title: Individual actin filaments in a microfluidic flow reveal the mechanism of ATP hydrolysis and give insight into the properties of profilin.
abstract: The hydrolysis of ATP associated with actin and profilin-actin polymerization is pivotal in cell motility. It is at the origin of treadmilling of actin filaments and controls their dynamics and mechanical properties, as well as their interactions with regulatory proteins. The slow release of inorganic phosphate (Pi) that follows rapid cleavage of ATP gamma phosphate is linked to an increase in the rate of filament disassembly. The mechanism of Pi release in actin filaments has remained elusive for over 20 years. Here, we developed a microfluidic setup to accurately monitor the depolymerization of individual filaments and determine their local ADP-Pi content. We demonstrate that Pi release in the filament is not a vectorial but a random process with a half-time of 102 seconds, irrespective of whether the filament is assembled from actin or profilin-actin. Pi release from the depolymerizing barbed end is faster (half-time of 0.39 seconds) and further accelerated by profilin. Profilin accelerates the depolymerization of both ADP- and ADP-Pi-F-actin. Altogether, our data show that during elongation from profilin-actin, the dissociation of profilin from the growing barbed end is not coupled to Pi release or to ATP cleavage on the terminal subunit. These results emphasize the potential of microfluidics in elucidating actin regulation at the scale of individual filaments.
authors: Laboratoire d'Enzymologie et Biochimie Structurales, Centre de Recherche de Gif, CNRS, Gif-sur-Yvette, France.
authors: Antoine
authors: A
authors: Jégou
authors: Thomas
authors: T
authors: Niedermayer
authors: József
authors: J
authors: Orbán
authors: Dominique
authors: D
authors: Didry
authors: Reinhard
authors: R
authors: Lipowsky
authors: Marie-France
authors: MF
authors: Carlier
authors: Guillaume
authors: G
authors: Romet-Lemonne
mesh_terms: D000199:Actins / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000465:Algorithms; D000818:Animals; D020240:Apraxia, Ideomotor; D006868:Hydrolysis; D051379:Mice; D046210:Microfluidic Analytical Techniques; D010710:Phosphates / Q000737:chemistry; D051304:Profilins / Q000737:chemistry*; D055503:Protein Multimerization; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D000199:Actins; C495739:Pfn1 protein, mouse; D010710:Phosphates; D051304:Profilins; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate