kdp-1 is not required for X chromosome pairing in meiosis. (A) Partial projection of kdp-1(RNAi) germline nuclei probed with a HIM-8 (red) antibody and stained with DAPI (blue). Proliferative zone nuclei are shown on the top row and pachytene nuclei on the bottom row. Scale bar: 2 μm. (B) Focal plane of a GFP::KDP-1 gonad stained with HIM-8 (red) and GFP (green) antibodies and DAPI (blue). No overlap is seen between GFP::KDP-1 and HIM-8 staining. Scale bar: 2 μm.

kdp-1(RNAi) causes germline defects. (A) Western blot of lysates from wild-type (WT; N2) or kdp-1(RNAi) mixed-stage animals probed with anti-KDP-1. Equal loading of the lysates was confirmed by staining with Coomassie blue. (B) Partial projections of DAPI-stained proliferative and transition zones of wild-type (untreated) and kdp-1(RNAi) gonads. Scale bar: 10 μm. (C) Partial projections of DAPI-stained gonads from kdp-1(RNAi), or sun-1(RNAi) animals. Arrows mark abnormal large chromatin masses. Outlines of the gonads have been drawn in grey. Scale bar: 10 μm. (D) Projections of deconvolved images of DAPI-stained diakinesis nuclei of the indicated genotypes. Scale bar: 2 μm. (E) Projection of a DAPI-stained untreated male gonad and a kdp-1(RNAi) male gonad. Scale bar: 10 μm.

GFP::KDP-1 localizes to the NE. (A) Single focal planes of fixed embryos or gonads expressing GFP::KDP-1 were probed with anti-GFP (first column and green in merge) and anti-lamin (second column and red in merge) antibodies and stained with DAPI to observe chromatin (third column and blue in merge). Images of an embryo are in the first row, pachytene regions of gonads in rows 2-3, and maturing oocytes in the proximal gonad in rows 4-5. Samples in rows 3 and 5 were from sun-1(RNAi) animals. Scale bar: 10 μm. (B) Single focal plane of a GFP::KDP-1 embryo probed with the GFP (green in merge) and KDP-1 (red in merge) antibodies and stained with DAPI (blue in merge). Scale bar: 10 μm. (C) Live larvae expressing GFP::KDP-1 in various tissues including (i) the ventral cord, (ii) hypodermal cells, (iii) body wall muscles, (iv) neurons and pharynx. NE enrichment is visible in the ventral cord and hypodermal cells. Scale bar: 10 μm.

kdp-1(RNAi) causes delays in the embryonic cell cycle. (A) Large chromatin masses are shown in a kdp-1(RNAi) embryo probed with a lamin antibody and stained with DAPI. Scale bar: 10 μm. (B) DIC and GFP fluorescence of TH32 (histone H2B::GFP, gamma tubulin::GFP) single-cell embryos at selected stages of the first mitotic cell cycle. Top two panels are the GFP and DIC frames from an untreated embryo. Bottom two frames are from a kdp-1(RNAi) embryo. Times are recorded in seconds, with time 0 being the moment the pronuclei meet. Scale bar: 10 μm. (C) GFP fluorescence kymographs of the first mitotic division of representative TH32 untreated and kdp-1(RNAi) embryos. The kymographs are vertically aligned at the moment the pronuclei meet. Each slice is 2 μm. Cell-cycle stages are labeled along the side as pnm (pronuclear migration), centration, pd (posterior displacement), or anaphase. Horizontal scale bar: 10 μm. Vertical scale bar: 60 seconds. (D) Comparison of the length of individual cell-cycle events in untreated and kdp-1(RNAi) embryos for the first three mitotic divisions. Centration was defined as the first frame in which the maternal and paternal pronuclei touched until centration was completed. NEBD was the moment the NE began to lose its clear definition by DIC. Furrow ingression was the first frame in which a furrow could be seen. Cytokinesis was the last frame in which the furrow advanced. Error bars denote s.e.m. P-values were calculated by performing a one-tailed unpaired t-test (^*P<0.05).

Antibodies against KDP-1 localize to the NE of germline nuclei. (A) Western blot of worm lysates from wild-type (N2) or transgenic (TG) worms expressing GFP::KDP-1. Blot was probed with pre-immune serum (left) or the affinity-purified KDP-1 antibody (right). Size in kDa is shown on the right. (B) Dissected adult hermaphrodite gonad stained with DAPI to show nuclei. The mitotic zone is the most distal part of the gonad (DG). Stages of meiotic prophase I are marked in orange. Individual bivalents can be seen in diakinesis oocytes at the proximal end of the gonad (PG). DTC is the region covered by the distal tip cell and PCD is the region of the gonad where most programmed cell death occurs. Image is a composite of three gonads; dashed lines correspond to intervening areas not covered by individual gonad images. Adapted from (Lints and Hall, 2005). (C) Single focal plane of fixed wild-type germline nuclei from the mitotic zone through early pachytene probed with anti-KDP-1 (row 1 and red in merge) and anti-nuclear pore complex (row 2 and green in merge) antibodies and stained with DAPI (row 3 and blue in merge). Scale bar: 10 μm. Box represents zoomed region to the right.

KDP-1 is a KASH protein that interacts with SUN-1 and UNC-84. (A) Schematic describing the MYTH screen. Left: The bait, a fusion of a transcription factor (TF; LexA-VP16), the C-terminal half of ubiquitin (Cub) and the transmembrane and C-terminal portion of UNC-84, is unable to bind to a random clone from the C. elegans cDNA library fused to a mutated form of the N-terminus of ubiquitin (NubG) that is not able to bind Cub on its own. Right: An interaction occurs between UNC-84 and the library clone, bringing NubG and Cub together in the cytoplasm. This results in the proteolytic cleavage of a transcription factor and subsequent activation of the reporter gene system in yeast. (B) Alignment of the KDP-1 KASH domain with other KASH domains. Boxed amino acids are identical to, and shaded amino acids are similar to, aligned residues in KDP-1. The percent identity to the KDP-1 KASH domain is noted to the right. (C) Schematic showing the size differences between some known KASH proteins: human Syne-1, Syne-2 and Nesprin-3, D. melanogaster Klarsicht, S. pombe Kms1p and C. elegans ANC-1, UNC-83, ZYG-12, and KDP-1. The KASH and transmembrane domains are in red. (D) L40 yeast transformed with the empty prey vector NubG, NubG::unc-83, or NubG::kdp-1 and the bait TF::Cub::unc-84 or TF::Cub::sun-1. Yeast were serial diluted on -Leu-Trp-His media supplemented with 75 mM 3-AT.