Publications by authors named "Angelika A Noegel"

130 Publications

A 24-generation-old founder mutation impairs splicing of RBBP8 in Pakistani families affected with Jawad syndrome.

Clin Genet 2021 Oct 16;100(4):486-488. Epub 2021 Jul 16.

Cologne Center for Genomics (CCG) and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany.

Jawad syndrome is a multiple congenital anomaly and intellectual disability syndrome with mutation in RBBP8 reported only in two families. Here, we report on two new families from Pakistan and identified a previously reported variant in RBBP8, NM_002894.3:c.1808-1809delTA. We could show that this mutation impairs splicing resulting in two different abnormal transcripts. Finally, we could verify a shared haplotype among all four families and estimate the founder event to have occurred some 24 generations ago.
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http://dx.doi.org/10.1111/cge.14028DOI Listing
October 2021

Biallelic variants in PCDHGC4 cause a novel neurodevelopmental syndrome with progressive microcephaly, seizures, and joint anomalies.

Genet Med 2021 Jul 9. Epub 2021 Jul 9.

Department of Bioinformatics & Biotechnology, Faculty of Basic and Applied Sciences, International Islamic University, Islamabad, Pakistan.

Purpose: We aimed to define a novel autosomal recessive neurodevelopmental disorder, characterize its clinical features, and identify the underlying genetic cause for this condition.

Methods: We performed a detailed clinical characterization of 19 individuals from nine unrelated, consanguineous families with a neurodevelopmental disorder. We used genome/exome sequencing approaches, linkage and cosegregation analyses to identify disease-causing variants, and we performed three-dimensional molecular in silico analysis to predict causality of variants where applicable.

Results: In all affected individuals who presented with a neurodevelopmental syndrome with progressive microcephaly, seizures, and intellectual disability we identified biallelic disease-causing variants in Protocadherin-gamma-C4 (PCDHGC4). Five variants were predicted to induce premature protein truncation leading to a loss of PCDHGC4 function. The three detected missense variants were located in extracellular cadherin (EC) domains EC5 and EC6 of PCDHGC4, and in silico analysis of the affected residues showed that two of these substitutions were predicted to influence the Ca-binding affinity, which is essential for multimerization of the protein, whereas the third missense variant directly influenced the cis-dimerization interface of PCDHGC4.

Conclusion: We show that biallelic variants in PCDHGC4 are causing a novel autosomal recessive neurodevelopmental disorder and link PCDHGC4 as a member of the clustered PCDH family to a Mendelian disorder in humans.
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http://dx.doi.org/10.1038/s41436-021-01260-4DOI Listing
July 2021

Nesprin-1 impact on tumorigenic cell phenotypes.

Mol Biol Rep 2020 Feb 18;47(2):921-934. Epub 2019 Nov 18.

Institute of Biochemistry I, Medical Faculty, University Hospital Cologne, Cologne, Germany.

The largest protein of the nuclear envelope (NE) is Nesprin-1 which forms a network along the NE interacting with actin, Emerin, Lamin, and SUN proteins. Mutations in the SYNE1 gene and reduction in Nesprin-1 protein levels have been reported to correlate with several age related diseases and cancer. In the present study, we tested whether Nesprin-1 overexpression can reverse the malignant phenotype of Huh7 cells, a human liver cancer cell line, which carries a mutation in the SYNE1 gene resulting in reduced Nesprin-1 protein levels, has altered nuclear shape, altered amounts and localization of NE components, centrosome localization and genome stability. Ectopic expression of a mini-Nesprin-1 led to an improvement of the nuclear shape, corrected the mislocalization of NE proteins, the centrosome positioning, and the alterations in the DNA damage response network. Additionally, Nesprin-1 had a profound effect on cellular senescence. These findings suggest that Nesprin-1 may be effective in tumorigenic cell phenotype correction of human liver cancer.
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http://dx.doi.org/10.1007/s11033-019-05184-wDOI Listing
February 2020

CRN2 binds to TIMP4 and MMP14 and promotes perivascular invasion of glioblastoma cells.

Eur J Cell Biol 2019 Dec 11;98(5-8):151046. Epub 2019 Oct 11.

Centre for Biochemistry, Institute of Biochemistry I, Medical Faculty, University of Cologne, 50931, Cologne, Germany; Institute of Neuropathology, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nürnberg, 91054, Erlangen, Germany; Center for Physiology and Pathophysiology, Institute of Vegetative Physiology, Medical Faculty, University of Cologne, 50931, Cologne, Germany. Electronic address:

CRN2 is an actin filament binding protein involved in the regulation of various cellular processes including cell migration and invasion. CRN2 has been implicated in the malignant progression of different types of human cancer. We used CRN2 knock-out mice for analyses as well as for crossbreeding with a Tp53/Pten knock-out glioblastoma mouse model. CRN2 knock-out mice were subjected to a phenotyping screen at the German Mouse Clinic. Murine glioblastoma tissue specimens as well as cultured murine brain slices and glioblastoma cell lines were investigated by immunohistochemistry, immunofluorescence, and cell biological experiments. Protein interactions were studied by immunoprecipitation, pull-down, and enzyme activity assays. CRN2 knock-out mice displayed neurological and behavioural alterations, e.g. reduced hearing sensitivity, reduced acoustic startle response, hypoactivity, and less frequent urination. While glioblastoma mice with or without the additional CRN2 knock-out allele exhibited no significant difference in their survival rates, the increased levels of CRN2 in transplanted glioblastoma cells caused a higher tumour cell encasement of murine brain slice capillaries. We identified two important factors of the tumour microenvironment, the tissue inhibitor of matrix metalloproteinase 4 (TIMP4) and the matrix metalloproteinase 14 (MMP14, synonym: MT1-MMP), as novel binding partners of CRN2. All three proteins mutually interacted and co-localised at the front of lamellipodia, and CRN2 was newly detected in exosomes. On the functional level, we demonstrate that CRN2 increased the secretion of TIMP4 as well as the catalytic activity of MMP14. Our results imply that CRN2 represents a pro-invasive effector within the tumour cell microenvironment of glioblastoma multiforme.
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http://dx.doi.org/10.1016/j.ejcb.2019.151046DOI Listing
December 2019

Depletion of Nesprin-2 is associated with an embryonic lethal phenotype in mice.

