First published online 3 June 2003
doi: 10.1242/jcs.00599
Effect of pathogenic mis-sense mutations in lamin A on its interaction with emerin in vivo
Ian Holt1,
Cecilia Östlund2,
Colin L. Stewart3,
Nguyen thi Man1,
Howard J. Worman2 and
Glenn E. Morris1,*
1 Biochemistry Group, North East Wales Institute, Wrexham LL11 2AW, UK
2 Departments of Medicine and of Anatomy and Cell Biology, College of Physicians
and Surgeons, Columbia University, New York, NY 10032, USA
3 Laboratory of Cancer and Developmental Biology, NCI-FCRDC, PO Box B,
Frederick, MD 21702-1201, USA
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Fig. 1. Specificity of antiserum for emerin in lmna-/- MEFs.
MEFs were extracted with a 1% SDS buffer and subjected to SDS-PAGE with
prestained molecular weight markers (Sigma), as previously described
(Manilal et al., 1996 ). The
western blot was developed with emerin anti-serum (1/1000),
peroxidase-labelled pig anti-rabbit Ig (DAKOpatts) and Supersignal
chemiluminescent substrate (Pierce). Captured images of prestained blue
markers (Mr; lane 1; sizes shown in kDa) and exposed film (Em; lane 2) were
aligned after capture. The emerin band at 34 kDa is indicated. There are no
higher molecular weight bands of other LEM domain proteins (LAP2 or MAN1).
Pre-immune rabbit serum gave no bands at all (not shown).
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Fig. 2. Effects of lamin A transfection on the distribution of endogenous emerin in
MEFs (lmna-/-). (A) Untransfected cells show most of the
emerin in the peripheral ER. A range of effects were observed for transfection
with wild-type lamin A on the endogenous emerin: little or no effect on the
distribution of emerin (B); partial relocation of emerin to the nucleus, with
significant amounts of emerin remaining in the ER (C,D); and efficient
relocation of emerin from the ER to the nucleus (E). All examples are
wild-type lamin A in the pSVK3 plasmid. Bars, 10 µm.
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Fig. 3. Some mutant lamins form nuclear foci in MEFs (lmna-/-)
and emerin co-localizes with foci formed by `rod' mutants but not `tail'
mutants. Transfection of MEFs (lmna-/-) with lamin A N195K
(A), E358K (B) or L85R (C) produced nuclear foci that were also positive for
emerin (L85R produced foci only rarely;
Table 1). Similar results were
obtained with R386K and M371K (not shown). Two of the three `tail' mutants,
R453W (D) and R527P (E), produced nuclear foci only rarely and these did not
accumulate emerin (arrows). Bars, 10 µm.
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Fig. 4. Horizontal and vertical sections through nuclear foci in MEFs
(lmna-/-) transfected with N195K. A transfected nucleus
with foci is shown from above (A) or in a side-on section (B) at the position
shown by the arrows. The side-on view was created by taking 15 confocal
sections 0.25 µm apart in a z-series and assembling them into a
three-dimensional image with LaserVox software. (A,B) Staining for transfected
lamin A detected with anti-FLAG mAb (red) and endogenous emerin detected with
anti-emerin serum (green). (C,D) Controls with identical laser settings, in
which cells were stained with anti-FLAG mAb against lamin A (red) and
pre-immune rabbit serum (green); in this case, the side-on view was built up
from 11 z-series sections instead of 15. (E-G) Confocal sections (top
to bottom) of a nucleus containing five foci. Two of these foci (asterisks in
E) appear to be attached to the nuclear rim, where emerin is concentrated. Of
the remaining three central foci visible in F, two appear to be attached to
the upper surface (E) and one to the lower surface (arrow in G). The inset
labelled LAP2 shows a double label with rabbit anti-FLAG for lamin A (green,
ALEXA488 anti-rabbit Ig) and a commercial mAb against LAP2 (red; ALEXA546
anti-mouse Ig). The inset labelled LAMB shows a double label with monoclonal
anti-FLAG for lamin A (green; FITC anti-mouse Ig) and a commercial goat
antibody against lamin B (red; TRITC anti-goat Ig). Bars, 10 µm.
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Fig. 5. Presence of emerin in nuclear foci in HeLa cells transfected with the N195K
lamin A mutant. Nuclear foci of transfected lamin A, detected with anti-FLAG
antibody, are shown to contain emerin using two different emerin-specific
monoclonal antibodies, MANEM1 (A) and MANEM8 (B). Bars, 10 µm.
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Fig. 6. Transfected lamin A colocalizes with endogenous lamin B1 at the nuclear rim
in MEFs (lmna-/-). Many nuclei in MEFs
(lmna-/-) showed `burst ends', or abnormal lamin B1
distribution, at one pole (A). Transfected lamin A colocalized with lamin B1
in these cells (B). Double label with anti-FLAG mAb (red) and rabbit
anti-lamin B1 (green). Bars, 10 µm.
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Fig. 7. Co-transfection with wild-type and mutant lamin A prevents or greatly
reduces nuclear focus formation. MEFs (lmna-/-) (A) and
COS-7 cells (B) were co-transfected with pSVK3/lamin A N195K and pcDNA4/lamin
A wild-type. The two lamins were detected with anti-FLAG polyclonal and
anti-Xpress monoclonal antibodies, respectively. Nuclear foci were not seen
with the MEFs (lmna-/-). Nuclear foci (arrows) were very
rarely seen with COS-7 cells (B) and these foci were positive for wild-type
and mutant lamin A. Bars, 10 µm.
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Fig. 8. Summary of results of transfection of MEFs with lamin A mutants. The 11-12
exons that encode lamins A and C are shown with amino acid numbers of
approximate exon boundaries. The rod region is encoded by exons 1-6 and the
common globular tail domain by exons 7-10. Shown below the diagram are
mutations that cause EDMD/CMD1A, divided into those that commonly form nuclear
foci (`FOCI') and those that rarely form nuclear foci (`RARE FOCI'). Those
that form emerin-positive foci are `em+', whereas those that form
emerin-negative foci are `em–' (foci were never seen for W520S). Above
the diagram is the FPLD mutant, which neither formed foci nor affected emerin
interaction. Mutants that retained the ability to recruit emerin to the
nuclear rim in MEFs are shown in boldface and underlined.
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© The Company of Biologists Ltd 2003
