Download conclusions

Atlastin-1 and trax:
binding partners?
Practical course in neurobiology
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
2
Trax
app. 32kDa
introduction Translin associated factor x
• translin is a protein of movement
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,
results
conclusions
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 the Transitional
Endoplasmic Reticulum ATPase, a Cytoskeletal ç Actin and Trax in Male Germ Cells and the Brain
Xin-Qi Wu et al.
practical course 16.01. - 24.02.2006 Eva Luther
3
Why all this?
Mutation of atlastin-1 causes hereditary spastic paraplegia
introduction
roadmap
results
conclusions
Understanding atlastin-1 functions by identifying
and characterizing its binding partners
Yeast-2-Hybrid screenings found trax as a potential
binding and interacting partner of atlastin-1
> Look for stronger evidence for the interaction
• Find endgenous interaction
practical course 16.01. - 24.02.2006 Eva Luther
4
Binding partners? How to find out:
1: Tissue test
introduction
drop
+
2: Check band sizes by
transfection of HEK and HeLa
roadmap
+
drop
3: Grow cell lines,
prepare extracts,
test for protein expression
-
results
drop
4: Co-Immunoprecipitation
-
conclusions
5: fractionation
-
practical course 16.01. - 24.02.2006 Eva Luther
+
+
(Check with 2nd cell line)
+
Variate conditions of Co-IP
6: backup
(2nd cell line)
Still to do
5
Test several tissues for atlastin and trax
introduction
roadmap
50 kDa
atlastin-1
30 kDa
trax
results
conclusions
practical course 16.01. - 24.02.2006 Eva Luther
6
Check band size
HEK
introduction
HeLa
pCDNA 3,1 a1 pCMV 1c
roadmap
results
atlastin-1
50 kDa
Neg. control
50 kDa
conclusions
30 kDa
practical course 16.01. - 24.02.2006 Eva Luther
atlastin-1
trax
7
Cell extract tests
SHEP-test
introduction
atlastin-1
50 kDa
Almost no atlastin-1
Co-IP would not be possible
37 kDa
trax
roadmap
results
SH-test
50 kDa
atlastin-1
37 kDa
trax
conclusions
practical course 16.01. - 24.02.2006 Eva Luther
Both protein are expressed
strong enough for trying a
Co-IP
8
Co-Immunoprecipitation of SH cells
Protein A
Protein G
introduction
roadmap
37 kDa
atlastin-1 (10% gel )
trax
50 kDa
results
50 kDa
atlastin-1 (12%gel )
conclusions
NO CO-IP
POSSIBLE
practical course 16.01. - 24.02.2006 Eva Luther
9
Fractionation scheme
Homogenate (total)
1000g, 10min
introduction
Pellet
roadmap
Nuclei and
debris
Supernatant
(post nuclear SN)
10.000g, 10min
results
Pellet
Supernatant
(mitochondrial pellet) (post mitochondrial SN)
Mitochondria,
synapotsomes
100.000g, 1hr
conclusions
Pellet
Supernatant
(Light membran fraction) (cytoplasmitic fraction)
ER, golgi
practical course 16.01. - 24.02.2006 Eva Luther
cytoplasm, endosomes,
ribosomes
10
Fractionation with SH cells
introduction
HSP60 (Mitochondria)
Synaptophysin (Synaptic vesicles)
roadmap
EEA1 (Endosomes)
results
S6 rib. Protein (Ribosomes)
Calnexin (ER)
conclusions
50 kDa
atlastin-1
30 kDa
trax
practical course 16.01. - 24.02.2006 Eva Luther
11
Fractionation with U343 cells
introduction
roadmap
50 kDa
atlastin-1
37 kDa
trax
Most of the atlastin-1 is
Found in the nuclear fraction
results
conclusions
Stopped fractionation before ultracentrifugation
practical course 16.01. - 24.02.2006 Eva Luther
12
Additional stuff
Transformation of E. coli with EGFP-N-atlastin-1
clones
introduction
M
M
C
1
2
3
EcoR1
roadmap
!
results
4
!
1 band
negative
2 bands
positive
!
Transfection of HEK and HeLa with EGFP-N-Atlastin-1
conclusions
Anti atlastin-1
Anti GFP
practical course 16.01. - 24.02.2006 Eva Luther
13
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
roadmap
• try again with changed conditions
• use fraction, that contain both proteins
results
Results of the fractionation were not completely conclusive
conclusions
•some protein bands that were expected did not
appear, maybe due to contamination,
maybe failures in processing
practical course 16.01. - 24.02.2006 Eva Luther
14
Thanks for your attention
practical course 16.01. - 24.02.2006 Eva Luther
15