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First published online September 19, 2007
doi: 10.1242/10.1242/jcs.014191

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Requirements for the localization of nesprin-3 at the nuclear envelope and its interaction with plectin

¹ Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
² Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO 63110, USA

View larger version (57K): [in this window] [in a new window]   Fig. 1. Nesprin-3 is displaced from the NE upon overexpression of nesprin-1 and nesprin-2. (A) NIH3T3 cells were transiently transfected with constructs encoding GFP-dnNesprin-1 or GFP-dnNepsrin-2, fixed in paraformaldehyde and stained for endogenous nesprin-3. Representative confocal images are shown. Bars, 10 µm. (B) Quantitative analysis of the effect demonstrated in (A). The average gray value of endogenous nesprin-3 at the NE was measured for individual transfected and untransfected cells. Results are shown as the mean gray value±s.e.m. (n=50). *, P<0.001.

View larger version (94K): [in this window] [in a new window]   Fig. 2. Nesprin-3 interacts with Sun proteins through its PPPX consensus sequence. (A) PA-JEB cells stably expressing GFP-nesprin-3 or GFP-nesprin-3PPPT were fixed in paraformaldehyde, stained for endogenous Sun1 and Sun2 and analyzed by confocal microscopy. Bar, 10 µm. (B) PA-JEB cells stably expressing GFP-nesprin-3PPPT were fixed in paraformaldehyde, stained for endogenous plectin (HD-121 antibody), the ER (PDI antibody) or the Golgi complex (Golgin97 antibody) and analyzed by confocal microscopy. Bar, 10 µm. (C) Ultrathin sections of PA-JEB cells stably expressing GFP-nesprin-3 or GFP-nesprin-3PPPT were labeled with a pAb against GFP, followed by an incubation with 10 nm colloidal-gold-conjugated protein A. Nesprin-3 associates with the ONM (arrowhead) and the ER (arrow). N, nucleus; C, cytoplasm; NE, nuclear envelope; ER, endoplasmic reticulum. Bars, 200 nm. (D) Whole-cell lysates (WCLs) from PA-JEB cells stably expressing GFP-nesprin-3 or GFP-nesprin-3PPPT were immunoprecipitated with the nesprin-3 mAb Nsp3. Immunoprecipitates (IPs) and WCLs were probed with antibodies directed against Sun1, Sun2, nesprin-3, and lamins A and C.

View larger version (92K): [in this window] [in a new window]   Fig. 3. Nesprin-3 is dependent on Sun1 and Sun2 for its localization at the NE. (A) PA-JEB cells stably expressing GFP-nesprin-3 were mock transfected or transfected with siRNA directed against lamin A/C (LmnA), Sun1, Sun2 or a combination of Sun1 and Sun2. 72 hours after transfection, cells were lysed and WCLs were analyzed for expression of the indicated proteins. (B) PA-JEB cells stably expressing GFP-nesprin-3 were mock transfected or transfected with siRNAs directed against LmnA, Sun1, Sun2 or a combination of Sun1 and Sun2. 72 hours after transfection, cells were fixed in paraformaldehyde, stained for the indicated proteins and analyzed by confocal microscopy. Bar, 10 µm. (C) Quantitative analysis of the effect shown in (B). The gray values of GFP-nesprin-3 at the nucleus and the complete cell were determined for individual siRNA- and mock-transfected cells. Results are shown as the mean ratio of the gray value in the nucleus to the gray value in the total cell±s.e.m. (n=50). *, P<0.001. (D) PA-JEB cells stably expressing GFP-nesprin-3 were transiently transfected with a Myc-tagged Sun2 lumenal domain construct carrying an ER-retention signal (Sun2L-ER). After 72 hours, cells were fixed in paraformaldehyde and stained for Myc. Representative confocal images are shown. Arrows indicate a displacement of GFP-nesprin-3 over the ER. Bar, 10 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 4. Nesprin-3 can form dimers. COS7 cells were transiently transfected with a construct encoding VSV-nesprin-3 (lanes 1-3, 5) and expression constructs for HA-nesprin-3 (lanes 1 and 4), HA-plectin 1C ABD (lanes 2 and 6) or HA--actinin ABD (lanes 3 and 7). The cells were lysed in RIPA buffer, and HA precipitates were probed for VSV glycoprotein (upper panel) and HA (third panel). WCLs were probed for the expression levels of VSV-nesprin-3 (second panel) and total nesprin-3 (lower panel).

View larger version (56K): [in this window] [in a new window]   Fig. 5. Plectin dimers are required for the interaction with nesprin-3 at the NE. (A) Schematic domain structure of a plectin molecule, containing an ABD (purple), a plakin domain (green), a rod domain (yellow) and a series of plakin repeats (blue). The length of the different plectin constructs used and their respective domains are indicated below. (B) PA-JEB cells stably expressing GFP-nesprin-3 or GFP-nesprin-3 were transiently transfected with HA-tagged constructs encoding plectin 1-399, plectin 1-606, plectin 1-2532 or full-length plectin. 48 hours after transfection, cells were fixed in paraformaldehyde, stained for HA and analyzed by confocal microscopy. Bar, 10 µm. (C) PA-JEB, MD-EBS-1 and MD-EBS-2 keratinocytes were lysed and WCLs were analyzed by western blot for the expression of plectin (clone 31). Myosin heavy chain (MHC) served as a loading control. (D) PA-JEB cells expressing GFP-nesprin-3 and MD-EBS-1 cells stably expressing GFP-nesprin-3 or GFP-nesprin-3 were fixed in paraformaldehyde, stained for plectin (clone 31) and analyzed by confocal microscopy. Arrows indicate the presence of plectin at the NE. Bar, 10 µm.

View larger version (51K): [in this window] [in a new window]   Fig. 6. Expression of the plectin ABD stabilizes the actin cytoskeleton and has a dominant-negative effect on the localization of endogenous plectin at the NE. (A) PA-JEB cells stably expressing GFP-nesprin-3 were transiently transfected with constructs encoding HA-plectin ABD or HA-plectin ABDE95S. 48 hours after transfection, cells were fixed in paraformaldehyde, stained for HA and endogenous plectin (HD-121 antibody) and analyzed by confocal microscopy. Bar, 10 µm. (B) PA-JEB cells stably expressing GFP-nesprin-3 were transiently transfected with a construct encoding the HA-plectin ABD. The cells were treated with cytochalasin D for 30 minutes, fixed in paraformaldehyde and stained for HA and either endogenous plectin (HD-121 antibody) or F-actin. Bar, 10 µm. (C) PA-JEB cells stably expressing GFP-nesprin-3 were treated with 100 nM jasplakinolide for two hours. Treated and untreated cells were fixed in paraformaldehyde and stained for F-actin and endogenous plectin (HD-121 antibody). Bar, 10 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 7. Model illustrating the nesprin-3 LINC complex. Nesprin-3 dimers (blue) are retained at the ONM through an interaction with Sun1 (brown) or Sun2 (green) protein dimers. The binding of nesprin-3 to dimers of plectin (yellow) connects the nucleus to the intermediate filament system. A potential interaction of Sun proteins with lamin A (red), indicated by a question mark, establishes an indirect link between the cytoskeleton and the nucleoskeleton: the nesprin-3 LINC complex. ONM, outer nuclear membrane; INM, inner nuclear membrane; PS; periplasmic space; IF, intermediate filaments.

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