Nucleus 2018 ;9(1):503-515

a Institute of Biochemistry I, Medical Faculty , University Hospital Cologne; Center for Molecular Medicine Cologne (CMMC) and Cologne Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD) , Koeln , Germany.

Nesprin-2 is a nuclear envelope component and provides a link between cytoskeletal components of the cytoplasm and the nucleoplasm. Several isoforms are generated from its gene Syne2. Loss of the largest isoform Nesprin-2 Giant in mice is associated with a skin phenotype and altered wound healing, loss of C-terminal isoforms in mice leads to cardiomyopathies and neurological defects. Here we attempted to establish mice with an inducible knockout of all Nesprin-2 isoforms by inserting shRNA encoding sequences targeting the N- and C-terminus into the ROSA26 locus of mice. This caused early embryonic death of the animals harboring the mutant allele, which was presumably due to leaky expression of the shRNAs. Mutant embryos were only observed before E13. They had an altered appearance and were smaller in size than their wild type littermates. From this we conclude that the Nesprin-2 gene function is crucial during embryonic growth, differentiation and organogenesis.
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http://dx.doi.org/10.1080/19491034.2018.1523664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6244730PMC
September 2019

Functional Analysis of LINC Complexes in the Skin.

Methods Mol Biol 2018 ;1840:295-306

Institute of Biochemistry I, Medical Faculty, University Hospital Cologne, Köln, Germany.

The genome in eukaryotic cells is encased by two intricate and interconnected concentric membranes, which together with the underlying nuclear lamina form the nuclear envelope (NE). Two fundamental macromolecular structures are embedded within the nuclear envelope: the nuclear pore (NPC) and the LINC complex. The former perforates the nucleus controlling biomolecule trafficking between the nucleoplasm and the cytoplasm, while the latter integrates the nucleus via the cytoskeleton to the extracellular matrix. LINC complex structural and functional integrity is of utmost importance for various fundamental cellular functions. Mechanical forces are relayed into the nuclear interior via the LINC complex, which controls lamina organization, chromosome dynamics, and genome organization and stability. Thus, LINC constituents play pivotal roles in cellular architecture including organelle positioning, cell movement, tissue assembly, organ homeostasis, and organismal aging. The LINC complex oligomeric core contains several multi-isomeric, multifunctional, and often tissue-specific proteins. Therefore, for a proper functional analysis, genetic mouse models are an invaluable resource. Herein, we focus on the LINC complex roles in the skin and describe methods that enable the successful isolation of primary embryonic fibroblast and newborn skin cells, which can be then investigated functionally in vitro.
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http://dx.doi.org/10.1007/978-1-4939-8691-0_20DOI Listing
April 2019

The regulatory subunit phr2AB of Dictyostelium discoideum phosphatase PP2A interacts with the centrosomal protein CEP161, a CDK5RAP2 ortholog.

Genes Cells 2018 Oct 21;23(10):923-931. Epub 2018 Sep 21.

Institute for Biochemistry I, Medical Faculty, University Hospital Cologne, Cologne, Germany.

phr2AB is the regulatory subunit of the Dictyostelium discoideum phosphatase PP2A and is the ortholog of the human B55 regulatory subunit of PP2A. phr2AB was isolated as a binding partner of the centrosomal protein CEP161, an ortholog of mammalian CDK5RAP2. CEP161 is presumably a phosphoprotein and a component of the Hippo pathway. The interaction site was located in the N-terminal half of CEP161 which encompasses the γTURC binding domain in CEP161. This binding domain is responsible for binding of the γ-tubulin ring complex which allows microtubule nucleation at the centrosome. GFP-tagged phr2AB is diffusely distributed throughout the cell and enriched at the centrosome. Ectopic expression of phr2AB as GFP fusion protein led to multinucleation, aberrant nucleus centrosome ratios and an altered sensitivity to okadaic acid. Some of these features were also affected in cells over-expressing domains of CEP161 and in cells from patients suffering from primary microcephaly, which carried a mutated CDK5RAP2 gene.
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http://dx.doi.org/10.1111/gtc.12637DOI Listing
October 2018

Coronin 1C promotes triple-negative breast cancer invasiveness through regulation of MT1-MMP traffic and invadopodia function.

Oncogene 2018 12 31;37(50):6425-6441. Epub 2018 Jul 31.

Institut Curie, PSL Research University, CNRS UMR144, Membrane and Cytoskeleton Dynamics group, 26 rue d'Ulm, F-75005, Paris, France.

