Practical course in neurobiology 16.01. – 24.02.2006

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


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


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


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


Test several tissues for atlastin and trax

atlastin-1ra

t bra

inU25

1SH SH1.3SK-N

-SA

SHEPOlig

oU34

3HEKHeL

aco

s

trax

50 kDa

30 kDa

introduction

roadmap

results

conclusions


human

brain

rat b

rain

SH SHEP

U343

trans

fected

HeL

a

trans

fected

HEK

50 kDa atlastin-1

atlastin-1

Neg. control

50 kDa

Check band size

HEK HeLapCDNA 3,1 a1 pCMV 1c

introduction

roadmap

results

conclusions

trax30 kDa


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


NO CO-IP POSSIBLE

Protein A

input

contr

olsa

mple

Protein G

atlastin-1 (10% gel )

trax

input

contr

olsa

mple

50 kDa

37 kDa

atlastin-1 (12%gel ) 50 kDa

Co-Immunoprecipitation of SH cells

introduction

roadmap

results

conclusions


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


Calnexin (ER)

S6 rib. Protein (Ribosomes)

EEA1 (Endosomes)

HSP60 (Mitochondria)

Synaptophysin (Synaptic vesicles)tot

alPos

t nuc

l. SN

Post m

ito. S

NMito

. pell

etCyto

pl. S

NLig

ht mem

br. p

ellet

traxatlastin-150 kDa

30 kDa

Fractionation with SH cells

introduction

roadmap

results

conclusions


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

total

Post n

ucl. S

N

Nucl. p

ellet

Post m

ito. S

N

Mito. p

ellet

atlastin-1

trax


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 t

r.

introduction

roadmap

results

conclusions

Transformation of E. coli with EGFP-N-atlastin-1


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


Thanks for your attention

Practical course in neurobiology 16.01. – 24.02.2006

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