Structural and cytochemical changes that precede, escort and succeed the irreverssible permeabilization of the inner mitochondrial membrane in apoptotic HeLa, COS-7 and PC-12 cells. a microscopic and cytometric study

• Grant number: 13/22816-2
• Support Opportunities: Regular Research Grants
• Start date: February 01, 2014
• End date: September 30, 2016
• Field of knowledge: Biological Sciences - Morphology - Cytology and Cell Biology
• Principal Investigator: Antonio Sesso
• Grantee: Antonio Sesso
• Host Institution: Instituto de Medicina Tropical de São Paulo (IMT). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated researchers: Noemi Nosomi Taniwaki ; Noemia Mie Orii ; Sylvia Mendes Carneiro

Abstract

There is no consensus on whether in apoptotic HeLa cells generated by staurosporine treatment, the release to the cytoplasm of apoptogenic proteins like cytochrome c that reside in the mitochondrial intermembrane space (MIS) precedes or not the loss of the selective permeability of the inner mitochondrial membrane (IMM). Observations done by us in 02/2013 showed that in staurosporine induced apoptotic HeLa cells mitochondria with ruptured outer membrane are found from the initial until the final apoptotic structural stages (Figs. 14-29). These results support the view that the release of cytochrome c is preceded by the collapse of the relatively impermeable IMM. We will reevaluate this issue on an ample structural and cytochemical scenario in HeLa and COS-7 cells exposed to staurosporine (0.1, 0.5 and 1.0 µM) and in PC-12 cells deprived of nutrients. The treated cells will be harvested every hour between 1 and 6 h after stimulation. Eventually, the samples will be collected every 30 minutes after stimulus until hour 6 (ADENDA p31). The morphological changes obtained in the transmission electron microscope (TEM) will be distributed in five successive apoptotic structural stages as previously defined (ea1-ea5) (ADENDA p28 and Figs. 2-13 and 14-29). Concomitant cytochemical analyses will be carried out with fluorescence microscopy and flow cytometry. The percentage of cells expressing the initial appearance of a cytochemical reaction that signals a given apoptotic event will be compared with the percentages of cells in each one of the five structural apoptotic stages in each one of the successively obtained samples. The idea is to try to recognize in which one of the 5 apoptotic stages a cytochemically characterized apoptotic event, starts and terminates. For example, in which of the structural stages a cell expresses initially active caspases-3/7, or stains with yo-pro-1 and with annexin V, or with annexin V and with propidium iodide, or only with propidium iodide?. The main corollaries from this study will be an answer to the question if the release of the cytochrome c from the mitochondria is preceded or not by the permeabilization of the inner mitochondrial membrane. This will be achieved by comparing in each structural apoptotic stage the percentages of cells having mitochondria with ruptured outer membrane and/or with crista remodeling with the percentages of cells with cytochemically evaluated release cytochrome c in the microscope and in the cytometer. The marked and peculiar modifications in the disposition of the mitochondrial cristae seen in HeLa apoptotic cells previously treated with staurosporine or teneposide was referred to as crista remodeling. These changes of the inner mitochondrial compartment have been correlated with the release of cytochrome c to the cytoplasm. Our group is among the few ones of Structural Biology on a global scale with conditions to undertake a multiparametric cytochemical study including a detailed monitoring by transmission electron microscopy, of the successive stages of the apoptotic process. This structural exam identifies unambiguously mitochondria whose inner membrane has irreversibly lost its selective permeability. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

SUN, WILLY W.; KRYSTOFIAK, EVAN S.; LEO-MACIAS, ALEJANDRA; CUI, RUNJIA; SESSO, ANTONIO; WEIGERT, ROBERTO; EBRAHIM, SEHAM; KACHAR, BECHARA. Nanoarchitecture and dynamics of the mouse enteric glycocalyx examined by freeze-etching electron tomography and intravital microscopy. COMMUNICATIONS BIOLOGY, v. 3, n. 1, JAN 7 2020. (13/22816-2)