Publications by authors named "Sigrid Hoyer-Fender"

23 Publications

  • Page 1 of 1

Expression of α-Tubulin Acetyltransferase 1 and Tubulin Acetylation as Selective Forces in Cell Competition.

Cells 2021 Feb 14;10(2). Epub 2021 Feb 14.

GZMB, Johann-Friedrich-Blumenbach-Institute of Zoology and Anthropology-Developmental Biology, Biological Faculty, Georg-August-Universität Göttingen, Ernst-Caspari-Haus, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany.

The wound healing response of fibroblasts critically depends on the primary cilium, a sensory organelle protruding into the environment and comprising a stable axonemal structure. A characteristic marker for primary cilia is acetylation of axonemal tubulin. Although formation of primary cilia is under cell cycle control, the environmental cues affecting ciliation are not fully understood. Our purpose was, therefore, to study the impact of culture conditions on cilia formation in NIH3T3 fibroblasts. We quantified ciliation in different NIH3T3 sub-cell lines and culture conditions by immunodetection of primary cilia and counting. Quantitative Western blotting, qRT-PCR, and proliferation assays completed our investigation. We observed large differences between NIH3T3 sub-cell lines in their ability to generate acetylated primary cilia that correlated with cytoplasmic tubulin acetylation. We found no increased activity of the major tubulin deacetylase, HDAC6, but instead reduced expression of the α-tubulin acetyltransferase 1 () as being causative. Our observations demonstrate that cells with reduced expression of and tubulin acetylation proliferate faster, eventually displacing all other cells in the population. Expression of and tubulin acetylation are therefore selective forces in cell competition.
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http://dx.doi.org/10.3390/cells10020390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918103PMC
February 2021

Transgenerational effect of drug-mediated inhibition of LSD1 on eye pigment expression in Drosophila.

BMC Ecol 2020 11 23;20(1):62. Epub 2020 Nov 23.

Johann-Friedrich-Blumenbach-Institute of Zoology and Anthropology-Developmental Biology, GZMB, Ernst-Caspari-Haus, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, Göttingen, Germany.

Background: The Drosophila melanogaster mutant white-mottled is a well-established model for position-effect variegation (PEV). Transposition of the euchromatic white gene into the vicinity of the pericentric heterochromatin caused variegated expression of white due to heterochromatin spreading. The establishment of the euchromatin-heterochromatin boundary and spreading of silencing is regulated by mutually exclusive histone modifications, i.e. the methylations of histone H3 at lysine 9 and lysine 4. Demethylation of H3K4, catalysed by lysine-specific demethylase LSD1, is required for subsequent methylation of H3K9 to establish heterochromatin. LSD1 is therefore essential for heterochromatin formation and spreading. We asked whether drug-mediated inhibition of LSD affects the expression of white and if this induced change can be transmitted to those generations that have never been exposed to the triggering signal, i.e. transgenerational epigenetic inheritance.

Results: We used the lysine-specific demethylase 1 (LSD1)-inhibitor Tranylcypromine to investigate its effect on eye colour expression in consecutive generations by feeding the parental and F1 generations of the Drosophila melanogaster mutant white-mottled. Quantitative Western blotting revealed that Tranylcypromine inhibits H3K4-demethylation both in vitro in S2 cells as well as in embryos when used as feeding additive. Eye colour expression in male flies was determined by optical measurement of pigment extracts and qRT-PCR of white gene expression. Flies raised in the presence of Tranylcypromine and its solvent DMSO showed increased eye pigment expression. Beyond that, eye pigment expression was also affected in consecutive generations including F3, which is the first generation without contact with the inhibitor.

Conclusions: Our results show that feeding of Tranylcypromine and DMSO caused desilencing of white in treated flies of generation F1. Consecutive generations, raised on standard food without further supplements, are also affected by the drug-induced alteration of histone modifications. Although eye pigment expression eventually returned to the basal state, the observed long-lasting effect points to a memory capacity of previous epigenomes. Furthermore, our results indicate that food compounds potentially affect chromatin modification and hence gene expression and that the alteration is putatively inherited not only parentally but transgenerationally.
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http://dx.doi.org/10.1186/s12898-020-00330-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7684728PMC
November 2020

The WD40-protein CFAP52/WDR16 is a centrosome/basal body protein and localizes to the manchette and the flagellum in male germ cells.

Sci Rep 2020 08 28;10(1):14240. Epub 2020 Aug 28.

Johann-Friedrich-Blumenbach-Institute of Zoology and Anthropology - Developmental Biology, GZMB, Ernst-Caspari-Haus, Justus-Von-Liebig-Weg11, Georg-August-Universität Göttingen, 37077, Göttingen, Germany.

