Ccdc124: A novel protein involved in cytokinesis.
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1 Ccdc124: A novel protein involved in cytokinesis. Uygar H. Tazebay Gebze Technical University Department of Molecular Biology and Genetics Gebze, Turkey International Conference on Science and Technology YTSAM, October 2016
2 Cytokinetic Abscission
3 Midbody: An organelle to be explored
4 The position of midbody in dividing cells
5 Blockage of abscission in response to Cep55 knock-down
6 Ccdc124 is encoded by a conserved eukaryotic gene CLUSTAL W (1.82) multiple sequence alignment human -MPKKFQGENTKSAAARARRAEAKA---AADAKKQKELEDAYWKDDDKHVMRKEQRKEEK 56 zebrafish -MPKKFQGENSKSATARARKAEAKA---VADARKQKELEDALWEDNDKRVVKKEQRKDDK 56 C.elegans -MPKKFASENPKVTAARDRKATAKK---DEADKKAKATEDAKWVDNDKLNNRKMQRKEDD 56 A.nidulans MGGKKGGGENSKKAAGNARKAEAAANKKAIEDQKRAAEEDKQWAKGAK----SSSKKEEA 56 **.**.* ::.. *:* * : * ** *.. *..:*:: human EKRRLDQLERKKETQRLLEEEDSKLKGGKAPRVATSSKVTRAQIEDTLRRDHQL-REAPD 115 zebrafish ERKRLEALERKRENQRLLEEEDSKIKGKQTK--EGPSKVTRAQIEETLQSKQNV-KEIK- 112 C.elegans EKKREEALRRKEENRKLAEEEMSSLGNKKPAG-AATQKVTRAHIHIRKEDEERINRELEE 115 A.nidulans EAKKAEAARKKAERDALLAAEEASQPSKPKNNKSAAKKNAPSRGTLNLD QLDD 109 * :: :.:* * * * :....* : ::. :. human TAEKAKSHLEVP---LEENVNRRVLEEGSVEARTIEDAIAVLSVAEEA-ADRHPERRMRA 171 zebrafish --EKEKSHLDVP---LEENVNRIVPEEGTVEARTIEDAIAVLSTKED--LDRHPERRMKA 165 C.elegans KRKQEAQKIEVAGDLLVENLNKLEVEEG--EARNVDDALKVLGEEKALDDDKHPEKRMRA 173 A.nidulans APSSRASALNAS GIDNALDALSLTSKDTSK-VDRHPERRYKA :: :. **: :. *:***:* :* human AFTAFEEAQLPRLKQENPNMRLSQLKQLLKKEWLRSPDNPMNQRAVPFNAPK zebrafish AYTAFEEANMPRVKMENPNMRLSQLKQQLKKEWTKSPENPLNQRAASYNTK C.elegans AYLAFEEARLPELKLNHPTFRLSQLKQILKKEWQKSPENPLNARLLALNS A.nidulans AYAAFEARRLPEIEAENPGLRRQQRIELVKKEFDKSPENPFNQVHVAFDASKEEIAAVRE 210 *: ***.:*.:: ::* :*.* : :***: :**:**:*. ::. human zebrafish C.elegans A.nidulans AERKKTEARLTR 222
7 Ccdc124 has high identity and similarity rates among eukaryotes Alignment Amino acid Length % Identity % Similarity Homo sapiens Pongo pygmaeus Canis familiaris Mus musculus Xenopus laevis Danio rerio Caenorhabditis elegans Drosophila melanogester Oryza sativa Aspergillus nidulans
8 Characterization of Ccdc124 at the protein level Mw kda FLAG- Ccdc124 EV Ccdc124- FLAG Mw kda
9 Prophase Metaphase Anaphase Ccdc124 DNA Merge
10 Ccdc124 is a novel centrosome protein Ccdc124 DAPI Aktin (Pha)
11 Centrosome structure
12 Interphase Prophase Metaphase Anaphase Cytokinesis Ccdc124 g-tubulin DNA Merge
13 Subcellular localization of Ccdc124 during cytokinetic abscission Ccdc124 g-tubulin Merge + DNA
14 Ccdc124 is a novel midbody protein Ccdc124 DAPI Aktin (Pha)
15 Cytokinetic abscission is blocked when Ccdc124 is knocked-down by RNAi
16 Genomic copies of Ccdc124 mutated in HEK-293 by CRISPR/Cas9 technique. Ccdc124 loci on Chromosome 19: WT 223 a.a CRISPR/Cas9 mutated copies: 91 nucleotide genomic deletion (Frameshift mutation) No protein Mut. 215 a.a 24 nucleotide (8 a.a) genomic deletion
17 Multi-nuclear cells in response to Ccdc124 mutations by CRISPR/Cas9 Ccdc124 g-tubulin DNA
18 Enlarged aberrant Ccdc124 +/- cell phenotypes
19 Time-lapse imaging of aberrant cell formation
20 Collective cell migration in development and epithelial mesenchymal transition Modeling and analysis of collective cell migration in an in vivo three-dimensional environment. Caia et al., 2015, P.N.A.S. USA, 113(5):E In vivo collective cell migration requires an LPAR2-dependent increase in tissue fluidity. Kuriyama et al., 2014, Journal of Cell Biology, 206 (1): Epithelial mesenchymal transition (EMT) is essential during embryo development and found in common pathologies such as organ fibrosis and in the initiation of metastasis for cancer progression.
