A Conserved Disruption of the Nuclear Permeability Barrier in Meiosis is Controlled by a Kinase-Phosphatase Pair in Saccharomyces cerevisiae

Abstract

In eukaryotic organisms, the nucleus is remodeled to accommodate the space required for chromosome segregation. Remodeling strategies range from closed division, where the nuclear envelope remains intact, to open divisions, where the nuclear envelope is temporarily disassembled. While the budding yeast Saccharomyces cerevisiae undergoes closed mitosis, its meiotic nuclear division strategy is less understood. Here we investigate nuclear permeability during meiosis in budding yeast and discover that meiosis II represents a semi-closed division marked by bidirectional mixing between the nucleus and cytoplasm. This includes nuclear entry of the Ran GTPase activating protein (RanGAP), typically cytoplasmic, although RanGAP relocalization appears to be a consequence, rather than a cause of permeability changes. This intercompartmental mixing occurs without nuclear envelope breakdown or dispersal of nucleoporins and is independent of known nuclear pore complex remodeling events. This phenomenon, termed virtual nuclear envelope breakdown (vNEBD), represents a unique mechanism distinct from other semi-closed divisions. We demonstrate that vNEBD is integrated into the meiotic program and regulated by the conserved meiotic kinase Ime2 and the meiosis-specific protein phosphatase 1 regulatory subunit, Gip1. Remarkably, the vNEBD event is conserved between S. cerevisiae and the distantly related Schizosaccharomyces pombe, indicating a conserved, critical role in meiosis.

Keywords: CDK; Glc7; Ime2; Kinase; Meiosis; Nuclear Envelope; Nuclear Permeability; Nuclear Pore Complex; PP1; Phosphatase; Ran.

Figure 1.. Budding yeast undergoes vNEBD during meiosis II.

A-B. Time-lapse microscopy of a cell progressing through meiosis. Open arrows indicate the start of vNEBD, and closed arrows represent the end of vNEBD. Maximum intensity projection shown. A. Cells contain fluorescently tagged histone Htb1-mCherry (pink) and nucleoplasmic protein GFP-Npl3 (green) (strain ÜB18509). B. Cells contain nucleoplasmic reporter 2XmCherry-SV40NLS (NLS, pink) and fluorescently-tagged histone Htb1-eGFP (green) (strain ÜB21380). C. The percent of cells that have initiated vNEBD (proteins disperse from the nucleus) over time (min) relative to the end of anaphase II (first time point of maximum chromatin separation). 2XmCherry-SV40NLS (pink), GFP-Npl3 (green), Trz1-GFP (blue), and Pus1-GFP (gray). Two replicates per strain, 65 ≤ n ≤ 140 cells per replicate. D. Duration of vNEBD (nuclear protein dispersal) measured via live time-lapse microscopy (two replicates per strain, 65 ≤ n ≤ 140 cells per replicate). Line represents mean duration (2XmCherry-SV40NLS = 45.5 min, GFP-Npl3 = 43.2 min, Trz1-GFP = 71.2 min, Pus1-GFP = 26.3 min). E. (Left) Live Airyscan microscopy of cells during meiosis II (MII) or during post-meiotic spore development expressing histone marker Htb1-mCherry (pink) and PKI-NES-3XeGFP (NES, green) (strain ÜB38411). Single Z-slice shown. (Middle) Line scans from the cells depicted to the left of raw fluorescence intensities (arbitrary units). Purple lines represent scans from each nucleus in the MII cell and teal lines represent scans from each nucleus in the cell with spores. Distance across the nucleus is measured in μm with the gray box representing the approximate nuclear boundary. (Right) Quantification of nuclear NES-3XeGFP signal in cells staged at late MII (n = 42 cells) or immature (pre-ascal collapse) spores (n = 45 cells). Line scans measured signal intensity across each nucleus, and the ratio of minimum to maximum intensity per nucleus was averaged for each cell. Mann-Whitney test, p<0.001. Scale bar is 2 μm for all images shown. Full sample size information can be found in the Materials and Methods.