Development of spermatozoa requires remodelling and formation of particular structures. In elongating spermatids, the transient microtubular manchette contributes to the formation of the head-tail coupling apparatus (HTCA) and the sperm tail. The HTCA derives from the centrosome in that the proximal centriole inserts into the nuclear indentation and the distal centriole gives rise to the sperm flagellum. Although impairments in the formation of HTCA and sperm tail cause male infertility their molecular constituents are only partially known. The WD40-protein CFAP52 is implicated in motile cilia, but its relevance for male germ cell differentiation is not known. Here we show that CFAP52 is widespread expressed and localizes to a subset of microtubular structures. In male germ cells, CFAP52 is a component of the transient manchette and the sperm tail. However, expression of Cfap52 is not restricted to motile cilia-bearing cells. In NIH3T3 cells, CFAP52 localizes to the centrosome, the basal body, and the mitotic spindle poles, but not to the primary cilium. Our results demonstrate that CFAP52 is not restricted to motile cilia but instead most likely functions in constituting the centrosome/basal body matrix and the sperm tail.
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http://dx.doi.org/10.1038/s41598-020-71120-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7455747PMC
August 2020

CCDC42 Localizes to Manchette, HTCA and Tail and Interacts With ODF1 and ODF2 in the Formation of the Male Germ Cell Cytoskeleton.

Front Cell Dev Biol 2019 14;7:151. Epub 2019 Aug 14.

Johann-Friedrich-Blumenbach-Institute of Zoology and Anthropology - Developmental Biology, Göttingen Center for Molecular Biosciences (GZMB), Georg-August-University of Göttingen, Göttingen, Germany.

Terminal differentiation of male germ cells into functional spermatozoa requires shaping and condensation of the nucleus as well as the formation of sperm-specific structures. A transient microtubular structure, the manchette, is mandatory for sperm head shaping and the development of the connecting piece and the sperm tail. The connecting piece or head-to-tail coupling apparatus (HTCA) mediates the tight linkage of sperm head and tail causing decapitation and infertility when faulty. Using mice as the experimental model, several proteins have already been identified affecting the linkage complex, manchette or tail formation when missing. However, our current knowledge is far too rudimentary to even draft an interacting protein network. Depletion of the major outer dense fiber protein 1 (ODF1) mainly caused decapitation and male infertility but validated binding partners collaborating in the formation of sperm-specific structures are largely unknown. Amongst all candidate proteins affecting the HTCA when missing, the structural protein CCDC42 attracted our attention. The coiled-coil domain containing 42 (CCDC42) is important for HTCA and sperm tail formation but is otherwise largely uncharacterized. We show here that CCDC42 is expressed in spermatids and localizes to the manchette, the connecting piece and the tail. Beyond that, we show that CCDC42 is not restricted to male germ cells but is also expressed in somatic cells in which it localizes to the centrosome. Although centrosomal and sperm tail location seems to be irrespective of ODF1 we asked whether both proteins may form an interacting network in the male germ cell. We additionally considered ODF2, a prevalent protein involved in the formation of spermatid-specific cytoskeletal structures, as a putative binding partner. Our data depict for the first time the subcellular location of CCDC42 in spermatids and deepen our knowledge about the composition of the spermatid/sperm-specific structures. The presence of CCDC42 in the centrosome of somatic cells together with the obvious restricted male-specific phenotype when missing strongly argues for a compensatory function by other still unknown proteins most likely of the same family.
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http://dx.doi.org/10.3389/fcell.2019.00151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702985PMC
August 2019

ODF2 maintains centrosome cohesion by restricting β-catenin accumulation.

J Cell Sci 2018 10 24;131(20). Epub 2018 Oct 24.

Johann-Friedrich-Blumenbach-Institute of Zoology and Anthropology-Developmental Biology, GZMB, Ernst-Caspari-Haus, Justus-von-Liebig-Weg 11, Georg-August-Universität Göttingen, 37077 Göttingen, Germany

The centrosome, as the main microtubule-organizing center, safeguards chromosome segregation by supporting the bipolar spindle. Centrosome aberrations are causally related to chromosome segregation disorders, both characterizing cancer cells. Thus, a restriction to only having one centrosome per cell and cell cycle-dependent duplication of the centrosome is mandatory. Duplicated centrosomes remain physically connected, in order to function as a single entity, until onset of mitosis when centrosome disjunction is licensed by disassembly of linker proteins and accumulation of β-catenin. The crucial role β-catenin plays in centrosome disjunction inevitably demands for restricting its premature accumulation. ODF2 (also known as cenexin) is an essential centrosomal component, but its relevance for the interphase centrosome has not been elucidated. We show here that ODF2 plays a central role in centrosome cohesion. Depletion of ODF2 induces premature centrosome splitting and formation of tripolar spindles that are likely caused by the observed accumulation of centrosomal β-catenin. Our data collectively indicate that ODF2 restricts β-catenin accumulation at the centrosome, thus preventing premature centrosome disjunction.
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http://dx.doi.org/10.1242/jcs.220954DOI Listing
October 2018

Ultra-structure of the sperm head-to-tail linkage complex in the absence of the spermatid-specific LINC component SPAG4.

Histochem Cell Biol 2018 Jul 16;150(1):49-59. Epub 2018 Apr 16.

