalent chromosomes. Time is in seconds relative to metaphase I. Scale bar, 5 μm. (Bottom) Plot of mean homologous chromosome separation over time (relative to metaphase I), measured for the central bivalent as indicated in the schematic (box). Error bars represent SD and n the number of embryos analyzed.
(E) (Top) Schematic of MEL-28 domain organization and position of stop codon in t1684. (Bottom) Representative images from time-lapse sequences in heterozygous and homozygous mel-28(t1684) oocytes. Scale bar, 5 μm.
See also Figure S1.
Developmental Cell  , DOI: ( /j.devcel )
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4. Figure 2
MEL-28 Acts after Anaphase Onset to Segregate Homologous Chromosomes
(A) Representative images of the oocyte meiosis I spindle region from a time-lapse sequence of GFP::MEL-28 and KNL-1::mCherry. Scale bar, 5 μm.
(B) Fixation and immunofluorescence analysis of MEL-28 localization during anaphase I. MEL-28 is detected using an antibody to GFP (green) and microtubules labeled using an antibody to α-tubulin (red). DNA was visualized using Hoechst (blue). Scale bar, 2 μm.
(C) Analysis of securinIFY-1::GFP loss. (Top) Representative images of securinIFY-1::GFP in a control meiosis I embryo. (Bottom) Plot of mean cytoplasmic securinIFY-1::GFP fluorescence intensity over time, relative to intensity at 100 s prior to metaphase I. n represents the number of embryos analyzed and error bars the SD. Scale bar, 20 μm.
(D) (Left) Images of the meiosis I spindle region from time-lapse sequences of embryos expressing GFP::β-tubulin and mCherry::H2b. (Right) Representative 5-pixel-wide linescans (drawn as indicated in schematic) of GFP::β-tubulin fluorescence intensity at indicated times in control and mel-28(RNAi). Scale bar, 5 μm.
See also Figure S2.
Developmental Cell  , DOI: ( /j.devcel )
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5. Figure 3
The Region of MEL-28 Required for Meiotic Chromosome Segregation Contains a Docking Motif for Protein Phosphatase 1
(A) Summary of analysis of meiosis I chromosome segregation for the indicated MEL-28 truncations, expressed from single-copy transgene insertions, following endogenous MEL-28 depletion. Six or more oocytes were filmed per truncation.
(B) Representative images from oocyte meiosis I time-lapse sequences of mCherry::H2b for the indicated conditions. Color-merge panels on the right depict metaphase GFP signal of indicated fusions. Scale bar, 5 μm.
(C) Sequence alignment of the region of MEL-28 required for oocyte meiotic chromosome segregation in nematodes (upper set) and vertebrates (lower set). The bipartite PP1 docking motif is indicated. Residues altered to create the SARA mutant are boxed in red.
(D) Yeast two-hybrid analysis of a WT or SARA mutant MEL-28 fragment with C. elegans PP1c orthologs GSP-1 and GSP-2. KNL-1 serves as a positive control.
(E) Biochemical analysis of MEL-28-GSP-2 interaction. MBP fused to amino acids 1,047–1,098 or 954–1,189 of MEL-28 (WT or SARA) was bound to amylose beads and used to pull down GST-fused GSP-2; MBP alone-coated beads served as a negative control. Bead eluates were analyzed by SDS-PAGE and Coomassie staining.
(F) Representative metaphase I images of GFP::MEL-28 (WT or SARA) following endogenous MEL-28 depletion. Scale bar, 2 μm. (Bottom plot) Mean ± SD integrated GFP intensity in the spindle region at metaphase I; values were normalized to the WT mean value. (Right plot) Embryo viability (mean ± SD) following endogenous MEL-28 depletion. n represents number of worms; >1,000 embryos were scored per condition. n.s., not significant.
(G) (Left) Images from time-lapse sequences of oocyte meiosis I monitored using mCherry::H2b, for the indicated conditions. (Right) Plot of mean separation of the homologous chromosomes in the central bivalent, measured 300 s after metaphase I. n represents the number of embryos analyzed and error bars the SD. Scale bar, 2 μm.
See also Figure S3.
Developmental Cell  , DOI: ( /j.devcel )
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6. Figure 4
The PP1c Docking Motif in MEL-28 Recruits PP1c/GSP-2 to Chromosomes in Anaphase and Is Required for Meiotic Segregation
(A) Localization of GSP-2::GFP (left) or GFP::MEL-28 (right) and chromosomes (mCh::H2b) during meiosis I. GFP::MEL-28 images are from a strain homozygous for the t1684 mutant of mel-28. Scale bars, 2 μm.