21
22 New (fair..!) city border proposed by Uygar
23 Acknowledgements People Hani Alotaibi (IBG-DEU) Pelin Telkoparan (YIU) Elif Yaman (Leica) Merve Tuzlakoğlu Öztürk (GTÜ) Ömer Güllülü (GTÜ) Sinem Gül (GTÜ) Özge Arslan Eltan (GTÜ) Batu Erman (SU) Fred Wittinghofer (Dortmund) Johannes L. Bos (Utrecht) Lars Meijer (Utrecht) Agencies and Foundations Turkish Scientific and Technological Research Council (TÜBİTAK) 114-Z-349 Turkish Ministry of Development 2014-KB Feyzi Akkaya Scientific Research Funds (FABED) EMBO Short Term Fellowships Program European Science Foundation (ESF) Funct. Genomics
24
25 Rap2 is involved in: Cellular polarization Cell-cell adhesion Cell-ECM attachment Cellular migration
26 Interaction Partners of Ccdc124
27 Interaction Partners of Ccdc124 Fusion of biotin ligase to Ccdc myc-bira Bio- Bio+ myc-bira-ccdc anti-ccdc124 b-actin Wb: anti-myc Antibody
28 Myc-BirA-Ccdc124 Monitoring cellular localization of Myc-BirA-Ccdc124 Anti-MYC Anti-g-TUBULIN MERGED + DNA
29 LC-MS/MS analysis of Myc-BirA-Ccdc124 biotinylated cellular factors.
30 Interpretation of initial MS data ~2000 proteins (total number of proteins) 330 protein (unique to Ccdc124-Bioid after NT and EV-Mock corrected) 212 strong candidates after assessing unique/total peptide ratios ( 0,20) 58 priority interactors of Ccdc124 after comparing previous MS data Validations by co-ip experiments
31 Candidate interaction partners of Ccdc124 RNA processing Cell division and cytoskeleton component Others* *Viral life cycle, primary cilium assembly, regulation of gene expression, protein complex subunit organization etc.
32
33 Ccdc124 interacts with components of RNA maturation and decay IP: anti-ha ; WB: anti-flag STAU2- Flag + pcdna 3.1 STAU2- Flag + pmex- HA STAU2- Flag + CCDC12 4-HA Untrans fected CCDC12 4-HA + pcdna 3.1 CCDC12 4-HA + 3X Flag CMV10 STAU1- Flag + pcdna 3.1 STAU1- Flag + pmex- HA STAU1- Flag + CCDC12 4-HA MR 250 Supernatant STAU2- Flag + pcdna 3.1 (SUP.) STAU2- Flag + pmex- HA (SUP.) STAU2- Flag + CCDC12 4-HA (SUP.) Untrans fected (SUP.) CCDC12 4-HA + pcdna 3.1 (SUP.) CCDC12 4-HA + 3X Flag CMV10 (SUP.) STAU1- Flag + pcdna 3.1 (SUP.) STAU1- Flag + pmex- HA (SUP.) STAU1- Flag + CCDC12 4-HA (SUP.) MR Staufen2 functions in Staufen1-mediated mrna decay by binding to itself and its paralog and promoting UPF1 helicase but not ATPase activity. Park E, Gleghorn ML, and Maquat LE. Proc Natl Acad Sci U S A Jan 8;110(2): doi: /pnas Epub 2012 Dec 20.
34 Ccdc124; a new RNA metabolism protein? (2014)
35 Ccdc124 is ubiquitously expressed in human tissues [kb] 1.0 CCDC124 b-actin GAPDH
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