Motor coordination deficits in Alpk1 mutant mice with the inserted piggyBac transposon

<p>Abstract</p> <p>Background</p> <p>ALPK1 (α-kinase 1) is a member of an unconventional alpha-kinase family, and its biological function remains largely unknown. Here we report the phenotypic characterization of one mutant line, in which the <it>piggyBac </it>(<it>PB</it>) transposon is inserted into the <it>Alpk1 </it>gene.</p> <p>Results</p> <p>The <it>piggyBac</it>(<it>PB</it>) insertion site in mutants was mapped to the first intron of the <it>Alpk1 </it>gene, resulting in the effective disruption of the intact <it>Alpk1 </it>transcript expression. The transposon-inserted <it>Alpk1 </it>homozygous mutants (<it>Alpk1^PB/PB</it>) displayed severe defects in motor coordination in a series of behavioral analysis, including dowel test, hanging wire test, rotarod analysis and footprint analysis. However, the cerebellar architecture, Purkinje cell morphology and electrophysiology of the Purkinje cells appeared normal in mutants. The motor coordination deficits in the <it>Alpk1^PB/PB</it>mice were rescued by transgenic mice expressing the full-length <it>Alpk1</it>-coding sequence under the control of the ubiquitous expression promoter.</p> <p>Conclusions</p> <p>Our results indicate that ALPK1 plays an important role in the regulation of motor coordination. <it>Alpk1^PB/PB</it>mice would be a useful model to provide a clue to the better understanding of the cellular and molecular mechanisms of ALPK1 in the control of fine motor activities.</p

Analysis of Meiosis in SUN1 Deficient Mice Reveals a Distinct Role of SUN2 in Mammalian Meiotic LINC Complex Formation and Function

LINC complexes are evolutionarily conserved nuclear envelope bridges, composed of SUN (Sad-1/UNC-84) and KASH (Klarsicht/ANC-1/Syne/homology) domain proteins. They are crucial for nuclear positioning and nuclear shape determination, and also mediate nuclear envelope (NE) attachment of meiotic telomeres, essential for driving homolog synapsis and recombination. In mice, SUN1 and SUN2 are the only SUN domain proteins expressed during meiosis, sharing their localization with meiosis-specific KASH5. Recent studies have shown that loss of SUN1 severely interferes with meiotic processes. Absence of SUN1 provokes defective telomere attachment and causes infertility. Here, we report that meiotic telomere attachment is not entirely lost in mice deficient for SUN1, but numerous telomeres are still attached to the NE through SUN2/KASH5-LINC complexes. In Sun12/2 meiocytes attached telomeres retained the capacity to form bouquetlike clusters. Furthermore, we could detect significant numbers of late meiotic recombination events in Sun12/2 mice. Together, this indicates that even in the absence of SUN1 telomere attachment and their movement within the nuclear envelope per se can be functional. Author summary: Correct genome haploidization during meiosis requires tightly regulated chromosome movements that follow a highly conserved choreography during prophase I. Errors in these movements cause subsequent meiotic defects, which typically lead to infertility. At the beginning of meiotic prophase, chromosome ends are tethered to the nuclear envelope (NE). This attachment of telomeres appears to be mediated by well-conserved membrane spanning protein complexes within the NE (LINC complexes). In mouse meiosis, the two main LINC components SUN1 and SUN2 were independently described to localize at the sites of telomere attachment. While SUN1 has been demonstrated to be critical for meiotic telomere attachment, the precise role of SUN2 in this context, however, has been discussed controversially in the field. Our current study was targeted to determine the factual capacity of SUN2 in telomere attachment and chromosome movements in SUN1 deficient mice. Remarkably, although telomere attachment is impaired in the absence of SUN1, we could find a yet undescribed SUN1-independent telomere attachment, which presumably is mediated by SUN2 and KASH5. This SUN2 mediated telomere attachment is stable throughout prophase I and functional in moving telomeres within the NE. Thus, our results clearly indicate that SUN1 and SUN2, at least partially, fulfill redundant meiotic functions

Inner Nuclear Envelope Proteins SUN1 and SUN2 Play a Prominent Role in the DNA Damage Response

