Melittin-induced alterations in morphology and deformability of human red blood cells using quantitative phase imaging techniques

Joonseok Hur,Sangyun Lee,Yongkeun Park,Hyunjoo Park,Kyoohyun Kim

doi:10.1038/s41598-017-08675-7

Abstract

Here, the actions of melittin, the active molecule of apitoxin or bee venom, were investigated on human red blood cells (RBCs) using quantitative phase imaging techniques. High-resolution real-time 3-D refractive index (RI) measurements and dynamic 2-D phase images of individual melittin-bound RBCs enabled in-depth examination of melittin-induced biophysical alterations of the cells. From the measurements, morphological, intracellular, and mechanical alterations of the RBCs were analyzed quantitatively. Furthermore, leakage of haemoglobin (Hb) inside the RBCs at high melittin concentration was also investigated.

Highlights

Melittin, the active molecule of apitoxin or bee venom, is a transmembrane protein, and it forms small pores on the cell membrane[1]
Melittin-induced morphological, intracellular, and mechanical changes in red blood cells (RBCs) were investigated with real-time measurements from 3-D RI tomograms and 2-D dynamic phase maps using common-path diffraction optical tomography (cDOT)
Geometrical parameters were extracted from the 3-D RI tomograms; cell volumes and surface areas exhibited decreases as the melittin concentration increased while the sphericities increased indicating the progression of echinocytosis and spherocytosis

Summary

Discussion and Conclusion

Melittin-induced morphological, intracellular, and mechanical changes in RBCs were investigated with real-time measurements from 3-D RI tomograms and 2-D dynamic phase maps using cDOT. There are studies which observed the morphological change in RBCs in hypertonic conditions[41, 51] It could be a sign of the osmotic effect that cell volume decreased and Hb concentration increased while dry mass was maintained as melittin of increasing concentrations was introduced (Figs 2 and 3). Considering the pore-forming effect of melittin[1] and the exponential decay of dry mass as well as Hb concentration, the leakage of Hb should be outward diffusion of intracellular Hb through melittin-induced pores in the membrane because the diffusion current is proportional to the concentration contrast between the cells and medium. Where jd is the current density of diffusion; D is the diffusion coefficient; Δc is the Hb mass concentration contrast between the cytoplasm and medium, and δ is the effective thickness of a diffusion layer (i.e., membrane of RBC), we could establish a rough estimation as follows: dm dt

Apore jd

Methods

Author Contributions

Findings

Additional Information