Trapping of normal EB1 ligands in aggresomes formed by an EB1 deletion mutant.
BACKGROUND
EB1 is a microtubule tip-associated protein that interacts with the APC tumour suppressor protein and the p150glued subunit of dynactin. We previously reported that an EB1 deletion mutant that retains both of these interactions but does not directly associate with microtubules (EB1-DeltaN2-GFP) spontaneously formed perinuclear aggregates when expressed in COS-7 cells.


RESULTS
In the present study live imaging indicated that EB1-DeltaN2-GFP aggregates underwent dynamic microtubule-dependent changes in morphology and appeared to be internally cohesive. EB1-DeltaN2-GFP aggregates were phase-dense structures that displayed microtubule-dependent accumulation around the centrosome, were immunoreactive for both the 20s subunit of the proteasome and ubiquitin, and induced the collapse of the vimentin cytoskeleton. Fractionation studies revealed that a proportion of EB1-DeltaN2-GFP was detergent-insoluble and ubiquitylated, indicating that EB1-DeltaN2-GFP aggregates are aggresomes. Immunostaining also revealed that APC and p150glued were present in EB1-DeltaN2-GFP aggregates, whereas EB3 was not. Furthermore, evidence for p150glued degradation was found in the insoluble fraction of EB1-DeltaN2-GFP transfected cultures.


CONCLUSIONS
Our data indicate that aggresomes can be internally cohesive and may not represent a simple "aggregate of aggregates" assembled around the centrosome. Our observations also indicate that a partially misfolded protein may retain the ability to interact with its normal physiological ligands, leading to their co-assembly into aggresomes. This supports the idea that the trapping and degradation of co-aggregated proteins might contribute to human pathologies characterised by aggresome formation.
Riess
Nick P
NP
CRUK Clinical Centre at Leeds, Division of Cancer Medicine Research, St James's University Hospital, Leeds LS9 7TF, UK. nick.riess@talk21.com
Milward
Kelly
K
Lee
Tracy
T
Adams
Matthew
M
Askham
Jon M
JM
Morrison
Ewan E
EE
D000818:Animals; D019556:COS Cells; D002458:Cell Fractionation; D048430:Cell Shape; D018385:Centrosome; D002522:Chlorocebus aethiops; D019281:Dimerization; D017353:Gene Deletion; D006801:Humans; D008024:Ligands; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D014162:Transfection; D025801:Ubiquitin
C405804:EB1 microtubule binding proteins; D008024:Ligands; D008869:Microtubule-Associated Proteins; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex