Biomedical subjects

N L Allbritton

Publications and source records attributed to N L Allbritton.

At least 19 recordsLinked to original sources

PubMed2001

Spatial control of cellular measurements with the laser micropipet.

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.

Amino Acid Sequence↗

PubMed2001

Improved capillary electrophoresis conditions for the separation of kinase substrates by the laser micropipet system.

Phosphorylated and nonphosphorylated forms of peptide substrates for protein kinase C (PKC) and calcium-calmodulin activated kinase II (CamKII) were separated by capillary zone electrophoresis. Electrophoresis of the peptide substrates and products in biologic buffer solutions in uncoated capillaries yielded asymmetric analyte peaks with substantial peak tailing. Some of the peptides also exhibited broad peaks with unstable migration times. To improve the electrophoretic separation of the peptides, several strategies were implemented: extensive washing of the capillary with a base, adding betaine to the electrophoretic buffer, and coating the capillaries with polydimethylacrylamide (PDMA). Prolonged rinsing of the capillaries with a base substantially improved the migration time reproducibility and decreased peak tailing. Addition of betaine to the electrophoretic buffer enhanced both the migration time stability as well as the theoretical plate numbers of the peaks. Finally PDMA-coated capillaries brought about significant improvements in the resolving power of the separations. These modifications all utilized an electrophoretic buffer that was compatible with a living biologic cell. Consequently they should be adaptable for the new capillary electrophoresis-based methods to measure kinase activation in single cells.

Amino Acid Sequence↗

PubMed2001

Sperm factor induces intracellular free calcium oscillations by stimulating the phosphoinositide pathway.

Injection of a porcine cytosolic sperm factor (SF) or of a porcine testicular extract into mammalian eggs triggers oscillations of intracellular free calcium ([Ca(2+)](i)) similar to those initiated by fertilization. To elucidate whether SF activates the phosphoinositide (PI) pathway, mouse eggs or SF were incubated with U73122, an inhibitor of events leading to phospholipase C (PLC) activation and/or of PLC itself. In both cases, U73122 blocked the ability of SF to induce [Ca(2+)](i) oscillations, although it did not inhibit Ca(2+) release caused by injection of inositol 1,4,5-triphosphate (IP(3)). The inactive analogue, U73343, had no effect on SF-induced Ca(2+) responses. To determine at the single cell level whether SF triggers IP(3) production concomitantly with a [Ca(2+)](i) rise, SF was injected into Xenopus oocytes and IP(3) concentration was determined using a biological detector cell combined with capillary electrophoresis. Injection of SF induced a significant increase in [Ca(2+)](i) and IP(3) production in these oocytes. Using ammonium sulfate precipitation, chromatographic fractionation, and Western blotting, we determined whether PLCgamma1, PLCgamma2, or PLCdelta4 and/or its splice variants, which are present in sperm and testis, are responsible for the Ca(2+) activity in the extracts. Our results revealed that active fractions do not contain PLCgamma1, PLCgamma2, or PLCdelta4 and/or its splice variants, which were present in inactive fractions. We also tested whether IP(3) could be the sensitizing stimulus of the Ca(2+)-induced Ca(2+) release mechanism, which is an important feature of fertilized and SF-injected eggs. Eggs injected with adenophostin A, an IP(3) receptor agonist, showed enhanced Ca(2+) responses to CaCl(2) injections. Thus, SF, and probably sperm, induces [Ca(2+)](i) rises by persistently stimulating IP(3) production, which in turn results in long-lasting sensitization of Ca(2+)-induced Ca(2+) release. Whether SF is itself a PLC or whether it acts upstream of the egg's PLCs remains to be elucidated.

Adenosine↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed2000

Characterization of a biological detector cell for quantitation of inositol 1,4,5-trisphosphate.

Prior strategies to measure inositol 1,4,5-trisphosphate (IP(3)) in single cells either have been qualitative or have had a limited spatial resolution. Capillary electrophoresis combined with a biological detector cell has been used to quantitate IP(3) in small regions of a Xenopus oocyte. To improve the detection limits of this method, we elucidated the experimental parameters which influenced the sensitivity and reliability of the IP(3)-detector cell coupled to capillary electrophoresis. The variables which influenced the detector cell were the magnitude of the voltage drop across the detector cell, the duration of this voltage drop, the direction of fluid flow in the capillary, the concentration of free Ca(2+) around the detector cell, and the presence of protease inhibitors during permeabilization of the detector cell. For the sample volumes imposed by the capillary diameter, the detector cell acted primarily as an IP(3) mass detector rather than a concentration detector. Characterization of the experimental variables influencing the sensitivity and reliability of this detector cell has the potential to enhance other analyte measurements performed by mating capillary electrophoresis with a biological detector cell.

