Practical course in neurobiology 16.01. – 24.02.2006

1. Practical course in neurobiology16.01. – 24.02.2006 Atlastin-1 and trax: binding partners? Eva Luther

2. Atlastin-1 2 TM domains, C- and N-termini are exposed to the cytoplasm Oligomer in vivo, most likely a tetramer introduction Functions as a novel GTPase roadmap May be involved in Golgi membrane dynamics or vesicle Trafficking Peng-Peng Zhu et al., 2003 www.nature.com/ng/journal/v29/n3/images/ng758-F5.gif results app. 55 - 62 kDa conclusions Mutations in amino acids in an N-terminal region that contains the conserved GTPase domain, cause ADHSP practical course 16.01. - 24.02.2006 Eva Luther

3. „Trax contains a putative bipartite nuclear targeting sequence, suggesting that trax could facilitate the movement of protein into nuclei.“ Mice Deficient for Testis-Brain RNA-Binding Protein Exhibit aCoordinate Loss of TRAX, Reduced Fertility, Altered Gene´Expression in the Brain, and Behavioral Changes Vargheese Chennathukuzhi et al., 2003 „Trax is a member of the TB-RBP (testis/brain RNA-binding protein) gene family. It associates with TB-RBP in both nuclear and cytoplasmic complexes from neural sources.“ Protein-Protein Interactions between the Testis Brain RNA-Binding Protein and theTransitional Endoplasmic Reticulum ATPase, a Cytoskeletal ç Actin and Trax inMale Germ Cells and the Brain Xin-Qi Wu et al. Trax app. 32kDa • Translin associated factor x • translin is a protein of movement introduction roadmap results conclusions practical course 16.01. - 24.02.2006 Eva Luther

4. Why all this? Mutation of atlastin-1 causes hereditary spastic paraplegia introduction Understanding atlastin-1 functions by identifying and characterizing its binding partners roadmap Yeast-2-Hybrid screenings found trax as a potential binding and interacting partner of atlastin-1 results conclusions > Look for stronger evidence for the interaction • Find endgenous interaction practical course 16.01. - 24.02.2006 Eva Luther

5. drop 2: Check band sizes by transfection of HEK and HeLa drop 3: Grow cell lines, prepare extracts, test for protein expression 4: Co-Immunoprecipitation drop + + + + + (Check with 2nd cell line) - - - - - Still to do Binding partners? How to find out: 1: Tissue test introduction roadmap results conclusions 5: fractionation Variate conditions of Co-IP 6: backup (2ndcell line) practical course 16.01. - 24.02.2006 Eva Luther

6. SK-N-SA rat brain SHEP SH1.3 Oligo HeLa U343 U251 HEK 50 kDa cos SH atlastin-1 30 kDa trax Test several tissues for atlastin and trax introduction roadmap results conclusions practical course 16.01. - 24.02.2006 Eva Luther

7. transfected HeLa transfected HEK human brain rat brain SHEP U343 SH atlastin-1 50 kDa 30 kDa Neg. control atlastin-1 50 kDa Check band size introduction roadmap results conclusions trax practical course 16.01. - 24.02.2006 Eva Luther

8. SHEP-test atlastin-1 50 kDa 50 kDa 37 kDa trax 37 kDa SH-test atlastin-1 trax Cell extract tests introduction Almost no atlastin-1 Co-IP would not be possible roadmap results Both protein are expressed strong enough for trying a Co-IP conclusions practical course 16.01. - 24.02.2006 Eva Luther

9. Protein A Protein G 50 kDa 50 kDa atlastin-1 (10% gel ) 37 kDa sample sample control control input input trax atlastin-1 (12%gel ) Co-Immunoprecipitation of SH cells introduction roadmap results conclusions NO CO-IP POSSIBLE practical course 16.01. - 24.02.2006 Eva Luther

10. Homogenate (total) 1000g, 10min Pellet Nuclei and debris Supernatant (post nuclear SN) 10.000g, 10min Pellet (mitochondrial pellet) Mitochondria, synapotsomes Supernatant (post mitochondrial SN) 100.000g, 1hr Pellet (Light membran fraction) ER, golgi Supernatant (cytoplasmitic fraction) cytoplasm, endosomes, ribosomes Fractionation scheme introduction roadmap results conclusions practical course 16.01. - 24.02.2006 Eva Luther

11. Light membr. pellet Post mito. SN Post nucl. SN Mito. pellet Cytopl. SN total EEA1 (Endosomes) 50 kDa Synaptophysin (Synaptic vesicles) HSP60 (Mitochondria) S6 rib. Protein (Ribosomes) Calnexin (ER) atlastin-1 trax 30 kDa Fractionation with SH cells introduction roadmap results conclusions practical course 16.01. - 24.02.2006 Eva Luther

12. Post mito. SN Post nucl. SN Nucl. pellet Mito. pellet total 50 kDa 37 kDa Fractionation with U343 cells introduction atlastin-1 Most of the atlastin-1 is Found in the nuclear fraction roadmap trax results conclusions Stopped fractionation before ultracentrifugation practical course 16.01. - 24.02.2006 Eva Luther

13. Anti atlastin-1 clones 2 M M C 1 3 4 Anti GFP ! ! ! Additional stuff Transformation of E. coli with EGFP-N-atlastin-1 introduction roadmap results Transfection of HEK and HeLa with EGFP-N-Atlastin-1 HeLa tr. HEK tr. HeLa HEK conclusions practical course 16.01. - 24.02.2006 Eva Luther

14. conclusion Even if trax is reported to be enriched in brain, it could not be detected in rat brain introduction • Co-IP of trax and atlastin was not possible • try again with changed conditions • use fraction, that contain both proteins roadmap results • Results of the fractionation were not completely conclusive • some protein bands that were expected did not • appear, maybe due to contamination, • maybe failures in processing conclusions practical course 16.01. - 24.02.2006 Eva Luther

15. Thanks for your attention practical course 16.01. - 24.02.2006 Eva Luther