Figure 2.. RanGAP relocalization to the nucleus does not drive vNEBD.

A. Schematic representing how RanGEF and RanGAP establish asymmetric populations of RanGTP and RanGDP essential for directional nucleocytoplasmic transport. NPCs are represented in gray. B. Time-lapse microscopy of a cell progressing through meiosis tagged with histone marker Htb1-mCherry (pink) and RanGEF Prp20-GFP (green) (strain ÜB20161). Maximum intensity projection shown. C. (Left) Live Airyscan microscopy of sporulating cells in MII (right cell) and immature spores (left cell) expressing histone marker Htb1-mCherry (pink) and RanGAP Rna1-3XeGFP (green) (strain ÜB20153). Single Z-slice shown. Arrowheads point to nuclei in-focus, with closed arrows indicating exclusion of Rna1 from the nucleus and open arrows indicating nuclear Rna1 signal. (Right) Quantification of nuclear Rna1 signal in cells staged at late MII (n = 33 cells) or immature (pre-ascal collapse) spores (n = 24 cells). Line scans measured signal intensity across each nucleus, and an average of the ratio of minimum to maximum intensity was taken for each cell, such that each point represents one cell. Mann-Whitney test, p<0.001. D. Schematic representing the experimental strategy to tether Rna1 outside of the nucleus. (Left) Tether protein (purple) is fused to an anti-GFP nanobody (black), which can bind to the 3XeGFP tag (green) fused to Rna1 (yellow). (Right) Schematic of the dividing nucleus with the NE in dark blue and developing gamete plasma membranes (teal) typical of late anaphase II. The Pil1 tether (purple) localizes to the cell periphery, outside of the developing gametes, whereas Nup159 is localized to the outside of the NE. E-F. Time-lapse microscopy of a cell progressing through meiosis tagged with histone marker Htb1-mCherry (pink) and RanGAP Rna1-3XeGFP (green) with either no tether protein (strain ÜB20155) (E), or both Pil1 and Nup159 tether proteins (strain ÜB36287) (F). Open arrows indicate the start of vNEBD, and closed arrows represent the end of vNEBD. The first two rows show a maximum intensity projection, and the last row shows a single z-slice. G. The percentage of cells that underwent meiosis II (MII) with vNEBD initiation and barrier return (black), vNEBD initiation with no reestablishment of the permeability barrier (gray), or no vNEBD initiation (white, no cells). Pil1+Nup159 tether refers to cells containing both the Pil1-nanobody and the Nup159-nanobody. Two replicates scored for each category (79 ≤ n ≤ 120 cells per replicate). H. Duration of vNEBD in minutes as assessed by 15-minute interval time-lapse microscopy. Each point represents one cell. Black line represents the mean vNEBD duration (no tether = 36.5 min, Pil1-tether = 64.1 min, Pil1- and Nup159-tether = 59.7 min). For each condition, two replicates were scored (79 ≤ n ≤ 120 cells per replicate). Scale bar is 2 μm for all images shown. Full sample size information can be found in the Materials and Methods.

Figure 3.. vNEBD is precisely timed relative to other meiotic events.