Animals↗

PubMed2000

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.

3T3 Cells↗

PubMed1999

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.

Amino Acid Sequence↗

PubMed1998

Laser-micropipet combination for single-cell analysis.

Due to its potential for exquisite mass detection limits and resolving power, capillary electrophoresis is used for biochemical measurements on single cells; however, accurate measurements of many physiological parameters require sampling strategies that are considerably faster than those presently available. We have developed a laser-based technique to lyse single, adherent, mammalian cells on millisecond time scales. The cellular contents are then introduced into a capillary where electrophoretic separation and detection are performed. Improved temporal resolution of biological measurements results from the extremely rapid lysis made possible by this method. Additionally, the cell is not perturbed by mechanical or electrical stresses prior to sampling. Such disturbances can alter cellular physiology, resulting in inaccurate measurements. The fast cell lysis, the absence of cellular stresses prior to lysis, and the application to adherent mammalian cells are significant refinements to CE-based measurements on single cells. With this laser-micropipet combination, it will be possible to measure the intracellular concentration of molecules that change on subsecond to second time scales, for example, substrates of many cellular enzymes.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

Metabolism of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate by the oocytes of Xenopus laevis.

The pathway and kinetics of inositol 1,4,5-trisphosphate (IP3) metabolism were measured in Xenopus laevis oocytes and cytoplasmic extracts of oocytes. Degradation of microinjected IP3 in intact oocytes was similar to that in the extracts containing comparable concentrations of IP3 ([IP3]). The rate and route of metabolism of IP3 depended on the [IP3] and the intracellular free Ca2+ concentration ([Ca2+]). At low [IP3] (100 nM) and high [Ca2+] (>/=1 microM), IP3 was metabolized predominantly by inositol 1,4, 5-trisphosphate 3-kinase (3-kinase) with a half-life of 60 s. As the [IP3] was increased, inositol polyphosphate 5-phosphatase (5-phosphatase) degraded progressively more IP3. At a [IP3] of 8 microM or greater, the dephosphorylation of IP3 was the dominant mode of IP3 removal irrespective of the [Ca2+]. At low [IP3] and low [Ca2+] (both </=400 nM), the activities of the 5-phosphatase and 3-kinase were comparable. The calculated range of action of IP3 in the oocyte was approximately 300 micron suggesting that IP3 acts as a global messenger in oocytes. In contrast to IP3, inositol 1,3,4, 5-tetrakisphosphate (IP4) was metabolized very slowly. The half-life of IP4 (100 nM) was 30 min and independent of the [Ca2+]. IP4 may act to sustain Ca2+ signals initiated by IP3. The half-life of both IP3 and IP4 in Xenopus oocytes was an order of magnitude or greater than that in small mammalian cells.

Animals↗

PubMed1997

Localized sampling of cytoplasm from Xenopus oocytes for capillary electrophoresis.

Continued progress in cellular physiology requires new measurement strategies which can be applied to solitary cells. Since many cellular signaling pathways act on time scales of a few seconds, there is a critical need for single-cell techniques with subsecond time resolution. Capillary electrophoresis shows great promise as a tool for the analysis of individual cells. In the present work, we describe a technique to load a capillary with picoliter to nanoliter volumes of cytoplasm and initiate electrophoresis in less than 500 ms. When cytoplasm was sampled from a Xenopus laevis oocyte previously loaded with fluorescein, calcium green, or a mixture of the two fluorophores, their fluorescent peaks were readily identifiable on the electropherogram. Since the volume of cytoplasm (< or = 30 nL) loaded into the capillary was much smaller than the 1 microL oocyte volume, spatially localized biochemical measurements were also possible. To demonstrate the utility of this new technique, the activity of the enzyme beta-galactosidase was measured in small regions of the Xenopus oocyte. Subcellular, subsecond sampling of oocyte cytoplasm will enable biochemical measurements with the resolution required to understand many cellular signal transduction pathways.

