Get Your kICS by Measuring Membrane Protein Dynamics

Abstract

Akin to thepuckon an ice hockey rink,the lateral dynamics of a representativemembrane protein exhibits rich be-havior(1).Dependingontheinteractionwiththehockeyplayersandotherobsta-cles (membrane constituents), the puck(protein)candart aroundthe ice(mem-brane surface) or be stalled momen-tarily (transient confinement zone). Ifwe watched the motion of a singlepuck, we would need to watch manyhockey games beforewe might eventu-allyworkouttherulesofthegame.Butwhat if we could watch many games inparallel? This is where the suite of im-age correlation spectroscopy (ICS)techniques and its newest member, k-space image correlation spectroscopy(kICS) comes in Abu-Arish et al. (2).ICSandkICSaremembersofafam-ily of techniques which began withfluorescence correlation spectroscopy(3). Fluorescence fluctuation analysisprovides the statistical mechanicalfoundation with correlations betweenfluorescence fluctuations measuredrelative to a given lag vector. Correla-tions can be made as a function oftime (fixed space) called FCS (3), fluc-tuationsinspace(fixedtime)calledICS(4), and space-time fluctuations calledSTICS (5). The ergodic principle en-sures equivalence between occupancyfluctuations whether in time or space.In temporal ICS or TICS, one essen-tially images in time (using a laserscanning fluorescence confocal micro-scope) many molecules in parallel asthey change positions from one imageto the next image. If the molecules donot move between the first image andthe second image, the spatial correla-tion between image one and imagetwo will be high. However, if move-ment does occur between image oneand imagetwo,there isaloss of spatialcorrelation. By analyzing the correla-tion between images collected atdifferent times, information on proteinmotioncouldbeobtained.Thismethodprovidesinformation from manymole-cules without the need for tracking in-dividual particles. A problem endemicto all these methods is that fluctuationsin fluorescence are measured asopposed to particle fluctuations whichare the desired quantities in evaluatingtransport properties.In kICS (6), images collected from atime-seriesarefirstFouriertransformedinto k-space before image cross-corre-lation is performed. This has twodistinct advantages over (real space)ICS. First, fluctuations from photophy-sics (e.g., cis-trans isomerization, (de)protonation, reversible dark statequenching, reversible photobleaching)do not contribute to the determinationof transport coefficients (6). Second,determinationofthelateralpointspreadfunction dimension is not required, un-like conventional fluorescence fluctua-tion approaches (6).In this issueof the Biophysical Jour-nal, the pioneer of ICS and kICS, PaulWisemanhasteamedupwithAsmahanAbu-Arish,ElvisPandzic,JulieGoepp,Elizabeth Matthes, and John Hanrahan(2) to gain new insights into the dy-namics of a biomedically importantmembrane protein, the cystic fibrosistransmembrane conductance regulator.Using the kICS technique, the authorshaveprovidedevidencefortwopopula-tions of cystic fibrosis transmembraneconductance regulator (CFTR) mole-cules, which differed in degree ofconfinement and lateral motion on thecell surface. Impressively, they wereable to extract information on the dy-namics of CFTR inside domains,CFTR dynamicsoutside(andbetween)domains, fractional populations, anddegree of confinement (within do-mains). ICS analysis delivered clusterdensities and mean number of mole-cules per cluster.The authors examined the effect ofcholesterol on dynamics and clusteringbehavior of CFTR. Cholesterol has aremarkable effect on membrane pro-tein assembly, dynamics and function(7–9). Depletion of cholesterol causedthe confinedfractionand averagenum-ber of CFTR molecules per cluster todecrease, whereas increase in choles-terol were found to be associated withincrease in clustering and increasedconfined fraction. Interestingly, viralinfection was shown to increase clus-tering further into larger platformswith reduced CFTR mobility. Theseobservations and analyses suggestthat cholesterol-influenced membranedomains play an important role in thecell surface behavior and pathologyof CFTR.Aside from the important new in-sights into this anion channel, the re-sults of this study revealed howcomplex cell surface dynamics andclustering can be measured usingpowerful fluorescence microscopytechniques. With these new methodsin hand, biophysicists can sit back,relax and enjoy the hockey game.REFERENCES