Although light has been used to manipulate physiology, it has rarely been used to control gene expression. Building on the inducible gene expression technology based on the ecdysone promoter system (No et al., 1996), Kao and colleagues have developed a method for using light to turn on transgene expression (Kao and Freilich, 2004). In this approach, components of the ecdysone-inducible expression system (the gene for the ecdysone steroid receptor and the transgene under control of the ecdysone-dependent promoter) are introduced into cells or tissues by standard transfection techniques or by virally mediated gene transfer. Application of steroid analogs of ecydysone (e.g., ponasterone) or nonsteroidal ecdysoids (NSEs) effectively activates transgene expression. The AM approach, widely used for loading fluorescent indicators into cells (Tsien, 1981), is used to load a caged NSE into transgenic cells at high concentrations (>0.1 mM) by passive incubation. Focal UV or two-photon photolysis generates active NSE intracellularly to activate the ecdysone receptor and turn on transgene expression. This is illustrated in Figure 6.
Detection and dissection of synaptic vesicle endocytosis using light
Defining the molecular mechanisms that control synaptic vesicle endocytosis has been challenging, in part because endocytosis is a rapid, compensatory process that follows exocytosis. To identify and characterize the molecular players in compensatory endocytosis, it is therefore necessary to use techniques that perturb specific proteins only during endocytosis, but not exocytosis. Because these two processes are temporally and spatially coupled, it is technically difficult to inhibit protein function during only one of these phases of the synaptic vesicle cycle. Furthermore, teasing out the role of a protein that may be involved in both exocytosis and endocytosis is especially difficult, because disruption of a protein involved in exocytosis would likely obscure analysis of its potential endocytic function.
Important advances in the study of endocytosis have come from the examination of temperature-sensitive mutations, such as shibire (dynamin), which cause specific inactivation of protein function at high temperatures (Koenig et al., 1983). However, even temperature-sensitive mutations have a fairly slow temporal resolution, and most proteins and individual protein domains are not accessible to this approach. Protein photoinactivation has the potential to combine high spatial resolution of inactivation with even better temporal resolution than temperature-sensitive mutations. Using photoinactivation, it may be possible to dissect out the precise exocytic and endocytic functions of a protein, as well as the function of a protein at discrete stages in the multistep process of synaptic vesicle endocytosis.
FlAsH [4',5'-bis(1,2,3-dithioarsolan-2-yl)fluorescein] is a membrane-permeant fluorescein derivative that fluoresces when bound to a short tetracysteine motif engineered into a protein of interest (Griffin et al., 1998; Gaietta et al., 2002). Graeme Davis and colleagues have shown that FlAsH can specifically label proteins in Drosophila in vivo and that a FlAsH-bound protein can be specifically photoinactivated in seconds in a dose-sensitive manner, with the spatial and temporal precision of light (Marek and Davis, 2002; Tour et al., 2003). Poskanzer et al. (2003) have combined this genetically encodable system of photoinactivation with use of the synapto-pHluorins, pH-sensitive green fluorescent protein (GFP)-tagged synaptic vesicle proteins that monitor synaptic vesicle recycling in real time (Miesenbock et al., 1998). In combination, these techniques enabled them to assay endocytosis while disrupting the function of specific proteins.
Synaptotagmin I (Syt I), a Ca2+ sensor for synaptic vesicle fusion, has been implicated previously in synaptic vesicle endocytosis. However, this hypothesis could not be tested directly, in part because neurotransmitter release is severely reduced in Syt I mutants. By FlAsH photoinactivating Syt I only during endocytosis and assaying vesicle recycling with the synapto-pHluorins, they have shown that this protein is necessary for synaptic vesicle endocytosis (Fig. 7) (Poskanzer et al., 2003). Thus, Syt I, during insertion in the synaptic plasma membrane during vesicle fusion, is poised to link the exocytosis of a synaptic vesicle with its subsequent internalization. They are using these techniques to define the function of Syt I in endocytosis more specifically and to explore how this protein coordinates different aspects of the synaptic vesicle cycle. These genetically encoded fluorescent tools should enable the analysis of other molecular interactions important in synaptic vesicle recycling, including those of other known exocytic proteins. In addition, this type of temporally and spatially specific light inactivation could be used in the future to dissect the mechanisms of other rapid cellular processes.