Biology subjects

Devin, A.

Publications and source records attributed to Devin, A..

6 recordsLinked to original sources

bioRxiv2026

From background to foreground: secondary antibodies coupled to lipophilic ATTO dyes enable high-density membrane labeling in super-resolution and expansion microscopy

Classical immunolabeling approaches can achieve homogeneous and continuous labeling of cellular membranes and organelles at wide-field and confocal resolution. In super-resolution and expansion microscopy, however, the lack of high-density labels hampers the localization of membrane proteins and protein complexes within their membrane context. Here we show that secondary antibodies coupled to the lipophilic dyes ATTO 647N or ATTO 550 brightly label the nuclear envelope, mitochondria, and endoplasmic reticulum of fixed, permeabilized cells, and that graded labelling intensities allow selective visualization of organelles and precise segmentation of mitochondria. Using state-of-the-art super-resolution and expansion microscopy, we achieve high-density labelling of nuclear and mitochondrial membranes, with targeting and density comparable to existing membrane-labelling approaches and a signal that can be further amplified with additional secondary antibodies. Finally, we show that these dye-conjugated IgG allow to resolve mitochondria-ER contacts and mitochondrial ultrastructure as well as precise visualization of the nuclear envelope and its invaginations. This study demonstrates that secondary antibodies conjugated to lipophilic fluorophores represent stable, convenient and affordable tools for organelle visualization in conventional microscopy and for high-density labeling of membranes in super-resolution and expansion microscopy.

cell biology↗

bioRxiv2024

Dissecting the nutritional regulations of a whole amino acid transporter family from a complex genome species: A holistic approach turning weaknesses into strengths

Amino acid transporters (AATs) are described as pivotal in maintaining circulating and cellular concentrations of AA via regulation of their expression in response to the cellular environment. Rainbow trout (RT), a complex genome species, is poorly described for AATs roles in controlling its predominant AA-based metabolism, despite representing a major challenge in the aquaculture nutrition field. Therefore, we identified the whole repertoire of AAT found in RT genome (>200), its expression in tissues and its nutritional regulations in vitro. Results garnered revealed the existence of different clusters of AATs, notably due to promoters bearing ATF4-related AA response elements. Moreover, the modeling of each AAT-specific cluster activities disclosed mTOR-related signaling functions of Ile and Phe, yet unknown in RT. Thus, this novel approach herein described should help to better grasp AA homeostasis in most organisms and topics such as fish nutrition and evolution.

cell biology↗

bioRxiv2024

The widely used cymoxanil fungicide impairs respiration in Saccharomyces cerevisiae via cytochrome c oxidase inhibition

Cymoxanil (CYM) is a synthetic acetamide fungicide that has been widely used to combat downy mildew diseases in grapevine cultures and late blight diseases in tomato and potato caused by the oomycetes Plasmopara viticola and Phytophthora infestans, respectively. Despite its extensive application, the biochemical mode of action of CYM remains elusive. Previous reports indicate that CYM affects growth, DNA and RNA synthesis in Phytophthora and inhibits cell growth, biomass production and respiration rate in the well-characterized fungal model Saccharomyces cerevisiae. We therefore used this model to further characterize the effect of CYM on mitochondria. We found that CYM inhibits oxygen consumption in whole cells after 3 h of exposure, which persists over time. Using isolated mitochondria, we demonstrated that CYM specifically inhibits cytochrome c oxidase (CcO) activity during oxidative phosphorylation. Based on molecular docking algorithms, we propose that CYM acts by blocking the interaction of cytochrome c (cyt c) with CcO, hampering electron transfer and inhibiting CcO catalytic activity. Although other targets cannot be excluded, our data offer valuable insights into the mode of action of CYM that can be instrumental to drive informed management of the use of this fungicide.

biochemistry↗

bioRxiv2021

A model of mitochondrial ATP synthase deficiencies. The role of mitochondrial carriers.

