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First published online 2 August 2005
doi: 10.1242/jcs.02484

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The Z-disc proteins myotilin and FATZ-1 interact with each other and are connected to the sarcolemma via muscle-specific filamins

¹ Department of Dermatology, University Hospital, HUG, Rue Micheli-du-Crest 24, 1211 Geneva 14 Switzerland
² Department of Pathology and Neuroscience Program, University of Helsinki and Helsinki University Hospital, Haartmaninkatu 8, 00014 Helsinki, Finland
³ Department of Pathology, University of Turku and Turku University Central Hospital, Kiinamyllynkatu 10, 20520 Turku, Finland
⁴ Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX Amsterdam, The Netherlands
⁵ International Center for Genetic Engineering and Biotechnology, Padriciano 99, Trieste, Italy

View larger version (166K): [in a new window]   Fig. 1. Expression and localization of myotilin and FATZ-1 in human skeletal muscle was detected using confocal microscopy of skeletal muscle cryosections. Myotilin was stained with a rabbit anti-serum against the N-terminus of myotilin (A) and FATZ-1 with a monoclonal rat antibody (B). The two proteins colocalize at the Z-discs along the skeletal muscle striations (C). Bar, 10 µm.

View larger version (68K): [in a new window]   Fig. 2. Expression of myotilin reorganizes FATZ-1 in double-transfected CHO cells. CHO cells were transfected with HA-tagged FATZ-1 (A,C), with T7-tagged myotilin (B,D) or with both constructs (E-H). Expressed proteins were detected with the corresponding anti-tag antibodies. In single-transfected CHO cells, FATZ-1 has a periodical staining pattern along the stress fibers (see inset in C), whereas myotilin decorates the filaments evenly and bundles them (B,D and inset). In double-transfected cells, in which FATZ-1 (E,G) and myotilin (F,H) were coexpressed, both proteins show a high degree of colocalization and the periodical staining of FATZ-1 is lost. Bar, 5 µm.

View larger version (37K): [in a new window]   Fig. 3. FATZ-1 forms a complex with myotilin in vitro. 35S-labeled HA-tagged recombinant form of FATZ-1 (A) and FATZ-2 (C) were generated by coupled in vitro transcription/translation and analyzed by SDS-PAGE and autoradiography. GST-fusion proteins of myotilin immobilized on glutathione-agarose beads were incubated with either 35S-labeled FATZ-1 (B) or FATZ-2 (D). Arrows indicate the protein of interest. After washing, bound proteins were analyzed by SDS-PAGE and autoradiography. Molecular mass markers are indicated in kDa.

View larger version (13K): [in a new window]   Fig. 4. Yeast two-hybrid survey of the sites in myotilin mediating interaction with FATZ-1. The PJ69-4A yeast strain was co-transformed with pACT2 plasmids encoding various deletion mutants of myotilin in fusion with GAL4-activation domain (AD) and pAS2-1 plasmids encoding FATZ-1 fused at its N-terminus with GAL4-DNA-binding domain (BD). The N-terminal serine-rich domain and the two Ig-like domains of myotilin are represented by a dark grey box and the black loops, respectively. Growth was estimated after 5 to 10 days of incubation at 30°C. +, growth; –, no growth, on selective media. The results indicate an interaction between myotilin1-498, myotilinS55F or myotilinT57I and FATZ-1, whereas shorter myotilin constructs did not interact. Transactivation controls were systematically performed for each construct with the opposite vectors without insert. Each experiment was repeated at least twice.

View larger version (10K): [in a new window]   Fig. 5. Yeast two-hybrid survey of the sites in FATZ-1 mediating interaction with myotilin. Experiments were performed as described in Materials and Methods and Fig. 4. CD1 and CD2 regions of FATZ-1 are represented by dark grey boxes, whereas the glycine-rich domain is depicted as a white box. +, growth; –, no growth, on selective media. FATZ-175-299 and FATZ-1163-299 caused auto-transactivation (TA) on both selective media. *FATZ-1163-299 as GAL4-AD fusion protein was not transactivating. The results indicate an interaction between FATZ-11-299 or FATZ-1163-299 and myotilin.

