Continued progress in understanding cellular physiology requires new strategies for biochemical measurements in solitary cells, multiple cells, and subcompartments of cells. Large spatial gradients in the concentrations of molecules and presumably the activities of enzymes can occur in cells. Consequently, there is a critical need for measurement techniques for mammalian cells with control over the numbers or regions of cells interrogated. In the present work, we developed a strategy to rapidly load the cytoplasmic contents of either multiple cells or a subregion of a single cell into a capillary. A single, focused pulse from a laser created a mechanical shock wave which disrupted a group of cells or a portion of a cell in the path of the shock wave. Simultaneously, the cytoplasm was loaded into a capillary for electrophoretic separation. The size of the region of cellular disruption (and therefore the volume of cytoplasm collected) was controlled by the amount of energy in the laser pulse. Higher energies could be used to sample groups of cells while much lower energies could be utilized to selectively sample the tip of a neuronal process. The feasibility of performing measurements on subcellular compartments was also demonstrated by targeting reporter molecules to these compartments. A reporter localized to the nucleus was detected on the electropherogram following laser-mediated disruption of the cell and the nucleus. Finally, we demonstrate that this method terminated cellular reactions with sufficient rapidity that cellular membrane repair mechanisms were not activated during cytoplasmic collection. The combined ability to preselect a spatial region of a cell or cells and to rapidly load that region into a capillary will greatly enhance the utility of CE in the biochemical analysis of cells.

The introduction of peptides into living cells for the purpose of manipulating cellular biochemistry has become widespread throughout biology. However, little is known about the behavior of these short sequences of amino acids within cells, particularly those used as substrates or inhibitors for kinases and other enzymes. We utilized a quantitative, single-cell assay to demonstrate that an 11-amino acid peptide was efficiently phosphorylated by intracellular protein kinase B (PKB) in fibrosarcoma cell line HT1080 and in NIH-3T3 cells. The phosphorylated peptide was also readily dephosphorylated by intracellular phosphatases. Assays of the peptide's phosphorylation in single, living cells measured the balance of the activities of PKB and phosphatases in that cell. At a peptide concentration below the K(M) of PKB and the phosphatases, the ratio of phosphorylated to nonphosphorylated peptide at the steady state was independent of the peptide concentration. A single-cell assay utilizing this peptide revealed the existence of two subpopulations of cells whose unique activities had hitherto been obscured by population averaging. Additional studies of cells stimulated by PDGF demonstrated that a quantitative analysis of PKB activation in response to a physiological stimulus was possible. These studies demonstrated that short peptides can remain specific within the complex intracellular milieu and function as sensitive reporters of the activation state of native kinases within live cells.

The conjugation of peptides derived from the HIV TAT protein to membrane-impermeant molecules has gained wide acceptance as a means for intracellular delivery. Numerous studies have addressed the mechanism of uptake and kinetics of TAT translocation, but the cytosolic concentrations and bioavailability of the transported cargo have not been well-characterized. The current paper utilizes a microanalytical assay to perform quantitative single-cell measurements of the concentration and accessibility of peptide-based substrates for protein kinase B (PKB) and Ca(2+)/calmodulin-activated kinase II. The substrate peptide and TAT were conjugated through a releasable linker, either a disulfide or photolabile bond. Free substrate peptide concentrations of approximately 10(-20)-10(-18) moles were attainable in a cell when substrates were delivered utilizing these conjugates. The substrate peptides delivered as a disulfide conjugate were often present in the cytosol as several oxidized forms. Brief exposure of cells loaded with the photolabile conjugates to UVA light released free substrate peptide into the cytosol. Substrate peptide delivered by either conjugate was accessible to cytosolic kinase as demonstrated by the efficient phosphorylation of the peptide when the appropriate kinase was active. After incubation of the conjugated substrate with cells, free, kinase-accessible substrate was detectable in less than 30 min. Release of the majority of loaded substrate peptide from sequestered organelles occurred within 1 h. The utility of the photocleavable conjugates was demonstrated by measuring the activation of PKB in 3T3 cells after addition of varying concentrations of platelet-derived growth factor.

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