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1.
Figure 2

Figure 2. From: Caenorhabditis elegans BAF-1 and its kinase VRK-1 participate directly in post-mitotic nuclear envelope assembly.

A functional NE is not formed in baf-1(t1639) or baf-1(RNAi) embryos. (A) 160 kDa fluorescent dextran was injected into gonads of worms and incorporated into oocytes and embryos. While the dextran was excluded from nuclei of a two-cell stage control embryo, 48 h RNAi against BAF-1 prevented formation of functional NEs. Arrows point to a nucleus in the baf-1(RNAi) embryo. (B–E) Transmission electron microscopy micrographs of nuclei from wild-type (B), baf-1(RNAi) 48 h (C), baf-1(RNAi) 72 h (D) and baf-1(t1639) at 25°C (E) embryos. Arrowheads point to NPCs. Arrows indicate intranuclear membranes; double arrows show parts of chromatin not covered by nuclear membranes. Bars; 10 μm (A); 1 μm (B–E, upper panels); 400 nm (B–E, lower panels).

Mátyás Gorjánácz, et al. EMBO J. 2007 January 10;26(1):132-143.
2.
Figure 7

Figure 7. From: Caenorhabditis elegans BAF-1 and its kinase VRK-1 participate directly in post-mitotic nuclear envelope assembly.

Depletion of VRK-1 abolishes mitotic release of LEM domain proteins and leads to defective assembly of nuclear membranes. (A) Time-lapse microscopy of control and vrk-1(RNAi) embryos demonstrated that depletion of VRK-1 led to a prominent association of GFP-LEM-2 with (pro-) nuclei (indicated by arrowheads; both nuclei were not always present in the same focal plane) throughout the cell cycle. Bars, 10 μm. (B) Transmission electron microscopy micrographs of nuclei from control and vrk-1(RNAi) embryos. In the control embryo, many regularly spaced NPCs were observed in the NE (arrowheads in bottom panels), whereas vrk-1(RNAi) embryos contained highly condensed chromatin surrounded by nuclear membranes with no or few NPCs. Arrows point to examples of abundant intranuclear membrane structures in vrk-1(RNAi) embryos. Bars, 1 μm (top panels); 200 nm (bottom panels).

Mátyás Gorjánácz, et al. EMBO J. 2007 January 10;26(1):132-143.
3.
Figure 5

Figure 5. From: Caenorhabditis elegans BAF-1 and its kinase VRK-1 participate directly in post-mitotic nuclear envelope assembly.

VRK-1 is essential for NE formation. (A) The first mitotic division of control and vrk-1(RNAi) embryos expressing YFP-LMN-1 was recorded by time-lapse microscopy. Arrowheads point to (pro-) nuclei in the vrk-1(RNAi) embryo. Bars, 10 μm. (B) Western blot of extracts from control and vrk-1(RNAi) embryos with VRK-1 antibodies revealed three isoforms of VRK-1 with estimated molecular weights of ∼67, ∼80 and ∼100 kDa, which were all efficiently depleted by vrk-1 RNAi. A prominent cross-reacting band of ∼90 kDa was detected. (C) Immunofluorescence analysis with mAb414 (red in merge) and Nup96 antibodies (green in merge) showed nuclear rim staining in control but not in vrk-1(RNAi) embryos. DNA was visualized with Hoechst (blue in merge). Bars, 5 μm. (D) Nuclear exclusion assay using GFP-β-tubulin demonstrated that VRK-1 depletion prevented the formation of a functional NE. Images were taken 10 min after the first mitotic anaphase onset. Arrowheads point to nuclei in the vrk-1(RNAi) embryo. Bars, 10 μm.

Mátyás Gorjánácz, et al. EMBO J. 2007 January 10;26(1):132-143.
4.
Figure 3

Figure 3. From: Caenorhabditis elegans BAF-1 and its kinase VRK-1 participate directly in post-mitotic nuclear envelope assembly.

Temperature shifts demonstrate a direct role of BAF-1 in NE assembly. (A) Still images of control, baf-1(RNAi) and baf-1(t1639) GFP-β-tubulin-expressing embryos grown and recorded at the temperatures indicated. Arrows point to (pro-) nuclei that failed to exclude soluble GFP-β-tubulin. (B) GFP-β-tubulin-expressing baf-1(t1639) embryos shifted between 15 and 25°C during recordings at the time points are indicated. Upshift, from 15 to 25°C; downshift, from 25 to 15°C. (C–F) Nuclear exclusion was quantified from time-lapse recordings by dividing nuclear fluorescence intensity by cytoplasmic fluorescence intensity after background subtraction. Time is indicated relative to anaphase onset. TS, temperature shift. For each condition, 3–4 embryos were analyzed. In (E) and (F), red and green bars represent the time period of temperature upshifts corresponding to the appropriate red and green graphs. (G) Localization of GFP-LEM-2 analyzed in wild-type and baf-1(t1639) embryos. Embryos were kept stably at 15°C or shifted from 15 to 25°C, either 80 or 280 s after anaphase onset. Bars, 10 μm.

