Measurement of kinase activation in single mammalian cells.

We demonstrate a new method for the simultaneous measurement of the activation of key regulatory enzymes within single cells. To illustrate the capabilities of the technique, the activation of protein kinase C (PKC), protein kinase A (PKA), calcium-calmodulin activated kinase II (CamKII), and cdc2 protein kinase (cdc2K) was measured in response to both pharmacological or physiological stimuli. This assay strategy should be applicable to a broad range of intracellular enzymes, including phosphatases, proteases, nucleases, and other kinases.

Characterization of cellular optoporation with distance.

We have developed and characterized cellular optoporation with visible wavelengths of light using standard uncoated glass cover slips as the absorptive media. A frequency-doubled Nd:YAG laser pulse was focused at the interface of the glass surface and aqueous buffer, creating a stress wave and transiently permeabilizing nearby cells. Following optoporation of adherent cells, three spatial zones were present which were distinguished by the viability of the cells and the loading efficiency (or number of extracellular molecules loaded). The loading efficiency also depended on the concentration of the extracellular molecules and the molecular weight of the molecules. In the zone farthest from the laser beam (> 60 microns under these conditions), nearly all cells were both successfully loaded and viable. To illustrate the wider applicability of this optoporation method, cells were loaded with a substrate for protein kinase C and the cellular contents then analyzed by capillary electrophoresis. In contrast to peptides loaded by microinjection, optoporated peptide showed little proteolytic degradation, suggesting that the cells were minimally perturbed. Also demonstrating the potential for future work, cells were optoporated and loaded with a fluorophore in the enclosed channels of microfluidic devices.

Localized measurement of kinase activation in oocytes of Xenopus laevis.

We have combined a rapid cytoplasmic sampling technique with capillary electrophoresis to measure the activation of protein kinase C (PKC) in a small region (approximately 60 microm) of a Xenopus oocyte. The phosphorylation of a fluorescent PKC substrate was measured following addition of a pharmacological or physiological stimulus to an oocyte. When substrates for cdc2 kinase (cdc2K), PKC, and protein kinase A (PKA) were comicroinjected into an oocyte, all three substrates could be identified on the electropherogram after cytoplasmic sampling. With this new method, it should be possible to measure simultaneously the activation of multiple different kinases in a single cell, enabling the quantitative dissection of signal transduction pathways.

The physiologic concentration of inositol 1,4,5-trisphosphate in the oocytes of Xenopus laevis.

To measure the concentration of inositol 1,4,5-trisphosphate ([IP3]) in small regions of single Xenopus oocytes, a biological detector cell was combined with capillary electrophoresis. This method is 10, 000 times more sensitive than all existing assays enabling subcellular measurement of [IP3] in Xenopus oocytes. Upon addition of lysophosphatidic acid to an oocyte, [IP3] increased from 40 to 650 nM within 2 min. IP3 concentrations as high as 1.8 microM were measured after activation with lysophosphatidic acid, suggesting that the physiologic concentration of IP3 ranges from the tens of nanomolar to a few micromolar in Xenopus oocytes. Since the IP3 receptor in Xenopus oocytes is nearly identical to the type I receptor of mammalian cells, the range of [IP3] in most mammalian cells is likely to be similar to that in the oocyte. By selecting or engineering the appropriate detector cell, this strategy should be applicable to cyclic adenosine diphosphate ribose and nicotinic acid adenine dinucleotide phosphate, and to the discovery of new Ca2+-releasing second messengers.