Department of Developmental Biology, Johann-Friedrich-Blumenbach-Institute of Zoology and Anthropology, GZMB, Ernst-Caspari-Haus, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, Göttingen, Germany.

Tight connection between sperm head and tail is crucial for the transport of the male genome and fertilization. The linkage complex, the sperm head-to-tail coupling apparatus (HTCA), originates from the centrosome and anchors to the nuclear membrane. In contrast to its ultra-structural organization, which is already well known for decades, its protein composition largely still awaits future deciphering. SUN-domain proteins are essential components of a complex that links the cytoskeleton to the peripheral nucleoskeleton, which is the nuclear lamina. Here, we studied the impact of the SUN protein SPAG4/SUN4 on the formation of the HTCA. SPAG4/SUN4 is specifically expressed in haploid male germ cells showing a polarized distribution towards the posterior pole in late spermatids that corresponds to the tail attachment site. SPAG4-deficient male mice are infertile with compromised manchette formation and malformed sperm heads. Nonetheless, sperm tails are present demonstrating dispensability of a proper manchette for their formation. Ultra-structural analyses revealed that the development of the sperm head-to-tail linkage complex in the absence of SPAG4 resembles that in the wild type. However, in SPAG4-deficient sperm, the attachment site is diminished with obvious lateral detachment of the HTCA from the nucleus. Our results thus indicate that SPAG4, albeit not essential for the formation of the HTCA per se, is, nevertheless, required for tightening the sperm head-to-tail anchorage by provoking the correct attachment of the lateral parts of the basal plate to the implantation fossa.
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http://dx.doi.org/10.1007/s00418-018-1668-7DOI Listing
July 2018

Effects of repeated long-term psychosocial stress and acute cannabinoid exposure on mouse corticostriatal circuitries: Implications for neuropsychiatric disorders.

CNS Neurosci Ther 2018 06 31;24(6):528-538. Epub 2018 Jan 31.

Department of Psychiatry and Psychotherapy, University of Göttingen, Göttingen, Germany.

Introduction: Vulnerability to psychiatric manifestations is achieved by the influence of genetic and environment including stress and cannabis consumption. Here, we used a psychosocial stress model based on resident-intruder confrontations to study the brain corticostriatal-function, since deregulation of corticostriatal circuitries has been reported in many psychiatric disorders. CB receptors are widely expressed in the central nervous system and particularly, in both cortex and striatum brain structures.

Aims And Methods: The investigation presented here is addressed to assess the impact of repeated stress following acute cannabinoid exposure on behavior and corticostriatal brain physiology by assessing mice behavior, the concentration of endocannabinoid and endocannabinoid-like molecules and changes in the transcriptome.

Results: Stressed animals urinated frequently; showed exacerbated scratching activity, lower striatal N-arachidonylethanolamine (AEA) levels and higher cortical expression of cholinergic receptor nicotinic alpha 6. The cannabinoid agonist WIN55212.2 diminished locomotor activity while the inverse agonist increased the distance travelled in the center of the open field. Upon CB activation, N-oleoylethanolamide and N-palmitoylethanolamide, two AEA congeners that do not interact directly with cannabinoid receptors, were enhanced in the striatum. The co-administration with both cannabinoids induced an up-regulation of striatal FK506 binding protein 5. The inverse agonist in controls reversed the effects of WIN55212.2 on motor activity. When Rimonabant was injected under stress, the cortical levels of 2-arachidonoylglycerol were maximum. The agonist and the antagonist influenced the cortical expression of cholinergic receptor nicotinic alpha 6 and serotonin transporter neurotransmitter type 4 in opposite directions, while their co-administration tended to produce a null effect under stress.

Conclusions: The endocannabinoid system had a direct effect on serotoninergic neurotransmission and glucocorticoid signaling. Cholinergic receptor nicotinic alpha-6 was shown to be deregulated in response to stress and following synthetic cannabinoid drugs thus could confer vulnerability to cannabis addiction and psychosis. Targeting the receptors of endocannabinoids and endocannabinoid-like mediators might be a valuable option for treating stress-related neuropsychiatric symptoms.
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http://dx.doi.org/10.1111/cns.12810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5969305PMC
June 2018

Pax6 controls centriole maturation in cortical progenitors through Odf2.

Cell Mol Life Sci 2015 May 29;72(9):1795-809. Epub 2014 Oct 29.

Research Group of Molecular Developmental Neurobiology, Department Molecular Cell Biology, Max-Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077, Göttingen, Germany.

