Abstract
Exposure to extracellular 5′-adenosine triphosphate (ATP) is known to induce membrane blebbing. In this study, we investigated the subcellular distribution of the cytoskeletal adaptor protein paxillin in primary bovine osteoblasts upon stimulation with ATP. Cells expressing a fusion protein of green fluorescent protein (GFP) and paxillin were followed by time-lapse video-microscopy after stimulation with 100 μM ATP. Within 100 s, GFP-paxillin became incorporated in numerous de novo formed focal aggregates localized at the cell periphery. The assembly of individual paxillin-containing aggregates occurred with a mean half-life time of <60 s, whereas their disassembly lasted twice as long. Despite the ongoing presence of ATP, the formation of paxillin aggregates was self-limiting within 25 min. Paxillin clustering was preceded by a transient rise in cytoplasmic calcium transients, which peaked already 20 s after adding ATP. The high mobility of paxillin was confirmed by measuring the dissociation rate of GFP-paxillin at mature focal adhesions, demonstrating the presence of a highly mobile fraction with a mean recovery half-life of 8.2 ± 1.2 s, followed by a slower phase (53 ± 20 s). Thus, both the exchange of paxillin at mature focal adhesions and the increase in intracellular calcium concentrations upon ATP stimulation are very rapid processes, which override the time course of ATP-induced paxillin membrane clustering by one to two orders of magnitude. Our data demonstrate that the transient recruitment of paxillin in membrane protuberances is based on the high intracytoplasmic mobility of unbound paxillin molecules and their rapid focal accumulation.
Highlights
Over the past years numerous extracellular signalling molecules have been identified to play pivotal roles in the regulation of bone turnover, among them are secreted nucleotides
We investigated the subcellular distribution of the cytoskeletal adaptor protein paxillin in primary bovine osteoblasts upon stimulation with adenosine triphosphate (ATP)
Extracellular 50-adenosine triphosphate (ATP) and other nucleotides signal through P2 receptors, formerly termed purinoceptors, which comprise a diverse group of receptors subdivided into P2Y G-protein coupled receptors and P2X ligand-gated ion channels (Bowler et al 2001; Gallagher and Buckley 2002; Li et al 2005; Orriss et al 2007, 2010; Panupinthu et al 2007; Kaunitz and Yamaguchi 2008)
Summary
Over the past years numerous extracellular signalling molecules have been identified to play pivotal roles in the regulation of bone turnover, among them are secreted nucleotides. A growing body of evidence suggested that purinergic stimulation increases proliferation of osteoblasts, suppresses bone mineralization and enhances the bone-resorbing activity of osteoclast (Nakamura et al 2000; Bowler et al 2001; Buckley et al 2002; Orriss et al 2006, 2007). Hypoxia has been shown to affect purinergic signalling by increasing vesicular ATP release (Lew and White 1987; Leung et al 1989; Orriss et al 2009). Nucleotides may be released constitutively by osteoblasts in a non-lytic manner such as inclusion in exocytotic vesicles (Lew and White 1987; Bowler et al 1998; Lazarowski et al 2000; Buckley et al 2002)
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