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transcript
Practical course in neurobiology16.01. – 24.02.2006
Eva Luther
Atlastin-1 and trax:
binding partners?
practical course 16.01. - 24.02.2006 Eva Luther 2
www.nature.com/ng/journal/v29/n3/images/ng758-F5.gif
Atlastin-1
Mutations in amino acids in an N-terminal region that contains the conserved GTPase domain, cause ADHSP
2 TM domains, C- and N-termini are exposed to the cytoplasm
Oligomer in vivo, most likely a tetramer
Functions as a novel GTPase
May be involved in Golgi membrane dynamics or vesicle Trafficking Peng-Peng Zhu et al., 2003
introduction
roadmap
results
conclusions
app. 55 - 62 kDa
practical course 16.01. - 24.02.2006 Eva Luther 3
Trax
introduction
roadmap
results
conclusions „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.
„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
Translin associated factor x• translin is a protein of movement
app. 32kDa
practical course 16.01. - 24.02.2006 Eva Luther 4
Why all this?
Mutation of atlastin-1 causes hereditary spastic paraplegia
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
introduction
roadmap
results
conclusions > Look for stronger evidence for the interaction• Find endgenous interaction
practical course 16.01. - 24.02.2006 Eva Luther 5
1: Tissue test
Variate conditions of Co-IP
5: fractionation
6: backup(2nd cell line)
(Check with 2nd cell line)
3: Grow cell lines, prepare extracts, test for protein expression
drop
2: Check band sizes by transfection of HEK and HeLa
drop
4: Co-Immunoprecipitationdrop
Still to do
Binding partners? How to find out:
+-
+-
+-
+-
+-
introduction
roadmap
results
conclusions
practical course 16.01. - 24.02.2006 Eva Luther 6
Test several tissues for atlastin and trax
atlastin-1ra
t bra
inU25
1SH SH1.
3SK-N
-SA
SHEPO
ligo
U343
HEKHeL
aco
s
trax
50 kDa
30 kDa
introduction
roadmap
results
conclusions
practical course 16.01. - 24.02.2006 Eva Luther 7
hum
an b
rain
rat b
rain
SH SHEP
U343
trans
fect
ed H
eLa
trans
fect
ed H
EK
50 kDa atlastin-1
atlastin-1
Neg. control
50 kDa
Check band size
HEK HeLa
pCDNA 3,1 a1 pCMV 1cintroduction
roadmap
results
conclusions
trax30 kDa
practical course 16.01. - 24.02.2006 Eva Luther 8
Almost no atlastin-1Co-IP would not be possible
50 kDa
37 kDa
SHEP-test
trax
atlastin-1
Both protein are expressedstrong enough for trying a Co-IP
atlastin-1
trax
50 kDa
37 kDa
SH-test
Cell extract tests
introduction
roadmap
results
conclusions
practical course 16.01. - 24.02.2006 Eva Luther 9
NO CO-IP POSSIBLE
Protein A
inpu
t
cont
rol
sam
ple
Protein G
atlastin-1 (10% gel )
trax
inpu
t
cont
rol
sam
ple
50 kDa
37 kDa
atlastin-1 (12%gel ) 50 kDa
Co-Immunoprecipitation of SH cells
introduction
roadmap
results
conclusions
practical course 16.01. - 24.02.2006 Eva Luther 10
Homogenate (total)
1000g, 10min
PelletNuclei and
debris
Supernatant (post nuclear SN)
Pellet(mitochondrial pellet)
Mitochondria,synapotsomes
Supernatant(post mitochondrial SN)
100.000g, 1hr
Pellet(Light membran fraction)
ER, golgi
Supernatant(cytoplasmitic fraction)cytoplasm, endosomes,
ribosomes
10.000g, 10min
Fractionation scheme
introduction
roadmap
results
conclusions
practical course 16.01. - 24.02.2006 Eva Luther 11
Calnexin (ER)
S6 rib. Protein (Ribosomes)
EEA1 (Endosomes)
HSP60 (Mitochondria)
Synaptophysin (Synaptic vesicles)to
tal
Post n
ucl.
SNPos
t mito
. SN
Mito
. pel
let
Cytop
l. SN
Ligh
t mem
br. p
elle
ttrax
atlastin-150 kDa
30 kDa
Fractionation with SH cells
introduction
roadmap
results
conclusions
practical course 16.01. - 24.02.2006 Eva Luther 12
Fractionation with U343 cells
Stopped fractionation before ultracentrifugation
Most of the atlastin-1 is Found in the nuclear fraction
introduction
roadmap
results
conclusions
50 kDa
37 kDa
tota
lPos
t nuc
l. SN
Nucl.
pelle
t
Post m
ito. S
N
Mito
. pel
let
atlastin-1
trax
practical course 16.01. - 24.02.2006 Eva Luther 13
Additional stuff
Anti atlastin-1
Anti GFP
Transfection of HEK and HeLa with EGFP-N-Atlastin-1
clones
1 2 3 4CM M
! !!
EcoR1
1 band negative
2 bands positiveHEK
HEK tr.
HeLa
HeLa
tr.
introduction
roadmap
results
conclusions
Transformation of E. coli with EGFP-N-atlastin-1
practical course 16.01. - 24.02.2006 Eva Luther 14
conclusion
Co-IP of trax and atlastin was not possible
• try again with changed conditions
• use fraction, that contain both proteins
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
Even if trax is reported to be enriched in brain, it could not
be detected in rat brainintroduction
roadmap
results
conclusions
practical course 16.01. - 24.02.2006 Eva Luther 15
Thanks for your attention