Membrane type 1-matrix metalloproteinase (MT1-MMP), a membrane-tethered protease, is key for matrix breakdown during cancer invasion and metastasis. Assembly of branched actin networks by the Arp2/3 complex is required for MT1-MMP traffic and formation of matrix-degradative invadopodia. Contrasting with the well-established role of actin filament branching factor cortactin in invadopodia function during cancer cell invasion, the contribution of coronin-family debranching factors to invadopodia-based matrix remodeling is not known. Here, we investigated the contribution of coronin 1C to the invasive potential of breast cancer cells. We report that expression of coronin 1C is elevated in invasive human breast cancers, correlates positively with MT1-MMP expression in relation with increased metastatic risk and is a new independent prognostic factor in breast cancer. We provide evidence that, akin to cortactin, coronin 1C is required for invadopodia formation and matrix degradation by breast cancer cells lines and for 3D collagen invasion by multicellular spheroids. Using intravital imaging of orthotopic human breast tumor xenografts, we find that coronin 1C accumulates in structures forming in association with collagen fibrils in the tumor microenvironment. Moreover, we establish the role of coronin 1C in the regulation of positioning and trafficking of MT1-MMP-positive endolysosomes. These results identify coronin 1C as a novel player of the multi-faceted mechanism responsible for invadopodia formation, MT1-MMP surface exposure and invasiveness in breast cancer cells.
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http://dx.doi.org/10.1038/s41388-018-0422-xDOI Listing
December 2018

Functional analyses of Pericentrin and Syne-2 interaction in ciliogenesis.

J Cell Sci 2018 08 17;131(16). Epub 2018 Aug 17.

Animal Physiology, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen, Germany

Pericentrin (Pcnt) is a multifunctional scaffold protein and mutations in the human gene are associated with several diseases, including ciliopathies. Pcnt plays a crucial role in ciliary development in olfactory receptor neurons, but its function in the photoreceptor-connecting cilium is unknown. We downregulated Pcnt in the retina and via a virus-based RNA interference approach to study Pcnt function in photoreceptors. ShRNA-mediated knockdown of Pcnt impaired the development of the connecting cilium and the outer segment of photoreceptors, and caused a nuclear migration defect. In protein interaction screens, we found that the outer nuclear membrane protein Syne-2 (also known as Nesprin-2) is an interaction partner of Pcnt in photoreceptors. Syne-2 is important for positioning murine photoreceptor cell nuclei and for centrosomal migration during early ciliogenesis. CRISPR/Cas9-mediated knockout of Syne-2 in cell culture led to an overexpression and mislocalization of Pcnt and to ciliogenesis defects. Our findings suggest that the Pcnt-Syne-2 complex is important for ciliogenesis and outer segment formation during retinal development and plays a role in nuclear migration.
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http://dx.doi.org/10.1242/jcs.218487DOI Listing
August 2018

Nesprin-2 Interacts with Condensin Component SMC2.

Int J Cell Biol 2017 27;2017:8607532. Epub 2017 Dec 27.

Institute of Biochemistry I, Medical Faculty, University Hospital Cologne, Joseph-Stelzmann-Str. 52, 50931 Cologne, Germany.

The nuclear envelope proteins, Nesprins, have been primarily studied during interphase where they function in maintaining nuclear shape, size, and positioning. We analyze here the function of Nesprin-2 in chromatin interactions in interphase and dividing cells. We characterize a region in the rod domain of Nesprin-2 that is predicted as SMC domain (aa 1436-1766). We show that this domain can interact with itself. It furthermore has the capacity to bind to SMC2 and SMC4, the core subunits of condensin. The interaction was observed during all phases of the cell cycle; it was particularly strong during S phase and persisted also during mitosis. Nesprin-2 knockdown did not affect condensin distribution; however we noticed significantly higher numbers of chromatin bridges in Nesprin-2 knockdown cells in anaphase. Thus, Nesprin-2 may have an impact on chromosomes which might be due to its interaction with condensins or to indirect mechanisms provided by its interactions at the nuclear envelope.
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http://dx.doi.org/10.1155/2017/8607532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5763115PMC
December 2017

The function of the inner nuclear envelope protein SUN1 in mRNA export is regulated by phosphorylation.

Sci Rep 2017 08 22;7(1):9157. Epub 2017 Aug 22.

Institute of Biochemistry I, Medical Faculty, University Hospital Cologne, Center for Molecular Medicine Cologne (CMMC) and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Joseph-Stelzmann-Strasse 52, 50931, Cologne, Germany.

SUN1, a component of the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, functions in mammalian mRNA export through the NXF1-dependent pathway. It associates with mRNP complexes by direct interaction with NXF1. It also binds to the NPC through association with the nuclear pore component Nup153, which is involved in mRNA export. The SUN1-NXF1 association is at least partly regulated by a protein kinase C (PKC) which phosphorylates serine 113 (S113) in the N-terminal domain leading to reduced interaction. The phosphorylation appears to be important for the SUN1 function in nuclear mRNA export since GFP-SUN1 carrying a S113A mutation was less efficient in restoring mRNA export after SUN1 knockdown as compared to the wild type protein. By contrast, GFP-SUN1-S113D resembling the phosphorylated state allowed very efficient export of poly(A)+RNA. Furthermore, probing a possible role of the LINC complex component Nesprin-2 in this process we observed impaired mRNA export in Nesprin-2 knockdown cells. This effect might be independent of SUN1 as expression of a GFP tagged SUN-domain deficient SUN1, which no longer can interact with Nesprin-2, did not affect mRNA export.
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http://dx.doi.org/10.1038/s41598-017-08837-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5567243PMC
August 2017

A tripeptidyl peptidase 1 is a binding partner of the Golgi pH regulator (GPHR) in .

Dis Model Mech 2017 07 25;10(7):897-907. Epub 2017 May 25.