Animals↗

PubMed1996

Cell-to-cell scanning in capillary electrophoresis.

A widespread limitation in using cell-based biosensors for repetitive chemical analysis is loss of agonist-induced response caused by receptor desensitization. We overcome this problem by scanning an array of immobilized cells underneath a capillary electrophoresis column outlet. In this way, electrophoretically fractionated components that exit the separation capillary are always directed onto cells previously unexposed to receptor agonists. To demonstrate this concept of response recovery using a scanning format, we have chosen the bradykinin B2 receptor system in the NG108-15 cell line, which is known to undergo desensitization. Whereas four subsequent injections of 250 microM bradykinin separated by 120 s are found to reduce the NG108-15 cell response markedly, scanning to new cells can fully restore the response during the separation. Furthermore, by pretesting individual NG108-15 cells for an agonist response and then later scanning back to the same cell, we achieved a 100% success rate in detecting bradykinin in subsequent electrophoretic separations.

Animals↗

PubMed1996

Evidence for a conformational change in a class II major histocompatibility complex molecule occurring in the same pH range where antigen binding is enhanced.

Many class II histocompatibility complex molecules bind antigenic peptides optimally at low pH, consistent with their exposure to antigen in acidic endosomal compartments. While it has been suggested that a partially unfolded state serves as an intermediate involved in peptide binding, very little evidence for such a state has been obtained. In this report, we show that the murine class II molecule IE becomes increasingly less stable to sodium dodecyl sulfate-induced dissociation since the pH is decreased in the same range that enhances antigenic peptide binding. Furthermore, at mildly acidic pH levels, IEk binds the fluorescent dye 1-anilino-naphthalene-8-sulfonic acid (ANS), a probe for exposed nonpolar sites in proteins, suggesting that protonation produces a molten globule-like state. The association of IEk with a single high-affinity peptide had only a small effect in these two assays, indicating that the changes that occur are distal to the peptide-binding groove. Circular dichroism analysis shows that a pH shift from neutral to mildly acidic pH causes subtle changes in the environment of aromatic residues but does not grossly disrupt the secondary structure of IEk. We propose a model in which perturbations in interdomain contacts outside the peptide-binding domain of IEk occur at acidic pH, producing a partially unfolded state that facilitates optimal antigen binding.

Animals↗

PubMed1995

Single cells as biosensors for chemical separations.

A biosensor system based on the response of living cells was demonstrated that can detect specific components of a complex mixture fractionated by a microcolumn separation technique. This system uses ligand-receptor binding and signal-transduction pathways to biochemically amplify the presence of an analyte after electrophoretic separation. The transduced signal was measured by means of two approaches: (i) fluorescence determination of intracellular calcium concentrations in one or more rat PC-12 cells and (ii) measurement of transmembrane current in a Xenopus laevis oocyte microinjected with messenger RNA that encodes a specific receptor. This analysis system has the potential to identify biologically active ligands present in a complex mixture with exceptional sensitivity and selectivity.

Acetylcholine↗

PubMed1995

Regulation of nuclear calcium concentration.

Transient increases in nuclear calcium concentration have been shown to activate gene expression and other nuclear processes. It has been suggested that nuclear calcium signals are controlled by a mechanism that is independent of calcium signalling in the cytosol. This would be possible if calcium diffusion is slow and a separate calcium release mechanism is localized to the nuclear region. Alternatively, the nuclear envelope could act as a diffusion barrier for calcium ions released either inside or outside the nucleus. It has also been proposed that inositol 1,4,5-trisphosphate (InsP3) can be generated inside the nucleus and that there are calcium release channels in the inner membrane of the nuclear envelope. Most of the experimental evidence supporting these hypotheses is based on the calibration of nuclear and cytosolic calcium concentrations. However, recent studies suggest that the local calibration of calcium indicators may not be accurate. We propose that nuclear calcium signals can be investigated by a different approach that does not rely on accurate calibration of indicators. We have developed calcium indicators that minimize facilitated calcium diffusion and are localized to either the nucleus or the cytosol. Using the diffusion coefficient of calcium ions, and measuring the delay between cytosolic and nuclear calcium increases, we show that the nuclear envelope is not a substantial barrier for calcium ions in PC12 (phaeochromocytoma) cells. This suggests that nuclear and cytosolic calcium signals equilibrate rapidly in these cells.

