Imaginary Imaging

Most of us enjoy special effects in a movie theater, but it's no fun if our eyes fool our brain when it comes to research. Unfortunately, Jaiswal et al,. report that an accepted method of visualizing exocytosis is liable to mislead researchers. In the technique, the dye acridine orange (AO) is loaded into cells, where it accumulates in exocytic vesicles to the degree that it self-quenches. When the vesicle fuses with the plasma membrane and releases its contents, there is a flash of signal as first the dye molecules spread out beyond quenching range and then the fluorescence dissipates with the further dilution of the dye. It's all very dramatic—and utterly artifactual, as the authors discovered while collecting data on Ca²⁺-triggered exocytosis in astrocytes. As they looked more closely, they found some very distressing behavior. First, the AO flash depended on illumination. Second, this supposed marker of exocytosis occurred whether or not the vesicle under observation was near the plasma membrane. Third, as “exocytic” events increased, the AO began accumulating in the nuclei. The conclusion? Far from being a marker of exocytosis, the striking AO flash was nothing more than an indicator of vesicle photolysis. Happily, the authors do not leave us with that dispiriting conclusion, but also describe methods that can be used to reliably track exocytosis. They found that fluorescein isothiocyanate (FITC)-dextran, though prone to photobleaching, does not disrupt vesicles. In addition, the authors show that carefully monitoring the signal intensity from a fluorescently labeled membrane protein can indicate if bona fide exocytosis is occurring. Specifically, due to the properties of the microscope's excitatory field, the total signal should increase as the vesicle changes shape while nearing the plasma membrane. Then, after membrane fusion, the labeled protein should diffuse laterally through the plasma membrane. Significantly, the authors show that if the membrane protein is labeled with the Cerulean cyan fluorescent protein (CFP) variant, this allows simultaneous two-color imaging with AO. By carefully observing the relative signals over time for these two dyes, it is possible to distinguish whether the AO flash is a true harbinger of exocytosis or a cruel trick of the eyes.

- Jaiswal et al. 2007. Resolving vesicle fusion from lysis to monitor calcium-triggered lysosomal exocytosis in astrocytes. Proceedings of the National Academy of Sciences 104:14151-14156.

Plugged In

The macro world might be obsessed with going wireless, but the same is not necessarily true at the nanoscale level. A new report in Biosensors and Bioelectronics describes peptide nanowires and their application to nanoelectrode arrays. The peptide nanowire is a 33-amino acid transcription factor domain that has been engineered to bind cobalt ions at its surface, thus giving it conductive ability. Just like traditional wires, the peptide nanowire joins two entities: on one side, a carbon nanotube element of a nanoelectrode array, and on the other, a redox protein. Although nanotubes have been directly biofunctionalized with proteins, there are advantages in adding in a conductive linker. First, the nanowire spaces the protein from the nanotube, minimizing interference from surface effects. At the same time, the nanowire allows a means to more intimately connect the enzyme's active site to the electrode: the narrow helix of the connecting peptide can penetrate the globular protein and productively link the active site to the electrode. In the procedure shown by Yeh et al,., a thiolate of the peptide nanowire is used in combination with the sulfenic acid of a cysteine residue in the secondary redox center of NADH peroxidase to connect the wire to the biosensing protein. The peptide wire was then “metallized,” such that three metal ions were bound per peptide strand. Finally, this bioassembly was then affixed to an ion-etched carbon nanotube via a two-step conjugation procedure. Confirmation of conductance was obtained via electrochemical measurements revealing robust cyclic voltammetry signals. This implies there's every reason to expect that these tiny nanowires will be plugging into other bionanoelectrode systems in the coming years.

- Yeh et al. 2007. Peptide nanowires for coordination and signal transduction of peroxidase biosensors to carbon nanotube electrode arrays. Biosensors and Bioelectronics [Epub ahead of print, July 31, 2007].

Muscles in musculus

If you're going to study muscle movement, it makes sense to do so in a freely moving animal. To date, however, no in vivo imaging method has allowed that kind of flexibility in monitoring the signals that trigger muscle movement. Now, Rogers et al,. describe a method for dynamically tracking Ca²⁺ uptake into mitochondria, a process that rapidly follows the ion's release from the sarcoplasmic reticulum during contraction of skeletal muscle. The technique uses a mitochondrially targeted green fluorescent protein (GFP)-aequorin that's ubiquitously expressed in transgenic mice. The result is a bioluminescent Ca²⁺ sensor that is primed by injection of the aequorin substrate coelenterazine and triggered by mitochondrial uptake of the calcium ions. Although in vivo imaging of muscle-related Ca²⁺ flux is not unprecedented, previous studies have not been able to examine the entire muscle, let alone the whole animal. Rogers et al. were able to monitor whole newborn mice for mitochondrial Ca²⁺ signals, directing their attention toward recording recognized movement patterns associated with particular phases of sleep. Muscle activity could be tracked just 15 minutes after coelenterazine injection and followed for up to 10 hours. This flexibility, combined with the high signal-to-noise ratio (while mammalian tissues exhibit background fluorescence, they do not have inherent bioluminescence), makes this technique ideal for monitoring movement in behavioral studies. The downside is suboptimal spatiotemporal resolution; for detailed studies of particular muscle fibers, invasive studies like two-photon microscopy remain an appropriate choice. Nevertheless, the report from Rogers et al. represents the first holistic view of intramuscular Ca²⁺ signaling in an unrestrained mammal and will be of particular interest to researchers studying motor development and movement-related behaviors.

- Rogers et al. 2007. Non-invasive in vivo imaging of calcium signaling in mice. PLoS ONE 2:e974.

See Span

The value of visualizing exactly which residues of a transmembrane protein bridge the lipid bilayer is not hard to understand; the way to get this information is less obvious. Even a high-quality crystal structure may be useless, since most lipids do not survive purification or crystallization. Likewise, though hydropathy plots can give clues about transmembrane α-helices, they can give misleading predictions too, particularly if there are charged residues that are not extramembranous. Clues about the environment in which an aromatic amino acid finds itself can be gained by measuring fluorescence emission; this has been described for tryptophan residues. However, for this approach to work, only one tryptophan can be present in the protein, and therefore heavy mutagenesis can be required. Since cysteine is generally infrequent in membrane proteins and can be fluorescently labeled, it might be a better choice. Jittikoon et al,. apply this premise to the study of diacylglycerol kinase, which has three α-helices that span the lipid bilayer. The protein contains only two cysteines and has been shown to be fully active when these are mutated to alanines. Therefore, the double-alanine mutant can be used as the basis for a series of mutants in which cysteine is independently introduced at each position that might be located within the transmembrane domain. The authors collected kinetic data for each of the mutant proteins, but even those that had reduced catalytic activity were shown by cross-linking studies to form trimers (the physiological form of the wild-type sequence). By measuring the emission maxima of the different fluorescently labeled mutants and by performing quenching experiments to gauge solvent access, a strong case could be made for having appropriately defined the identities of the boundary residues of the protein's first transmembrane domain. This fluorescence method may therefore be a useful means to refine hydropathy plots in situations where charged residues may throw off the prediction.

- Jittikoon et al. 2007. A fluorescence method to define transmembrane alpha-helices in membrane proteins: studies with bacterial diacylglycerol kinase. Biochemistry 46:10950-10959.