Institute of Biochemistry I, Medical Faculty, University Hospital Cologne, Center for Molecular Medicine Cologne, University of Cologne, Joseph-Stelzmann-Str. 52, Köln 50931, Germany

Mutations in tripeptidyl peptidase 1 () have been associated with late infantile neuronal ceroid lipofuscinosis (NCL), a neurodegenerative disorder. TPP1 is a lysosomal serine protease, which removes tripeptides from the N-terminus of proteins and is composed of an N-terminal prodomain and a catalytic domain. It is conserved in mammals, amphibians, fish and the amoeba harbors at least six genes encoding TPP1, to We identified TPP1F as binding partner of GPHR (Golgi pH regulator), which is an evolutionarily highly conserved intracellular transmembrane protein. A region encompassing the DUF3735 (GPHR_N) domain of GPHR was responsible for the interaction. In TPP1F, the binding site is located in the prodomain of the protein. The gene is transcribed throughout development and translated into a polypeptide of ∼65 kDa. TPP1 activity was demonstrated for TPP1F-GFP immunoprecipitated from cells. Its activity could be inhibited by addition of the recombinant DUF3735 domain of GPHR. Knockout mutants did not display any particular phenotype, and TPP1 activity was not abrogated, presumably because compensates as it has the highest expression level of all the genes during growth. The GPHR interaction was not restricted to TPP1F but occurred also with TPP1B. As previous reports show that the majority of the TPP1 mutations in NCL resulted in reduction or loss of enzyme activity, we suggest that could be used as a model system in which to test new reagents that could affect the activity of the protein and ameliorate the disease.
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http://dx.doi.org/10.1242/dmm.029280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5536908PMC
July 2017

CDK5RAP2 interaction with components of the Hippo signaling pathway may play a role in primary microcephaly.

Mol Genet Genomics 2017 Apr 21;292(2):365-383. Epub 2016 Dec 21.

Institute of Biochemistry I, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, 50931, Köln, Germany.

Autosomal recessive primary microcephaly (MCPH) is characterized by a substantial reduction in brain size but with normal architecture. It is often linked to mutations in genes coding for centrosomal proteins; however, their role in brain size regulation is not completely understood. By combining homozygosity mapping and whole-exome sequencing in an MCPH family from Pakistan, we identified a novel mutation (XM_011518861.1; c.4114C > T) in CDK5RAP2, the gene associated with primary microcephaly-3 (MCPH3), leading to a premature stop codon (p.Arg1372*). CDK5RAP2 is a component of the pericentriolar material important for the microtubule-organizing function of the centrosome. Patient-derived primary fibroblasts had strongly decreased CDK5RAP2 amounts, showed centrosomal and nuclear abnormalities and exhibited changes in cell size and migration. We further identified an interaction of CDK5RAP2 with the Hippo pathway components MST1 kinase and the transcriptional regulator TAZ. This finding potentially provides a mechanism through which the Hippo pathway with its roles in the regulation of centrosome number is linked to the centrosome. In the patient fibroblasts, we observed higher levels of TAZ and YAP. However, common target genes of the Hippo pathway were downregulated as compared to the control with the exception of BIRC5 (Survivin), which was significantly upregulated. We propose that the centrosomal deficiencies and the altered cellular properties in the patient fibroblasts can also result from the observed changes in the Hippo pathway components which could thus be relevant for MCPH and play a role in brain size regulation and development.
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http://dx.doi.org/10.1007/s00438-016-1277-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5357305PMC
April 2017

The C-Terminal SynMuv/DdDUF926 Domain Regulates the Function of the N-Terminal Domain of DdNKAP.

PLoS One 2016 20;11(12):e0168617. Epub 2016 Dec 20.

Institute of Biochemistry I, Medical Faculty, Center for Molecular Medicine Cologne (CMMC), Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), University of Cologne, Cologne, Germany.

NKAP (NF-κB activating protein) is a highly conserved SR (serine/arginine-rich) protein involved in transcriptional control and splicing in mammals. We identified DdNKAP, the Dictyostelium discoideum ortholog of mammalian NKAP, as interacting partner of the nuclear envelope protein SUN-1. DdNKAP harbors a number of basic RDR/RDRS repeats in its N-terminal domain and the SynMuv/DUF926 domain at its C-terminus. We describe a novel and direct interaction between DdNKAP and Prp19 (Pre mRNA processing factor 19) which might be relevant for the observed DdNKAP ubiquitination. Genome wide analysis using cross-linking immunoprecipitation-high-throughput sequencing (CLIP-seq) revealed DdNKAP association with intergenic regions, exons, introns and non-coding RNAs. Ectopic expression of DdNKAP and its domains affects several developmental aspects like stream formation, aggregation, and chemotaxis. We conclude that DdNKAP is a multifunctional protein, which might influence Dictyostelium development through its interaction with RNA and RNA binding proteins. Mutants overexpressing full length DdNKAP and the N-terminal domain alone (DdN-NKAP) showed opposite phenotypes in development and opposite expression profiles of several genes and rRNAs. The observed interaction between DdN-NKAP and the DdDUF926 domain indicates that the DdDUF926 domain acts as negative regulator of the N-terminus.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0168617PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5173251PMC
June 2017

A set of genes conserved in sequence and expression traces back the establishment of multicellularity in social amoebae.

BMC Genomics 2016 11 4;17(1):871. Epub 2016 Nov 4.

Institute for Biochemistry I, Medical Faculty, University of Cologne, Cologne, Germany.

