Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

University of Cincinnati College of Medicine

The aggregation of beta-lactoglobulin (BLG) at ambient temperature was studied using turbidimetry and dynamic light scattering in the range 3.8<pH<5.2 in 0.0045 M NaCl, and in the ionic strength range 0.0045-0.5 M at fixed pH=5.0. The initial rate of aggregation, taken as the initial slope of turbidity vs time, (dtau/dt)0, indicated maximum aggregation near pH 4.6 (below the isoelectric point of 5.2), but the dependence of the initial rate of aggregation on pH was highly asymmetric. At pH 5.0, (dtau/dt)0 strongly increased with a decrease in ionic strength I from 0.1 to 0.0045 M and was found to be nearly linear with 1/I. DLS measurements revealed an increase in particle size with time, with the appearance of bimodal distributions in which the fast and slow modes corresponded, respectively, to a BLG dimer and to larger aggregates in the 100-800 nm range. At conditions of slower aggregation, DLS revealed the consumption of dimers to form higher order aggregates with no intermediate species. Computer modeling (Delphi) was used to visualize the electrostatic potential around the dimer to elucidate the pH and ionic strength dependence of the initial aggregation rates. The aggregation process appears to comprise an initial fast consumption of the dimer, whose dependence on pH and I arises from the interaction of the positive and negative domains of interacting dimers, followed by the slow formation of much larger aggregates with relatively little sensitivity to pH and I. The open-ended nature of BLG aggregation is thought to arise from the asymmetry of the dimer charge distribution.

Electrostatically driven protein aggregation: beta-lactoglobulin at low ionic strength.

The aggregation of insulin near its isoelectric point and at low ionic strength was suppressed in the presence of heparin. To understand this effect, we used turbidimetry and stopped-flow to study the pH- and ionic strength ( I)-dependence of the aggregation of heparin-free insulin. The results supported the role of interprotein electrostatic interactions, contrary to the commonly held view that such forces are minimized at pH = pI. Electrostatic modeling of insulin (DelPhi) revealed that attractive interactions arise from the marked charge anisotropy of insulin near pI. We show how screening of the interprotein attractions by added salt lead to maximum aggregation near I = 0.01 M, corresponding to a Debye length nearly equal to the diameter of the insulin dimer, consistent with a dipole-like protein charge distribution. This analysis is also consistent with suppression of aggregation by heparin, a strong polyanion that by binding to the positive domain of one protein, inhibits its interaction with the negative domain of another.

Suppression of insulin aggregation by heparin.

Cell membranes are continually undergoing a wide range of shape transformations. Here, we demonstrate the formation of several structures in supported bilayers, including tubules, caps, and giant multivesicular structures. The key elements required for these transformations are osmotic pressure imbalances, insertion of lipids with positive curvature, and lipids whose curvature is dependent on the screening environment. With these elements, a wide variety of transformations can be achieved in the absence of protein.

Formation of complex three-dimensional structures in supported lipid bilayers.

The cytoplasmic domain of band 3 serves as a center of erythrocyte membrane organization and constitutes the major substrate of erythrocyte tyrosine kinases. Tyrosine phosphorylation of band 3 is induced by several physiologic stimuli, including malaria parasite invasion, cell shrinkage, normal cell aging, and oxidant stress (thalassemias, sickle cell disease, glucose-6-phosphate dehydrogenase deficiency, etc). In an effort to characterize the biologic sequelae of band 3 tyrosine phosphorylation, we looked for changes in the polypeptide's function that accompany its phosphorylation. We report that tyrosine phosphorylation promotes dissociation of band 3 from the spectrin-actin skeleton as evidenced by: (1) a decrease in ankyrin affinity in direct binding studies, (2) an increase in detergent extractability of band 3 from ghosts, (3) a rise in band 3 cross-linkability by bis-sulfosuccinimidyl-suberate, (4) significant changes in erythrocyte morphology, and (5) elevation of the rate of band 3 diffusion in intact cells. Because release of band 3 from its ankyrin and adducin linkages to the cytoskeleton can facilitate changes in multiple membrane properties, tyrosine phosphorylation of band 3 is argued to enable adaptive changes in erythrocyte biology that permit the cell to respond to the above stresses.

Regulation of membrane-cytoskeletal interactions by tyrosine phosphorylation of erythrocyte band 3.

The network of erythrocyte cytoskeletal proteins significantly influences erythrocyte physical and biological properties. Here we show that the kinetics of erythrocyte lysis during exposure to an electric field is sensitively correlated with defects in the cytoskeletal network. Histograms compiled from single-cell electrical lysis data show characteristics of erythrocyte populations that are deficient in a specific cytoskeletal protein, revealing the presence of cell subpopulations.

Single-cell electrical lysis of erythrocytes detects deficiencies in the cytoskeletal protein network.

Current models of the erythrocyte membrane depict three populations of band 3: (i) a population tethered to spectrin via ankyrin, (ii) a fraction attached to the spectrin-actin junctional complex via adducin, and (iii) a freely diffusing population. Because many studies of band 3 diffusion also distinguish three populations of the polypeptide, it has been speculated that the three populations envisioned in membrane models correspond to the three fractions observed in diffusion analyses. To test this hypothesis, we characterized band 3 diffusion by single-particle tracking in wild-type and ankyrin- and adducin-deficient erythrocytes. We report that ∼40% of total band 3 in wild-type murine erythrocytes is attached to ankyrin, whereas ∼33% is immobilized by adducin, and ∼27% is not attached to any cytoskeletal anchor. More detailed analyses reveal that mobilities of individual ankyrin- and adducin-tethered band 3 molecules are heterogeneous, varying by nearly 2 orders of magnitude and that there is considerable overlap in diffusion coefficients for adducin and ankyrin-tethered populations. Taken together, the data suggest that although the ankyrin- and adducin-immobilized band 3 can be monitored separately, significant heterogeneity still exists within each population, suggesting that structural and compositional properties likely vary considerably within each band 3 complex.

Analysis of the mobilities of band 3 populations associated with ankyrin protein and junctional complexes in intact murine erythrocytes.

Aurora A is overexpressed in majority of breast carcinomas. With the exception of BRCA1 and PHLDA1, no oncogenic Aurora A substrates are known in breast cancer. In this study, a chemical genetic approach was used to identify malignant targets of Aurora A, which revealed LIMK2 as a novel Aurora A substrate. Aurora A regulates LIMK2 kinase activity, subcellular localization and protein levels by direct phosphorylation at S283, T494 and T505. In response, LIMK2 also positively regulates the level of Aurora A, thereby engaging in a positive-feedback loop, promoting Aurora-A-mediated oncogenic pathways. Most importantly, LIMK2 ablation fully abrogates Aurora-A-mediated tumorigenesis in nude mice, suggesting that LIMK2 is a key oncogenic effector of Aurora A. Furthermore, LIMK2 ablation acts synergistically with inhibition of Aurora A in promoting cell death. Finally, Aurora-A-mediated upregulation of LIMK2 appears to be a common mechanism in many cancers. LIMK2 inhibition or ablation is therefore an alternative approach for modulating Aurora A deregulation in cancer.

Katie Giger

Cancer and Blood Diseases Institute,
Cincinnati Children's Hospital Medical Center,
Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center

University of Cincinnati College of Medicine

Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Katie Giger

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University of Cincinnati College of Medicine

Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Cancer and Blood Diseases Institute,
Cincinnati Children's Hospital Medical Center,
Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center