Cortical glutamatergic neurons are generated by radial glial cells (RGCs), specified by the expression of transcription factor (TF) Pax6, in the germinative zones of the dorsal telencephalon. Here, we demonstrate that Pax6 regulates the structural assembly of the interphase centrosomes. In the cortex of the Pax6-deficient Small eye (Sey/Sey) mutant, we find a defect of the appendages of the mother centrioles, indicating incomplete centrosome maturation. Consequently, RGCs fail to generate primary cilia, and instead of staying in the germinative zone for renewal, RGCs detach from the ventricular surface thus affecting the interkinetic nuclear migration and they exit prematurely from mitosis. Mechanistically, we show that TF Pax6 directly regulates the activity of the Odf2 gene encoding for the appendage-specific protein Odf2 with a role for the assembly of mother centriole. Our findings demonstrate a molecular mechanism that explains important characteristics of the centrosome disassembly and malfunctioning in developing cortex lacking Pax6.
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http://dx.doi.org/10.1007/s00018-014-1766-1DOI Listing
May 2015

Haplo-deficiency of ODF1/HSPB10 in mouse sperm causes relaxation of head-to-tail linkage.

Reproduction 2014 Nov 12;148(5):499-506. Epub 2014 Aug 12.

Johann-Friedrich-Blumenbach-Institute of Zoology and Anthropology - Developmental BiologyGZMB, Ernst-Caspari-Haus, Justus-von-Liebig-Weg 11, Georg-August-Universität Göttingen, 37077 Göttingen, GermanyInstitute of Human GeneticsUniversity Medicine, Heinrich-Düker-Weg 12, Georg-August-Universität Göttingen, 37077 Göttingen, GermanyDepartment of Genetics and EvolutionInstitute of Zoology, Jagiellonian University, Gronostajowa 9, 30-387 Kraków, Poland andDepartment of Anatomy and Cell BiologyAulweg 123, Justus-Liebig-University Gießen, 35392 Gießen, Germany

The small heat shock protein ODF1/HSPB10 is essential for male fertility in mice. Targeted deletion of Odf1 resulted in acephalic sperm in homozygous mice of mixed background (C57BL/6J//129/Sv), whereas heterozygous animals are fully fertile. To further elucidate the function of ODF1, we generated incipient congenic mice with targeted deletion of Odf1 by successive backcrossing on the 129/Sv background. We observed that fecundity of heterozygous Odf1(+/-) male mice was severely reduced over backcross generations. However, neither aberrant sperm parameters nor sperm anomalies could be observed. Ultra-structural analyses of sperm from incipient congenic heterozygous Odf1(+/-) males of backcross generation N7 revealed no obvious pathological findings. However, we observed an enlargement of the distance between nuclear membrane and capitulum, indicating a weakening of the sperm head-to-tail coupling. Severe male subfertility provoked by haplo-deficiency of ODF1 is therefore most probably caused by impaired head-to-tail coupling that eventually might induce sperm decapitation on the specific conditions of in vivo fertilisation. As subfertility in haplo-deficient ODF1 male mice could not be diagnosed by semen analysis, it seems to be a paradigm for unexplained infertility that is a frequent diagnosis for male fertility impairment in humans.
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http://dx.doi.org/10.1530/REP-14-0370DOI Listing
November 2014

Transcriptional activation of Odf2/Cenexin by cell cycle arrest and the stress activated signaling pathway (JNK pathway).

Biochim Biophys Acta 2013 Jun 28;1833(6):1338-46. Epub 2013 Feb 28.

Johann-Friedrich-Blumenbach Institute for Zoology and Anthropology, Developmental Biology, Göttingen, Germany.

The centrosome/basal body protein ODF2/Cenexin is necessary for the formation of the primary cilium. Primary cilia are essential organelles that sense and transduce environmental signals. Primary cilia are therefore critical for embryonic and postnatal development as well as for tissue homeostasis in adulthood. Impaired function of primary cilia causes severe human diseases. ODF2 deficiency prevents formation of the primary cilium and is embryonically lethal. To explore the regulation of primary cilia formation we analyzed the promoter region of Odf2 and its transcriptional activity. In cycling cells, Odf2 transcription is depressed but becomes up-regulated in quiescent cells. Low transcriptional activity is mediated by sequences located upstream from the basal promoter, and neither transcription factors with predicted binding sites in the Odf2 promoter nor Rfx3 or Foxj, which are known to control ciliary gene expression, could activate Odf2 transcription. However, co-expression of either C/EBPα, c-Jun or c-Jun and its regulator MEKK1 enhances Odf2 transcription in cycling cells. Our results provide the first analysis of transcriptional regulation of a ciliary gene. Furthermore, we suggest that transcription of even more ciliary genes is largely inhibited in cycling cells but could be activated by cell cycle arrest and by the stress signaling JNK pathway.
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http://dx.doi.org/10.1016/j.bbamcr.2013.02.023DOI Listing
June 2013

The small heat shock protein ODF1/HSPB10 is essential for tight linkage of sperm head to tail and male fertility in mice.

Mol Cell Biol 2012 Jan 28;32(1):216-25. Epub 2011 Oct 28.

Johann Friedrich Blumenbach Institute of Zoology and Anthropology-Developmental Biology, GZMB, Georg-August-Universität Göttingen, Göttingen, Germany.