Background: The developmental cycle of Dictyostelid amoebae represents an early form of multicellularity with cell type differentiation. Mutant studies in the model Dictyostelium discoideum revealed that its developmental program integrates the actions of genes involved in signal transduction, adhesion, motility, autophagy and cell wall and matrix biosynthesis. However, due to functional redundancy and fail safe options not required in the laboratory, this single organism approach cannot capture all essential genes. To understand how multicellular organisms evolved, it is essential to recognize both the conserved core features of their developmental programs and the gene modifications that instigated phenotypic innovation. For complex organisms, such as animals, this is not within easy reach, but it is feasible for less complex forms, such as the Dictyostelid social amoebas.

Results: We compared global profiles of gene expression during the development of four social amoebae species that represent 600 mya of Dictyostelia evolution, and identified orthologous conserved genes with similar developmental up-regulation of expression using three different methods. For validation, we disrupted five genes of this core set and examined the phenotypic consequences.

Conclusion: At least 71 of the developmentally regulated genes that were identified with all methods were likely to be already present in the last ancestor of all Dictyostelia. The lack of phenotypic changes in null mutants indicates that even highly conserved genes either participate in functionally redundant pathways or are necessary for developmental progression under adverse, non-standard laboratory conditions. Both mechanisms provide robustness to the developmental program, but impose a limit on the information that can be obtained from deleting single genes.
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http://dx.doi.org/10.1186/s12864-016-3223-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5097433PMC
November 2016

Neuronal Actin Dynamics, Spine Density and Neuronal Dendritic Complexity Are Regulated by CAP2.

Front Cell Neurosci 2016 26;10:180. Epub 2016 Jul 26.

Institute of Biochemistry I, Medical Faculty, University of Cologne, CologneGermany; Center for Molecular Medicine Cologne, University of Cologne, CologneGermany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, CologneGermany.

Actin remodeling is crucial for dendritic spine development, morphology and density. CAP2 is a regulator of actin dynamics through sequestering G-actin and severing F-actin. In a mouse model, ablation of CAP2 leads to cardiovascular defects and delayed wound healing. This report investigates the role of CAP2 in the brain using Cap2(gt/gt) mice. Dendritic complexity, the number and morphology of dendritic spines were altered in Cap2(gt/gt) with increased number of excitatory synapses. This was accompanied by increased F-actin content and F-actin accumulation in cultured Cap2(gt/gt) neurons. Moreover, reduced surface GluA1 was observed in mutant neurons under basal condition and after induction of chemical LTP. Additionally, we show an interaction between CAP2 and n-cofilin, presumably mediated through the C-terminal domain of CAP2 and dependent on cofilin Ser3 phosphorylation. In vivo, the consequences of this interaction were altered phosphorylated cofilin levels and formation of cofilin aggregates in the neurons. Thus, our studies identify a novel role of CAP2 in neuronal development and neuronal actin dynamics.
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http://dx.doi.org/10.3389/fncel.2016.00180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4960234PMC
August 2016

Coronin 1C-free primary mouse fibroblasts exhibit robust rearrangements in the orientation of actin filaments, microtubules and intermediate filaments.

Eur J Cell Biol 2016 Aug 4;95(8):239-51. Epub 2016 May 4.

Center for Biochemistry, Institute of Biochemistry I, Medical Faculty, University of Cologne, 50931 Cologne, Germany. Electronic address:

Coronin 1C is an established modulator of actin cytoskeleton dynamics. It has been shown to be involved in protrusion formation, cell migration and invasion. Here, we report the generation of primary fibroblasts from coronin 1C knock-out mice in order to investigate the impact of the loss of coronin 1C on cellular structural organisation. We demonstrate that the lack of coronin 1C not only affects the actin system, but also the microtubule and the vimentin intermediate filament networks. In particular, we show that the knock-out cells exhibit a reduced proliferation rate, impaired cell migration and protrusion formation as well as an aberrant subcellular localisation and function of mitochondria. Moreover, we demonstrate that coronin 1C specifically interacts with the non-α-helical amino-terminal domain ("head") of vimentin. Our data suggest that coronin 1C acts as a cytoskeletal integrator of actin filaments, microtubules and intermediate filaments.
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http://dx.doi.org/10.1016/j.ejcb.2016.04.004DOI Listing
August 2016

Novel Coronin7 interactions with Cdc42 and N-WASP regulate actin organization and Golgi morphology.

Sci Rep 2016 05 4;6:25411. Epub 2016 May 4.

Center for Biochemistry, Medical Faculty, University of Cologne, 50931 Cologne, Germany.

The contribution of the actin cytoskeleton to the unique architecture of the Golgi complex is manifold. An important player in this process is Coronin7 (CRN7), a Golgi-resident protein that stabilizes F-actin assembly at the trans-Golgi network (TGN) thereby facilitating anterograde trafficking. Here, we establish that CRN7-mediated association of F-actin with the Golgi apparatus is distinctly modulated via the small Rho GTPase Cdc42 and N-WASP. We identify N-WASP as a novel interaction partner of CRN7 and demonstrate that CRN7 restricts spurious F-actin reorganizations by repressing N-WASP 'hyperactivity' upon constitutive Cdc42 activation. Loss of CRN7 leads to increased cellular F-actin content and causes a concomitant disruption of the Golgi structure. CRN7 harbours a Cdc42- and Rac-interactive binding (CRIB) motif in its tandem β-propellers and binds selectively to GDP-bound Cdc42N17 mutant. We speculate that CRN7 can act as a cofactor for active Cdc42 generation. Mutation of CRIB motif residues that abrogate Cdc42 binding to CRN7 also fail to rescue the cellular defects in fibroblasts derived from CRN7 KO mice. Cdc42N17 overexpression partially rescued the KO phenotypes whereas N-WASP overexpression failed to do so. We conclude that CRN7 spatiotemporally influences F-actin organization and Golgi integrity in a Cdc42- and N-WASP-dependent manner.
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http://dx.doi.org/10.1038/srep25411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4855144PMC
May 2016

Nesprin-2 mediated nuclear trafficking and its clinical implications.

