Multidrug extrusion systems have been reported for both prokaryotes and eukaryotes [62-64]. Membrane permeability and active extrusion systems appear to play a crucial role in universal cellular defense mechanisms. For instance, the efflux pump of Pseudomonas aeruginosa can extrude a variety of structurally and functionally diverse antibiotics . It is very likely that membrane proteins are triggered by substrates to assemble membrane transporters optimizing for the extrusion of encountering substrates. Thus, the study of the real-time size and the dynamics of membrane transporters will lead to a better understanding of the multidrug resistance mechanism.
X-ray crystallographic methods have been used to determine the sizes of membrane transporters and the cellular transport mechanism. This method is limited by the difficulties of crystallization of membrane proteins and this method cannot provide the real-time dynamics of live cells [65-69]. The metal nanoparticle with the help of molecular dynamic microscopy solves this problem to a large extent . The optical properties of the noble metal nanoparticles (SEGNPs) are attributed to its surface plasmon resonance and show size dependence [19-21]. Thus, we used the color index of nanoparticles as the size index to study the real-time size transformation of membrane transporters in live cells (Figs. 1-3). The sizes of the nanoparticles transported through the live-cell membranes can be measured by directly visualizing the color of individual nanoparti-cles, which are correlated with the size of the nanoparti-cles. This study has demonstrated that nanoparticle optics showed the possibility of real-time live-cell imaging with sub-100-nm spatial resolution and millisecond time resolution. Furthermore, unlike conventional fluorescent dyes or QDs, noble metal nanoparticles resist photobleaching and blinking. These unique features allow these nanoparticles to be used for continuously monitoring the size transformation of membrane transporters and efflux dynamics in live cells for an unlimited time. Nanoparticle optics is superior to TEM and scanning probe microscopy (SPM) for the study of sub-cellular dynamics in live cells because TEM cannot provide real-time measurements of dynamics events in live cells and SPM offers lower temporal resolution and cannot monitor nanoparticles inside live cells or many single nanoparticles simultaneously.
Three strains of P. aeruginosa [70-72], wild-type (PA04290, a wild-type cell level expression of MexAB-OprM), A ABM (TNP076, deletion of MexAB-OprM, derivative of PA04290), and nalB-1 (TNP030#1, overexpression of all MexA, MexB, and OprM, derivative of PA04290), have been constructed and studied. The MexAB-OprM membrane pump was used as a working model. We investigated the roles of these membrane proteins on the extrusion system by measuring the sizes and dynamics of substrates (nanoparticles) transported by mutants of overexpression or deletion of these subunits. The sizes of single substrates (nanoparticles) transported through live-cell membranes were measured by directly visualizing the color of the individual nanoparticles and hence the sizes of the extrusion pump were determined in real time at the nanometer scale (Figs. 1-3).
Based on random-walk theory [73, 74], the transport of nanoparticles through the live-cell membrane (in a buffer solution, on the cell membrane, and inside the cells) was determined . Representative snapshots of real-time images of single nanoparticles transported across live-cell membranes in Figure 2 shows all three strains of P. aeruginosa were uptake and efflux both blue (~50 ± 5 nm) and green (~70 ± 5 nm), and, occasionally, yellow nanoparticles (~75 ± 5 nm). The red nanoparticles (~95 ± 5 nm) tended to remain outside the cells on the cell membranes and were rarely observed inside the cells (Fig. 2). These results suggest that the membrane of three mutants of P. aeruginosa permeated and extruded substrates up to 80 nm in diameter.
The accumulated nanoparticles by cells showed time and concentration dependence (Fig. 3). The uptake and efflux dynamics of nanoparticles by three mutants of P. aeruginosa differ at 100 min of incubation time. A greater number of nalB-1 cells took up and retained nanoparticles during the first 100 min in comparison with AABM (MexAB-OprM-deficient mutants) and WT. At 100 min, nalB-1 cells began to extrude the nanoparticles and the number of cells with nanoparticles decreased whereas AABM and WT have distinguished uptake and efflux characteristics. The observation of uptake and efflux of nanoparticles by AABM implies that nanoparticles (substrate) may trigger the assembly of membrane transporters optimized for the extrusion of encountered substrates.
This work demonstrates the possibility of real-time tracking of membrane permeability and the observation of pump machinery of single live cells at the single-extrusion-pump level and may lead to a better understanding of multidrug resistance and designing of pump inhibitors. Furthermore, alternative molecular pump designs may be explored so that pumps may one day be engineered to selectively transport specific molecules into and out of live cells for a variety of applications (e.g., intelligent drug delivery).
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