Abstract
It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn't need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on µm-sized membrane regions in the micro-to-milli-second time range.
Highlights
Starting from the original “fluid mosaic” model by Singer and Nicolson, the picture of cellular plasma membrane has been continuously updated during the last decades in order to include the emerging role of cytoskeleton and lipid domains[1,2].The first observations were obtained by fluorescent recovery after photobleaching (FRAP) unveiling that a significant fraction of membrane proteins is immobile[3,4,5]
The same physical quantities of STED-based microscopy can be obtained by a modified spatio-temporal image correlation spectroscopy (STICS32,33) method that is suitable for the study of the dynamics of fluorescently-tagged membrane proteins and/or lipids in live cells and by a commercial microscope
The peak at about 180 DL is due to the camera response to no photon, and it represents the contribution of Analog Digital (AD) converter
Summary
Starting from the original “fluid mosaic” model by Singer and Nicolson, the picture of cellular plasma membrane has been continuously updated during the last decades in order to include the emerging role of cytoskeleton and lipid domains[1,2]. The same physical quantities of STED-based microscopy can be obtained by a modified spatio-temporal image correlation spectroscopy (STICS32,33) method that is suitable for the study of the dynamics of fluorescently-tagged membrane proteins and/or lipids in live cells and by a commercial microscope. By fitting the series of correlation functions, the molecular ‘diffusion law’ can be obtained directly from imaging in the form of an apparent diffusivity (Dapp)-vs-average displacement plot This plot critically depends on the environment explored by the molecules and allows recognizing directly the actual diffusion modes of the lipid/protein of interest. The camera will read a reduced number www.jove.com of lines increasing the time resolution In this readout regime the frame time would be limited by the time required to shift the charges from the exposure to the readout chip on the camera and is usually in the order of milliseconds for 512 x 512 pixel EMCCD. In the case of ATTO488-PPE tpdhrifiefsuvasioipoupnsr6loy.aMrcehoprocearotnevdes3ru,0c,i3nc5e.thsBesyfulcaloltytnertrreaccsaotsv,eeTrfitRai-snGpaFolmPssosisbhtloecwotsonsaqtuadanentctDriefyaaptpshianesgloaDcfaauplpndcaitfsifouansifoounfnaccvtoieonrnsatgoaefnatdvaisenprdalagtcheeedmaisevpneltraaicngedemicceaontnitnf,ignseuamgmgeeonsstttialnyrgefrapeaeorvtdieaifrlflumys-acioonnny,fmianseicdrons on the membrane plane
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