Biconcave shape of human red-blood-cell ghosts relies on density differences between the rim and dimple of the ghost's plasma membrane

Abstract

The shape of the human red blood cell is known to be a biconcave disk. It is evident from a variety of theoretical work that known physical properties of the membrane, such as its bending energy and elasticity, can explain the red-blood-cell biconcave shape as well as other shapes that red blood cells assume. But these analyses do not provide information on the underlying molecular causes. This paper describes experiments that attempt to identify some of the underlying determinates of cell shape. To this end, red-blood-cell ghosts were made by hypotonic hemolysis and then reconstituted such that they were smooth spheres in hypo-osmotic solutions and smooth biconcave discs in iso-osmotic solutions. The spherical ghosts were centrifuged onto a coated coverslip upon which they adhered. When the attached spheres were changed to biconcave discs by flushing with an iso-osmotic solution, the ghosts were observed to be mainly oriented in a flat alignment on the coverslip. This was interpreted to mean that, during centrifugation, the spherical ghosts were oriented by a dense band in its equatorial plane, parallel to the centrifugal field. This appears to be evidence that the difference in the densities between the rim and the dimple regions of red blood cells and their ghosts may be responsible for their biconcave shape.red-blood-cell ghosts | membrane/cytoskeletal complex | biconcave discs | spheres T his paper is concerned with identifying a possible determinant responsible for the biconcave shape of human red blood cells (RBCs). Although RBCs ("globules") were first discovered in the latter part of the 17th century (1), it was not until 1827 that they were definitively shown by Hodgkin and Lister (2) to be biconcave discs. Since that time much has been learned about RBC composition (3), in particular identification of its membrane/cytoskeletal (M/CS) elements and structure (4). Our particular interest focuses on whether the symmetry of the M/CS is the same or different between the cell's dimple and rim regions. If different, such an observation might provide insight into the structural basis for the cell's biconcave shape. The approach used was to centrifuge hypotonically sphered red-blood-cell ghosts onto a coverslip upon which they adhered. The question was, what was the orientation of the stuck ghosts when they were made into biconcave discs upon exposure to an isotonic solution?It is important to mention that others have used a quite different approach to understand the basis for the red blood cell's biconcave shape. These types of studies have used solutions of theoretical models that are mainly based on the red blood cell's known physical parameters, such as the membrane's bending energy and elastic properties (e.g., 5-11) in addition to lateral inhomogeneities in the M/CS (12). These analyses have remarkably generated not only the cell's biconcave shape but also many other types of shapes that the cells are known to assume. Thus, they provide insight into and understanding of the types of forces that characteri...

Evidence that asymmetry of the membrane/cytoskeletal complex in human red blood cell ghosts is responsible for their biconcave shape

“…The results, using continuous microscopic imaging, as reported in this article, provide clear and direct evidence that the transition of the same group of spherical ghosts to biconcave discs occurs by the ghosts directly shrinking/ collapsing onto the coverslip without any lateral displacement/ movement. This behavior confirms the interpretation, previously suggested (6), that the sphered ghosts during their centrifugation become oriented with a dense band around their equatorial plane parallel to the direction of the centrifugal force. This dense outer band after the ghost's shrinkage represents the rim in the resulting biconcave disk.…”

“…The main purpose of the present article is to provide results that extend those reported before (6). In the previous paper, it was interpreted that spherical human red blood cell ghosts, which were attached to a coverslip after being centrifuged in a hypotonic solution, became directly flattened to biconcave discs when they were subsequently shrunk by exposure to an isotonic solution.…”

“…The biochemical/structural basis responsible for the biconcave shape of human red blood cells has eluded resolution since its definitive shape was first discerned. In contrast, various theoretical analyses of the physical forces involved that underlie and characterize the red cell biconcave shape have been remarkably successful and extensively described, as referred to before (6). It was also suggested in the previous paper that the density [i.e., mass differences in the membrane/cytoskeletal (M/CS) complex between the dimple and the rim] was critical in providing insight into the forces/molecular scaffolding responsible for the cell's biconcave shape.…”

“…1 for an overview of the protocol with the resultant horizontal band around the equatorial plane of the sphered ghosts (modified from ref. 6). At the present time, there is no theoretical basis available to understand what force or forces act to promote this unique orientation during the ghost's centrifugation.…”

Non-uniform distribution of myosin-mediated forces governs red blood cell membrane curvature through tension modulation

“…The idea of the asymmetrical distribution of the membrane skeleton and its components in the dimple and rim areas of RBCs was initially introduced by Hoffman, although no direct evidence for this was obtained [110,111] . Recently, Svetina et al modeled RBC volume regulation according to the permeability of the Piezo1 channel.…”