Sperm motility and hence male fertility strictly depends on proper development of the sperm tail and its tight anchorage to the head. The main protein of sperm tail outer dense fibers, ODF1/HSPB10, belongs to the family of small heat shock proteins that function as molecular chaperones. However, the impact of ODF1 on sperm tail formation and motility and on male fecundity is unknown. We therefore generated mutant mice in which the Odf1 gene was disrupted. Heterozygous mutant male mice are fertile while sperm motility is reduced, but Odf1-deficient male mice are infertile due to the detachment of the sperm head. Although headless tails are somehow motile, transmission electron microscopy revealed disturbed organization of the mitochondrial sheath, as well as of the outer dense fibers. Our results thus suggest that ODF1, besides being involved in the correct arrangement of mitochondrial sheath and outer dense fibers, is essential for rigid junction of sperm head and tail. Loss of function of ODF1, therefore, might account for some of the cases of human infertility with decapitated sperm heads. In addition, since sperm motility is already affected in heterozygous mice, impairment of ODF1 might even account for some cases of reduced fertility in male patients.
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http://dx.doi.org/10.1128/MCB.06158-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3255718PMC
January 2012

SPAG4L/SPAG4L-2 are testis-specific SUN domain proteins restricted to the apical nuclear envelope of round spermatids facing the acrosome.

Mol Hum Reprod 2011 Apr 15;17(4):207-18. Epub 2010 Dec 15.

Johann-Friedrich-Blumenbach Institute of Zoology and Anthropology-Developmental Biology, Georg-August-Universität Göttingen, GZMB, Ernst-Caspari-Haus, Justus-von-Liebig-Weg 11, Göttingen, Germany.

SUN domain proteins are integral proteins of the inner nuclear membrane and functions in linkage of the nuclear lamina to the cytoskeleton. Moreover, SUN domain proteins seem to mediate the tethering of the centrosome to the nuclear membrane, and they are involved in telomere attachment to the nuclear envelope in meiotic cells, and in germ cell development in invertebrates. In contrast to the widely expressed SUN domain proteins in mammals, SUN1 and SUN2, which have been analysed in great detail, there is virtually nothing known about testicular SUN domain proteins. Since a hallmark of male germ cell development is the profound remodelling of the nuclear envelope, emphasized, for example, by the reshaping of the nucleus during spermiogenesis, and the biogenesis of its tightly associated acrosome, SUN domain proteins might be engaged in these processes. We have isolated a novel SUN domain protein, SPAG4L-2, that differs from SPAG4L by an N-terminal insertion of 25 amino acids. Spag4l and Spag4l-2 are exclusively expressed in testis at about equimolar amounts, and show elevated transcription during ongoing spermiogenesis coincident with the appearance of round spermatids. Molecular dissection of the protein followed by cytological and biochemical investigations revealed that SPAG4L-2 and SPAG4L are transmembrane proteins that localize to the nuclear envelope. SPAG4L/4L-2 are restricted to the apical nuclear region of round spermatids that face the acrosomic vesicle, and thus are most probably involved in linkage of the acrosomic vesicle to the spermatid nucleus, and in acrosome biogenesis.
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http://dx.doi.org/10.1093/molehr/gaq099DOI Listing
April 2011

CHD8 interacts with CHD7, a protein which is mutated in CHARGE syndrome.

Hum Mol Genet 2010 Jul 7;19(14):2858-66. Epub 2010 May 7.

Institute of Human Genetics, University of Göttingen, 37073 Göttingen, Germany.

CHARGE syndrome is an autosomal dominant disorder caused in about two-third of cases by mutations in the CHD7 gene. For other genetic diseases e.g. hereditary spastic paraplegia, it was shown that interacting partners are involved in the underlying cause of the disease. These data encouraged us to search for CHD7 binding partners by a yeast two-hybrid library screen and CHD8 was identified as an interacting partner. The result was confirmed by a direct yeast two-hybrid analysis, co-immunoprecipitation studies and by a bimolecular fluorescence complementation assay. To investigate the function of CHD7 missense mutations in the CHD7-CHD8 interacting area on the binding capacity of both proteins, we included three known missense mutations (p.His2096Arg, p.Val2102Ile and p.Gly2108Arg) and one newly identified missense mutation (p.Trp2091Arg) in the CHD7 gene and performed both direct yeast two-hybrid and co-immunoprecipitation studies. In the direct yeast two-hybrid system, the CHD7-CHD8 interaction was disrupted by the missense mutations p.Trp2091Arg, p.His2096Arg and p.Gly2108Arg, whereas in the co-immunoprecipitation studies disruption of the CHD7-CHD8 interaction by the mutations could not be observed. The results lead to the hypothesis that CHD7 and CHD8 proteins are interacting directly and indirectly via additional linker proteins. Disruption of the direct CHD7-CHD8 interaction might change the conformation of a putative large CHD7-CHD8 complex and could be a disease mechanism in CHARGE syndrome.
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http://dx.doi.org/10.1093/hmg/ddq189DOI Listing
July 2010

Pelota interacts with HAX1, EIF3G and SRPX and the resulting protein complexes are associated with the actin cytoskeleton.

BMC Cell Biol 2010 Apr 20;11:28. Epub 2010 Apr 20.

