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Myne-1, a spectrin repeat transmembrane protein of the myocyte inner nuclear membrane, interacts with lamin A/C

¹ Department of Pathology, The University of Chicago, Chicago, IL 60637, USA
² Department of Medicine, Section of Cardiology, The University of Chicago, Chicago, IL 60637, USA
³ Department of Human Genetics, The University of Chicago, Chicago, IL 60637, USA

View larger version (19K): [in a new window]   Fig. 1. Domain structure of myne-1. (A) The predicted domain structure of myne-1 is shown. It includes seven spectrin repeats (large gray bars) and a transmembrane domain (black bar) at the C-terminus. Between the fifth and sixth spectrin repeat is a region (small gray bars) with homology to the LEM domain found in LAP2, emerin and MAN1. The LEM domain of myne-1 is interrupted in its midportion by an -helical domain (white bar). Just before the transmembrane domain is a serine-rich domain (hexagon). (B) This region is not present in one splice form of myne. (C) The amino-acid sequence of the interrupted LEM domain of myne-1 is shown. The alignment of the LEM domains of human MAN1, emerin, LAP2, and two C. elegans homologs (F42H11.2/CAEEL; W01G7.5/CAEEL) is shown. Light gray represents homology; dark gray represents identity.

View larger version (33K): [in a new window]   Fig. 2. Expression and localization of myne-1. (A) A human multi-tissue northern blot hybridized to the 3' UTR of myne-1 is shown. Lane 1, heart; lane 2, brain; lane 3, placenta; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, pancreas. The major transcript is 4.2 kb. (B) An immunoblot from murine tissues using affinity-purified AM1 antibody is shown. G, gastrocnemius; Q, quadriceps; H, heart; B, brain; K, kidney; S, stomach; Bl, bladder. Loading control panels are shown below. (C) Immunolocalization using AM1 in heart and quadriceps muscle shows that myne-1 is at the nuclear membrane. Double-labeling with DAPI and AM1 is shown. The left-hand panels represent nuclei visualized with DAPI staining. The right-hand panels are stained with AM1. H, heart; S, skeletal muscle. Bars represent 20 µm.

View larger version (26K): [in a new window]   Fig. 3. Subcellular localization of myne-1. Adult mouse skeletal muscle was fractionated and immunoblotted with AM1. The protocol for cell-membrane fractionation is shown in A. The corresponding fractions are shown in lanes 1, 2 and 3. Enrichment of myne-1 is shown in the crude nuclear fraction 3 (lane 3). The protocol for nuclear membrane and nucleoplasm separation is shown in B. The corresponding fractions are shown in lanes 1, 2 and 3. Myne-1 is enriched in the nuclear membrane fraction 3 (lane 3).

View larger version (91K): [in a new window]   Fig. 4. Expression of myne-1 in smooth muscle. A-D represent sections from the stomach, whereas E-H are from the bladder. A and E show DAPI-stained nuclei as blue. B and F represent smooth muscle actin (green). C and G show myne-1 staining (red). D represents the merged images from A, B and C. H represents the merged images from E, F and G. Myne-1 is most abundantly expressed in smooth muscle. Bars represent 100 µm.

View larger version (94K): [in a new window]   Fig. 5. Colocalization of myne-1, lamin A/C and emerin in skeletal muscle. Transverse sections of quadriceps muscle were stained with AM1 (A,C,D,E,G and H). Staining with lamin A antibody is shown in B, and double staining with AM1 and lamin is shown in C and D. Staining with emerin is shown in F, and double staining with AM1 and emerin is shown in G and H. D shows a higher magnification view of the boxed region in C. Similarly, H shows the boxed region in G at higher magnification. Note colocalization of myne-1 and lamin A appears as yellow staining in C and D. In contrast, in G and H, some nuclei that express myne-1, but not emerin, are shown as red. The arrow in H indicates a cell within the artery wall that expressed myne-1 but not emerin. Low magnification bars represent 100 µm. High magnification bars represent 25 µm.

View larger version (56K): [in a new window]   Fig. 6. Immunolocalization of myne-1 in differentiating C2C12 cells. A,C,E,G and I represent nuclei stained with DAPI. AM1 labeling is shown in B,D,F,H and J. UN indicates undifferentiated C2C12 cells. D1, D3, D5 andD9 indicate 1 day, 3 days, 5 days and 9 days of differentiation, respectively. During differentiation, myne-1 is localized to discrete intranuclear foci, whereas late in differentiation, it becomes localized to the nuclear membrane. The bar represents 30 µm.

View larger version (70K): [in a new window]   Fig. 7. Immunolocalization of myne-1, lamin A/C and LAP2ß in C2C12 cells. (A) Fully differentiated C2C12 stained with lamin A/C is shown. (B) The same cells stained with AM1 demonstrating the coexpression and colocalization of myne-1 and lamin A is shown. (C) A partially differentiated C2C12 culture stained with AM1 is shown; (D) shows the same cells stained with LAP2ß demonstrating the coexpression and colocalization of myne-1 and LAP2ß. Bars represent 20 µm.

View larger version (24K): [in a new window]   Fig. 8. Co-immunoprecipitation of myne-1 and lamin A/C. Fully differentiated C2C12 cells were lysed and the soluble fraction was processed for immunoprecipitations using myne-1-specific antibody AM1 coupled to Protein A/G sepharose beads. The precipitate was separated by electrophoresis on parallel SDS-PAGE gels. One was stained with Coomassie blue (A), and the second was immunoblotted with a lamin-A/C-specific antibody XB10 (B). Lane 1, total cell lysate; lane 2, lysate precipitated with Protein A/G sepharose beads lacking AM1; lane 3, AM1 immunoprecipitated with Protein A/G sepharose in the absence of lysate; lane 4, lysate immunoprecipitated with AM1 on Protein A/G sepharose beads.