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Select cytoplasmic and membrane proteins increase the percentage of immobile integrins but do not affect the average diffusion coefficient of mobile integrins

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Abstract

Integrins are ubiquitous adhesion receptors that are important for signaling and integrating the extracellular matrix and cytoskeleton. The role of cytoplasmic proteins vinculin, focal adhesion kinase (FAK), integrin-linked kinase (ILK), and membrane proteins epidermal growth factor receptor (EGFR) and Notch in altering αPS2CβPS integrin lateral diffusion was measured using single particle tracking (SPT) and RNA interference (RNAi). SPT measures heterogeneous diffusion properties, and RNAi selectively reduces the concentration of a target protein. After systematically reducing the concentration of vinculin, FAK, ILK, EGFR, or Notch, there was a 31 to 80 % increase in the mobile integrin fraction, indicating that these five targeted proteins (or assemblies that contain these proteins) are responsible for immobilizing a fraction of the integrins when all proteins are present at native concentrations. The average diffusion coefficient of all mobile integrins did not change after any of the RNAi treatments, and the percentage of Brownian, directed, or anomalous/constrained trajectories relative to total mobile trajectories did not change after vinculin or EGFR RNAi. However, the fraction of anomalous/constrained trajectories relative to the total mobile trajectories increased 9 to 19 % after FAK, ILK, and Notch RNAi, when the concentration of these proteins was reduced. In the case of FAK, ILK, and Notch, native concentrations of these proteins simultaneously increase the immobile fraction of integrins but decrease the diffusion constraints to those integrins that remain mobile. Comparisons of single receptor and ensemble measurements of diffusion and what is known about the effect of these proteins in altering integrin clustering are discussed.

Membrane and Cytoplasmic proteins that affect the heterogeneous diffusion properties of integrin receptors can be elucidated using RNA interference to reduce the concentration of a single protein combined with single particle tracking measurements at native or reduced protein concentrations. Trajectories exhibit directed, Brownian, or anomalous motion; there is also a fraction of immobile receptor.

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Acknowledgments

This work is supported by the National Science Foundation under Grant CHE-0845236.

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  1. Department of Chemistry, Iowa State University, 1605 Gilman Hall, Ames, IA, 50011, USA

    Dipak Mainali & Emily A. Smith

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  1. Dipak Mainali
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Correspondence to Emily A. Smith.

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Mainali, D., Smith, E.A. Select cytoplasmic and membrane proteins increase the percentage of immobile integrins but do not affect the average diffusion coefficient of mobile integrins. Anal Bioanal Chem 405, 8561–8568 (2013). https://doi.org/10.1007/s00216-013-7279-1

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