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Mapping the apoptotic process with super resolution microscopy in kidney cells challenged with high glucose
Author(s) -
Bernhem Kristoffer,
Zhang Liang,
Fontana Jacopo,
Nilsson Linnéa,
Scott Lena,
Brismar Hjalmar,
Aperia Anita
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.876.3
Subject(s) - mitochondrion , microbiology and biotechnology , apoptosis , cytoplasm , mitochondrial apoptosis induced channel , voltage dependent anion channel , transfection , biology , programmed cell death , staurosporine , chemistry , inner mitochondrial membrane , cell culture , biochemistry , bacterial outer membrane , signal transduction , genetics , protein kinase c , escherichia coli , gene
The role of the Bcl‐family proteins in the mitochondrial apoptotic process is well described with biochemical and molecular methods as well as with studies of isolated mitochondria and transfected cell lines. There is yet no proof of principle that this description of apoptosis conforms with the in vivo process in primary mammalian cells. Here we have used Stimulated Emission Depletion (STED) microscopy to study the apoptotic process in immune‐stained rat renal epithelia cells exposed to 20 mM glucose (HG) and to study its rescue by ouabain. To assess distance between Bcl proteins, we used the nearest‐neighbor algorithm. The anti‐apoptotic protein Bcl‐xl was predominantly expressed on mitochondria in control cells, and remained so throughout the process, although its abundance decreased. After 2h HG the apoptosis‐inducing protein BAD had translocated from the cytoplasm to the mitochondria where it clustered with Bcl‐xL. This occurred before an increase in reactive oxygen species and was dependent on activation of the PI3K –AKT pathway. According to current concepts, Bcl‐xl interacts with the apoptotic protein Bax on the mitochondria under control conditions to translocate Bax back to the cytosol. We found that Bax started to accumulate on the mitochondria after 4h HG and, surprisingly, that the interaction between Bcl‐xL and Bax became more pronounced during the course of the apoptotic process. After 6h HG Bax also interacted with the non‐specific ion transporter VDAC; an interaction described to lead to penetration of the inner mitochondrial membrane and mark the point of no return. Our group has previously identified an anti‐apoptotic signaling pathway that is triggered by ouabain binding to Na,K‐ATPase and activates PI3K and AKT. Here we show that 10 nM ouabain rescues from apoptosis by counteracting the AKT dependent inhibition of BAD activation. This study reveals new information about interaction between Bcl family of proteins during the apoptotic process and about mechanism and rescue from kidney cell death in poorly controlled diabetes.