Differential actin organization by vinculin isoforms: implications for cell type‐specific microfilament anchorage

Abstract

Vinculin is found in all adherens junctions, while metavinculin, a larger splice variant, is coexpressed with vinculin only in smooth and cardiac muscle. To understand the significance of metavinculin expression, we compared ligand binding between turkey vinculin and metavinculin. Residues 12 58 were found essential for head-tail interactions in both proteins. The tail domains (VT and MVT, respectively) both bind to F-actin. However, while VT bundles F-actin, MVT generates highly viscous F-actin webs. In transfected PtK2 cells, VT causes F-actin needles or coils, while MVT-expressing cells display a diffuse F-actin distribution. Thus, the MVT-specific insert induces an F-actin supraorganization different from the VTbased form, suggesting that metavinculin has a specific role in muscle.z 1998 Federation of European Biochemical Societies.

Comparative Biochemical Analysis Suggests That Vinculin and Metavinculin Cooperate in Muscular Adhesion Sites

“…Our biochemical studies demonstrate that both vinculin and metavinculin are regulated by an intramolecular head-tail interaction, as had already been suggested from yeast two-hybrid studies (32). However, the binding kinetics of the respective tail domains to the head domain differ to a considerable extent.…”

“…So far, no metavinculin-specific ligand has been identified, but there is strong evidence that the metavinculin-insert has a modulatory influence on the interaction with ligands of the tail domain. For example, the tail domains of vinculin and metavinculin (MVT) display different F-actin bundling properties (32), and a recently identified novel ligand protein, raver1, shows a higher affinity for metavinculin than for vinculin (33). Because neither binding to actin nor to raver1 directly involves the metavinculin insert, these data argue for a more complex effect of the insert, possibly on the conformation of the vinculin molecule.…”

Metavinculin Mutations Alter Actin Interaction in Dilated Cardiomyopathy

“…Whereas centrifugation at high speed (100 000g) pellets all filamentous actin and associated proteins, centrifugation at 12 000g only sediments actin filaments that are organized into F-actin suprastructures such as filamentous networks or bundles comprising cross-linked filaments. 15 Increasing the molar ratio of any MVT construct with respect to actin caused an increase in the amount of actin that was recovered in the pellet after low-speed centrifugation, indicating that all MVT proteins were able to crosslink actin filaments. Again, no significant differences among the 4 proteins were observed in densitometric analysis of Coomassie Blue-stained gels (data not shown).…”

“…Previous studies demonstrated that N-terminal His-tagging of vinculin and metavinculin tail domains does not affect their interaction with actin filaments. 15 Batch purification on Nickel-NTA sepharose was performed according to the manufacturer's instructions (Qiagen). Subsequently, proteins were purified on a MonoS column (Amersham Pharmacia Biotech) in S-buffer (50 mmol/L sodium phosphate, pH 6.5, 0.5 mmol/L EGTA, 15 mmol/L KCl) and eluted with S-buffer containing 1 mol/L NaCl.…”

“…The identical head portions of each isoform interact with membrane-associated ligand proteins, while their unique tail fragments harbor actin filament binding sites. Previous in vitro studies demonstrated differential geometric organization of actin filaments by the tail domain of each isoform, 15 suggesting metavinculin may have a unique role in anchoring actin filaments to intercalated discs in the heart.…”