immunostaining-based-detection-dynamic-alterations-red-blood-cell

Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins

Antibody labeling of red blood cell (RBC) proteins is a commonly used, semi-quantitative method to detect changes in overall protein content or acute ...

Griffith University Gold Coast

The classic view of the red blood cell (RBC) presents a biologically inert cell that upon maturation has limited capacity to alter its physical properties. This view developed largely because of the absence of translational machinery and inability to synthesize or repair proteins in circulating RBC. Recent developments have challenged this perspective, in light of observations supporting the importance of posttranslational modifications and greater understanding of ion movement in these cells, that each regulate a myriad of cellular properties. There is thus now sufficient evidence to induce a step change in understanding of RBC: rather than passively responding to the surrounding environment, these cells have the capacity to actively regulate their physical properties and thus alter flow behavior of blood. Specific evidence supports that the physical and rheological properties of RBC are subject to active modulation, primarily by the second-messenger molecules nitric oxide (NO) and calcium-ions (Ca). Furthermore, an isoform of nitric oxide synthase is expressed in RBC (RBC-NOS), which has been recently demonstrated to have an active role in regulating the physical properties of RBC. Mechanical stimulation of the cell membrane activates RBC-NOS, leading to NO generation, which has several intracellular effects, including the -nitrosylation of integral membrane components. Intracellular concentration of Ca is increased upon mechanical stimulation via the recently identified mechanosensitive cation channel piezo1. Increased intracellular Ca modifies the physical properties of RBC by regulating cell volume and potentially altering several important intracellular proteins. A synthesis of recent advances in understanding of molecular processes within RBC thus challenges the classic view of these cells and rather indicates a highly active cell with self-regulated mechanical properties.

Active modulation of human erythrocyte mechanics.

Red blood cells (RBC) are constantly exposed to varying mechanical forces while traversing the cardiovascular system. Upon exposure to mechanical stimuli (e.g., shear stress), calcium enters the cell and prompts potassium-efflux. Efflux of potassium is accompanied by a loss of intracellular fluid; thus, the volume of RBC decreases proportionately (i.e., 'Gárdos effect'). The mechanical properties of the cell are subsequently impacted due to complex interactions between cytosolic viscosity (dependent on cell hydration), the surface-area-to-volume ratio, and other molecular processes. The dynamic effects of calcium on RBC mechanics are yet to be elucidated, although accumulating evidence suggests a vital role. The present study thus examined the effects of calcium on contemporary biomechanical properties of RBC in conjunction with high-precision geometrical analyses with exposure to shear. Mechanical stimulation of RBC was performed using a co-axial Couette shearing system to deform the cell membrane; intracellular signaling events were observed via fluorescent imaging. Calcium was introduced into RBC using ionophore A23187. Increased intracellular calcium significantly impaired RBC deformability; these impairments were mediated by a calcium-induced reduction of cell volume through the Gárdos channel. Extracellular calcium in the absence of the ionophore only had an effect under shear, not at stasis. Under low shear, the presence of extracellular calcium induced progressive lysis of a sub-population of RBC; all remaining RBC exhibited exceptional capacity to deform, implying preferential removal of potentially aged cells. Collectively, we provide evidence of the mechanism by which calcium acutely regulates RBC mechanical properties.

Lennart Kuck

Biorheology Research Laboratory,
Menzies Health Institute,
Biorheology Research Laboratory, Menzies Health Institute

Griffith University Gold Coast

Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins

Lennart Kuck

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Griffith University Gold Coast

Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins

Biorheology Research Laboratory,
Menzies Health Institute,
Biorheology Research Laboratory, Menzies Health Institute