SummaryThe DNA damage response (DDR) and DNA repair are critical for maintaining genomic stability and evading many human diseases [1, 2]. Recent findings indicate that accumulation of SUN1, a nuclear envelope (NE) protein, is a significant pathogenic event in Emery-Dreifuss muscular dystrophy and Hutchinson-Gilford progeria syndrome, both caused by mutations in LMNA [3, 4]. However, roles of mammalian SUN proteins in mitotic cell division and genomic stability are unknown. Here we report that the inner NE proteins SUN1 and SUN2 may play a redundant role in DDR. Mouse embryonic fibroblasts from Sun1−/−Sun2−/− mice displayed premature proliferation arrest in S phase of cell cycle, increased apoptosis and DNA damage, and decreased perinuclear heterochromatin, indicating genome instability. Furthermore, activation of ATM and H2A.X, early events in DDR, were impaired in Sun1−/−Sun2−/− fibroblasts. A biochemical screen identified interactions between SUN1 and SUN2 and DNA-dependent protein kinase (DNAPK) complex that functions in DNA nonhomologous end joining repair and possibly in DDR [2, 5, 6]. Knockdown of DNAPK reduced ATM activation in NIH 3T3 cells, consistent with a potential role of SUN1- and SUN2-DNAPK interaction during DDR. SUN1 and SUN2 could affect DDR by localizing certain nuclear factors to the NE or by mediating communication between nuclear and cytoplasmic events

KASH protein Syne-2/Nesprin-2 and SUN proteins SUN1/2 mediate nuclear migration during mammalian retinal development

Nuclear movement relative to cell bodies is a fundamental process during certain aspects of mammalian retinal development. During the generation of photoreceptor cells in the cell division cycle, the nuclei of progenitors oscillate between the apical and basal surfaces of the neuroblastic layer (NBL). This process is termed interkinetic nuclear migration (INM). Furthermore, newly formed photoreceptor cells migrate and form the outer nuclear layer (ONL). In the current study, we demonstrated that a KASH domain-containing protein, Syne-2/Nesprin-2, as well as SUN domain-containing proteins, SUN1 and SUN2, play critical roles during INM and photoreceptor cell migration in the mouse retina. A deletion mutation of Syne-2/Nesprin-2 or double mutations of Sun1 and Sun2 caused severe reduction of the thickness of the ONL, mislocalization of photoreceptor nuclei and profound electrophysiological dysfunction of the retina characterized by a reduction of a- and b-wave amplitudes. We also provide evidence that Syne-2/Nesprin-2 forms complexes with either SUN1 or SUN2 at the nuclear envelope to connect the nucleus with dynein/dynactin and kinesin molecular motors during the nuclear migrations in the retina. These key retinal developmental signaling results will advance our understanding of the mechanism of nuclear migration in the mammalian retina

Meiotic recombination in SUN1 deficient oocytes.

<p>Representative chromosome spreads of oocytes from 19.5<i>Sun1^{+/+(Δex10-11)}</i> and <i>Sun1^{−/−(Δex10-11)}</i> females labeled with anti-SYCP3, anti-SYCP1 and anti-MLH1 antibodies. Complete pairing of all homologous chromosomes as judged by the co-localization of SYCP3 and SYCP1 is observed in heterozygous control pachytene oocytes (A). As expected the homolog pairs exhibit 1–2 distinct MLH1 foci each. In SUN1 deficient pachytene-like oocytes (B, C) only some chromosome stretches and few homologous chromosomes are fully paired. Frequent defects in synapsis formation and many univalent chromosomes can be detected, labeled only by SYCP3. However, distinct MLH1 foci can be observed where SYCP3 and SYCP1 co-localize, (arrowheads in B and C). See also inset in B; magnified by a factor of 2. Scale bars 10 µm.</p

KASH5 localization in SUN1 deficient males.

<p>Representative spermatocytes in paraffin sections of <i>Sun1^{+/+(Δex10-11)}</i> and <i>Sun1^{−/−(Δex10-11)}</i> testis stained for SYCP3 and KASH5. In the wildtype (A) the expected KASH5 localization at the distal ends of synaptonemal complex axes can clearly be observed. In <i>Sun1^{−/−(Δex10-11)}</i> spermatocytes (A′–A″) the KASH5 signal, although weaker, is also clearly detectable. As seen in the wildtype, distinct KASH5 foci also co-localize with the ends of synaptonemal complex axes. Scale bars 10 µm.</p

Presence of meiotic telomere attachment in <i>Sun1^−/−</i> mouse strains.

