Trapping of normal EB1 ligands in aggresomes formed by an EB1 deletion mutant

Nick P Riess,Matthew Adams,Jon M Askham,Ewan E Morrison,Tracy Lee,Kelly Milward

doi:10.1186/1471-2121-6-17

Nick P Riess, Matthew Adams + Show 4 more

Open Access

https://doi.org/10.1186/1471-2121-6-17

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Abstract

BackgroundEB1 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-ΔN2-GFP) spontaneously formed perinuclear aggregates when expressed in COS-7 cells.ResultsIn the present study live imaging indicated that EB1-ΔN2-GFP aggregates underwent dynamic microtubule-dependent changes in morphology and appeared to be internally cohesive. EB1-ΔN2-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-ΔN2-GFP was detergent-insoluble and ubiquitylated, indicating that EB1-ΔN2-GFP aggregates are aggresomes. Immunostaining also revealed that APC and p150glued were present in EB1-ΔN2-GFP aggregates, whereas EB3 was not. Furthermore, evidence for p150glued degradation was found in the insoluble fraction of EB1-ΔN2-GFP transfected cultures.ConclusionOur 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.

Highlights

EB1 is a microtubule tip-associated protein that interacts with the adenomatous polyposis coli (APC) tumour suppressor protein and the p150glued subunit of dynactin
Removal of the N-terminal 100aa of EB1 to generate EB1-∆N2-GFP neatly removes one of the major structural features of the protein, a calponin homology (CH) domain implicated in the microtubule-binding ability of EB1 [3,9,10,11], while leaving the region that mediates the interactions with APC and p150glued intact
We first examined the behaviour of EB1-∆N2-GFP using time-lapse fluorescence microscopy in living COS-7 cells

Summary

Introduction

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-∆N2-GFP) spontaneously formed perinuclear aggregates when expressed in COS-7 cells. In a previous study of a series of EB1 deletion mutants we noted that an EB1 protein lacking its N-terminal 100aa and fused at its C-terminus to GFP (EB1-∆N2GFP) spontaneously formed perinuclear aggregates in transfected COS-7 cells fixed and examined by immunostaining [3]. As EB1∆N2-GFP retains the ability to directly interact with p150glued, an examination of the aggregation of this protein in transfected cells might yield further insight into the process of aggresome formation. We reasoned that detailed examination of EB1-∆N2-GFP aggregation might shed further light on the normal function of this interaction within cells

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