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Bulkescher, R.

Publications and source records attributed to Bulkescher, R..

2 recordsLinked to original sources

Induction of mitochondrial heat shock proteins and mitochondrial biogenesis in endothelial cells upon acute methylglyoxal stress: Evidence for hormetic autofeedback

Increased metabolic flux produces potentially harmful side-products, such as reactive dicarbonyl and oxygen species. The reactive dicarbonly methylglyoxal (MG) can impair oxidative capacity, which is downregulated in type 2 diabetes. Heat shock proteins (HSPs) of subfamily A (Hsp70s) promote ATP-dependent processing of damaged proteins during MG exposure which also involve mitochondrial proteins. Since the protection of mitochondrial proteins could promote higher production of reactive metabolites due to increased substrate flux, tight regulation of HspA-mediated protein handling is important. We hypothesized that stress-inducible HspAs (HspA1A/HspA1B) are pivotal for maintaining mitochondrial biogenesis during acute MG-stress. To analyze the role of stress-inducible HspA1A/HspA1B for maintenance of mitochondrial homeostasis during acute MG exposure, we knocked out HSPA1A/HSPA1B in mouse endothelial cells. HSPA1A/HSPA1B KO cells showed upregulation of the mitochondrial chaperones HspA9 (mitochondrial Hsp70/mortalin) and HspD1 (Hsp60) as well as induction of mitochondrial biogenesis upon MG exposure. Increased mitochondrial biogenesis was reflected by elevated mitochondrial branching, total count and area as well as by upregulation of mitochondrial proteins and corresponding transcription factors. Our findings suggest that mitochondrial HspA9 and HspD1 promote mitochondrial biogenesis during acute MG stress, which is counterregulated by HspA1A/HspA1B to prevent mitochondrial overstimulation and to maintain balanced oxidative capacity under metabolic stress conditions. These data support an important role of HSPs in MG-induced hormesis.

cell biology↗

Automated screening by 3D light-sheet microscopy with high spatial and temporal resolution reveals mitotic phenotypes

3D cell cultures enable the in vitro study of dynamic biological processes such as the cell cycle, but their use in high-throughput screens remains impractical with conventional fluorescent microscopy. Here, we present a screening workflow for the automated evaluation of mitotic phenotypes in 3D cell cultures by light-sheet microscopy. After sample preparation by a liquid handling robot, three-dimensional cell spheroids are imaged for 24 hours in toto with a dual inverted selective plane illumination (diSPIM) microscope with a much improved signal-to-noise ratio, higher imaging speed, isotropic resolution and reduced light exposure compared to a spinning disc confocal microscope. A dedicated high-content image processing pipeline implements convolutional neural network based phenotype classification. We illustrate the potential of our approach by siRNA knock-down and epigenetic modification of 28 mitotic target genes for assessing their phenotypic role in mitosis. By rendering light-sheet microscopy operational for high-throughput screening applications, this workflow enables target gene characterization or drug candidate evaluation in tissue-like 3D cell culture models.

cell biology↗