Search

c-Cbl-associated protein (CAP) is an important cytoskeletal adaptor protein involved in the regulation of adhesion turnover. The interaction between CAP and vinculin is critical for the recruitment of CAP to focal adhesions. The tandem SH3 domains (herein termed SH3a and SH3b) of CAP are responsible for its interaction with vinculin. However, the structural mechanism underlying the interaction between CAP and vinculin is poorly understood. In this manuscript, we report the solution structure of the tandem SH3 domains of CAP. Our NMR and ITC data indicate that the SH3a and SH3b domains of CAP simultaneously bind to a long proline-rich region of vinculin with different binding specificities. Furthermore, the crystal structures of the individual SH3a and SH3b domains complexed with their substrate peptides indicate that Q807(SH3a) and D881(SH3b) are the critical residues determining the different binding specificities of the SH3 domains. Based on the obtained structural information, a model of the SH3ab-vinculin complex was generated using MD simulation and SAXS data., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The use of cardiomyocyte cell culture models allows the identification of various cell mediators that bring about changes in subcellular structures and gene expression associated with hypertrophy. The effects of insulin-like growth factor-I (IGF-I), basic fibroblast growth factor (bFGF), and triiodothyronine (T3) on gene expression and on the structural organization of myofibrillar and cytoskeletal proteins were compared in adult atrial (aARC) and ventricular (vARC) as well as in neonatal ventricular rat cardiomyocytes (vNRC) in long-term culture. Structural changes were evaluated by confocal microscopy and correlated to biochemical alterations. In vARC, IGF-I enhanced myofibrillar growth, whereas bFGF or T3 restricted sarcomere assembly to the central cell area, forming a sharp boundary in more than 50% of the cells. However, myosin occurred both in the cross-striated myofibrillar structures and in patches running along the nonsarcomeric fibrillar structures (also called stress fiber-like structures) in the cell periphery. In cells treated with either bFGF or T3, the expression of alpha-smooth muscle actin (alpha-sm actin) was greatly increased. This actin isoform was incorporated mainly into the nonsarcomeric contractile structures outside the area where myofibrils ended abruptly. alpha-sm actin protein increased up to 14- to 17-fold while the mRNA showed a moderate increase of 2- to 4-fold. This suggests that alpha-sm actin is mainly regulated at the translational or posttranslational level. In contrast, the cytoskeletal proteins alpha-actinin and vinculin increased only moderately (less than 2-fold) but also showed a relocalization in cells with restricted myofibrils. In aARC and in vNRC, alpha-sm actin was only moderately upregulated by bFGF or T3 and no drastic morphological changes were observed. In conclusion, IGF-I, bFGF, and T3 induced characteristic structural phenotypes depending on the type of cardiomyocyte. Large amounts of alpha-sm actin as expressed in bFGF and T3 treated vARC seem to be incompatible with sarcomere assembly., (Copyright 1998 Academic Press.)

Neonatal cardiac myocytes continue to undergo nuclear division, but lose their ability to complete cell division between 3 and 4 days of age. To examine cytoskeletal organisation of cardiac myocytes during mitosis, freshly isolated cardiac myocytes from 2-, 4-, 6- and 8-day-old rats were fixed and labeled with anti-tubulin, vinculin, desmin and sarcomeric alpha-actinin antibodies. The central, nuclear region of cardiac myocytes is expanded to form a balloon-like structure when they entered prophase. The organisation of microtubules, vinculin and desmin in mitotic myocytes from 4-, 6- and 8-day-old rats was identical to that in dividing myocytes from 2-day-old animals. Microtubules emanating from the nuclear membrane mainly ran along the longitudinal axis of cardiac myocytes in interphase. Microtubules were disassembled and reorganised into the mitotic spindle during mitosis. Desmin was disassembled, either diffusely distributed in the cytoplasm or formed spotty cytoplasmic aggregates during mitosis. Vinculin was disassembled in prometaphase, diffusely distributed in the cytoplasm and associated with cell membranes. During telophase it concentrated in the equator of mitotic spindles. Sarcomeric alpha-actinin became dispersed in the cytoplasm of mitotic myocytes from 2-day-old rats in prometaphase. It remained diffusely distributed in the cytoplasm and associated with cell membranes until the completion of cytokinesis. However, sarcomeric alpha-actinin was only partially disassembled in 4-, 6- and 8-day-old myocytes. Striations of alpha-actinin with full sarcomere length were observed in the cytoplasm as well as in the region of furrow formation. Thus, incomplete disassembly and presence of myofibrils in the equator region where cleavage furrows from may physically impede the furrowing of sarcolemma driven by the contractile ring, resulting in the formation of binucleated cardiac myocytes.

We have used high-resolution electron spectroscopic imaging to study the ultrastructure of negatively stained chicken gizzard vinculin. A careful examination of uranium salt-stained molecules revealed a high versatility of the overall shape of vinculin, for which an element of high flexibility is mainly responsible. This neck region links the vinculin head, probably consisting of the biochemically defined 90-kDa N-terminal fragment, to the rod-like tail. The hinge allows for sharp kinks in the molecule, so that head and tail structures can contact each other. By electron spectroscopic imaging, we were able to reveal substructural components in both head and tail regions. The head resembles a cloverleaf-like structure, consisting of three globular centers of mass, surrounding a protein-deficient center in a planar arrangement and of a short, stem-like fragment. The tail contains four spherical protein masses arranged like pearls on a string. Our data reveal a substructural organization of vinculin which is consistent with its presumed function as a structural component of microfilament attachment sites and support the concept of cryptic ligand-binding domains, previously based on biochemical evidence.