Figure 1.
Nesprin-2 SR52,53 (aa 6146–6347) interacts with lamin A residues extending from position 403 to 425.
(A) Schematic of GST Nesprin-2 fusion proteins used in this study. Aa positions refer to Nesprin-2 giant. (B) Schematic of His tag lamin A fusion proteins. (C) Bacterially expressed lamin A proteins bound to beads were incubated with lysates of COS7 cells expressing GFP Nesprin-2 SR52-56. The polypeptides used for pull down are shown in the upper panel. GFP-tagged Nesprin-2 SR52-56 was detected with mAb K3-184 (WB: anti GFP). The molecular weight markers are indicated on the left. GFP-tagged Nesprin-2 SR52-56 precipitates with lamin A polypeptides 345–425. No signals were detected for lamin A 264–402. For this reason we used these two lamin A proteins for our following experiments, one as a positive control and one as a negative control, to narrow the binding site of Nesprin-2 to lamin A. (D) Identification of the lamin A binding site in Nesprin-2. GFP Nesprin-2 polypeptides harboring individual SRs were expressed as GFP tagged proteins in COS7 cells (lower panels) and incubated with lamin A fusion proteins (upper panel, Coomassie Blue stained SDS PAGE, 15% acrylamide). (E) Interaction of GST Nesprin-2 fusion proteins bound to beads with GFP Lamin A aa 403–425. (F) Determination of the Nesprin-2 SR domain for interaction with lamin. Nesprin-2 SR52 and SR53 were expressed as GST fusion proteins and binding to GFP lamin A 403–425 was probed. For all experiments the use of equal protein amounts is demonstrated by Coomassie Blue stained SDS-PAGE. Equal amounts of GFP fusion proteins are shown by western blots of supernatants after coupling the GST and GFP fusion proteins. Nes-2 SR – Nesprin-2 Spectrin Repeats, LA – lamin A, WB – western blot, SPN – supernatant, P – pellet.
Figure 2.
Schematic of lamin A mutations.
(A) aa residues 399 to 434 of WT lamin A are shown. The interaction site of lamin A to Nesprin-2 is given in green. Below, the WT sequence mutations analysed in the present study are highlighted. Similarities to the WT sequence are shown as black lines, exchanged aa are indicated in red. The mutation Q432X leads to a stop codon (X). (B) Western blot analysis of GFP lamin A fusion proteins. The proteins were transiently expressed in COS7 cells, lysates were analysed by SDS-PAGE followed by western blot with mAb K3-184 that confirmed the predicted molecular weights (given in kDa on the left). WB – western blot.
Figure 3.
Most mutations in lamin A do not alter the distribution of Nesprin-2.
The distribution of Nesprin-2 in transiently GFP-lamin A wild type (WT) and mutant protein expressing COS7 and HaCaT cells was analysed by immunofluorescence. For GFP lamin A Q432X two panels are shown indicating different distribution and presence of smaller and larger GFP lamin A Q432X aggregates. Upper panel, asterisks points out cells in which lamin A Q432X proteins localize indistinguishable from the WT proteins. Lamin A Q432X proteins form aggregates with varying extend. In COS7 cells endogenous Nesprin-2 is sequestered into strong aggregates (arrowhead) whereas smaller aggregates show no obvious Nesprin-2 accumulation (bold arrow). In HaCaT cells Nesprin-2 is largely absent from the aggregates (HaCaT, Q432X, lower panel). Aggregates are present along the NE (COS7, lower panel, thin arrow). Merged pictures contain overlays of single stainings and DAPI. Scale bar, 10 µm.
Figure 4.
The interaction site of lamin A to Nesprin-2 is in a loop and mutations in LMNA modulate the interaction to Nesprin-2.
(A) Amino acid alignment of a human lamin A/C amino acids 351–490 (HsLaminA; uniprot accession number P02545) and the corresponding sequence in human lamin B (HsLamin; P20700) and Mus musculus lamin A (MmLaminA; P48678). Secondary structure elements are shown on top of the alignment and the lamin A region that interacts with Nesprin-2 is boxed in green. Conserved residues are highlighted black, similar residues are boxed. (B) Prediction of the three dimensional structure of human lamin A aa 351–490. The three dimensional structure of human Lamin A351–490 was predicted by using multiple PDB structures as templates (1UFGA, 1IFRA, and 2LLA) in the MULTICOM server [41]. The lamin A interaction site aa 403–425 to Nesprin-2 is highlighted in green and aa 403 and 425 are pointed out in blue. Amino acids that targets for the mutations analysed here are highlighted in red. The structure prediction was generated by using pyMOL v1.3. (C) The binding properties between WT GST Nesprin-2 SR52,53 and GFP lamin A mutations were analysed by pull down experiments. COS7 cells expressing WT or mutated GFP lamin A proteins were lysed and incubated with recombinant GST Nesprin-2 SR52,53 proteins. GST Nesprin-2 SR55,56 proteins were used as negative controls. (D) WT and mutant GFP lamin A proteins show distinct binding properties to WT GST Nesprin-2 SR52,53. The zero baseline represents the 100% binding affinity between WT GST Nesprin-2 SR52,53 and GFP lamin A (for details see materials and methods). Deviations caused by distinct mutations in LMNA are given in percent. Each mutation was analysed by four to seven independent experiments.
Figure 5.
Lamin A mutations V415I and Q432X trigger nuclear deformations in heat shock experiments.
C2C12 murine myoblast cells transiently expressing GFP WT, V415I or Q432X lamin A were exposed to a 15 minute heat shock at 42°C. After fixation the nuclear morphology was analysed by immunofluorescence (A) and statistically evaluated (B). Cells expressing GFP lamin A WT were used for reference. 600 nuclei each were analysed. Cells expressing GFP lamin A Q432X showed significant nuclear deformations already before the heat shock. P-Values of less than 0,001 are defined as highly significant (**).
Figure 6.
The perinuclear actin cap is not altered in the presence of GFP lamin A Q432X or V415I.
C2C12 cells transiently expressing WT GFP lamin A, the mutant proteins Q432X or V415I were stained with DAPI and TRITC phalloidin. The stainings were documented by using a confocal microscope and pictures were taken at the apical plane (red), the mid plane (yelow) and the basal plane (gray). Actin cap fibers are present in control or mutant lamin A expressing cells (long arrow). TRITC phalloidin was present in some GFP lamin A Q432X aggregates (short arrow).
Figure 7.
GFP lamin A Q432X aggregates displace chromatin and sequester SREBP1.
COS7 cells transiently expressing GFP lamin A Q432X were stained with DAPI (A–A) and SREBP1 and DAPI (B–B). (A) At sides of strong aggregates the DAPI staining showed gaps (A, arrow). (B) Endogenous SRBP1 was sequestered into strong aggregates (B, arrow), rather than into smaller aggregates (B, thin arrow). Scale Bar: 10 µm.