La familia perciben a los nombrados PcD como carentes y enfermos

of mobility of molecules [183-185].

Single particle tracking

Signaling events at the plasma membrane are initiated via interactions between individual molecules, and information about these interactions can be obtained by monitoring the lateral dynamic behavior of proteins within or proximal to the plasma membrane. Since these interactions can be highly dynamic and tightly regulated in space and time, bulk studies such as protein biochemistry or conventional confocal microscopy will only yield information on the average properties of the dynamic behaviors and are compromised by poor time resolution. SPT in living cells is a key approach to directly monitor dynamics of proteins within their natural environment. In SPT the centre of mass of the emission peaks fluorescent molecules are determined with a positional accuracy up to 10 nm, depending on the number of photons detected. However, in order to study protein mobility it is critical to monitor individual spots which requires labeling at suboptimal conditions (Fig. 3) [186]. In addition, studying protein mobility requires high temporal resolution and therefore camera based microscopy approaches are the method of choice, because they allow simultaneous imaging of the whole field of view. Consequently, the time resolution of SPT is only limited by the acquisition speed of the camera system and the fluorescence properties of the labeled species. TIRF microscopy is advantageous over conventional wide field microscopy, since it limits the excitation volume to a region close to the coverslip surface thereby reducing out-of-focus noise resulting from autofluorescence or fluorescence from internalized fluorophores, thus improving spatial resolution without compromising acquisition speed. However, TIRF-based SPT is limited to imaging dynamics at the ventral plasma membrane, where protein mobility might be hindered by adhesions or substrate anchors. Therefore, the choice of wide field versus TIRF to perform SPT depends on the biological questions one wants to address, and appropriate labeling techniques have to be chosen with stronger fluorophores, such as quantum dots, for conventional, wide field imaging, to overcome out-of-focus noise. Analysis of the molecule tracks obtained with SPT (Fig. 3B) provides unprecedented detailed information about mobility, membrane-binding lifetimes and domain formation, all important determinants of signaling function (Fig. 3C) [88, 187, 188]. For example, the heterogeneous nanoscale organization of the plasma membrane imparts different types of motion onto proteins, like immobility, free Brownian diffusion, transient confinement in dynamic domains, hop diffusion across membrane picket fences and directed flow [189]. Therefore, the analysis of the trajectories and subsequent quantification of the mode of diffusion and the diffusion coefficient of molecules can give insight into the influence of the membrane nanoenvironment on signaling function [190].

The power of SPT for signal transduction research has been convincingly demonstrated since the beginning of the century, and SPT has yielded new insights into the dynamical processes occurring in receptor signaling. SPT perfomed on live cells showed that

amplification in signal transduction occurs, for example, through the epidermal growth factor (EGF)-induced clustering of the EGF receptor [191] or via increased mobility of chemotaxis receptor [88] in combination with faster cycling of the ligand [192] at the leading edge of migrating cells. SPT of the FcRI receptor demonstrated that steps in receptor signaling occur changes between protein aggregation states [90, 193]. These studies have also elucidated mechanisms that control protein mobility, such as compartmentalization caused by cytoskeletal contacts via the actin cytoskeleton [91, 194] or microtubules [89, 92]. In addition, the lipid environment [195] and protein-protein interactions [196, 197] were shown to regulate receptor mobility. These mechanisms enable a cell to fine-tune its response through local changes in the rate, duration, and extent of signaling.

Although SPT analysis of the behavior of one molecule within the plasma membrane elucidates signaling events occurring through one protein, the direct visualization of signaling interaction events requires dual-color tracking of two proteins. However, due to the requirement of suboptimal labeling this is very challenging. Nevertheless, studies have shown tracking of two spectrally distinct fluorophores to determine for example receptor dimerization [187, 198]. In addition, methods are used to overcome the suboptimal labeling problem, like single-molecule FRET, were one can track the donor and/or acceptor [199] and measure the mobility of a protein when it is interacting with another protein. Currently, multicolor single particle tracking is developed on a hyperspectral microscope, where up to 8 colors of quantum dots can be tracked in hyperspectral images [200].

Fluorescence correlation spectroscopy

Among the methods to study dynamics of cell membrane components, fluorescence correlation spectroscopy (FCS) has been extensively used to monitor mobility of lipids and proteins in living cells. In general in FCS, the sample is illuminated by a laser beam focused to a very small observation volume (Fig. 4A). For membrane measurements the focus of a laser spot is a 2D Gaussian detection area. The intensity of fluorescent molecules within this volume is monitored over time (Fig. 4B). Both the diffusion of fluorescent species in and out of the observation volume and photochemical processes of the fluorescent dye will give rise to intensity fluctuations in time (Fig. 4B). Since the photochemical processes, such as blinking and bleaching occur at much shorter time scales than the diffusion, these processes can be separated. To analyze the dynamic properties of the fluorescent species, the autocorrelation function (ACF) is used, which describes the self-similarity of the signal as a function of time lag τ. The ACF bears information about both the number of molecules and the average time the species resides within the observation volume (Figure 4C), and by fitting the ACF with an appropriate diffusion model these quantitative parameters can be extracted. Advantages of FCS include sensitivity for low concentrations, non-invasiveness due to low excitation energies, and accurate measurements of lipid and protein dynamics thanks to robust

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