ALBERT

Description: It has been hypothesized that some of the irreversible microrheologic abnormalities of sickle red blood cell (RBC) membranes could result from autoxidative perturbation. To model this possibility, we used micromechanical manipulation to examine the static extensional rigidity and inelastic or plastic behavior of normal RBCs exposed to phenazine methosulfate (PMS), an agent that generates superoxide from within the cell. In response to this stress, RBC membranes became stiff as evidenced by increasing extensional rigidity. At 50 mumol/L PMS they were as stiff as the membranes of most dense, dehydrated sickle RBCs; and at 25 mumols/L PMS the membranes were similar to somewhat less dense sickle RBCs. When examined for inelastic behavior, RBCs exposed to PMS even at 10 mumols/L showed hysteresis in loading and unloading phases of the curve relating aspiration length to suction pressure, and they developed membrane bumps that persisted after RBC release from the pipette. Examination of single cells in both isotonic and hypotonic buffers showed that the effect of PMS on RBC microheology is not mediated by cellular dehydration. Independent confirmation of the membrane stiffening effect of PMS was obtained by ektacytometric analysis of resealed RBC ghosts, with sickle-like increases in membrane rigidity observed between 50 and 100 mumol/L PMS. The rigidity of these ghosts was partially ameliorated by exposure to a thiol reductant. In terms of biochemical abnormalities, treated RBCs became significantly different from control RBCs at 25 mumol/L PMS, at which point they just began to enter the sickle range for amounts of membrane thiol oxidation and membrane-associated heme. The sickle average was achieved at 50 mumol/L PMS (for thiol oxidation) to 100 mumol/L PMS (for membrane heme). Thus, micromolar concentrations of PMS induce abnormalities of membrane microrheology that closely mimic those of unmanipulated sickle RBCs while reproducing similar degrees of oxidative biochemical change. We conclude that membrane protein oxidation could explain existence of an irreversible component to the abnormal rheology of the sickle membrane.

Description: Membrane skeletal and cytoskeletal remodeling occurs throughout erythroid maturation. Microtubules and microfilaments have been identified morphologically in the nucleated erythroblast but the functional capability of these cytoskeletal structures during reticulocyte maturation has not been studied. Reticulocytes are formed from orthochromatic normoblasts by the process of nuclear extrusion. Two recognizable stages of reticulocyte maturation follow. The least mature reticulocytes are motile and multilobular, while the more mature reticulocytes are cup-shaped and nonmotile. To study the respective roles of microtubules and microfilaments in nuclear extrusion and cell motility, experiments were performed with agents that perturb these structures. Following the injection into rats of colchicine, a microtubule-disrupting substance, the number of normoblasts arrested at the stage of nuclear extrusion increased linearly over four hours. Similar results were obtained when bone marrow cells were incubated in culture in the presence of colchicine. In contrast, cell motility was dramatically decreased by cytochalasin B, a microfilament-disrupting agent, but not by colchicine. These results imply that microtubules are essential for the nuclear extrusion process, while microfilaments are essential for cell motility. Simultaneous changes in membrane skeletal assembly were assessed by measuring membrane deformability and stability, two properties regulated by the skeletal proteins. In ektacytometric assays, membrane deformability and mechanical stability of immature reticulocytes were markedly decreased to approximately 10% of normal, while that of more mature reticulocytes were nearly normal. Since the skeletal protein organization regulates these membrane properties, our findings imply that substantial membrane skeletal remodeling occurs during reticulocyte maturation. Thus we have identified major remodeling of both skeletal and cytoskeletal components during reticulocyte maturation.

Description: Membrane skeletal and cytoskeletal remodeling occurs throughout erythroid maturation. Microtubules and microfilaments have been identified morphologically in the nucleated erythroblast but the functional capability of these cytoskeletal structures during reticulocyte maturation has not been studied. Reticulocytes are formed from orthochromatic normoblasts by the process of nuclear extrusion. Two recognizable stages of reticulocyte maturation follow. The least mature reticulocytes are motile and multilobular, while the more mature reticulocytes are cup-shaped and nonmotile. To study the respective roles of microtubules and microfilaments in nuclear extrusion and cell motility, experiments were performed with agents that perturb these structures. Following the injection into rats of colchicine, a microtubule-disrupting substance, the number of normoblasts arrested at the stage of nuclear extrusion increased linearly over four hours. Similar results were obtained when bone marrow cells were incubated in culture in the presence of colchicine. In contrast, cell motility was dramatically decreased by cytochalasin B, a microfilament-disrupting agent, but not by colchicine. These results imply that microtubules are essential for the nuclear extrusion process, while microfilaments are essential for cell motility. Simultaneous changes in membrane skeletal assembly were assessed by measuring membrane deformability and stability, two properties regulated by the skeletal proteins. In ektacytometric assays, membrane deformability and mechanical stability of immature reticulocytes were markedly decreased to approximately 10% of normal, while that of more mature reticulocytes were nearly normal. Since the skeletal protein organization regulates these membrane properties, our findings imply that substantial membrane skeletal remodeling occurs during reticulocyte maturation. Thus we have identified major remodeling of both skeletal and cytoskeletal components during reticulocyte maturation.

Description: It has been hypothesized that some of the irreversible microrheologic abnormalities of sickle red blood cell (RBC) membranes could result from autoxidative perturbation. To model this possibility, we used micromechanical manipulation to examine the static extensional rigidity and inelastic or plastic behavior of normal RBCs exposed to phenazine methosulfate (PMS), an agent that generates superoxide from within the cell. In response to this stress, RBC membranes became stiff as evidenced by increasing extensional rigidity. At 50 mumol/L PMS they were as stiff as the membranes of most dense, dehydrated sickle RBCs; and at 25 mumols/L PMS the membranes were similar to somewhat less dense sickle RBCs. When examined for inelastic behavior, RBCs exposed to PMS even at 10 mumols/L showed hysteresis in loading and unloading phases of the curve relating aspiration length to suction pressure, and they developed membrane bumps that persisted after RBC release from the pipette. Examination of single cells in both isotonic and hypotonic buffers showed that the effect of PMS on RBC microheology is not mediated by cellular dehydration. Independent confirmation of the membrane stiffening effect of PMS was obtained by ektacytometric analysis of resealed RBC ghosts, with sickle-like increases in membrane rigidity observed between 50 and 100 mumol/L PMS. The rigidity of these ghosts was partially ameliorated by exposure to a thiol reductant. In terms of biochemical abnormalities, treated RBCs became significantly different from control RBCs at 25 mumol/L PMS, at which point they just began to enter the sickle range for amounts of membrane thiol oxidation and membrane-associated heme. The sickle average was achieved at 50 mumol/L PMS (for thiol oxidation) to 100 mumol/L PMS (for membrane heme). Thus, micromolar concentrations of PMS induce abnormalities of membrane microrheology that closely mimic those of unmanipulated sickle RBCs while reproducing similar degrees of oxidative biochemical change. We conclude that membrane protein oxidation could explain existence of an irreversible component to the abnormal rheology of the sickle membrane.