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

2. 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
Peng-Peng Zhu et al., 2003
app kDa
conclusions
Mutations in amino acids in an N-terminal region that contains
the conserved GTPase domain, cause ADHSP
practical course Eva Luther

3. 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 Eva Luther

4. 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 Eva Luther

5. 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 Eva Luther

6. 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 Eva Luther

7. 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 Eva Luther

8. 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 Eva Luther

9. 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 Eva Luther

10. 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)
g, 1hr
(Light membran fraction)
ER, golgi
(cytoplasmitic fraction)
cytoplasm, endosomes,
ribosomes
10.000g, 10min
introduction
roadmap
results
conclusions
practical course Eva Luther

11. 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 Eva Luther

12. 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 Eva Luther

13. 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 Eva Luther

14. 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 Eva Luther

15. Thanks for your attention
practical course Eva Luther