Nesprin interchain associations control nuclear size

Abstract

Nesprins-1/-2/-3/-4 are nuclear envelope proteins, which connect nuclei to the cytoskeleton. The largest nesprin-1/-2 isoforms (termed giant) tether F-actin through their N-terminal actin binding domain (ABD). Nesprin-3, however, lacks an ABD and associates instead to plectin, which binds intermediate filaments. Nesprins are integrated into the outer nuclear membrane via their C-terminal KASH-domain. Here, we show that nesprin-1/-2 ABDs physically and functionally interact with nesprin-3. Thus, both ends of nesprin-1/-2 giant are integrated at the nuclear surface: via the C-terminal KASH-domain and the N-terminal ABD-nesprin-3 association. Interestingly, nesprin-2 ABD or KASH-domain overexpression leads to increased nuclear areas. Conversely, nesprin-2 mini (contains the ABD and KASH-domain but lacks the massive nesprin-2 giant rod segment) expression yields smaller nuclei. Nuclear shrinkage is further enhanced upon nesprin-3 co-expression or microfilament depolymerization. Our findings suggest that multivariate intermolecular nesprin interactions with the cytoskeleton form a lattice-like filamentous network covering the outer nuclear membrane, which determines nuclear size.

Fig. 1

a–g Nesprin C-termini are conserved and tethered at the NE by an identical mechanism. a Sequence alignment of mouse and human nesprin-1/-2/-3 KASH-domains, identical amino acids are depicted in black and similar residues in gray. b, b′ The expression of myc-tagged nesprin-3 (nes-3) KASH fusions exerts dominant negative effects on endogenous nesprin-2 proteins. Transiently transfected HaCaT cells were subjected to immunofluorescence using nesprin-2 C-terminal (Nes2CT) and anti-myc antibodies. Note the prominent nuclear rim staining of endogenous nesprin-2 in untransfected cells (b′, arrowheads) and the absence of nesprin-2 staining in myc-positive cells (b′, arrows). c Myc-tagged nes-3 KASH positive HaCaT cells subjected to immunofluorescence using nesprin-2N-terminal (Nes2NT) antibodies lack also nesprin-2 staining at the NE (arrows). d Histogram representing the nes-3 KASH displacement effects on the localization of endogenous nesprin-2 from the NE to the ER (endoplasmatic reticulum) in transfected HaCaT cells (300 transfected cells each were evaluated). e–f HA-tagged nes-3 FL transiently transfected HaCaT cells processed for immunofluorescence using anti-HA and either nesprin-2 C-terminal (Nes2CT, e, e′) or N-terminal antibodies (Nes2NT, f). Note that although nes-3 FL localized to the NE (e, arrows), its effects on nesprin-2 are heterogeneous. Some cells lack nesprin-2 (e′, f, asterisks) whereas some still retain staining at the NE (e′, f, arrowheads). DNA was stained with DAPI. Scale bar 10 μm. g Statistical evaluations of the diminished dominant negative effects of full-length nesprin-3 fusions on endogenous nesprin-2 (examples shown in e–f; 300 cells counted for each experiment)

Fig. 2

a–h Nesprin-3 associates with the nesprin-1/-2 ABDs in vitro and recruits the nesprin-2 ABD to the NE. a Immunofluorescence of GFP-tagged nesprin-2 (nes-2) ABD transfected COS7 cells stained with TRITC-phalloidin (Phall; reveals F-actin), indicates that the nesprin-2 ABD colocalizes with F-actin. b–b″ In a small number of GFP-tagged nes-2 ABD transfected COS7 cells the nesprin-2 ABD localizes at the NE (b, arrows); colocalization with actin filaments is also indicated (b–b″, arrowheads). c In HA-tagged nes-3 FL (nes-3 FL) transfected COS7 cells, pronounced HA-staining is found at the NE, which does not colocalize with phalloidin. d–d″ In most HA-tagged nes-3 FL and GFP-tagged nes-2 ABD co-transfected COS7 cells, the GFP-fusion decorated intensively the NE and colocalized with nesprin-3. e–e′′′ Phalloidin staining of HA-tagged nes-3 FL and GFP-tagged nes-2 ABD co-transfected cells, reveals prominent F-actin structures at the NE. f–f′′′ Similarly, co-transfected cells were treated with latrunculin B. F-actin disruption (absence of phalloidin stain, f′′) did not affect the persistence of nes-2 ABD at the nuclear surface (f′). Scale bars 10 μm. g Histogram representing a statistical evaluation (percentage of transfected cells, n = 300) of the GFP-tagged nes-2 ABD localization profiles to the cytoplasm (Cyto) and the NE, in single and in HA-tagged nes-3 FL co-transfected COS7 cells. h COS7 cell lysates expressing GFP-tagged nesprin-3 ΔC (GFP-nes-3 ΔC) were incubated with immobilized His-tagged fusion proteins (His-nes-1 ABD, His-nes-2 ABD) as indicated. COS7 cell lysates expressing GFP alone were used as control. The cell lysates (Input) and the specifically bound proteins (Pellet) were separated by SDS-PAGE and subjected to western blot analysis using GFP specific antibodies (upper panels) or were directly stained with Coomassie Blue to demonstrate equal recombinant protein usage (lower panel)