Institute of Human Genetics, Georg-August-University, Göttingen, Germany.

Background: Pelota (PELO) is an evolutionary conserved protein, which has been reported to be involved in the regulation of cell proliferation and stem cell self-renewal. Recent studies revealed the essential role of PELO in the No-Go mRNA decay, by which mRNA with translational stall are endonucleotically cleaved and degraded. Further, PELO-deficient mice die early during gastrulation due to defects in cell proliferation and/or differentiation.

Results: We show here that PELO is associated with actin microfilaments of mammalian cells. Overexpression of human PELO in Hep2G cells had prominent effect on cell growth, cytoskeleton organization and cell spreading. To find proteins interacting with PELO, full-length human PELO cDNA was used as a bait in a yeast two-hybrid screening assay. Partial sequences of HAX1, EIF3G and SRPX protein were identified as PELO-interacting partners from the screening. The interactions between PELO and HAX1, EIF3G and SRPX were confirmed in vitro by GST pull-down assays and in vivo by co-immunoprecipitation. Furthermore, the PELO interaction domain was mapped to residues 268-385 containing the c-terminal and acidic tail domain. By bimolecular fluorescence complementation assay (BiFC), we found that protein complexes resulting from the interactions between PELO and either HAX1, EIF3G or SRPX were mainly localized to cytoskeletal filaments.

Conclusion: We could show that PELO is subcellularly localized at the actin cytoskeleton, interacts with HAX1, EIF3G and SRPX proteins and that this interaction occurs at the cytoskeleton. Binding of PELO to cytoskeleton-associated proteins may facilitate PELO to detect and degrade aberrant mRNAs, at which the ribosome is stalled during translation.
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http://dx.doi.org/10.1186/1471-2121-11-28DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2867792PMC
April 2010

Mouse Odf2 localizes to centrosomes and basal bodies in adult tissues and to the photoreceptor primary cilium.

Cell Tissue Res 2009 Nov 10;338(2):295-301. Epub 2009 Sep 10.

Johann Friedrich Blumenbach Institute of Zoology and Anthropology - Developmental Biology, GZMB, Georg August University of Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany.

Odf2 (outer dense fiber 2) is the major protein of the cytoskeleton of the sperm tail. In somatic cells, it is a component of the centrosome in which it is located in the appendages of the mother centriole. Additionally, as shown previously by forced expression in cultured cells, Odf2 localizes to centrioles, basal bodies, and primary cilia, which are all structurally and functionally interconnected. The importance of Odf2 has become obvious by the absence of primary cilia in Odf2-deficient cells and by the embryonic lethality of the Odf2 gene trap insertional mouse. However, nothing is known about the endogenous localization of Odf2 in the tissues of adult mice. We show here that Odf2 protein localizes to centrosomes, to photoreceptor primary cilia, and to basal bodies of ciliated cells of the respiratory epithelium and of the kidney. Our results thus suggest that Odf2 contributes to assorted ciliopathies.
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http://dx.doi.org/10.1007/s00441-009-0861-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2766462PMC
November 2009

Centriole maturation and transformation to basal body.

Semin Cell Dev Biol 2010 Apr 10;21(2):142-7. Epub 2009 Jul 10.

Johann-Friedrich-Blumenbach-Institute of Zoology and Anthropology - Developmental Biology, GZMB, Ernst-Caspari-Haus, Justus-von-Liebig-Weg 11, Georg-August-University of Göttingen, 37077 Gottingen, Germany.

Centrioles and basal bodies are fascinating and mysterious organelles. They interconvert and seem to be crucial for a wide range of crucial cellular processes. However, intense research over the last years suggested that centrioles/basal bodies are essential mainly for the generation of cilia. Although a neglected organelle over a long time, interest in the primary cilia was recently rekindled by the notion that they are affected in a number of human diseases. Cilia formation is an intricate process that starts with the transformation of centrioles to basal bodies and their docking to the apical plasma membrane. Disturbance of basal body formation thus might cause ciliopathies. This review focuses on the formation of basal bodies in mammalian cells with an emphasis on basal bodies sprouting a primary cilium.
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http://dx.doi.org/10.1016/j.semcdb.2009.07.002DOI Listing
April 2010

Molecular dissection of ODF2/Cenexin revealed a short stretch of amino acids necessary for targeting to the centrosome and the primary cilium.

Eur J Cell Biol 2008 Mar 2;87(3):137-46. Epub 2008 Jan 2.

Johann-Friedrich-Blumenbach-Institut für Zoologie, Anthropologie und Entwicklungsbiologie, Universität Göttingen, GZMB, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany.