Nucleus 2015 8;6(6):479-89. Epub 2015 Dec 8.

a Institute for Biochemistry I; Medical Faculty; University of Cologne ; Köln , Germany.

Nuclear translocation of proteins has a crucial role in the pathogenesis of cancer, Alzheimer disease and viral infections. A complete understanding of nuclear trafficking mechanisms is therefore necessary in order to establish effective intervention strategies. Here we elucidate the role of Nesprin-2 in Ca(2+)/Calmodulin mediated nuclear transport. Nesprin-2 is an actin-binding nuclear envelope (NE) protein with roles in maintaining nuclear structure and location, regulation of transcription and mechanotransduction. Upon depletion of Nesprin-2 using shRNA, HaCaT cells show abnormal localization of the shuttling proteins BRCA1 and NF-κB. We show that their nuclear transport is unlikely due to the canonical RAN mediated nuclear import, but rather to a RAN independent Ca(2+)/Calmodulin driven mechanism involving Nesprin-2. We report novel interactions between the actin-binding domain of Nesprin-2 and Calmodulin and between the NLS containing region of BRCA1 and Calmodulin. Strikingly, displacing Nesprins from the NE resulted in increased steady state Ca(2+) concentrations in the cytoplasm suggesting a previously unidentified role of Nesprins in Ca(2+) regulation. On comparing Nesprin-2 and BRCA1 localization in the ovarian cancer cell lines SKOV-3 and Caov-3, Nesprin-2 and BRCA1 were localized to the NE envelope and the nucleus in SKOV-3, respectively, and to the cytoplasm in Caov-3 cells. Fibroblasts obtained from EDMD5 (Emery Dreifuss muscular dystrophy) patients showed loss of Nesprin-2 from the nuclear envelope, corresponding reduced nuclear localization of BRCA1 and enhanced cytoplasmic Ca(2+). Taken together, the data suggests a novel role of Nesprin-2 in Ca(2+)/Calmodulin mediated nuclear trafficking and provides new insights which can guide future therapies.
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http://dx.doi.org/10.1080/19491034.2015.1128608DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915507PMC
November 2016

The Physarum polycephalum Genome Reveals Extensive Use of Prokaryotic Two-Component and Metazoan-Type Tyrosine Kinase Signaling.

Genome Biol Evol 2015 Nov 27;8(1):109-25. Epub 2015 Nov 27.

Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg.

Physarum polycephalum is a well-studied microbial eukaryote with unique experimental attributes relative to other experimental model organisms. It has a sophisticated life cycle with several distinct stages including amoebal, flagellated, and plasmodial cells. It is unusual in switching between open and closed mitosis according to specific life-cycle stages. Here we present the analysis of the genome of this enigmatic and important model organism and compare it with closely related species. The genome is littered with simple and complex repeats and the coding regions are frequently interrupted by introns with a mean size of 100 bases. Complemented with extensive transcriptome data, we define approximately 31,000 gene loci, providing unexpected insights into early eukaryote evolution. We describe extensive use of histidine kinase-based two-component systems and tyrosine kinase signaling, the presence of bacterial and plant type photoreceptors (phytochromes, cryptochrome, and phototropin) and of plant-type pentatricopeptide repeat proteins, as well as metabolic pathways, and a cell cycle control system typically found in more complex eukaryotes. Our analysis characterizes P. polycephalum as a prototypical eukaryote with features attributed to the last common ancestor of Amorphea, that is, the Amoebozoa and Opisthokonts. Specifically, the presence of tyrosine kinases in Acanthamoeba and Physarum as representatives of two distantly related subdivisions of Amoebozoa argues against the later emergence of tyrosine kinase signaling in the opisthokont lineage and also against the acquisition by horizontal gene transfer.
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http://dx.doi.org/10.1093/gbe/evv237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4758236PMC
November 2015

Balanced cortical stiffness is important for efficient migration of Dictyostelium cells in confined environments.

Biochem Biophys Res Commun 2015 Nov 19;467(4):730-5. Epub 2015 Oct 19.

Department of Cell Biology (Anatomy III), Biomedical Center, Ludwig Maximilian University of Munich, 82152, Planegg-Martinsried, Germany. Electronic address:

Dictyostelium discoideum cells resemble in many aspects human leukocytes and serve as a model to study actin cytoskeleton dynamics and cell migration of highly motile cells. Dictyostelium cells deficient in the actin-binding protein filamin (ddFLN) showed a surprisingly subtle change in phenotype with no or only minor effects in single cell motility. These findings were in contrast to the strong actin-crosslinking activities measured for filamin in vitro. In the present study, we set out to revisit the role of ddFLN in cell migration. For this purpose, we examined migration of wild-type, ddFLN-null and ddFLN-overexpressing cells under different conditions. In addition to cyclic-AMP chemotaxis assays using micropipettes, we explored cell migration under more confined conditions: an under-agarose 2D assay and a 3D assay employing a collagen matrix that was adapted from assays for leukocytes. Using 3D migration conditions, cells deficient in ddFLN displayed only a minor impairment of motility, similar to the results obtained for migration in 2D. However, cells overexpressing ddFLN showed a remarkable decrease in the speed of migration in particular in 3D environments. We suggest that these results are in line with an increased stiffening of the cortex due to the crosslinking activity of overexpressed ddFLN. Our conclusion is that the absolute level of ddFLN is critical for efficient migration. Furthermore, our results show that under conditions of increased mechanical stress, Dictyostelium cells, like leukocytes, switch to a bleb-based mode of movement.
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http://dx.doi.org/10.1016/j.bbrc.2015.10.073DOI Listing
November 2015

Inner nuclear envelope protein SUN1 plays a prominent role in mammalian mRNA export.

