Both emerin and lamin C depend on lamin A for localization at the nuclear envelope
O. Anthony Vaughan1,
Mauricio Alvarez-Reyes1,
Joanna M. Bridger2,
Jos L. V. Broers3,
Frans C. S. Ramaekers3,
Manfred Wehnert4,
Glen E. Morris5,
William G. F. Whitfield6 and
Christopher J. Hutchison1,*
1
Department of Biological Sciences, The University of Durham, South Road,
Durham, DH1 3LE, UK
2
Department of Biology and Biochemistry, The University of Brunel, Middlesex,
UB8 3PH, UK
3
Department of Molecular Cell Biology and Genetics, University Maastricht, PO
Box 616, Maastricht, 6200 MD, The Netherlands
4
Ernst-Moritz-Arndt-University, Institute of Human Genetics, Fleischmannstrasse
42-44, 17487 Greifswald, Germany
5
MRIC, North East Wales Institute, Plas Coch, Mold Road, Wrexham, LL11 2AW,
Wales
6
Department of Biological Sciences, The University of Dundee, Dundee, DD1 4HN,
Scotland
*
Author for correspondence (e-mail:
c.j.hutchison{at}durham.ac.uk
)
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Fig. 1. Co-immunoprecipitation of in vitro translated lamins and emerin. Human
lamins A (lamA), B1 (lamB), C (lamC), human Nup153 (Nup) and emerin
were translated as 35S-met labelled proteins in rabbit reticulocyte
lysates. Lysates were mixed in the following combinations to give
approximately equal starting amounts of radiolabelled protein: (A,B)
Emerin+lamin C+Nup; emerin+lamin A+Nup; emerin+lamin B+Nup. (C,D) Emerin+lamin
C+lamin B; emerin+lamin A+lamin B; emerin+lamin A+lamin C. (F) Lamin A, lamin
B1, lamin C and emerin translated separately and not mixed. E shows
a lower exposure of a lamin A+lamin B+emerin co-immunoprecipitation. The area
corresponding to the lamin A and lamin B bands is presented. Two bands are
clearly visible. Immunoprecipitations were performed with MANEM3 and -5 in
combination (MANEM pull downs (A,C,E)). B and D show starting mixtures.
Immunoprecipitates or samples of starting lysates were resolved on 8% SDS PAGE
and fluorographed.  indicates the position of lamin A; — indicates
the position of lamin B; + indicates the position of lamin C; = indicates the
position of emerin; * indicates the position of Nup153.
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Fig. 2. Comparison of emerin and lamin C distributions in SW13 and HeLa cells. The
level of expression of different lamins was compared in HeLa (A) and SW13
cells (B) by immunoblotting. Nuclei isolated from 106 cells were
resolved on 10% SDS-PAGE, transferred to nitrocellulose and immunoblotted with
mAbs JoL2 (lamin A/C), JoL4 (lamin A), LN43 (lamin B2), rabbit
polyclonal anti-lamin C or goat anti-lamin B1 using a multi-blot
apparatus. The distribution of lamin C (blocks C and D), lamin A (blocks C and
D), emerin (blocks E and F) and LAP2ß (blocks E and F) in HeLa cells
(blocks C and E) and SW13 cells (blocks D and F) were investigated by indirect
immunofluorescence using polyclonal anti-lamin C, JoL4, MANEM5 and LAP17,
respectively. In all samples the distribution of DNA was detected with DAPI.
Bars, 10 µm.
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Fig. 3. Co-localisation of emerin and calreticulin in SW13 cells. The distribution
of emerin (MANEM3) was compared with the distribution of calreticulin (ER) in
SW13 cells by indirect immunofluorescence and confocal microscopy. The
distribution of DNA was detected using DAPI. Bar, 10 µm.
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Fig. 4. GFP-emerin forms aggregates within a sub-domain of the ER in SW13 cells.
GFP-emerin was transiently expressed in SW13 cells (rows A-D) or HeLa cells
(row E) and its distribution was compared with lamin C (row A), lamin
B1 (row B), lamin B2 (row C) or calreticulin (row D, ER)
after fixation. In each sample the distribution of DNA was revealed with DAPI.
Images are displayed as individual black and white panels and as three-colour
merged images. In merged images `yellow' indicates spectral overlap between
red and green signals. Arrowheads (row D) indicate the position of emerin
aggregates within the ER. Bars, 10 µm.
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Fig. 5. The distribution of lamin C and emerin in lymphoblastoid cell lines.