The outer dense fiber protein ODF2 is the major component of the sperm tail cytoskeleton and a critical component of the mature centriole of the centrosome. Centriole maturation involves the formation of appendages and the recruitment of ODF2/Cenexin. ODF2 and Cenexin are alternative splice variants that differ in a short stretch of amino acids at their N-terminal regions encoded by exon 3b. Whereas Cenexin is ubiquitously expressed, Odf2 is the predominant transcript of testes [Hüber, D., Hoyer-Fender, S., 2007. Alternative splicing of exon 3b gives rise to ODF2 and Cenexin. Cytogenet. Genome Res. 119, doi:10.1159/000109621]. Here, we show that testicular expression of Odf2 correlates with spermiogenesis and ongoing sperm tail formation thus implicating functional differences between ODF2 and Cenexin. By generation of a series of ODF2/Cenexin deletion constructs fused to GFP and inspection of their subcellular localization in transfected NIH3T3 cells we found that a peptide of 42 amino acids specific for Cenexin is necessary for targeting ODF2/Cenexin to the centrosome and the primary cilium. Additionally, this region is also necessary for the formation of ODF2/Cenexin fibers that are associated with acetylated microtubules. Centrosomal targeting of ODF2/Cenexin does not depend on dynein-mediated transport further supporting an alternative targeting mechanism. However, part of the C-terminal coiled-coil region of ODF2 is also important in centrosomal/ciliary targeting and fiber formation presumably by supporting self-association and the formation of higher-order structures.
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http://dx.doi.org/10.1016/j.ejcb.2007.10.004DOI Listing
March 2008

Reduction of spermatogenesis but not fertility in Creb3l4-deficient mice.

Mol Cell Biol 2005 Sep;25(17):7657-64

Institute of Human Genetics, University of Göttingen, Germany.

Creb3l4 belongs to the CREB/ATF family of transcription factors that are involved in mediating transcription in response to intracellular signaling. This study shows that Creb3l4 is expressed at low levels in all organs and in different stages of embryogenesis but is present at very high levels in the testis, particularly in postmeiotic male germ cells. In contrast to CREB3L4 in the human prostate, of which specific expression was detected, Creb3l4 transcripts in the mouse prostate could be detected only by RT-PCR. To identify the physiological function of Creb3l4, the murine gene was inactivated by replacement with the gene encoding green fluorescent protein. Surprisingly, Creb3l4-deficient mice were born at expected ratios, were healthy, and displayed normal long-term survival rates. Despite a significant reduction in the number of spermatozoa in the epididymis of Creb3l4(-)(/)(-) mice, the breeding of mutant males with wild-type females was productive and the average litter size was not significantly altered in comparison to wild-type littermates. Further analyses revealed that the seminiferous tubules of Creb3l4(-)(/)(-) mice contained all of the developmental stages, though there was evidence for increased apoptosis of meiotic/postmeiotic germ cells. These results suggest that Creb3l4 plays a role in male germ cell development, but its loss is insufficient to completely compromise the production of spermatozoa.
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http://dx.doi.org/10.1128/MCB.25.17.7657-7664.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1190296PMC
September 2005

Outer dense fibre protein 2 (ODF2) is a self-interacting centrosomal protein with affinity for microtubules.

J Cell Sci 2004 Sep 31;117(Pt 20):4643-51. Epub 2004 Aug 31.

Göttinger Zentrum für Molekulare Biowissenschaften, Entwicklungsbiologie, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany.

Outer dense fibre protein 2 (ODF2) is a major protein of sperm tail outer dense fibres which are prominent sperm tail-specific cytoskeletal structures. Moreover, ODF2 was also identified as a widespread component of the centrosomal scaffold and was found to associate preferentially with the appendages of the mother centriole [Nakagawa, Y., Yamane, Y., Okanoue, T., Tsukita, S. and Tsukita, S. (2001) Mol. Biol. Cell 12, 1687-1697]. Secondary structure predictions indicated ODF2 as an overall coiled-coil protein with a putative fibre forming capacity. To investigate its potential functions in generating the centrosomal scaffold and in microtubule nucleation we asked whether ODF2 is able to form a fibrillar structure by self-association in vivo and if it interacts with microtubules. By cytological investigation of transfected mammalian cells expressing ODF2-GFP fusion proteins and in vitro coprecipitation assays we could demonstrate that ODF2 is a self-interacting protein that forms a fibrillar structure partially linked to the microtubule network. Microtubule cosedimentation and coprecipitation assays indicated ODF2 as a microtubule-associated protein. However, we could not demonstrate a direct interaction of ODF2 with tubulin, suggesting that binding of endogenous ODF2 to the axonemal as well as to centrosomal microtubules may be mediated by, as yet, unknown proteins.
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http://dx.doi.org/10.1242/jcs.01303DOI Listing
September 2004

Localisation of histone macroH2A1.2 to the XY-body is not a response to the presence of asynapsed chromosome axes.

