The cellular fate of mutant rhodopsin: quality control, degradation and aggresome formation
Richard S. Saliba1,
Peter M. G. Munro2,
Philip J. Luthert1 and
Michael E. Cheetham1,*
1 Division of Pathology, Institute of Ophthalmology, University College London,
London, UK
2 Electron Microscopy, Institute of Ophthalmology, University College London,
London, UK
View larger version (48K):
[in a new window]
|
Fig. 1. Mutant opsin accumulates in a structure close to the Golgi apparatus. The
cellular localisations of opsin in transiently transfected COS-7 cells
expressing WT opsin and WT opsin with a C-terminal GFP tag (WT-GFP) were
compared with mutant opsin (P23H-opsin) and GFP-tagged mutant opsin (P23H-GFP)
by confocal immunofluorescence. The intracellular opsin colocalised with the
Golgi marker ß-COP; however, in cells with mutant opsin inclusions, the
ß-COP staining is dispersed. Bar, 10 µm.
|
|
View larger version (49K):
[in a new window]
|
Fig. 2. Mutant opsin does not accumulate in the Golgi. Cells expressing WT (WT-GFP)
and mutant (P23H-GFP) opsin tagged with GFP were treated with BFA (20 µg/ml
for 15 minutes prior to fixation). BFA dispersed the Golgi (ß-COP) and
the WT-GFP opsin that was trafficking through the Golgi but did not disperse
the P23H-GFP opsin from its juxtanuclear position. Bar, 10 µm.
|
|
View larger version (52K):
[in a new window]
|
Fig. 3. Mutant opsin inclusions have the characteristics of an aggresome. Mutant
opsin (P23H-GFP) colocalised with c-myctagged ubiquitin when co-transfected in
COS-7 cells. The P23H-GFP opsin inclusions led to the disruption of the
intermediate filament network (Vimentin). The cytoplasmic chaperone Hsc70 was
recruited to the P23H-GFP inclusions. Bar, 10 µm.
|
|
View larger version (163K):
[in a new window]
|
Fig. 4. Ultrastructure of opsin aggresomes. Electron micrograph of a COS-7 cell
with a P23H-GFP mutant aggresome. (i) The aggresome (A) forms near the nucleus
(N) and centrosome (C) and is surrounded by mitochondria (M). (ii) At higher
magnification the intermediate filaments (IF) that surround the aggresome can
be seen. (iii) The aggresome also has a high membrane content and in places
appears to be surrounded by membrane (arrowheads). Bar, 1 µm.
|
|
View larger version (29K):
[in a new window]
|
Fig. 5. Co-transfection of normal and mutant opsin leads to the recruitment of the
normal protein to the mutant opsin aggresome. (A) GFP-tagged WT opsin in pEGFP
was co-transfected with untagged WT opsin in pMT3 (WT-GFP/WT-opsin) at a
vector ratio of 1:10. Cells showed no increase in aggresome formation compared
with WT-GFP alone, whereas co-transfection of GFP-tagged WT opsin in pEGFP
with untagged P23H opsin in pMT3 (WT-GFP/P23H-opsin) at a vector ratio of 1:10
increased the percentage of WT opsin recruited to aggresomes, as judged by GFP
fluorescence. Bar, 10 µm. (B) Quantification of co-transfection aggresome
formation after 24 hours of opsin expression. The percentage of cells with
WT-GFP fluorescence aggresomes was counted double blindly from four separate
experiments (error bars±2 s.d.). BFA (20 µg/ml for 15 minutes prior
to fixation) was added to both experiments to reduce confusion over WT opsin
in the secretory pathway.
|
|
View larger version (75K):
[in a new window]
|
Fig. 6. Inhibition of the proteasome leads to an increase of mutant opsin ER
staining. Confocal immunofluorescence shows that inhibition of the proteasome
machinery with MG-132 (5 µM for 8 hours) did not affect the processing of
WT opsin. The treatment led to an increase in the level of P23H and K296E
mutant opsin in the ER, as shown by colocalisation with the ER-resident Hsp70,
BiP. Bar, 10 µm.
|
|
View larger version (51K):
[in a new window]
|
Fig. 8. Inhibition of N-linked glycosylation leads to an increase of mutant opsin
ER staining. The colocalisation of WT (WT-opsin) and mutant opsins (P23H-opsin
and K296E-opsin) with the ER marker calnexin in untreated cells is shown in
the top row (Untreated). Inhibition of protein glycosylation with tunicamycin
(0.8 µg/ml for 8 hours) led to an increase in the level of mutant protein
retained within the ER. The trafficking of the WT protein (WT-opsin) to the
plasma membrane was not affected by the treatment with tunicamycin and showed
no increase in ER staining. By contrast, the mutant proteins (P23H-opsin and
K296E-opsin) accumulated in the ER as shown by colocalisation with the
ER-membrane-bound lectin chaperone calnexin. Bar, 10 µm.
|
|
© The Company of Biologists Ltd 2002
40 kDa) (arrowhead); and immature forms (>41 kDa);
deglycosylated form (
