Biology subjects

Plessis, A.

Publications and source records attributed to Plessis, A..

5 recordsLinked to original sources

bioRxiv2024

Direct observation of fluorescent proteins in gels: a rapid, cost-efficient and quantitative alternative to immunoblotting

The discovery of Green Fluorescent Protein (GFP) and its derivatives has revolutionized cell biology. These fluorescent proteins (FPs) have enabled the real-time observation of protein localization and dynamics within live cells. Applications of FP vary from monitoring gene/protein expression patterns, visualizing protein-protein interactions, measuring protein stability, assessing protein mobility and creating biosensors. The utility of FPs also extends to biochemical approaches through immunoblotting and proteomic analyses, aided by anti-FP antibodies and nanobodies. FPs are notoriously robust proteins with a tightly folded domain that confers a strong stability and a relative resistance to degradation and denaturation. In this study, we report that various green, orange and red FPs can be maintained in a native, fluorescent state during the entire process of protein sample extraction, incubation with sample buffer, loading and migration on SDS-PAGE with only minor adaptations of traditional protocols. This protocol results in the ability to detect and quantify in-gel fluorescence (IGF) of endogenously-expressed proteins tagged with FPs directly after migration, using standard fluorescence-imaging devices. This approach eliminates the need for antibodies and chemiluminescent reagents, as well as the time-consuming steps inherent in immunoblotting such as transfer onto a membrane and antibody incubations. Overall, IGF detection provides clearer data with less background interference, a sensitivity comparable or better to antibody-based detection, a better quantification and a broader dynamic range. After fluorescence imaging, gels can still be used for other applications such as total protein staining or immunoblotting if needed. It also expands possibilities by allowing the detection of FPs for which antibodies are not available. Our study explores the feasibility, limitations, and applications of IGF for detecting endogenously expressed proteins in cell extracts, providing insights into sample preparation, imaging conditions, and sensitivity optimizations, and potential applications such as co-immunoprecipitation experiments.

biochemistry↗

bioRxiv2024

Cardiaca adipokinetic hormone and hedgehog signaling combine to generate intracellular waves of Ca++ in starved Drosophila melanogaster fat body

The Drosophila melanogaster fat body combines the functions of the vertebrate liver and fat. It plays a central role in metabolism where it integrates information about nutritional status to regulate fat utilization. During feeding, signaling through the Insulin Receptor causes lipogenesis, while fasting leads to signaling through the cardiaca Adipokinetic Hormone Receptor (AKHR) and mobilization of lipid stores. Here we examine intracellular calcium levels in the fat body during fasting. In fasting early third instar larvae, spikes of intracellular calcium are generated in the fat body lobes on either side of the brain. These spikes propagate through a narrow connection into the main lobes of the fat body that lie along the length of the larva. The spikes of intracellular Ca++ are dependent on the corpora cardiaca AKH expressing neurons and AKHR. Unexpectedly, the spikes also require Hedgehog (Hh) signaling from the midgut enterocytes and within the fat body. When Hh signaling is blocked, the Ca++ levels in the fat body are elevated and the spiking behavior lost. Hh signaling appears to regulate fat body intracellular Ca++ using both the transcription factor Cubitus interruptus and the trimeric G protein Gi. AKH/Hh signaling in the fat body lobes on either side of the brain appears to function as a pulse generator to initiate Ca++ spikes that then propagate through the main lobes of the fat body. These studies show how signaling from the brain and the midgut and within the fat body are integrated to regulate a key intracellular second messenger.

physiology↗

bioRxiv2022

High Hedgehog signaling is transduced by a multikinase-dependent switch controling the apico-basal distribution of the GPCR Smoothened.

The oncogenic GPCR Smoothened is a key transducer of the Hedgehog morphogen, which plays essential roles in the patterning of epithelial structures. Here, we examine how Hedgehog controls Smoothened subcellular localization and activity in a polarized epithelium using the Drosophila wing imaginal disc as a model. We provide evidence that Hedgehog promotes the stabilization of Smoothened by switching its fate after endocytosis toward recycling. This effect involves the sequential and additive action of Protein Kinase A, Casein Kinase I and the Fused kinase. Moreover, in the presence of very high levels of Hedgehog, a second effect of Fused leads to the local enrichment of Smoothened in the most basal domain of the cell membrane. Together, these results link the morphogenetic effects of Hedgehog to the apico-basal distribution of Smoothened and provide a novel mechanism for regulation of a GPCR by plasma membrane subcompartimentalisation.

developmental biology↗

bioRxiv2021

A large disordered region confers a wide spanning volume to vertebrate Suppressor of Fused as shown in a trans-species solution study.

Hedgehog (Hh) pathway inhibition by the conserved protein Suppressor of Fused (SuFu) is crucial to vertebrate development. By constrast, SuFu removal has little effect in drosophila. Previous publications showed that the crystal structures of human and drosophila SuFu consist of two ordered domains that are capable of breathing motions upon ligand binding. However, the crystal structure of human SuFu does not give information about 20 N-terminal residues (IDR1) and an eighty-residue-long disordered region (IDR2) in the C-terminus, whose function is important for the pathway repression. These two IDRs are species-dependent. We studied SuFus structure in solution, both with circular dichroism and small angle X-ray scattering, comparing drosophila, zebrafish and human species, to better understand this considerable difference. Our studies show that, in spite of similar crystal structures restricted to ordered domains, drosophila and vertebrate SuFu have very different structures in solution. The IDR2 of vertebrates spans a large area, thus enabling it to reach for partners and be accessible for post-translational modifications. Furthermore, we show that the IDR2 region is highly conserved within phyla but varies in length and sequence, with insects having a shorter disordered region while that of vertebrates is broad and mobile. This major variation may explain the different phenotypes observed upon SuFu removal.

biophysics↗

bioRxiv2020

Smaug membraneless organelles regulate mitochondrial function

Smaug is a conserved translational repressor that recognizes specific RNA motifs in a large number of mRNAs, including nuclear transcripts that encode mitochondrial enzymes. Smaug orthologs have been shown to form membraneless organelles (MLOs) in several organisms and cell types. Using single-molecule FISH we show here that SDHB and UQCRC1 mRNAs associate with Smaug1 MLOs in the human cell line U2OS. Simultaneous loss of function of Smaug1 and Smaug2 affects both mitochondrial respiration and mitochondrial network morphology. Deletion of specific Smaug1 protein regions resulted in impaired MLO formation that correlates with mitochondrial defects. In addition, rotenone but not the respiratory chain uncoupling agent CCCP rapidly induces Smaug1 MLO dissolution. Finally, metformin elicits a similar effect on Smaug1 MLOs and provokes the release of bounded mRNAs. We propose that mitochondrial activity affects Smaug1 MLO dynamics, thus allowing for regulation of nuclear mRNAs that encode key mitochondrial proteins.

cell biology↗