Protein sorting represents a potential point of regulation in neurotransmission because it dictates the protein composition of synaptic vesicles, the organelle that mediates transmitter release. Although the average number of most vesicle proteins has been estimated using bulk biochemical approaches (Takamori et al., 2006), no information exists on the intervesicle variability of protein number, and thus on the precision with which proteins are sorted to vesicles. To address this, we adapted a single molecule quantification approach (Mutch et al., 2007) and used it to quantify both the average number and variance of seven integral membrane proteins in brain synaptic vesicles. We report that four vesicle proteins, SV2, the proton ATPase, Vglut1, and synaptotagmin 1, showed little intervesicle variation in number, indicating they are sorted to vesicles with high precision. In contrast, the apparent number of VAMP2/synaptobrevin 2, synaptophysin, and synaptogyrin demonstrated significant intervesicle variability. These findings place constraints on models of protein function at the synapse and raise the possibility that changes in vesicle protein expression affect vesicle composition and functioning.

This protocol describes a method to determine both the average number and variance of proteins in the few to tens of copies in isolated cellular compartments, such as organelles and protein complexes. Other currently available protein quantification techniques either provide an average number but lack information on the variance or are not suitable for reliably counting proteins present in the few to tens of copies. This protocol entails labeling the cellular compartment with fluorescent primary-secondary antibody complexes, TIRF (total internal reflection fluorescence) microscopy imaging of the cellular compartment, digital image analysis, and deconvolution of the fluorescence intensity data. A minimum of 2.5 days is required to complete the labeling, imaging, and analysis of a set of samples. As an illustrative example, we describe in detail the procedure used to determine the copy number of proteins in synaptic vesicles. The same procedure can be applied to other organelles or signaling complexes.

This article describes two complementary techniques, single-particle tracking and correlation spectroscopy, for accurately sizing nanoparticles confined within picoliter volume aqueous droplets. Single-particle tracking works well with bright particles that can be continuously illuminated and imaged, and we demonstrated this approach for sizing single fluorescent beads. Fluorescence correlation spectroscopy detects small intensity bursts from particles or molecules diffusing through the confocal probe volume, which works well with dim and rapidly diffusing particles or molecules; we demonstrated FCS for sizing synaptic vesicles confined in aqueous droplets. In combination with recent advances in droplet manipulations and analysis, we anticipate this capability to size single nanoparticles and molecules in free solution will complement existing tools for probing cellular systems, subcellular organelles, and nanoparticles.

This paper describes a method by which molecules that are impermeable to cells are encapsulated in dye-sensitized lipid nanocapsules for delivery into cells via endocytosis. Once inside the cells, the molecules are released from the lipid nanocapsules into the cytoplasm with a single nanosecond pulse from a laser in the far red (645nm). We demonstrate this method with the intracellular release of the second messenger IP 3 in CHO-M1 cells, and report that calcium responses from the cells changed from a sustained increase to a transient spike when the average number of IP 3 released is decreased below 50 molecules per nanocapsule. We also demonstrate the delivery of a 23 kDa AGT fusion protein into Ba/F3 cells to inhibit a key player BCR-ABL in the apoptotic pathway. We show that an average of ~ 8 molecules of the inhibitor is sufficient to induce apoptosis in the majority of Ba/F3 cells. KeywordsLipids; Dye-Sensitized; Nanocapsule; Intracellular; Photolysis To uncover the spatiotemporal dynamics of cellular function, we need techniques that allow us to perturb cells over space and time in a controlled fashion. The most common approach for the delivery of bioactive molecules to cells in a highly spatiotemporally-resolved fashion is the two-photon release of a chemically-caged compound. 1-5 Caged molecules, however, suffer from a number of drawbacks. The design and synthesis of a new caged compound can be tedious and the caging of large protein molecules can be difficult, if not impossible. For intracellular delivery, the caged molecules must be permeable to the cell; this requirement, in turn, makes it difficult to control the precise concentration of the caged molecules inside the cell because of their preferential partitioning into different cellular organelles and membranes. These drawbacks of caged compounds have prompted us to develop lightaddressable lipid nanocapsules as a general platform for caging a wide range of bioactive molecules. 6-12 Nanocapsules based on lipid vesicles represent an emerging class of physical cages,, some of which also have been used recently for intracellular release. [13][14][15][16][17][18] Our first nanocapsules were lipid vesicles which we manipulated with optical tweezers and photolyzed using a single nanosecond laser pulse in the UV. 6,8,10 This capability was sufficient for delivering molecules extracellularly in a cell-culture setting. Unfortunately, the lipid vesicles were fragile and could not be stored in solution for more than a few days. This * To whom correspondence should be addressed chiu@chem.washington.edu. Supporting Information