J Cell Sci 2004 Jan;117(Pt 2):189-98

III. Department of Zoology-Developmental Biology, University of Göttingen, Humboldtallee 34A, 37073 Göttingen, Germany.

Histone macroH2A1.2 and the murine heterochromatin protein 1, HP1 beta, have both been implicated in meiotic sex chromosome inactivation (MSCI) and the formation of the XY-body in male meiosis. In order to get a closer insight into the function of histone macroH2A1.2 we have investigated the localisation of macroH2A1.2 in surface spread spermatocytes from normal male mice and in oocytes of XX and XYTdym1 mice. Oocytes of XYTdym1 mice have no XY-body or MSCI despite having an XY chromosome constitution, so the presence or absence of 'XY-body' proteins in association with the X and/or Y chromosome of these oocytes enables some discrimination between potential functions of XY-body located proteins. We demonstrate here that macroH2A1.2 localises to the X and Y chromatin of spermatocytes as they condense to form the XY-body but is not associated with the X and Y chromatin of XYTdym1 early pachytene oocytes. MacroH2A1.2 and HP1 beta co-localise to autosomal pericentromeric heterochromatin in spermatocytes. However, the two proteins show temporally and spatially distinct patterns of association to X and Y chromatin.
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http://dx.doi.org/10.1242/jcs.00851DOI Listing
January 2004

Disruption of the pelota gene causes early embryonic lethality and defects in cell cycle progression.

Mol Cell Biol 2003 Feb;23(4):1470-6

Institute of Human Genetics, University of Göttingen, 37073 Göttingen, Germany.

Mutations in either the Drosophila melanogaster pelota or pelo gene or the Saccharomyces cerevisiae homologous gene, DOM34, cause defects of spermatogenesis and oogenesis in Drosophila, and delay of growth and failure of sporulation in yeast. These phenotypes suggest that pelota is required for normal progression of the mitotic and meiotic cell cycle. To determine the role of the pelota in mouse development and progression of cell cycle, we have established a targeted disruption of the mouse PELO: Heterozygous animals are variable and fertile. Genotyping of the progeny of heterozygous intercrosses shows the absence of Pelo(-/-) pups and suggests an embryo-lethal phenotype. Histological analyses reveal that the homozygous Pelo deficient embryos fail to develop past day 7.5 of embryogenesis (E7.5). The failure of mitotic active inner cell mass of the Pelo(-/-) blastocysts to expand in growth after 4 days in culture and the survival of mitotic inactive trophoplast indicate that the lethality of Pelo-null embryos is due to defects in cell proliferation. Analysis of the cellular DNA content reveals the significant increase of aneuploid cells in Pelo(-/-) embryos at E7.5. Therefore, the percent increase of aneuploid cells at E7.5 may be directly responsible for the arrested development and suggests that Pelo is required for the maintenance of genomic stability.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC141158PMC
http://dx.doi.org/10.1128/mcb.23.4.1470-1476.2003DOI Listing
February 2003

The Hook1 gene is non-functional in the abnormal spermatozoon head shape (azh) mutant mouse.

Hum Mol Genet 2002 Jul;11(14):1647-58

Institute of Human Genetics, Department of Zoology and Developmental Biology, University of Göttingen, 37073 Göttingen, Germany.

In mice carrying the autosomal recessive mutation 'abnormal spermatozoon head shape' (azh) all spermatozoa display a highly abnormal head morphology that differs drastically from the compact and hook-shaped head of the normal murine sperm. Moreover, the azh mutation causes tail abnormalities often resulting in coiled sperm tails or in the decapitation of the sperm head from the flagellum. We have isolated and characterized murine Hook1 cDNA and analyzed the corresponding genomic structure. Furthermore, the Hook1 gene was mapped to the same region on chromosome 4 to which the azh locus was previously linked. The Hook1 gene is predominantly expressed in haploid male germ cells, and immunohistochemical analysis revealed that Hook1 is responsible for the linkage of the microtubular manchette and the flagellum to cellular structures. Here, we report that the azh mutation is due to a deletion of exons 10 and 11 in the murine Hook1 gene leading to a non-functional protein. Our results indicate that loss of Hook1 function results in ectopic positioning of microtubular structures within the spermatid and causes the azh phenotype. Therefore, the human HOOK1 gene could serve as a candidate gene for male infertility due to teratozoospermia or decapitation defects.
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http://dx.doi.org/10.1093/hmg/11.14.1647DOI Listing
July 2002

Molecular cloning of Odf3 encoding a novel coiled-coil protein of sperm tail outer dense fibers.

Mol Reprod Dev 2002 Jan;61(1):102-12

III. Zoologisches Institut-Entwicklungsbiologie, Georg-August-Universität, Göttingen, Germany.

The outer dense fibers (ODF) are the main cytoskeletal structures of the sperm tail found in animals with internal fecundation. They consist of at least 14 polypeptides from which only a few are identified due to difficulties in isolation of the protein components. Here we report the isolation and molecular characterization of Odf3, encoding a novel protein of rat sperm ODF. Odf3 is transcribed in testes and more specifically in spermatids but it is also expressed in epididymides and brain suggesting a possible involvement in building of the cellular cytoskeleton. Odf3 encodes a putative protein of approximately 110 kDa. Secondary structure predictions indicated that ODF3 is a coiled-coil protein. The identification of coiled-coil proteins as constituents of outer dense fibers reveals a model for ODF formation.
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http://dx.doi.org/10.1002/mrd.1136DOI Listing
January 2002