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First published online January 10, 2008
doi: 10.1242/10.1242/jcs.022020

Journal of Cell Science 121, 215-225 (2008)
Published by The Company of Biologists 2008
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Filaments made from A- and B-type lamins differ in structure and organization

Martin W. Goldberg1,*, Irm Huttenlauch2, Christopher J. Hutchison1 and Reimer Stick2

1 School of Biological and Biomedical Sciences, The University of Durham, South Road, Durham DH1 3LE, UK
2 Department of Cell Biology, University of Bremen, PO Box 33 04 40, 28334 Bremen, Germany


Figure 1
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  		Fig. 1. Field-emission scanning electron microscopy (feSEM) of spreads of non-injected control Xenopus nuclear envelopes. (A) Nucleoplasmic face of a nuclear envelope. (B) An area of parallel filaments, indicated by top small horizontal arrows in A is shown at higher magnification. (C) Area of highly ordered lamin lattice at high magnification. (D) Nuclear envelopes treated with 0.5% Triton X-100 before fixation to remove membranes. Some filaments that run in parallel are marked by small arrows. Arrowheads in A and D mark lamin-free membrane areas. (E-G) Further examples, from (E) the same and (F,G) different oocytes, showing arrays of parallel filaments. The arrow in E indicates where one set of parallel filaments bifurcates.

 

Figure 2
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  		Fig. 2. (A) Same area as Fig. 1B showing the position of parallel filaments (large arrows) and cross-connections (small arrows). (B) our interpretation of the filament and cross-connection arrangement, with filaments coloured red and cross-connections yellow. (C) Proposed model for the arrangement of lamin LIII filaments.

 

Figure 3
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  		Fig. 3. feSEM and TEM of lamin LIII-induced intranuclear membrane arrays. FLAG-tagged lamin LIII was expressed by nuclear injection of plasmid DNA. (A) Nuclear envelope of an oocyte overexpressing lamin LIII, shown at low magnification. Lamin-induced intranuclear membrane arrays (arrows) are distributed throughout the nuclear envelope. (B) Lamin LIII-induced membrane arrays sparsely covered by lamin filaments. Arrows mark filament-free areas of the extra membrane. (C-E) Lamin LIII-induced membrane arrays densely packed with filaments. Nuclear envelopes were either (C) fixed immediately or (D,E) treated with 0.5% Triton X-100 before fixation to remove membranes. In several areas of the membrane arrays, the LIII filaments are organized in a lattice-like arrangement. Note the pronounced repeat pattern along the LIII filament axis in D. The inset in D shows repeats at higher magnification. The line of arrowheads in the inset highlights the repeats. (F) TEM section of an isolated oocyte nucleus expressing lamin LIII. (G,H) Immuno-feSEM detection of FLAG-tagged lamin LIII on intranuclear membrane arrays using FLAG-specific antibodies. The immuno-gold labels are indicated by gold spots in G and H.

 

Figure 4
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  		Fig. 4. feSEM and TEM of lamin B2-induced intranuclear membrane arrays. FLAG-tagged lamin B2 was expressed by nuclear injection of plasmid DNA. (A) Nuclear envelope of an oocyte overexpressing lamin B2, shown at low magnification. (B) Membrane arrays covered by irregular wavy lamin B2 filaments. (C) A more-ordered package of lamin B2 filaments on a membrane array. Note the more-or-less parallel alignment of B2 filaments. (D) TEM section of an isolated oocyte nucleus expressing lamin B2. (E,F) Immuno-feSEM detection of lamin B2 on membrane arrays using the mAb L7-8C6 that is specific for Xenopus lamin B2 (E) and antibodies against the FLAG epitope (F). The immuno-gold labels are indicated by gold spots in E and F.

 

Figure 5
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  		Fig. 5. feSEM of nuclear envelope spreads of oocytes expressing lamin A. FLAG-tagged lamin A was expressed by nuclear injection of a small amount (0.38 ng) of plasmid DNA. (A-C) Small filamentous aggregates are marked by arrows in A and C, and immuno-gold labels against FLAG are indicated by gold spots in B. (D,E) NPC baskets are indicated by arrowheads in D, and a multilayer filament region is indicated by large arrows in D and E. Image E is a red-green 3D stereo pair of an area similar to that in D and requires red-green glasses.

 

Figure 6
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  		Fig. 6. FeSEM of isolated nuclear envelopes from oocytes expressing high levels of lamin A after injection of 1.5 ng of plasmid DNA. (A,B) Lamin A filaments that are arranged in layered bundles are indicated by small arrows; they surround, but appear not to interact with, structures that are consistent with NPC baskets. In general, the distal basket ring is visible (large arrows), and sometimes basket filaments can be seen (arrowheads). Oocytes expressing lamin A were also analysed by TEM thin sections and compared with controls. (C,D) The lamina is hardly discernible in (C) controls but forms a thick electron-dense layer in (D) oocytes expressing lamin A, which leaves the NPCs clear (arrows).

 

Figure 7
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  		Fig. 7. Comparison of filaments formed by endogenous LIII (`endo LIII'), overexpressed LIII, lamin B2 and lamin A, and short filaments of lamin A obtained at low levels of expression (`short A'; inset). All filaments are shown at the same magnification. The chart shows the mean diameter, including the chromium coat, with standard deviation bars for each filament type (n>100).

 

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