(B) Kymographs of chromosomal GSP-2::GFP and mCherry::H2b during oocyte meiosis I for indicated conditions. Mean integrated GSP-2::GFP intensity in the spindle region at indicated time points is plotted on the right. Intensity variation due to depth was corrected using the mCherry::H2b signal (Figures S4A and S4B) and the control metaphase I mean value was set to 100 a.u. Error bars represent SD.
(C) Representative images of GSP-2 localization in WT versus SARA forms of MEL-28 at indicated time points. Scale bar, 5 μm. (Right) Mean GSP-2::GFP integrated intensity, measured as in (B), at the indicated time points for WT versus SARA forms of GFP::MEL-28. Error bars represent SD. n.s., not significant.
See also Figure S4.
Developmental Cell  , DOI: ( /j.devcel )
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7. Figure 5
PP1c Docking on MEL-28 Directs Chromosome Segregation by Promoting Kinetochore Disassembly
(A) Kinetochore disassembly, monitored using kymographs in a strain expressing GFP::KNL-3 and mCherry::H2b, for control and mel-28(RNAi).
(B) Mean integrated GFP::KNL-3 fluorescence intensity over time in the spindle region, in control and mel-28(RNAi). Signal variation due to depth was corrected using the mCherry::H2b signal (Figures S4A and S4B) and the control metaphase I mean value was set to 100 a.u. Error bars represent SD.
(C) Images of meiosis I chromosome segregation in the indicated conditions, monitored using time-lapse imaging of GFP::β-tubulin and mCherry::H2b. Panels to the right are kymographs of the mCherry::H2b signal. Scale bars, 2 μm.
(D) Plot of mean separation of the homologous chromosomes in the central bivalent, measured 300 s after metaphase I, for the indicated conditions. n represents number of embryos analyzed and error bars the SD.
See also Figure S5.
Developmental Cell  , DOI: ( /j.devcel )
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8. Figure 6
Docking of PP1c on MEL-28 Is Required for Nuclear Assembly in One-Cell Embryos
(A) MEL-28 localization in live (top) and fixed (bottom) one-cell embryos. Endogenous MEL-28 was depleted in the live imaging analysis. Scale bars, 5 μm.
(B) (Top) Images from a time-lapse sequence illustrating pronuclear expansion and migration, monitored using mCherry::H2b, in one-cell embryos. Times are relative to full regression of the pseudocleavage furrow (white arrow). (Bottom) Representative image of a mel-28(RNAi) one-cell embryo and plot of mean sperm nucleus diameter 300 s prior to pseudocleavage furrow. n represents number of embryos analyzed and error bars the SD. Scale bar, 5 μm.
(C) Representative images of one-cell embryos with magnified views of sperm nuclei and plot of mean sperm nucleus diameter 300 s prior to full regression of the pseudocleavage furrow. Error bars are SD. Scale bars, 5 μm.
(D) Immunofluorescence images of one-cell embryos for the indicated conditions labeled with mAb414 and Hoechst dye (to detect DNA). The mAb414 signal of the sperm nucleus is magnified to the right of each embryo panel. Scale bars, 2 μm.
(E) (Top) Images of the sperm nucleus from time-lapse sequences of one-cell embryos expressing endogenous locus-tagged GFP::GSP-2. Five-pixel-wide linescans through the center of the nucleus at each time point are shown below. Time is relative to complete regression of the pseudocleavage furrow. (Bottom) Images show GFP::GSP-2 and mCh::H2b signals for the indicated conditions 400 s prior to full regression of the pseudocleavage furrow. Scale bar, 5 μm.
(F) Representative images from a time-lapse sequence of the nuclear/spindle region in one-cell embryos expressing GFP::GSP-2 and mCherry::H2b. Scale bar, 5 μm.
See also Figure S6.
Developmental Cell  , DOI: ( /j.devcel )
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9. Figure 7
A Kinetochore Nucleoporin-Docked Phosphatase Pool Promotes Kinetochore Disassembly and Nuclear Assembly during M-Phase Exit
Schematic summarizing the two major roles proposed for MEL-28/ELYS-docked PP1c during M-phase exit. See text for details.
Developmental Cell  , DOI: ( /j.devcel )
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