A. Timing of key meiotic events relative to vNEBD initiation (left) and conclusion (right). Events timed around vNEBD onset are NPC sequestration to the GUNC (pink), mitochondrial collapse onto the nucleus (orange), Cdc14 release from the nucleolus (yellow), the end of anaphase II (green), nuclear basket return (light blue), and meiosis II spindle breakdown (dark blue). Two replicates each strain with 81≤ n ≤ 154 cells per replicate. Events timed around vNEBD end are the closure of the prospore membranes (black) and return of Cdc14 to the nucleolus (purple). Two replicates are measured for each strain with 111 ≤ n ≤ 137 cells per replicate. B-E. Time-lapse microscopy of a cell progressing through meiosis. Open arrows indicate the start of vNEBD, and closed arrows represent the end of vNEBD. Maximum intensity projection shown. Scale bar is 2 μm. B. Cells contain reporter 2XmCherry-SV40NLS (pink) and fluorescently tagged channel FG-nucleoporin Nup49-GFP (green) (strain ÜB18513). Asterisk marks the onset of NPC sequestration away from the forming gamete nuclei. C. Cells contain reporter 2XmCherry-SV40NLS (pink) and GFP-tagged nuclear basket protein Nup60-GFP (green) (strain ÜB44905). Asterisk marks the first time point of nuclear basket return to the gamete nuclei. D. Cells contain reporter 2XmCherry-SV40NLS and exogenous fluorescently tagged tubulin Tub1-GFP (strain ÜB33507). Asterisk marks first time of meiosis II spindle breakdown. E. Cells contain reporter 2XmCherry-SV40NLS (pink) and prospore membrane marker (PSM; spo20(51-91)-eGFP) (strain ÜB34583). Asterisk marks the first time point of PSM closure. F. Schematic showing the relative order of meiotic events to-scale using the time difference of each relative to vNEBD onset or end. Numbers represent the mean time difference from vNEBD onset (t = 0) in minutes. Full sample size information can be found in the Materials and Methods.

Figure 4.. vNEBD can be uncoupled from known NPC remodeling events.

A. Schematic diagramming the two NPC remodeling events in meiosis II. (Left) Most NPCs (blue) are excluded from growing prospore membranes (PSM; purple), resulting in their sequestration to the GUNC (orange arrow). (Right) Following sequestration to the GUNC, the nuclear basket (pink) detaches from the NPC core and returns to the gamete nuclei, where they associate with the nuclear periphery. B, D-E. Time-lapse microscopy of a cell progressing through meiosis. Open arrows indicate the start of vNEBD, and closed arrows represent the end of vNEBD. Maximum intensity projection shown. Scale bar 2 μm. B. (Left) Cells contain fluorescently tagged histone Htb1-mCherry (pink) and nucleoplasmic protein GFP-Npl3 in the mutant spo21Δ background (strain ÜB21612). (Right) Cartoon showing that NPCs fail to be sequestered to the GUNC when PSMs do not form in spo21Δ mutants. C. The percentage of cells that underwent meiosis II (MII) with vNEBD initiation and barrier return (black), vNEBD initiation with no reestablishment of the permeability barrier (gray), or no vNEBD initiation (white) for various mutant backgrounds. NLS ctrl (strain ÜB21380) and nup60Δ (strain ÜB25072) both contain Htb1-eGFP and 2XmCherry-SV40NLS. GFP-NPL3 ctrl (strain ÜB18509), spo21Δ (strain ÜB36318), and nup60ΔAH (strain ÜB25839) all contain Htb1-mCherry and GFP-Npl3. Two replicates used for each strain (62 ≤ n ≤ 118 cells per replicate). D. (Top) A cell containing reporter 2XmCherry-SV40NLS (pink) and fluorescently tagged Htb1-eGFP (green) in the mutant nup60Δ background (strain ÜB25072). (Bottom) Cartoon representing the state of the nuclear basket in wild-type NUP60 cells (left) with an intact basket or nup60Δ cells (right), which cannot assemble four of the five basket nucleoporins. E. (Top) A cell containing fluorescently tagged histone Htb1-mCherry (pink) and nucleoplasmic protein GFP-Npl3 (green) in the mutant nup60-ΔAH background (strain ÜB25839). (Bottom) Cartoon representing the state of the nuclear basket in nup60-ΔAH cells, in which the nuclear basket cannot return to the nuclear periphery following meiosis. Full sample size information can be found in the Materials and Methods.

Figure 5.. vNEBD initiation is regulated by meiotic cell cycle regulators Ndt80 and Ime2.