Fig. 3

a–f Nesprin-3 recruits nesprin-1 ABD-containing isoforms to the NE. a–a′′′ Endogenous nesprin-1 colocalizes with F-actin in HaCaT cells. The cells were stained with nesprin-1 ABD directed antibodies (nes1NT; Fig. S1) and TRITC-phalloidin (Phall). b–b′′′ HaCaT cells transiently expressing HA-tagged nes-3 FL were processed for immunofluorescence using anti-HA and nesprin-1 (nes1NT) antibodies. Endogenous nesprin-1 colocalizes with both nesprin-3 and F-actin at the NE (arrows). c, c′ Ectopically expressed GFP-tagged Enaptin-165 localizes to actin-based structures (arrowheads) in COS7 cells. d, d′ In GFP-Enaptin-165/HA-nes-3 FL co-transfected cells, GFP-Enaptin-165 was recruited to the NE (arrows). Scale bars 10 μm. e, f Histograms representing the percentage of cells exhibiting (gray bars) or lacking (white bars) NE-staining: for endogenous nesprin-1 (e) in untransfected (Control) or HA-nes-3 FL transfected COS7 cells and for GFP-Enaptin-165 (f) in single or HA-nes-3 FL double transfected cells, respectively

Fig. 4

a, b The nesprin-2 ABD associates to nesprin-3 in the presence of F-actin in vitro. Recombinantly expressed and purified His-nes-2 ABD protein was incubated either with GST (samples 1, 3) or GST-nes-3 SR1,2,3 (samples 2, 4) coupled Sepharose-beads in the absence (a) or presence of F-actin (b). Samples were then subjected to low speed centrifugation. The resulting supernatants from each experiment were ultra-centrifuged (see “Materials and methods” for more detail). The supernatants (S), pellets (P) from the ultra-centrifugation, and the pellets from the GST pull-down assays (PD; first centrifugation step) were analyzed by SDS-PAGE and stained with Coomassie Blue. Note that the presence of filamentous actin does not preclude the nesprin-2 ABD from binding the nes-3 SR1,2,3 recombinant protein (samples 4)

Fig. 5

a, b Nesprin-3 interacts in vitro and in vivo with plectin and the nesprin-1/-2 ABDs. a COS7 cells were transiently transfected with GFP-tagged nesprin-1 (nes-1), nesprin-2 (nes-2), or plectin ABDs and lysed in RIPA-buffer. Equal amounts of each lysate were incubated with either immobilized GST-fused nesprin-3 (nes-3) SR1, SR1,2, SR1,2,3, or GST alone (negative control). Specifically bound proteins were analyzed by SDS-PAGE and subjected to western blot analysis using either GFP (top panel) or GST specific antibodies (bottom panel). b Nesprin-2 giant co-immunoprecipitates with nesprin-3 and plectin. COS7 cells were transiently transfected with either empty myc-tag expression vector or myc-tagged nesprin-3 (nes-3) FL. Cells were lysed in RIPA-buffer and subjected to anti-myc immunoprecipitation (IP). Total cell lysates and myc-IPs were separated by SDS-PAGE and either stained directly with silver (left panel), or processed further for immunoblotting (right panel), using specific nesprin-2 (nes2CT), plectin and myc antibodies. Gel loading information: lane 1 total COS7 cell lysate expressing empty myc-tag vector; lane 2 total COS7 cell homogenate expressing myc-tagged nesprin-3 (nes-3) FL; lane 3 myc only immunoprecipitate; lane 4 myc-nesprin-3 FL immunoprecipitates. Silver staining is used as a control to demonstrate equal cell lysate loading and IP specificity