View larger version (30K): [in a new window]   Fig. 6. Binding of filamin isoforms and variants to GST-myotilin and GST-FATZ-1 fusion proteins. (A) HA-tagged recombinant form of filamin-A (FLN-A), filamin-B (FLN-B), filamin-ßVar-1 (FLN-Bvar-1) and filamin-C (FLN-C), containing repeats 19-24 were generated by coupled in vitro transcription/translation and analyzed by SDS-PAGE and autoradiography. The positions of FLN-A, FLN-B and FLN-Bvar-1 (arrowhead), and FLN-C (arrow) are indicated. (B,C) GST-fusion proteins immobilized on glutathione-agarose beads were incubated with the various radiolabeled filamin recombinants. After washing, bound proteins were analyzed by SDS-PAGE and autoradiography. Filamin-A, filamin-B, filamin-Bvar-1 as well as filamin-C containing repeats 19-24 bind to GST-myotilin but not to GST alone (B). Filamin-A, filamin-B, filamin-Bvar-1 as well as filamin-C containing repeats 19-24 bind to FATZ-1 but not to GST alone (C). The grouping of images was arranged from two different gels in B and in C. Note that filamin-B in the presence of FATZ-1 showed a tendency to proteolytic degradation under the conditions of the GST pull down experiments. Molecular mass markers are indicated in kDa.

View larger version (14K): [in a new window]   Fig. 7. Characterization of the interaction of different filamin isoforms with myotilin, FATZ-1 and the ß1A integrin subunit in yeast. +, growth; –, no growth, on selective media, tested as described in Materials and Methods and Fig. 4. Ig-like repeats are depicted as light grey boxes. H2 box corresponds to the second hinge region. Both myotilin and FATZ-1 interact with the C-terminal region of all the filamins tested. ß1A interacts with all filamins except filamin-A.

View larger version (8K): [in a new window]   Fig. 8. Characterization of the interaction of the C-terminal region of filamin-C with deletion mutants of myotilin and FATZ-1 in yeast. +, growth; –, no growth, on selective media, tested as described in Materials and Methods and Fig. 4. The N-terminal regions of both myotilin and FATZ-1 interact with the C-terminal region of filamin-C. *As FATZ163-299 fused with the GAL4-BD caused auto-transactivation on both selective media, binding of FATZ163-299 to filamin-C was verified utilizing GAL4-AD protein of FATZ-1.

View larger version (36K): [in a new window]   Fig. 9. Binding of the C-terminal region of filamin-C with the cytoplasmic domain of the ß1A integrin subunit in yeast two-hybrid assays and pull-down experiments. (A) +, growth; –, no growth, on selective media in yeast two-hybrid assays, tested as described in Materials and Methods and Fig. 4. Although filamin-C (19-21) and filamin C (20-21) bound to ß1A, filamin C (21-23) did not. (B) Detection by immunoblotting of the expressed products from cDNA constructs for HA-tagged filamin-C (19-21) and filamin-C (21-23) in lysates of transiently transfected COS-7 cells. Pull-down assay with GST-ß1A or GST immobilized on glutathione beads containing either HA-tagged filamin C (19-21) (C) or filamin-C (21-23) (D) fusion proteins. Immunoblot detection of bound filamin-C (19-21) to GST-ß1A. No binding was observed with filamin-C (21-23).

View larger version (37K): [in a new window]   Fig. 10. Schematic representation of two potential mechanical linkages between the contractile apparatus and the sarcolemma. The C-terminal region of filamin-C (FLN-C, represented as tail-to-tail associated dimer) binds to the ß1 integrin cytoplasmic domain and both and sarcoglycans associated with the dystroglycan complex at the sarcolemma. The N-terminal region of FLN-C contains an actin-binding domain, which may associate with both sarcomeric actin filaments and cortical actin in the sub-sarcolemmal region, serving thus as a nexus between actin filaments and costameric sites. A number of Z-disc proteins are depicted. Myotilin represented hypothetically as an antiparallel dimer can bind to FATZ-1, FLN-C and -actinin 2 (ACTN2). FATZ-1 can also bind to myotilin, FLN-C and ACTN2. It should be noted that in some cases, where there is more than one protein partner, competition and/or simultaneous binding could occur. Furthermore, it is likely that certain interactions occur only at distinct stages of development.