EBV-transformed lymphoblastoid cell lines were obtained from a donor control
(row A) and from an AD EDMD patient with a single point mutation giving rise
to an amino acid substitution at position T528K (row B). Each cell line was
co-stained with rabbit anti-lamin C (Lamin C) and MANEM5 (emerin). The
distribution of DNA in each sample was detected with DAPI. Panels show
individual black and white micrographs or three-colour merged images (merge)
in which the distribution of DAPI is shown in blue, lamin C in green and
emerin in red/orange. In merged images, `yellow' indicates spectral overlap
between red and green signals. Bar, 10 µm.
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Fig. 6. The distribution of emerin and lamin C in SW13 cells stably transfected
with GFP-lamin A (SW13/20). The distribution of lamin C, emerin and LAP2ß
in SW13 cells was investigated by antibody staining using rabbit anti-lamin C
(Lamin C, row A), MANEM5 (emerin, row B) and LAP17 (LAP2, row C),
respectively. In each case, antibody distribution was compared with the
distribution of GFP-lamin A (GFP). The distribution of DNA in each sample was
detected with DAPI. Each panel displays individual black and white images and
three-colour merged images (merge) in which DAPI is displayed in blue,
GFP-lamin A in green and antibody staining in red. In merged images `yellow'
indicates spectral overlap between red and green signals. Bars, 10 µm.
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Fig. 7. Transient transfection of SW13 with GFP-lamin A, but not GFP-lamin C,
rescues endogenous emerin and lamin C distributions. The distribution of lamin
C and emerin was investigated in SW13 cells following transient transfection
with GFP-lamin A (rows A,B) or GFP-lamin C (rows C,D). The distribution of
endogenous lamin C and emerin was detected by immunofluorescence as described
in Materials and Methods. The distribution of DNA was detected with DAPI. In
each panel the distributions of DNA, GFP or antibody staining are presented as
individual black and white images or as three-colour merged images in which
antibody staining is shown in red, GFP-lamin A is shown in green and DAPI is
shown in blue. In merged images `yellow' indicates spectral overlap between
red and green signals. The arrows in A indicate the position of a mitotic
cell. Bars, 10 µm.
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Fig. 8. The effects of a dominant negative mutant of lamin B1 on lamin
distribution in HeLa cells. A GFP-fusion of a dominant negative lamin
B1 mutant (GFP-Delta 2+) was transiently transfected into HeLa
cells. The distribution of lamin A (row A), lamin C (row B), lamin
B1 (row C) and lamin B2 (row D) in transfected cells was
compared with untransfected cells by antibody staining with JoL2, rabbit
anti-lamin C, goat anti-lamin B1 and LN43, respectively. The
distribution of DNA was detected with DAPI. Each panel displays either
individual black and white images or three-colour merged images in which DAPI
is shown in blue, GFP-Delta 2+ in green and antibody staining in red. Bar, 10
µm.
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Fig. 9. The influence of GFP-Delta 2+ on the distribution of emerin and LAP2ß
in HeLa cells. HeLa cells were transiently transfected with GFP-delta 2+ and
the distributions of emerin (row A) and LAP2ß (row B) were compared in
transfected and untransfected cells by immunofluorescence using MANEM5
(emerin) and LAP17 (LAP2). The distribution of DNA was detected with DAPI.
Panel displays individual black and white micrographs or three-colour merged
images in which DAPI is shown in blue, GFP-Delta 2+ in green and antibody
staining in red. In merged images `yellow' indicates spectral overlap between
red and green images. Bar, 10 µm.
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Fig. 10. Emerin is located in aggregates within the ER in HeLa cells transfected
with GFP-delta 2+. The distribution of emerin was compared with the
distribution of calreticulin in cells that were transiently transfected with
GFP-delta 2+ by four-channel fluorescence. Transfected cells were stained with
DAPI to reveal the distribution of DNA; with MANEM5 (Cy5) to reveal the
distribution of emerin; and with rabbit anti-calreticulin (TRITC) to reveal
the distribution of calreticulin within the ER. Images are displayed either as
individual black and white panels or a four-colour merged image in which DAPI
is displayed in blue, GFP-delta 2+ in green, calreticulin in red, and emerin
in white. It should be noted that when calreticulin staining was omitted no
bleed through between the Cy5 and the TRITC filters was observed. Bars, 10
µm.
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© The Company of Biologists Ltd 2001
