Direct measurement of sizes and dynamics of single living membrane transporters using nanooptics

Sizes and dynamics of single membrane transporters on single living cells, Pseudomonas aeruginosa, were directly measured showing transported substrates up to 80 nm in diameter, which is one order magnitude larger than the reported exclusion limit of the outer membrane, over times of seconds to hours. The uptake and efflux dynamics depend on size and concentration of substrates, incubation time, and mutants. The strikingly active efflux of substrates by mutants devoid of pump proteins was observed, suggesting that substrates trigger the assembly of a new extrusive system. This new observation is in excellent agreement with the recent results of genome project analysis showing that P. aeruginosa encode more than a dozen unidentified efflux pump proteins. An extraordinary heterogeneity of size and dynamics of membrane transport mechanisms was observed, demonstrating real-time transformation of membrane permeability and an active extrusion system. This constitutes the first direct observation of living membrane transportation triggered by substrates with a variety of sizes at the single membrane pump level and opens up the new possibility of direct measurements of membrane active extrusion mechanisms for advancing the understanding of multidrug resistance and living molecular pumps.