Structure and function in native and pathological erythrocytes: a quantitative view from the nanoscale

Abstract

The red blood cells (RBCs) are among the most simple and less expensive cells to purify; for this reason and for their physiological relevance, they have been extensively studied with a variety of techniques. The picture that results is that these cells have several peculiarities including extreme mechanical performances, relatively simple architecture, biological relevance and predictable behavior that make them a perfect laboratory of testing for novel techniques, methodologies and ideas. These include the re-evaluation of old concepts, such as the relationship between structure and function (which is one of the guideline of this report) but considered at the cellular level. The studies reported on this paper, indeed, exploit the full potential of an high resolution quantitative microscopy such as the atomic force microscopy (AFM) to investigate different aspect of the erythrocytes' life, death and interaction with the environment. Indeed, the erythrocytes have a special relationship with the environment that is able to deeply influence their morphology as consequence of alteration of their biochemical or biophysical status. In this context the conditions under which the erythrocytes can be considered as biochemically programmable systems have been investigated by analyzing different environmentally induced alteration of the cell's morphology and comparing the results with naturally occurring pathological morphologies. This class of studies takes great advantage by the additional consideration of the nanomechanical properties of the cells. These latter are particularly important for the cell functionality and are shown to be of practical usefulness to discriminate and partition environmental effects charging different cellular structure (e.g. membrane or membrane-skeleton). Moreover, the development of novel morphological parameter can be important to push the level of investigation on the RBCs' status towards the molecular level. In particular, we describe the introduction and use of the plasma membrane roughness as a morphometric parameter of simple derivation from the AFM images and that results sensitive to the structural integrity of the cells' membrane-skeleton. This offer a remarkable opportunity to investigate the relationship between structure and function in normal and pathological cells by using a morphometric parameter that probes the cell surface at the nanoscale level. At last, a complex but physio-pathologically important phenomenon such as the erythrocytes aging was considered. To properly analyze the many variation that the cells experience during the whole aging path we used all the parameters that the AFM can provides: quantitative imaging, analysis of the membrane roughness and local measure of the nanomechanical properties analyzed together with biochemical parameter such as the ATP content. The picture that emerged is that the aging path is triggered by the ATP intracellular concentration that influence the membrane-skeleton structure and the support exerted on the plasma membrane. The consequences of the membrane-skeleton involvement can be monitored by AFM and showed the occurrence of peculiar morphologies and morphological defects that appear in the very place where the membrane-skeleton contact with the membrane became loose. As a whole, the collected data enable to describe the entire phenomenon as a sequence of morphological intermediates following one another along the aging path.