Fig. 6

a–g Nesprin-3 simultaneously recruits both plectin and the nesprin-2 ABD to the NE. a COS7 cells were stained for endogenous plectin (rabbit Ab). Note the absence of plectin enrichment at the NE and the profound filamentous cytoplasmic plectin staining. b–b′′ In HA-tagged nesprin-3 (nes-3) FL transfected COS7 cells, plectin was recruited to the NE. c–c′′′ Both plectin and GFP-nesprin-2 (nes-2) ABD accumulated at the NE in GFP-nes-2 ABD/HA-nes-3 FL co-transfected cells. d Histogram representing the statistical evaluation of the NE plectin localization profiles in untransfected, in HA-nes-3 FL transfected and in GFP-nes-2 ABD/HA-nes-3 FL co-transfected COS7 cells. Note that nes-2 ABD recruitment by nesprin-3 does not have consequences on the plectin accumulation at the NE. Scale bars 10 μm. e Control (1) and plectin (2) siRNA treated COS7 lysates were analyzed by SDS-PAGE and either stained directly with Coomassie Blue (left panel; to demonstrate equal loading) or subjected to immunoblotting (right panels), using specific tubulin and plectin (to demonstrate silencing efficacy) antibodies. f Control siRNA treated COS7 cells, transiently expressing GFP-nes-2 ABD, exhibit profound nes-2 ABD filaments in the cytoplasm and plectin staining (Ab C-20). Scale bar 5 μm. g–g′′ Plectin siRNA treated COS7 cells that transiently co-express nes-3 FL/nes-2 ABD constructs, lack endogenous plectin staining and possess prominent NE-associated ABD structures. Insets in g′′, are higher magnifications of dotted boxed areas in g and g′. Scale bar 10 μm

Fig. 7

a–h Nesprin-2 and neprin-3 associations synergistically control nuclear morphology. a The nucleus of an untransfected HaCaT cell stained with DAPI is shown. HaCaT cells expressing transiently GFP-nes-2 ABD (b-b′) or GFP-nes-2 KASH (c, c′), display increased nuclear areas. In contrast, the nuclei of GFP-nes-2 mini transfected (d, d′) or GFP-Nes-2-mini/HA-Nes-3 FL co-transfected (e–e″) HaCaT cells are smaller than those of untransfected cells. DNA was stained with DAPI. Scale bars 10 μm. f Histogram indicates the normalized nuclear areas of untransfected and GFP-nes-2 ABD, GFP-nes-2 KASH, GFP-nes-2 mini, GFP-nes-2 mini/HA-nes-3 FL, GFP-SR-KASH, GFP-SR-KASH/HA-nes-3 FL expressing HaCaT cells. The mean nuclear area of untransfected HaCaTs was normalized to 1. The normalized mean nuclear area was 1.43 ± 0.03 for GFP-nes-2 ABD, 1.40 ± 0.03 for GFP-nes-2 KASH, 0.87 ± 0.02 for GFP-nes-2 mini, 0.77 ± 0.03 for GFP-nes-2 mini/HA-nes-3 FL, 1.29 ± 0.1 for GFP-SR-KASH, and 1.30 ± 0.08 for GFP-SR-KASH/HA-nes-3 FL cotransfected HaCaT cells. G Histogram indicating that, GFP-nes-2 ABD, nes-2 KASH overexpression leads to cellular enlargement while nes-2 mini yields smaller cells; * indicates statistically significant differences obtained by Student’s t test (200 cells counted for each experiment). Values in (f, g) are the mean ± SD. h Western blot analysis of equal amounts of untransfected (1), GFP (2), GFP-nes-2 ABD (3), GFP-nes-2 KASH (4), and GFP-nes-2 mini (5) HaCaT lysates shows that cytoskeletal and vinculin protein expression is not altered; * in top panel indicates GFP-nes-2 mini

Fig. 8

a–e Nes-2 mini and the cytoskeleton regulate nuclear shape. a–a′′′ Representative GFP-nes-2 mini induced nuclear and cellular shrinkage effects in untreated HaCaTs. Note that effects of nes-2 mini expression on F-actin (Phall) and microtubule structures are not obvious. b, b′ Latrunculin B (LatB) induced F-actin depolymerization yields smaller and highly irregular nuclei. c, c′ Colchicine (Colch) induced microtubule depolymerization enlarges nuclei. d, d′′′ Latrunculin B and colchicine co-treated cells harbor smaller nuclei. Note that under all examined conditions that nes-2 mini expressing nuclei are more compact than control nuclei. DAPI was used to stain nuclei. Scale bar 10 μm. e Histogram representing the nuclear areas of untreated, latrunculin B, colchicine treated, and co-treated, control (untransfected) and nes-2 mini expressing HaCaT cells. Values are the mean ± SEM; * indicates statistically significant differences obtained by Student’s t test (100 nuclei counted for each condition)

Fig. 9

Model illustrating nesprin-1/-2/-3 interchain interactions and links with the cytoskeleton at the nuclear surface. Nesprins-1/-2 giant isoforms are drawn to align the NE perimeter. Nesprin-1/-2 ABDs interact with F-actin and nesprin-3, the latter is shown to interact also with the cytolinker plectin. Thus, nesprin-cytoskeleton complexes form a 3-D filamentous network that covers the NE exterior, which functions as a molecular “belt” (see “Discussion” for details). SUN-domain proteins are depicted as dimers and tetramers. IFs intermediate filaments, NPC nuclear pore complex