The m.8993T>G mutation of the mitochondrial MT-ATP6 gene is associated with NARP syndrome (Neuropathy, Ataxia and Retinitis Pigmentosa). The equivalent point mutation introduced in yeast Saccharomyces cerevisiae mitochondrial DNA considerably reduced the activity of ATP synthase and of cytochrome-c-oxidase preventing yeast growth on oxidative substrates. The over-expression of the mitochondrial oxodicarboxylate carrier (Odc1p) is able to rescue the growth on oxidative substrate by increasing the substrate-level phosphorylation of ADP coupled to conversion of -ketoglutarate (AKG) into succinate with an increase in Complex IV activity. Previous studies showed that equivalent point mutations in ATP synthase behave similarly and can be rescued by Odc1p overexpression and/or uncoupler of OXPHOS from ATP synthesis. In order to better understand the mechanism of ATP synthase mutation bypass, we developed a core model of mitochondrial metabolism based on AKG as respiratory substrate. We describe the different possible metabolite output and the ATP/O ratio values as a function of ATP synthase inhibition.

systems biology↗

bioRxiv2020

TMEM70 promotes ATP synthase assembly within cristae via transient interactions with subunit c

Mitochondrial ATP-synthesis is catalyzed by a F1Fo-ATP synthase, an enzyme of dual genetic origin enriched at the edge of cristae where it plays a key role in their structure/stability. The enzymes biogenesis remains poorly understood, both from a mechanistic and a compartmentalization point of view. The present study provides novel molecular insights into this process through investigations on a human protein called TMEM70 with an unclear role in the assembly of ATP synthase. A recent study has revealed the existence of physical interactions between TMEM70 and the subunit c (Su.c), a protein present in 8 identical copies forming a transmembrane oligomeric ring (c-ring) within the ATP synthase proton translocating domain (FO). Herein we analyzed the ATP-synthase assembly in cells lacking TMEM70, mitochondrial DNA or F1 subunits and observe a reciprocal dependence of TMEM70 and Su.c levels, regardless of the status of other ATP synthase subunits or of mitochondrial bioenergetics. Immunoprecipitation and two-dimensional blue-native/SDS-PAGE reveal that TMEM70 forms large oligomers composed of 8 TMEM70 dimers and that TMEM70 oligomers interact with Su.c not yet incorporated into ATP synthase complexes. Moreover, discrete TMEM70-Su.c complexes with increasing Su.c contents can be detected, suggesting a role for TMEM70 oligomers in the gradual assembly of the c-ring. Furthermore, we demonstrate using expansion super-resolution microscopy the specific localization of TMEM70 at the inner cristae membrane, distinct from the MICOS component MIC60. Taken together, our results show that TMEM70 oligomers provide a scaffold for c-ring assembly and that mammalian ATP synthase is assembled within inner cristae membranes.

cell biology↗

bioRxiv2020

Ascorbate maintains a low plasma low oxygen level

In human blood, oxygen is mainly transported by red blood cells. Accordingly, the oxygen level in plasma is expected to be limited, although it has not been quantified yet. Here, by developing dedicated methods and tools, we determined that human plasma pO2 = 8.4 mmHg (1.2% O2). Oxygen solubility in plasma was believed to be similar to water. Here we reveal that plasma has an additional ascorbate-dependent oxygen-reduction activity. Plasma oxygenation oxidizes ascorbate (49.5 M in fresh plasma vs <2 M in oxidized plasma) and abolishes this capacity, which is restored by ascorbate supplementation. We confirmed these results in vivo, showing that the plasma pO2 is significantly higher in ascorbate-deficient guinea pigs (plasma ascorbate < 2M), compared to control (plasma ascorbate > 15M). Plasma low oxygen level preserves the integrity of oxidation-sensitive components such as ubiquinol. Circulating leucocytes are well adapted to these conditions, since the abundance of their mitochondrial network is limited. These results shed a new light on the importance of oxygen exposure on leucocyte biological study, in regards with the reducing conditions they encounter in vivo; but also on the manipulation of blood products to improve their integrity and potentially improve transfusions efficacy. Key points

immunology↗