<p>(A) Telomere fluorescence in-situ hybridization (TeloFISH) in co-localization with Lamin B and SYCP3 immunofluorescence on representative 15 dpp (days past partum) <i>Sun1^{+/+(Δex10-11)}</i> and littermate <i>Sun1^{−/−(Δex10-11)}</i> testis sections (A, A′) and 17.5 dpf (days past fertilization) <i>Sun1^{+/+(Δex10-11)}</i> and littermate <i>Sun1^{−/−(Δex10-11)}</i> ovary sections (A″, A′″). In all WT sections investigated, attached telomeres appear embedded within the labeled lamina (white arrowheads in A and A″). All sections from knockout tissues clearly show both detached, internal telomere signals (yellow arrows in A′ and A′″) as well as attached, peripheral telomere signals (white arrowheads in A′ and A′″) in both oocytes and spermatocytes. Peripheral, attached telomeres in SUN1 deficient oocytes and spermatocytes are also seen at the ends of synaptonemal complex (SC) axes shown by SYCP3, as is the case in wildtype cells. Scale bar 10 µm. (B) Representative electron micrographs of spermatocytes from adult <i>Sun1^{+/+(Δex10-13)}</i> (B) and <i>Sun1^{−/−(Δex10-13)}</i> (B″) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004099#pgen.1004099-Ding1" target="_blank">[11]</a> mice and of spermatocytes from 15 dpp <i>Sun1^{+/−(Δex10-11)}</i> (B′) and <i>Sun1^{−/−(Δex10-11)}</i> (B′″) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004099#pgen.1004099-Chi1" target="_blank">[21]</a> mice. (C) Representative electron micrographs from E17.5 female <i>Sun1^+/+(Δ10-11)</i> (C–C′) and <i>Sun1^{−/−(Δ10-11)}</i>(C″–C′″) oocytes. In male wildtype meiocytes of both mouse strains and female wildtype meiocytes of the <i>SUN1^(Δex10-11)</i> strain, components of the SC and the telomere attachment plates (black arrowheads) are clearly visible. Meiocytes from all <i>Sun1^−/−</i> males (B″– B′″) as well as females (C″–C′″) also show the wildtype-like formation of telomere attachment sites. (D) Quantification of attached and non-attached telomeres in wildtype and knockout spermatocytes at different meiotic stages. Pre-leptotene/early leptotene spermatocytes from littermate 12 dpp mice (D), zygotene spermatocytes from littermate 12 dpp mice (D′) and spermatocytes from littermate 14 dpp mice in a pachytene or pachytene-like stage, respectively (D″). (12 dpp pre-leptotene/early leptotene: <i>Sun1^{+/+(Δex10-11)} n</i> = 16 spermatocytes, 772 telomeres; <i>Sun1^{−/−(Δex10-11)} n</i> = 13 spermatocytes, 645 telomeres. 12 dpp zygotene: <i>Sun1^{+/+(Δex10-11)} n</i> = 5 spermatocytes, 194 telomeres; <i>Sun1^{−/−(Δex10-11)} n</i> = 7 spermatocytes, 337 telomeres. 14 dpp pachytene: <i>Sun1^{+/+(Δex10-1)1} n</i> = 54 spermatocytes, 2138 telomeres; pachytene-like <i>Sun1^{−/−(Δex10-11)} n</i> = 31 spermatocytes, 1150 telomeres.) LE lateral element, CE central element, NE nuclear envelope, Nuc nucleoplasm, Cyt Cytoplasm.</p

Meiotic telomere clustering in the absence of SUN1.

<p>Representative projections of entire spermatocyte nuclei of <i>Sun1^{+/+(Δex10-11)}</i> and <i>Sun1^{−/−(Δex10-11)}</i> mice labeled by KASH5 and SYCP3. As expected non-clustered (A) and clustered (A′) telomere patterns are observed in wildtype spermatocytes. Similar non-clustered (A″) as well as clustered (A′″–A″″) telomere patterns could also be found in SUN1 deficient spermatocytes. All scale bars 5 µm.</p

Meiotic telomere tethering by LINC complex components in the absence of SUN1.

<p>(A,B) Representative meiocytes in paraffin sections of testis and ovary tissue of <i>Sun1^{+/+(Δex10-11)}</i> and <i>Sun1^{−/−(Δex10-11)}</i> mice labeled by anti-SUN2 and anti-SYCP3 antibodies. SUN2 foci, located at the end of synaptonemal complex axes, are present in both wildtype spermatocytes and oocytes (A, B). Similar SUN2 signals are also present in spermatocytes and oocytes of SUN1 deficient littermate mice (A′, A″, B′, B″). The nuclear envelope of somatic cells in the ovary tissue of both <i>Sun1^{+/+(Δex10-11)}</i> and <i>Sun1^{−/−(Δex10-11)}</i> females (B–B″) is also strongly labeled by SUN2. (C–C′) Spermatocytes in paraffin sections of testis tissue of <i>Sun1^{−/−(Δex10-11)}</i> males labeled by anti-SUN2 and anti-KASH5 antibodies. In SUN1 deficient spermatocytes KASH5 and SUN2 co-localize, both showing distinct foci at the nuclear periphery. DNA counterstained using Hoechst 33258. Scale bars 5 µm.</p