Protocol for evaluating mitochondrial morphology changes in response to CCCP-induced stress through open-source image processing software.

Mitochondrial morphology is an indicator of cellular health and function; however, its quantification and categorization into different subclasses is a complicated process. Here, we present a protocol for mitochondrial morphology quantification in the presence and absence of carbonyl cyanide m-chlorophenyl hydrazone stress. We describe steps for the preparation of cells for immunofluorescence microscopy, staining, and morphology quantification. The quantification protocol generates an aspect ratio that helps to categorize mitochondria into two clear subclasses. For complete details on the use and execution of this protocol, please refer to Nag et al.1.

PGAM5 is an MFN2 phosphatase that plays an essential role in the regulation of mitochondrial dynamics.

Mitochondrial morphology is regulated by the post-translational modifications of the dynamin family GTPase proteins including mitofusin 1 (MFN1), MFN2, and dynamin-related protein 1 (DRP1). Mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5) is emerging as a regulator of these post-translational modifications; however, its precise role in the regulation of mitochondrial morphology is unknown. We show that PGAM5 interacts with MFN2 and DRP1 in a stress-sensitive manner. PGAM5 regulates MFN2 phosphorylation and consequently protects it from ubiquitination and degradation. Further, phosphorylation and dephosphorylation modification of MFN2 regulates its fusion ability. Phosphorylation enhances fission and degradation, whereas dephosphorylation enhances fusion. PGAM5 dephosphorylates MFN2 to promote mitochondrial network formation. Further, using a Drosophila genetic model, we demonstrate that the MFN2 homolog Marf and dPGAM5 are in the same biological pathway. Our results identify MFN2 dephosphorylation as a regulator of mitochondrial fusion and PGAM5 as an MFN2 phosphatase.

A simple, inexpensive and multi-scale 3-D fluorescent test sample for optical sectioning microscopies.

Fluorescence standards allow for quality control and for the comparison of data sets across instruments and laboratories in applications of quantitative fluorescence. For example, users of microscopy core facilities can expect a homogenous and time-invariant illumination and an uniform detection sensitivity, which are prerequisites for imaging analysis, tracking or fluorimetric pH or Ca2+ -concentration measurements. Similarly, confirming the three-dimensional (3-D) resolution of optical sectioning microscopes calls for a regular calibration with a standardized point source. The test samples required for such measurements are typically different ones, they are often expensive and they depend much on the very microscope technique used. Similarly, the ever-increasing choice among microscope techniques and geometries increases the demand for comparison across instruments. Here, we advocate and demonstrate the multiple uses of a surprisingly versatile and simple 3-D test sample that can complement existing and much more expensive calibration samples: commercial tissue paper labeled with a fluorescent highlighter pen. We provide relevant sample characteristics and show examples ranging from the sub-µm to cm scale, acquired on epifluorescence, confocal, image scanning, two-photon (2P) and light-sheet microscopes.

Use of gene knockout to examine serotonergic control of ion uptake in zebrafish reveals the importance of controlling for genetic background: A cautionary tale.

Freshwater (FW) fishes inhabit dilute environments and must actively absorb ions in order to counteract diffusive salt loss. Neuroendocrine control of ion uptake in FW fishes is an important feature of ion homeostasis and several important neuroendocrine factors have been identified. The role of serotonin (5-HT), however, has received less attention despite several studies pointing to a role for 5-HT in the control of ion balance. Here, we used a gene knockout approach to elucidate the role of 5-HT in regulating Na+ and Ca2+ uptake rates in larval zebrafish. Tryptophan hydroxylase (TPH) is the rate-limiting step in 5-HT synthesis and we therefore hypothesized that ion uptake rates would be altered in zebrafish larvae carrying knockout mutations in tph genes. We first examined the effect of tph1b knockout (KO) and found that tph1bKO larvae, obtained from Harvard University, had reduced rates of Na+ and Ca2+ uptake compared to wild-type (WT) larvae from our institution (uOttawa WT), lending support to our hypothesis. However, further experiments controlling for differences in genetic background demonstrated that WT larvae from Harvard University (Harvard WT) had lower ion uptake rates than those of uOttawa WT, and that ion uptake rate between Harvard WT and tph1bKO larvae were not significantly different. Therefore, our initial observation that tph1bKO larvae (Harvard source) had reduced ion uptake rates relative to uOttawa WT was a function of genetic background and not of knockout itself. These data provide a cautionary tale of the importance of controlling for genetic background in gene knockout experiments.