Abstract

One of the main hallmarks of le necroptosis is the loss of normal cell morphology to a "balloon-like" morphology. The balloon-like morphology has been poorly studied to date. Also, it is unknown how spherical the cells are during the necroptotic process. At the same time, changes in the cytoskeleton or membrane proteins involved in this morphological change are unknown. Recently, we have reported that neuronal necroptosis can be induced by the oxidation of vitamin C. Using the oxidation model of vitamin C, and taking advantage of the high expression of glucose transporter GLUT1, we analyzed the balloon-like morphology during necroptosis in the cell line Neuro2a and HN33.11. With spectral confocal microscopy and 3D SIM super resolution, we generate 3D reconstructions using GLUT1 staining. At the same time, we used tubulin III as a cytoskeleton marker. The 3D reconstructions were analyzed using the Imaris tool of elliptical parameters: prolate (an elongated spheroid), oblate (a spheroid with flattened poles) and sphericity with the objective of determining that to which morphology corresponds the balloon-like during the necroptosis. On the other hand, the cellular dimensions were determined using the bounding-box tool of Imaris. Interestingly, we determined that although the cells acquire a balloon-like spherical morphology during necroptosis, the main morphological change corresponds to prolate. On the other hand, we found that in the Neuro2a cells a drastic redistribution of GLUT1 occurs towards the perinuclear zone, which is accompanied by changes in the tubulin cytoskeleton. These results propose that 3D analysis is necessary for the correct determination of morphological changes during necroptosis.