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Direct Monomer-By-Monomer Observation of Gelsolin-Mediated Actin Filament Nucleation
- Source :
- Biophysical Journal. (2):508a
- Publisher :
- Biophysical Society. Published by Elsevier Inc.
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Abstract
- Actin nucleation is a fundamental biological process involved in several cellular events. Actin filament nucleation requires several hundred nano-molars to micro-molar amounts of polymerizable actin monomers. This requirement makes the impossible use of standard single molecule techniques. Here, we use zero-mode waveguides to reduce the observation volume and monitor, with single molecule resolution, the initial steps in actin filament formation. We immobilized gelsolin, a capping and nucleating protein at the bottom of the waveguides and added up to several micro-molar amounts of fully labeled actin monomers in polymerization conditions. We observe the first steps during filament formation as step-wise increases in detected fluorescence intensity. Mean reaction rates are then estimated for each oligomer size. Arrival time distribution of individual steps reveals the presence of kinetic intermediates. Furthermore, analysis of oligomer size distribution as a function of actin concentration reveals transitions at around 600 and 900 nM where the system dynamics change concomitant with the appearance of filament elongation. The change in dynamics at the transition is associated with an increase in dissociation rates. Such transition is blocked by Latrunculin A, suggesting a functional role for ATP hydrolysis and/or monomer flattening. Our results point towards a gelsolin-mediated actin filament nucleation mechanism where protein-protein interactions trigger ‘nuclei’ instability necessary for filament elongation.
Details
- Language :
- English
- ISSN :
- 00063495
- Issue :
- 2
- Database :
- OpenAIRE
- Journal :
- Biophysical Journal
- Accession number :
- edsair.doi.dedup.....b190215cc8fbb990b1eb2760ef6d6653
- Full Text :
- https://doi.org/10.1016/j.bpj.2014.11.2781