Rapid vinculin exchange dynamics at focal adhesions in primary osteoblasts following shear flow stimulation

Abstract

The adaptor protein vinculin plays a key position in the formation of focal adhesions and regulates cell attachment. To study the turnover of vinculin in bone-derived cells, we expressed green fluorescent protein-tagged vinculin in primary bovine osteoblasts and examined the appearance of focal adhesions in cells exposed to laminar shear flow. Already 20 sec after application of shear stress fluorescently labelled focal adhesions became visible as small flashing dots at the periphery of cells. The number of newly formed focal adhesions per individual cell increased continuously over approximately 300 sec and then remained relatively stable. The assembly of focal adhesions in shear stress-stimulated osteoblasts was accompanied by a transient rise in intracellular calcium levels. The mean assembly time of an individual focal adhesion plaque was 32.2+/-2.2 sec and the mean disassembly time was 60.5+/-6.0 sec. The recruitment of vinculin to nascent focal adhesions was in the same range as the recovery half-life of GFP-vinculin at stable focal adhesions (13.0+/-2.0 sec). These data show that accumulation of GFP-vinculin in newly formed focal adhesions and its exchange from pre-existing, mature plaques are both rapid processes that occur in mechanically stimulated osteoblasts within less than one minute.