Nucleic Acids Res 2015 Nov 17;43(20):9874-88. Epub 2015 Oct 17.

Institute of Biochemistry I, Medical Faculty, Center for Molecular Medicine Cologne (CMMC) and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany

Nuclear export of messenger ribonucleoproteins (mRNPs) through the nuclear pore complex (NPC) can be roughly classified into two forms: bulk and specific export, involving an nuclear RNA export factor 1 (NXF1)-dependent pathway and chromosome region maintenance 1 (CRM1)-dependent pathway, respectively. SUN proteins constitute the inner nuclear envelope component of the l I: nker of N: ucleoskeleton and C: ytoskeleton (LINC) complex. Here, we show that mammalian cells require SUN1 for efficient nuclear mRNP export. The results indicate that both SUN1 and SUN2 interact with heterogeneous nuclear ribonucleoprotein (hnRNP) F/H and hnRNP K/J. SUN1 depletion inhibits the mRNP export, with accumulations of both hnRNPs and poly(A)+RNA in the nucleus. Leptomycin B treatment indicates that SUN1 functions in mammalian mRNA export involving the NXF1-dependent pathway. SUN1 mediates mRNA export through its association with mRNP complexes via a direct interaction with NXF1. Additionally, SUN1 associates with the NPC through a direct interaction with Nup153, a nuclear pore component involved in mRNA export. Taken together, our results reveal that the inner nuclear envelope protein SUN1 has additional functions aside from being a central component of the LINC complex and that it is an integral component of the mammalian mRNA export pathway suggesting a model whereby SUN1 recruits NXF1-containing mRNP onto the nuclear envelope and hands it over to Nup153.
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http://dx.doi.org/10.1093/nar/gkv1058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4787764PMC
November 2015

A resilient formin-derived cortical actin meshwork in the rear drives actomyosin-based motility in 2D confinement.

Nat Commun 2015 Sep 29;6:8496. Epub 2015 Sep 29.

Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, Hannover 30625, Germany.

Cell migration is driven by the establishment of disparity between the cortical properties of the softer front and the more rigid rear allowing front extension and actomyosin-based rear contraction. However, how the cortical actin meshwork in the rear is generated remains elusive. Here we identify the mDia1-like formin A (ForA) from Dictyostelium discoideum that generates a subset of filaments as the basis of a resilient cortical actin sheath in the rear. Mechanical resistance of this actin compartment is accomplished by actin crosslinkers and IQGAP-related proteins, and is mandatory to withstand the increased contractile forces in response to mechanical stress by impeding unproductive blebbing in the rear, allowing efficient cell migration in two-dimensional-confined environments. Consistently, ForA supresses the formation of lateral protrusions, rapidly relocalizes to new prospective ends in repolarizing cells and is required for cortical integrity. Finally, we show that ForA utilizes the phosphoinositide gradients in polarized cells for subcellular targeting.
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http://dx.doi.org/10.1038/ncomms9496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4598863PMC
September 2015

Coronin7 regulates WASP and SCAR through CRIB mediated interaction with Rac proteins.

Sci Rep 2015 Sep 28;5:14437. Epub 2015 Sep 28.

Center for Biochemistry, Medical Faculty, Center for Molecular Medicine Cologne (CMMC) and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 50931 Köln, Germany.

Coronin7 (CRN7) stabilizes F-actin and is a regulator of processes associated with the actin cytoskeleton. Its loss leads to defects in phagocytosis, motility and development. It harbors a CRIB (Cdc42- and Rac-interactive binding) domain in each of its WD repeat domains which bind to Rac GTPases preferably in their GDP-loaded forms. Expression of wild type CRN7 in CRN7 deficient cells rescued these defects, whereas proteins with mutations in the CRIB motifs which were associated with altered Rac binding were effective to varying degrees. The presence of one functional CRIB was sufficient to reestablish phagocytosis, cell motility and development. Furthermore, by molecular modeling and mutational analysis we identified the contact regions between CRN7 and the GTPases. We also identified WASP, SCAR and PAKa as downstream effectors in phagocytosis, development and cell surface adhesion, respectively, since ectopic expression rescued these functions.
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http://dx.doi.org/10.1038/srep14437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4585930PMC
September 2015

The centrosomal component CEP161 of Dictyostelium discoideum interacts with the Hippo signaling pathway.

Cell Cycle 2015 ;14(7):1024-35

a Institute of Biochemistry I; Medical Faculty University of Cologne ; Köln , Germany.

