Practical course in neurobiology 16.01. – 24.02.2006 Atlastin-1 and trax: binding partners? Practical course in neurobiology 16.01. – 24.02.2006 Eva Luther

practical course 16.01. - 24.02.2006 Eva Luther 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 results www.nature.com/ng/journal/v29/n3/images/ng758-F5.gif Peng-Peng Zhu et al., 2003 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

practical course 16.01. - 24.02.2006 Eva Luther Trax app. 32kDa Translin associated factor x translin is a protein of movement introduction roadmap „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 a Coordinate Loss of TRAX, Reduced Fertility, Altered Gene´Expression in the Brain, and Behavioral Changes Vargheese Chennathukuzhi et al., 2003 results „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 the Transitional Endoplasmic Reticulum ATPase, a Cytoskeletal ç Actin and Trax in Male Germ Cells and the Brain Xin-Qi Wu et al. conclusions practical course 16.01. - 24.02.2006 Eva Luther

practical course 16.01. - 24.02.2006 Eva Luther 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

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

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

practical course 16.01. - 24.02.2006 Eva Luther Check band size HEK HeLa pCDNA 3,1 a1 pCMV 1c introduction human brain rat brain SH SHEP U343 transfected HeLa transfected HEK 50 kDa atlastin-1 roadmap results atlastin-1 Neg. control 50 kDa Both plasmids were HA-tagged, but HA-antibody did not work conclusions trax 30 kDa practical course 16.01. - 24.02.2006 Eva Luther

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

practical course 16.01. - 24.02.2006 Eva Luther Co-Immunoprecipitation of SH cells Protein A input control sample Protein G atlastin-1 (10% gel ) trax 50 kDa 37 kDa introduction roadmap results >> trax is detected with both, protein A- and protein G- antibody >> Band in control A? - maybe immunoglobulin 12% gel was made to separate the band better, to see wether there is a difference in the bands of control and sample: >> both bands have the same size and are a bit smaller than the input. atlastin-1 (12%gel ) 50 kDa conclusions NO CO-IP POSSIBLE practical course 16.01. - 24.02.2006 Eva Luther

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

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

practical course 16.01. - 24.02.2006 Eva Luther Fractionation with U343 cells 50 kDa 37 kDa total Post nucl. SN Nucl. pellet Post mito. SN Mito. pellet 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

practical course 16.01. - 24.02.2006 Eva Luther Additional stuff Transformation of E. coli with EGFP-N-atlastin-1 clones 1 2 3 4 C M ! introduction EcoR1 1 band negative 2 bands positive roadmap results Transfection of HEK and HeLa with EGFP-N-Atlastin-1 HEK tr. HeLa tr. HEK HeLa conclusions Anti atlastin-1 Anti GFP practical course 16.01. - 24.02.2006 Eva Luther

practical course 16.01. - 24.02.2006 Eva Luther 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

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