QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 8 April 2003
doi: 10.1242/jcs.00425

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lu, S. Articles by Horowits, R. Search for Related Content PubMed PubMed Citation Articles by Lu, S. Articles by Horowits, R.

New N-RAP-binding partners -actinin, filamin and Krp1 detected by yeast two-hybrid screening: implications for myofibril assembly

¹ Laboratory of Muscle Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA
² Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK

View larger version (63K): [in a new window]   Fig. 1. Lysates from bait L40 yeast strains carrying plasmids encoding the indicated fusion proteins were analyzed for protein expression by immunoblot. LexA fusion proteins were detected using anti-LexA or anti-N-RAP antibodies, as indicated. The results show that the bait strain expressed an intact LexA-N-RAP fusion migrating at 170 kDa, whereas unfused LexA protein migrated at 26 kDa on SDS-PAGE. Also shown are results from a LexA-lamin fusion construct migrating at 35 kDa and used as a negative control for N-RAP binding experiments.

View larger version (13K): [in a new window]   Fig. 2. Schematic representation of human filamin-2 domain structure (Thompson et al., 2000; van der Ven et al., 2000b; Xie et al., 1998), indicating the 24 Ig repeats, the actin binding domain, and the hinge region. The regions encoded by the mouse filamin-2 cDNA clones isolated in the yeast two-hybrid screen for N-RAP binding partners are shown below. Restriction enzyme analysis placed the 15 clones into seven groups of 1-6 clones each, and a total of eight of these clones were end-sequenced. The shortest mouse clones align with Ig repeats 20-24 of the human filamin-2 protein.

View larger version (40K): [in a new window]   Fig. 3. (A) Schematic diagram showing the domain organization of N-RAP (top), along with the regions of N-RAP expressed as histidine-tagged fusion proteins. Numbers refer to amino acid residues from the full-length mouse N-RAP open reading frame (Luo et al., 1997). (B) -actinin binding to N-RAP recombinant proteins immobilized on Ni-NTA agarose beads were analyzed by SDS-PAGE. (C) Duplicate loadings were blotted and probed with -actinin antibody. The antibody specifically detects -actinin binding to N-RAP-IB and N-RAP-LIM, but does not detect any -actinin binding to N-RAP-SR or to the His-CAT control proteins.

View larger version (44K): [in a new window]   Fig. 4. (A,B) Filamin binding to blotted proteins. Duplicate loadings were used (A) for total protein detection with Coomassie blue and (B) for immunoblot detection of bound filamin. Lanes were loaded with purified proteins as indicated. The histidine-tagged CAT proteins (HIS-CAT-1, HIS-CAT-2) served as negative controls. Significant filamin binding to recombinant N-RAP-SR was observed. (C,D) Filamin binding to N-RAP recombinant proteins immobilized on Ni-NTA agarose beads. Duplicate loadings were used (C) for total protein detection with Coomassie blue and (D) for detection of bound filamin. Significant filamin binding to recombinant N-RAP-SR was observed.

View larger version (36K): [in a new window]   Fig. 5. (A,B) Purified Krp1. Duplicate loadings were used for detection (A) of purified proteins with Coomassie blue and (B) of Krp1 by immunoblot. Lanes were loaded with affinity purified GST-Krp1, and the purified Krp1 and GST following thrombin cleavage. (C,D) Krp1 binding to N-RAP recombinant proteins immobilized on Ni-NTA agarose beads. Duplicate loadings were used (C) for total protein detection with Coomassie blue and (D) for detection of bound Krp1. Significant Krp1 binding to recombinant N-RAP-SR and N-RAP-IB was observed.

View larger version (79K): [in a new window]   Fig. 6. Cultured embryonic chick cardiomyocytes double-stained for (A,E) -actinin and (B,F) N-RAP. (C,G) Composite images show both -actinin staining in red and N-RAP staining in green. (D,H) The multiplied products of the red and green channels emphasize regions containing both N-RAP and -actinin fluorescence intensity. (A-D) Arrowheads are adjacent to peripheral regions of punctate -actinin staining that are characteristic of premyofibrils forming near the cell membrane. N-RAP is found in these regions, but is not confined to the -actinin dots. (E-H) N-RAP is also concentrated in areas where more mature sarcomeres appear to be fusing laterally (arrows).

View larger version (71K): [in a new window]   Fig. 7. Cultured embryonic chick cardiomyocytes double-stained for (A,E) -actinin and (B,F) filamin. Composite images (C,G) show both -actinin staining in red and filamin staining in green. (D,H) The multiplied products of the red and green channels emphasize regions containing both filamin and -actinin fluorescence intensity. (A-D) Large regions of premyofibrils are characterized by punctate -actinin staining along the cell preiphery (asterisks); filamin staining exhibits a more diffuse, mesh-like appearance in these regions. More centrally located mature myofibrils exhibit broad periodic striations of filamin staining that co-localize with the -actinin staining in Z-lines (arrowheads). (E-H) Some myofibril precursors are characterized by near continuous -actinin and filamin staining (arrows). As sarcomeres mature and -actinin spacing increases, filamin staining is still observed between the newly formed Z-lines (arrowheads). In fully mature regions, filamin staining is restricted to the Z-lines (asterisks).

View larger version (74K): [in a new window]   Fig. 8. Krp1 expression in mouse muscle and chick cardiomyocytes. Duplicate loadings were used (left) for detection of total proteins with coomassie blue and (right) of Krp1 by immunoblot. Lanes were loaded with total homogenates from mouse skeletal muscle (M) and heart (H), as well as lystates from cultured embryonic chick cardiomyocytes (C). The Krp1 antibody specifically detects a 68 kDa band in mouse skeletal muscle and chick cardiomyocytes, but no Krp1 is detected in mouse heart tissue.

View larger version (143K): [in a new window]   Fig. 9. Cultured embryonic chick cardiomyocytes double-stained for -actinin (red) and Krp1 (green). (A) Krp1 staining is diffuse during early stages of myofibril assembly. (B-D) In more mature cells, Krp1 often outlines the periphery of longitudinally oriented myofibrils (arrows). The boxed area in (D) is shown at higher magnification in (E), and (F) shows the multiplied product of the red and green channels, which emphasizes regions containing both -actinin and Krp1 fluorescence intensity. (E,F) Krp1 staining is often punctate and periodic, exhibiting spacings from 0.5 to 1.0 µm (arrowheads). This example also shows Krp1 localized near narrow myofibrils that appear to be fusing laterally with more mature striations (arrows), but no Krp1 staining in a region of the same myofibril that appears to have already undergone this fusion process (asterisk).