Chapter Five - Single-Particle Tracking Photoactivated Localization Microscopy for Mapping Single-Molecule Dynamics
Introduction
Dissecting the molecular mechanisms for biological processes lends great insight into how these systems function, both in health and disease states. Likewise, fluorescence imaging of specific proteins allows for the identification of dynamic processes and causal relationships. Single-molecule measurements aim to exploit and expand upon both of these powerful avenues by elucidating the dynamics of specific proteins at the molecular scale. By following the motions of single molecular motors such as myosin or kinesin with high spatial and temporal resolution, the mechanics of their work cycle have been revealed (Svoboda et al., 1993, Yildiz et al., 2003). For many biological processes, including receptor trafficking, cytoskeletal dynamics, and cellular signaling, the collective behaviors of molecules may be important, and it would be interesting to extend these measurements to many molecules. Moreover, obtaining information on statistically rare events requires the acquisition of data on large numbers of single molecules, ideally under multiple conditions. However, traditional single-molecule measurements using fluorescence imaging require an extremely low labeling density. This is due to the diffraction of light when passing through a lens, which spreads the fluorescence of a single molecule into an Airy disk nearly 100 times the size of the molecule itself. Thus, in the case of closely packed membrane proteins, only one protein in thousands can be labeled to distinguish individual molecules. Since many small structures such as clathrin-coated pits and viruses typically contain only 1000 copies or fewer of a single protein, the extension of single-molecule imaging to their study was not evident until the recent advent of photoswitchable fluorescent probes (Ando et al., 2002, Patterson & Lippincott-Schwartz, 2002). In this era of large data sets, new advances in particle tracking combined with photoactivation of fluorescence have enabled the study of the dynamics of these previously inaccessible systems. Here, we describe the method of single-particle tracking combined with single-molecule localization using photoactivated localization microscopy (PALM) (Betzig et al., 2006), called single-particle tracking PALM (sptPALM) (Manley et al., 2008). This approach permits the study of protein dynamics in the context of living cells where proteins exist in dense populations.
Section snippets
Description of the sptPALM Method
The technological basis underlying sptPALM is the localization of the positions of specific proteins at near-molecular spatial resolution. This method further exploits the switchable properties of photoactivatable fluorescent proteins (PA-FPs; Ando et al., 2002, Patterson & Lippincott-Schwartz, 2002, Wiedenmann et al., 2004) used in PALM, although the same principle (termed STORM) has been applied to imaging with antibody-targeted photoswitchable synthetic labels (Rust et al., 2006). In PALM (
Labeling with Photoactivatable Fluorescent Probes
There are two primary means of labeling proteins for measurements with sptPALM, these are genetic encoding and expression of a PA-FP chimera, or targeting with an antibody or small molecule coupled to photocaged or photoswitchable dyes. In both cases, precautions must be taken to ensure minimal perturbations due to the labels. This generally means selecting cells for imaging with low expression levels, and demonstrating preservation of protein function in the chimeric form.
Tracking Single Molecules
As indicated in Fig. 5.2B, there are two analyses involved in recreating multiple single-molecule trajectories. In the first, single molecules are identified and located. In the second, locations are connected to form trajectories.
Experimental Example: sptPALM on a Membrane Protein
In the example presented here, we begin by plating cells onto a glass coverslip. Coverglass thickness and index of refraction should be chosen for compatibility with your objective. For the purposes of studying the motion of membrane proteins, use of TIRF is implemented to reduce background light. Use of a camera with single-photon sensitivity is also necessary to collect single-molecule fluorescence emission.
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Plate cells on a glass coverslip.
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Transfect cells ~ 18 h before measuring. Grow to 80–90%
Conclusions
The method of sptPALM represents a step toward a statistical description of single proteins in cells, where information on thousands of molecules provides a more complete picture of the wide variety of molecular motions taking place in living cells. Thus far, the method of sptPALM has been used to study the diffusion of membrane proteins (VSVG), membrane-binding proteins (Gag), and cytoskeletal proteins (actin and FtsZ). These studies have revealed new information about these systems. In the
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