A conserved KASH domain protein associates with telomeres, SUN1, and dynactin during mammalian meiosis

Abstract

In yeasts and worms, KASH (Klarsicht/ANC-1/Syne/homology) domain and SUN (Sad-1/UNC-84) domain nuclear envelope (NE) proteins play a crucial role in meiotic chromosome movement and homologue pairing. However, although the vertebrate SUN domain protein SUN1 is involved in these processes, its partner has remained identified. Based on subcellular localization screening in mouse spermatocytes, we identified a novel germ cell-specific protein, KASH5, that localized exclusively at telomeres from the leptotene to diplotene stages in both spermatocytes and oocytes. KASH5 possesses hitherto unknown KASH-related sequences that directly interacted with SUN1 and mediated telomere localization. Thus, KASH5 is a mammalian meiosis-specific KASH domain protein. We show that meiotic chromosome movement depended on microtubules and that KASH5 interacted with the microtubule-associated dynein-dynactin complex. These results suggest that KASH5 connects the telomere-associated SUN1 protein to the cytoplasmic force-generating mechanism involved in meiotic chromosome movement. Our study strongly suggests that the meiotic homologue-pairing mechanism mediated by the SUN-KASH NE bridge is highly conserved among eukaryotes.

Figure 1.

Identification of a novel mammalian KASH protein. (A) Total testis extracts were loaded, and Western blotting was performed with KASH5 serum. A single band was detected corresponding to the predicted size (72 kD). (B) Chromosome spreads from spermatocytes were stained with KASH5 and CENP-C antibodies. (C) Chromosome spreads from spermatocytes were stained with KASH5 and TRF2 antibodies. Magnified images of the boxed area are shown on the right. (D) Amino acid sequence alignment of KASH proteins. Identical amino acids are shaded in black, and similar amino acids are shaded in gray. Dm, Drosophila melanogaster; Mm, Mus musculus; Ce, C. elegans; Sp, S. pombe. (E and F) mRNA expression in various tissues was analyzed by RT-PCR. E, embryonic day; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Bars, 3 µm.

Figure 2.

KASH5 localizes at telomeres with SUN1 during meiotic prophase. (A) Spermatocytes were cultured with EdU and stained with Alexa Flour 488 azide and antibodies against SCP3 and KASH5 (left) or SUN1 (right). L, leptotene; P, pachytene. (B) Chromosome spreads from spermatocytes were stained with KASH5 and SCP3 antibodies. (C) Chromosome spreads from spermatocytes were stained with KASH5, SUN1, and SCP3 antibodies. Bars, 5 µm (unless otherwise indicated).

Figure 3.

KASH5 interacts with SUN1/2 via the KASH domain. (A) Immunoprecipitates (IP) from mouse testis using a SUN1 antibody (Ab.) or control (Cont.) IgG were analyzed by immunoblot using the indicated antibodies. Total soluble extract (0.1%) was loaded as input. (B) Yeast two-hybrid assay using KASH5 fragments. T-antigen (T) and p53 act as a positive control. C, C-terminal region; M, middle region; N, N-terminal region; TM, transmembrane.

Figure 4.

The KASH domain of KASH5 and inner NE protein SUN1 are required for telomere localization of KASH5. (A) Schematic representations of GFP-tagged KASH5 mutants. TM, transmembrane. (B) Spermatocytes from the testis transfected with GFP-tagged KASH5 mutants were stained with GFP and SCP3 antibodies. (C) Pachytene spermatocytes of wild-type (WT) and SUN1 KO mice were stained by the indicated antibodies. The top images show the localization of KASH5 signals (anti-KASH5 antibody) in the areas boxed in the bottom images. Note that KASH5 localization to telomeres was abolished in SUN1 KO mice. Bars, 5 µm.

Figure 5.

Microtubule-dependent meiotic nuclear movement and the interaction between KASH5 and dynein. (A) Immunoprecipitates (IP) from mouse testis using a KASH5 antibody or control (Cont.) IgG were analyzed by immunoblot using the indicated antibodies. Total soluble extract (0.05%) was loaded as input. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (B) Time-lapse images of a mitotic cell or pachytene spermatocytes treated with or without nocodazole (Noc) were collected for 5 min at 30-s intervals (Video 1–3). Note that TRF1 signals distribute randomly in the nucleus of mitotic cells, whereas TRF1 signals localize near the NE of pachytene spermatocytes. Bar, 5 µm. (C) A model for mechanical bridging of the NE by SUN1 and KASH5 proteins during mammalian meiotic prophase. SUN1 and KASH5 locate at the INM and ONM, respectively, of the NE and interact across the lumen between the membranes. KASH5 associates with the dynein–dynactin complexes, whereas SUN1 may associate with telomeres on the opposite outsides of the NE, thus transferring the driving force generated by cytoplasmic microtubules to telomeres in the nucleus.