CEP161 is a novel component of the Dictyostelium discoideum centrosome which was identified as binding partner of the pericentriolar component CP250. Here we show that the amino acids 1-763 of the 1381 amino acids CEP161 are sufficient for CP250 binding, centrosomal targeting and centrosome association. Analysis of AX2 cells over-expressing truncated and full length CEP161 proteins revealed defects in growth and development. By immunoprecipitation experiments we identified the Hippo related kinase SvkA (Hrk-svk) as binding partner for CEP161. Both proteins colocalize at the centrosome. In in vitro kinase assays the N-terminal domain of CEP161 (residues 1-763) inhibited the kinase activity of Hrk-svk. A comparison of D. discoideum Hippo kinase mutants with mutants overexpressing CEP161 polypeptides revealed similar defects. We propose that the centrosomal component CEP161 is a novel player in the Hippo signaling pathway and affects various cellular properties through this interaction.
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http://dx.doi.org/10.1080/15384101.2015.1007015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4614953PMC
December 2015

CAP2 is a regulator of the actin cytoskeleton and its absence changes infiltration of inflammatory cells and contraction of wounds.

Eur J Cell Biol 2015 Jan 3;94(1):32-45. Epub 2014 Nov 3.

Institute of Biochemistry I, Medical Faculty, University of Cologne, Cologne, Germany; Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany. Electronic address:

Cyclase associated protein (CAP) is a highly conserved protein with roles in actin dynamics and many cellular processes. Two isoforms exist in higher eukaryotes, CAP1 and CAP2. CAP1 is ubiquitously expressed whereas CAP2 shows restricted tissue distribution. In mice, ablation of CAP2 leads to development of cardiomyopathy. CAP2 is expressed in skin. In human skin its expression is increased in wounds. To elucidate the role of CAP2 in skin upon injury, we studied the wound healing in CAP2 deficient mice and found altered wound healing response presumably resulting from reduced levels of α-SMA, decreased macrophage infiltration and slower neovascularization. In vitro cultured Cap2 deficient keratinocytes showed reduced velocity and a delay in scratch closure. The analysis of primary mutant fibroblasts also showed reduced velocity and less contractibility. They had extended protrusions and more focal adhesions. In addition the F-actin content was increased keeping the total actin content unaltered. Mutant fibroblasts furthermore exhibited an altered response during recovery from drug-induced disruption of the actin cytoskeleton. Interestingly, CAP1 was upregulated in knockout unwounded skin and in wounds which might partially compensate for the loss of CAP2. Taken together, our studies reveal a role for CAP2 in wound healing which may be based on its function as a regulator of the actin cytoskeleton.
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http://dx.doi.org/10.1016/j.ejcb.2014.10.004DOI Listing
January 2015

The Dictyostelium discoideum GPHR ortholog is an endoplasmic reticulum and Golgi protein with roles during development.

Eukaryot Cell 2015 Jan 7;14(1):41-54. Epub 2014 Nov 7.

Institute of Biochemistry I, Medical Faculty, Center for Molecular Medicine Cologne (CMMC), and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany

Dictyostelium discoideum GPHR (Golgi pH regulator)/Gpr89 is a developmentally regulated transmembrane protein present on the endoplasmic reticulum (ER) and the Golgi apparatus. Transcript levels are low during growth and vary during development, reaching high levels during the aggregation and late developmental stages. The Arabidopsis ortholog was described as a G protein-coupled receptor (GPCR) for abscisic acid present at the plasma membrane, whereas the mammalian ortholog is a Golgi apparatus-associated anion channel functioning as a Golgi apparatus pH regulator. To probe its role in D. discoideum, we generated a strain lacking GPHR. The mutant had different growth characteristics than the AX2 parent strain, exhibited changes during late development, and formed abnormally shaped small slugs and fruiting bodies. An analysis of development-specific markers revealed that their expression was disturbed. The distributions of the endoplasmic reticulum and the Golgi apparatus were unaltered at the immunofluorescence level. Likewise, their functions did not appear to be impaired, since membrane proteins were properly processed and glycosylated. Also, changes in the external pH were sensed by the ER, as indicated by a pH-sensitive ER probe, as in the wild type.
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http://dx.doi.org/10.1128/EC.00208-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4279027PMC
January 2015

The Dictyostelium discoideum RACK1 orthologue has roles in growth and development.

Cell Commun Signal 2014 Jun 15;12:37. Epub 2014 Jun 15.

Institute of Biochemistry I, Medical Faculty, Center for Molecular Medicine Cologne (CMMC) and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 50931 Köln, Germany.

Background: The receptor for activated C-kinase 1 (RACK1) is a conserved protein belonging to the WD40 repeat family of proteins. It folds into a beta propeller with seven blades which allow interactions with many proteins. Thus it can serve as a scaffolding protein and have roles in several cellular processes.

Results: We identified the product of the Dictyostelium discoideum gpbB gene as the Dictyostelium RACK1 homolog. The protein is mainly cytosolic but can also associate with cellular membranes. DdRACK1 binds to phosphoinositides (PIPs) in protein-lipid overlay and liposome-binding assays. The basis of this activity resides in a basic region located in the extended loop between blades 6 and 7 as revealed by mutational analysis. Similar to RACK1 proteins from other organisms DdRACK1 interacts with G protein subunits alpha, beta and gamma as shown by yeast two-hybrid, pulldown, and immunoprecipitation assays. Unlike the Saccharomyces cerevisiae and Cryptococcus neoformans RACK1 proteins it does not appear to take over Gβ function in D. discoideum as developmental and other defects were not rescued in Gβ null mutants overexpressing GFP-DdRACK1. Overexpression of GFP-tagged DdRACK1 and a mutant version (DdRACK1mut) which carried a charge-reversal mutation in the basic region in wild type cells led to changes during growth and development.

Conclusion: DdRACK1 interacts with heterotrimeric G proteins and can through these interactions impact on processes specifically regulated by these proteins.
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http://dx.doi.org/10.1186/1478-811X-12-37DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4094278PMC
June 2014
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