Animals↗

PubMed1995

Localized calcium signals in early zebrafish development.

Activation of the phosphoinositide (PI) pathway has been shown to be involved in the compaction of blastomeres in mouse embryos and in embryonic axis formation in Xenopus and in zebrafish embryos. Here we investigate Ca2+ signals in individual blastomeres of zebrafish embryos with the goal to better understand the role of PI and Ca2+ signaling for early vertebrate embryogenesis. Initial studies showed that the inositol 1,4,5-trisphosphate (IP3) concentration increases after the 32-cell stage of development, suggesting that IP3-mediated Ca2+ signals may be present during the blastula stage. Ca2+ signals were measured by identifying individual cells using confocal imaging of a nuclear localized Ca2+ indicator. Using this in situ indicator, changes in Ca2+ concentration were measured over several hours in each cell of a series of sections through the developing embryo. Transient increases in Ca2+ concentration that lasted 20-50 sec (Ca2+ spikes) were first triggered during the 32- to 128-cell stage in cells of the outer embryonic cell layer. These cells develop epithelial characteristics and specialize into the enveloping layer (EVL). No Ca2+ activity was observed during the earlier cleavage cycles or in deep blastomeres. Ca2+ spikes remained restricted to the EVL until the end of the blastula stage. Ca2+ spikes in neighboring EVL cells often occurred in the same short time interval, indicating that small groups of EVL cells can synchronize their activity. When averaged over several cell cycles, Ca2+ activity showed an even distribution in the EVL and did not indicate future polarities.

Animals↗

PubMed1994

Source of nuclear calcium signals.

Transient increases of Ca2+ concentration in the nucleus regulate gene expression and other nuclear processes. We investigated whether nuclear Ca2+ signals could be regulated independently of the cytoplasm or were controlled by cytoplasmic Ca2+ signals. A fluorescent Ca2+ indicator that is targeted to the nucleus was synthesized by coupling a nuclear localization peptide to Calcium Green dextran, a 70-kDa Ca2+ indicator. Stimulation of rat basophilic leukemia cells by antigen or by photolytic uncaging of inositol 1,4,5-trisphosphate induced transient increases in nuclear and cytosolic Ca2+ concentrations. Elevations in the nuclear Ca2+ concentration followed those in the nearby perinuclear cytosol within 200 ms. Heparin-dextran, an inhibitor of the inositol 1,4,5-trisphosphate receptor that is excluded from the nucleus, was synthesized to specifically block the release of Ca2+ from cytosolic stores. Addition of this inhibitor suppressed Ca2+ transients in the nucleus and the cytosol. We conclude that the Ca2+ level in the nucleus is not independently controlled. Rather, nuclear Ca2+ increases follow cytosolic Ca2+ increases with a short delay most likely due to Ca2+ diffusion from the cytosol through the nuclear pores.

Amino Acid Sequence↗

PubMed1993

pH affects both the mechanism and the specificity of peptide binding to a class II major histocompatibility complex molecule.

We have compared the contribution of electrostatic forces in the binding of antigenic peptides to the class II MHC molecule, IEk, at weakly acidic (pH 5.4) and neutral (pH 7.5) pH values. The binding of specific moth cytochrome c (MCC) and hemoglobin (Hb) peptides to IEk is very sensitive to ionic strength at pH 7.5 but not at pH 5.4, indicating that the mechanism of peptide binding is pH-dependent. Substitution of the C-terminal Lys in MCC for an Ala residue selectively destroyed peptide binding at neutral pH and increased the dissociation rate at least 30-fold, implicating this residue in the pH-dependent electrostatic interaction. The presence of a C-terminal Lys in many of the peptides that are restricted to IEk suggests that this electrostatic interaction is widely used to bind peptides to this MHC molecule. We also probed the electrostatic environment of the peptide binding groove adjacent to the N-terminus of the bound peptide by rapid-diffusion fluorescence energy transfer using a terbium-labeled MCC peptide. In this region of the peptide binding groove, more negative charge is present at pH 7.5 than at pH 5.4. These findings indicate the importance of MHC carboxylates to the mechanism and specificity of peptide binding. The biological importance of having two distinct mechanisms of peptide binding at different pH may be that it acts to broaden the spectrum of antigenic peptides that can be presented to T-cells.

Amino Acid Sequence↗