Mátyás Gorjánácz, et al. EMBO J. 2007 January 10;26(1):132-143.
5.
Figure 6

Figure 6. From: Caenorhabditis elegans BAF-1 and its kinase VRK-1 participate directly in post-mitotic nuclear envelope assembly.

VRK-1 localizes to the NE and controls association of BAF-1 with chromatin. (A) Frames from time-lapse recording of an embryo expressing GFP-VRK-1 (top, inset: zoom × 3) and immunofluorescence analysis of fixed embryos (bottom) with mAb414 (red in merge) and VRK-1 (green in merge) together with Hoechst to visualize DNA (blue in merge). Timing in GFP images refer to anaphase onset in live recording; immunofluorescence images where chosen to represent similar time points. Bars, 5 μm. (B) Localization of GFP-BAF-1 and GFP-BAF-1 G38D in control, vrk-1(RNAi) and ran-1(RNAi) embryos was monitored by time-lapse microscopy (inset shows × 2 magnification of GFP-BAF-1 recruited to the chromatin ‘core' region in anaphase). Bar, 10 μm. (C) Embryonic lethality was determined after incubating hermaphrodites expressing GFP-histone H2B, GFP-BAF-1 or GFP-BAF-1 G38D for 72 h with control bacteria (green bars) or bacteria expressing dsRNA corresponding to the 3′UTR of the baf-1 transcript (red bars). Average values from 15 animals are shown; error bars indicate standard deviation. (D) Immunofluorescence analysis with mAb414 and BAF-1 antiserum showed enhanced BAF-1 association with anaphase chromatin (arrows) in vrk-1(RNAi) embryos. Bars, 10 μm.

Mátyás Gorjánácz, et al. EMBO J. 2007 January 10;26(1):132-143.
6.
Figure 1

Figure 1. From: Caenorhabditis elegans BAF-1 and its kinase VRK-1 participate directly in post-mitotic nuclear envelope assembly.

BAF-1 is required for normal nuclear appearance in C. elegans embryos. (A) Wild-type, baf-1(t1639) (upper, embryo grown at 15°C; lower, embryo at 25°C) and baf-1(RNAi) (72 h RNAi) embryos were stained with BAF-1 antiserum, mAb414 and Hoechst to visualize BAF-1, NPCs and chromatin, respectively. Arrows point to chromatin that has failed to segregate correctly. In this and all subsequent figures, embryos are oriented with anterior to the left. (B) Embryonic lethality was determined after 24, 48 and 72 h of RNAi against baf-1 (green bars) or for baf-1(t1639) animals grown at 15 or 25°C (red bars). Average values from 15 animals are shown. Error bars indicate standard deviation. (C) Western blotting was used to compare the amount of BAF-1 in baf-1(RNAi) embryos after 24 h (lane 4) and 72 h (lane 5) and BAF-1 G38D in baf-1(t1639) embryos grown at 15°C (lane 6) or 25°C (lane 7) with control embryos (lanes 1–3) using BAF-1 antiserum. α-Tubulin antibodies were used as a loading control. (D) Still images from recordings of wild-type and baf-1(t1639) embryos expressing YFP-LMN-1 and grown at 25°C. Time in this and subsequent time-lapse recordings is indicated in seconds relative to anaphase onset. Arrowheads point to nuclei with abnormal YFP-lamin distribution. (E) baf-1(t1639) embryos grown at 15 or 25°C were stained with LEM-2 antiserum, mAb414 and Hoechst. Bars, 10 μm.

Mátyás Gorjánácz, et al. EMBO J. 2007 January 10;26(1):132-143.
7.
Figure 4

Figure 4. From: Caenorhabditis elegans BAF-1 and its kinase VRK-1 participate directly in post-mitotic nuclear envelope assembly.

BAF-1 is phosphorylated by VRK-1. (A–F) A 10 μg portion of wild-type embryo extract was untreated (A), mock-treated (C) or λ protein phosphatase-treated (D). Similar extracts from baf-1(t1639) mutants grown at 15°C and upshifted to 25°C for 12 h (B) or constantly grown at 15°C (E), or from vrk-1(RNAi) embryos (F). The extracts were resolved in two dimensions and immunoblotted with BAF-1 antiserum. Arrows 14 indicate different isoforms of BAF-1. Spot 4 is decreased in (C) and (D) owing to slight differences in extract preparation and treatment required for λ protein phosphatase action (see Materials and methods). The linear pI gradient is indicated above the panels, whereas the molecular mass is indicated on the left side of each panel. (G) Wild-type embryo extract was incubated with beads containing either control IgG or VRK-1 antibody. A total of 0.4% of unbound and 33% of bound material was analyzed by immunoblotting with BAF-1 antiserum. (H) In vitro phosphorylation reactions containing GST-VRK-1-His and zz-BAF-1-His dimers were performed for 60 or 120 min and analyzed by SDS–PAGE and autoradiography.

Mátyás Gorjánácz, et al. EMBO J. 2007 January 10;26(1):132-143.

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