FUNDAMENTACIÓN CONCEPTUAL

Most research on the Tat pathway has focused on the biomolecular mecha- nism of translocation itself and the protein-protein interactions of translo- cation. In comparison, very little research has been done on the behaviour of the substrate targeted to the Tat pathway and there are many open ques- tions regarding the manner in which Tat substrates arrive at an appropriate site for translocation after all necessary modifications to the substrate.

Chloroplast protein targeting is an inherently spatial process and yet the majority of the research carried out is of a non-spatial, biochemical nature. We use a Tat signal peptide fused to a fluorescent protein to allow

in vivo visualization of the substrate population by fluorescence confocal

our spatio-temporal approach to Tat protein targeting. Ourin vivoresults complement thein vitroevidence for membrane-binding as an early step in the translocation process (di Cola et al.,2005;Hou et al.,2006) and address the importance of the signal peptide on the substrate dynamics before the translocation step.

We use fluorescence confocal microscopy as an analytical tool for ob- serving and perturbing the spatial distribution of fluorescent proteins over a short interval of time at the micron-scale in common with techniques shown in figure 1.1. Working at this length scale is challenging for light microscopy and we take care to avoid unwanted damage from prolonged imaging. This length scale is also too coarse to resolve structures visible by electron microscopy at the nano-scale and we are unable to resolve the highly convoluted thylakoid membrane structure and its influence on protein targeting. This study attempts to take anin vivoapproach without probing the movement of individual molecules as would be possible for single-particle tracking and other invasive techniques shown in table1.4.

1.3.1

Fluorescence Microscopy Techniques

Fluorescence microscopy is the use of fluorescent molecules, which emit light when excited with appropriately chosen electromagnetic radiation,

reflected or transmitted light in conventional light microscopy. Fluores- cence microscopy may use the same arrangement of mirrors and lenses as in conventional light microscopy but sophisticated electronics, and both computer hardware and software are usually present to allow precise con- trol of lasers and sensors in acquiring digital images from the analogue emission of fluorescence from fluorescent molecules.

Techniques for Localization and Measurement of Dynamics

The earliest use of fluorescence microscopy was with small molecules such as fluorescein which can be used to label a protein of interest directly or used to label antibodies which bind to a protein of interest. (Frye and Edidin,1970) More recently, molecules such as the Green Fluorescent Protein (GFP) (Tsien, 1998), from the jellyfish Aequorea victoria, and its deriatives have been used to study protein dynamicsin vivo. (Lippincott- Schwartz et al.,2001)

A number of fluorescence microscopy techniques have been employed ranging from observation of the intermixing of fluorescent molecules (Frye and Edidin,1970) to the use of an effect known as photobleaching. Photo- bleaching is the use of a high power laser setting to abolish the fluorescent property of a molecule and this effect can be used on steady-state distribu- tions to get more information by observation of fluorescence redistribution. (White and Stelzer,1999)

One of the earliest and most popular techniques of fluorescence mi- croscopy which uses the photobleaching effect is the Fluorescence Recov- ery After Photobleaching (FRAP) technique (Peters et al.,1974). A region of the sample is selected to be the bleach region and the average fluores- cence intensity is measured during the course of the experiment. A high intensity laser setting rapidly depletes the fluorescence in within the bleach region and fluorescence redistributes according to interactions within the sample of interest. Typically the biological data are compared to diffusion within idealized geometries, such as circles or spheres (Soumpasis, 1983), or more recently comparisons may be made to computer simulations, per- haps employing actual geometries from the data (Sbalzarini et al.,2005).

The Fluorescence Loss In Photobleaching (FLIP) technique again uses a high intensity laser to deplete fluorescence in a small region but the aim is to observe the redistribution of fluorescence in order to determine the connectivity of biological compartments (Cole et al.,1996). An alternative to the FLIP technique is the use of photoactivatable GFP (PA-GFP) allowing selective activation of a small population of PA-GFP using high intensity ultraviolet illumination so that the redistribution of the marked population may be observed (Patterson and Lippincott-Schwartz,2002).

Both FRAP and FLIP have counterparts, respectively fluorescence pho- tobleaching recovery (FPR) and continuous fluorescence microlysis (CFM)

CFM (Peters et al.,1981) and FLIP (Cole et al.,1996) techniques.

The tendency for fluorescence molecules to be influenced by their sur- roundings is used by the Fluorescence Lifetime Imaging (FLIM) technique to create a map of where fluorescence remains present for long lengths of time and regions where fluorescence is lost very quickly in order to infer properties about binding interactions and the local environment in the sam- ple (Lakowicz et al.,1992). Moving even further away from fluorescence microscopy for capturing static images, the Fluorescence Correlation Spec- troscopy (FCS) technique measures fluorescence fluctuations in femto-litre volumes and uses statistical physics to extract quantitative information about the dynamics of the fluorescent molecules under observation (Maiti et al.,1997;Haupts et al.,1998).

Techniques for Detecting Co-Localization and Proximity

When the interaction of two particular proteins is of interest, both proteins may be expressed fused with variants of GFP, such as Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP), so that the emission of the donor fluorophore, for example CFP, excites the acceptor fluorophore, for example YFP, in the Fluorescence Resonance Energy Transfer (FRET) technique which allows the proximity of the proteins of interest to be determined (Gadella et al.,1999).

Another approach to test for co-localization is the Bi-Fluorescence Com- plementation technique (BiFC) which detects co-location of two proteins of interest by fusing two non-fluorescent fragments of a fluorescent protein to the two proteins so that co-localization and interaction allows complemen- tation to result in a detectable fluorescent protein (Hu et al.,2002; Walter et al.,2004).

Whereas most photobleaching techniques measure in a small part of the domain, we will bleach in a spot and measure the depletion in the whole domain and we consider this a slight modification of FLIP as it has been described up to the present. Such a whole chloroplast measurement is vital given the heterogeneous nature of the chloroplast and the stromal compartment. In the mature techniques such as those in table 1.1there is often a collection of common analyses that may be applied, examples are shown in tables1.2and1.3.