Abstract
Desmin, the muscle-specific intermediate filament protein, surrounds the Z disks and links the entire contractile apparatus to the sarcolemmal cytoskeleton, cytoplasmic organelles, and the nucleus. In an attempt to explore the molecular mechanisms of these associations, we performed a yeast two-hybrid screening of a cardiac cDNA library. We showed that the desmin amino-terminal domain (N-(1-103)) binds to a 413-kDa TRIM-like protein, myospryn, originally identified as the muscle-specific partner of dysbindin, a component of the biogenesis of lysosome-related organelles complex 1 (BLOC-1). Binding of desmin with myospryn was confirmed with glutathione S-transferase pulldown assays and coimmunoprecipitation experiments. Western blot analysis revealed that the complex immunoprecipitated by desmin antibodies, in addition to myospryn, contained the BLOC-1 components dysbindin and pallidin. Deletion analysis revealed that only the (N-(1-103)) fragment of desmin binds to myospryn carboxyl terminus and that this association takes place through the 24-amino acid-long carboxyl-terminal end of the SPRY domain of myospryn. Using an antibody against the COOH terminus of myospryn, we demonstrated that myospryn colocalizes with desmin at the periphery of the nucleus, in close proximity to the endoplasmic reticulum, of mouse neonatal cardiomyocytes. In adult heart muscle, the two proteins colocalize, predominantly at intercalated disks and costameres. We also showed that myospryn colocalizes with lysosomes. Using desmin null hearts, we determined that desmin is required for both the proper perinuclear localization of myospryn, as well as the proper positioning of lysosomes, thus suggesting a potential role of desmin intermediate filaments in lysosomes and lysosome-related organelle biogenesis and/or positioning.
Highlights
Desmin, the muscle-specific member of the intermediate filament protein family, is one of the earliest known myogenic markers, both in heart and in somites [2,3,4]
Desmin Interacts with the tripartite motif (TRIM) Protein Myospryn support the involvement of intermediate filaments (IFs) in similar functions, including organelle positioning and protein targeting to the proper intracellular compartments [17, 18]
Dysbindin is a component of the biogenesis of lysosome-related organelles complex 1 (BLOC-1), which is involved in protein trafficking and organelle biogenesis
Summary
The procedures for the care and treatment of animals were according to institutional guidelines, which follow the guidelines of Association for the Assessment ad Accreditation of Laboratory Animal Care (AAALAC) and the recommendations of Federation of European Laboratory Animal Science Association (FELASA). Preparation of Heart Homogenates—Cardiac tissue from wild type and desmin null mice was homogenized in extraction buffer containing 10 mM Tris, pH 8.5, 0.01% (v/v) SDS, 20 mM NaCl, 0.01% (v/v) Nonidet P-40, 0.01% (v/v) DOC, 5 mM EDTA, 2 mM DTT, and protease inhibitors. The mixture was centrifuged at 1,000 ϫ g for 1 min, and the pellet was washed four times with 1ϫ PBS, 0.5% (v/v) Nonidet P-40, 0.25% (v/v) DOC and resuspended in SDS-PAGE sample buffer. GST-myospryn bound to a glutathione matrix, desmin was efficiently and absorbed, as detected by Western blot analysis using anti-desmin antibodies (Fig. 1I, A2, 2nd lane). Washed with PBS/Tween 20 (0.01%), mounted with fluores- labeled translated protein was immunoprecipitated with either cent mounting medium from DAKO (Carpinteria, CA), and anti-c-Myc or anti-HA antibody and analyzed by SDS-PAGE analyzed with a Leica confocal laser scanning microscope (Fig. 1II). LAS-AF, at 23–24 °C) equipped with ϫ63 NA 1.4 objective. itate desmin, whereas desmin alone cannot be precipitated using
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