A. Time-lapse microscopy of an arrested ndt80Δ cell. Cell contains fluorescently tagged histone Htb1-mCherry (pink) and nucleoplasmic protein GFP-Npl3 (green) (strain ÜB27968). The number of hours in sporulation media is indicated in minutes. B. Time-lapse microscopy of a cdc28-as cell treated with 1-NM-PP1 as in B. Cell contains reporter 2XmCherry-SV40NLS (pink) and fluorescently tagged histone Htb1-eGFP (green) (strain ÜB36314). C. Percentage of all cells in sporulation media with vNEBD initiation and barrier return (black), vNEBD initiation only (gray), or no vNEBD initiation (white). Either DMSO (vehicle) or chemical inhibitor 1-NM-PP1 was added to a final concentration of 1 μM at 5 hrs after moving cells to sporulation media (two replicates, each with 71 ≤ n ≤ 148 cells). ndt80Δ is strain ÜB27968, WT is strain ÜB21380, and cdc28-as is strain ÜB36314. D. Time-lapse microscopy of ime2-as cells treated with DMSO (left) or 1-NA-PP1 (right) to a final concentration of 20 μM at 5 hrs after moving cells to sporulation media. Cells contain reporter 2XmCherry-SV40NLS (pink) and fluorescently tagged histone Htb1-eGFP (green) in a mutant ime2-as background (strain ÜB25646). E. Percentage of cells that went through meiosis II (MII) with vNEBD initiation and barrier return (black), vNEBD initiation only (gray), or vNEBD initiation (white). Cells treated as in part D (45 ≤ n ≤ 118 cells). IME2 is strain ÜB21380 and ime2-as is strain ÜB25646. F. Time-lapse microscopy of cells with reporter 2XmCherry-SV40NLS (pink) and fluorescently tagged histone Htb1-eGFP (green) in a mutant ime2-st background (strain ÜB27197). Cells with a constitutively active allele of Ime2 (ime2-st) under control of an inducible promoter (pGAL) were treated with either a vehicle (+EtOH) or the inducer (+ß-estradiol) in prophase arrest (ndt80Δ). For all images, the maximum-intensity z-projection is shown, and the scale bar is 2 μm. Full sample size information can be found in the Materials and Methods.

Figure 6.. vNEBD completion depends on the Glc7 phosphatase regulator Gip1.

A-B. Time-lapse microscopy of a cell progressing through meiosis. Open arrows indicate the start of vNEBD, and closed arrows represent the end of vNEBD. Maximum intensity projection shown and the scale bar is 2 μm. A. Cells contain fluorescently tagged histone Htb1-mCherry (pink) and nucleoplasmic protein GFP-Npl3 (green) in an AMA1 background (left; strain ÜB18509) or a mutant ama1Δ background (right; strain ÜB20607). B. Cells contain reporter 2XmCherry-SV40NLS (pink) and fluorescently tagged histone Htb1-eGFP (green) in a GIP1 background (left; strain ÜB21380) or a mutant gip1Δ background (right; strain ÜB21614). C. Percentage of cells that went through meiosis II (MII) with vNEBD initiation and barrier return (black), vNEBD initiation only (gray), or no vNEBD initiation (white) for various mutant backgrounds. Npl3 ctrl (strain ÜB18509) and Npl3 ama1Δ (strain ÜB20607) contain Htb1-mCherry and GFP-Npl3. NLS ctrl (strain ÜB21380), spo12Δ (strain ÜB36318), and gip1Δ (strain ÜB21614) all contain Htb1-eGFP and 2XmCherry-SV40NLS. Two replicates used for each strain (53 ≤ n ≤ 123 cells per replicate). D. Duration of vNEBD for various mutant backgrounds. Two replicates per strain with 53 ≤ n ≤ 123 cells per replicate. The strains are the same as in A. Lines represent the mean duration. Full sample size information can be found in the Materials and Methods.