Biology subjects

Mely, Y.

Publications and source records attributed to Mely, Y..

7 recordsLinked to original sources

bioRxiv2023

PvdL Orchestrates the Assembly of the Nonribosomal Peptide Synthetases Involved in Pyoverdine Biosynthesis in Pseudomonas aeruginosa.

The pyoverdine siderophore is produced by Pseudomonas aeruginosa to access iron. Its synthesis involves the complex co-ordination of four nonribosomal peptide synthetases (NRPSs), responsible for assembling the pyoverdine peptide backbone. The precise cellular organization of these NRPSs and their mechanisms of interaction remain unclear. Here, we used a combination of several single-molecule microscopy techniques to to elucidate the spatial arrangement of NRPSs within pyoverdine-producing cells. Our findings revealed that PvdL differs from the three other NRPS in term of localization and mobility patterns. PvdL is predominantly located at the inner membrane, while the other also explore the cytoplasmic compartment. Leveraging the power of multicolor single-molecule localization, we further reveal co-localization between PvdL and the other NRPSs, suggesting a pivotal role for PvdL in orchestrating the intricate biosynthetic pathway. Our observations strongly indicates that PvdL serves as a central orchestrator in the assembly of NRPSs involved in pyoverdine biosynthesis, assuming a critical regulatory function.

microbiology↗

bioRxiv2023

The kinesin Kif21b regulates radial migration of cortical projection neurons through a noncanonical function on actin cytoskeleton.

Completion of neuronal migration is critical for brain development. Kif21b is a plus-end directed kinesin motor protein that promotes intracellular transport and controls microtubule dynamics in neurons. Here we report a physiological function of Kif21b during radial migration of projection neurons in the mouse developing cortex. In vivo analysis in mouse and live imaging on cultured slices demonstrate that Kif21b regulates the radial glia-guided locomotion of new-born neurons independently of its motility on microtubules. Unexpectedly we show that Kif21b directly binds and regulates the actin cytoskeleton both in vitro and in vivo in migratory neurons. We establish that Kif21b-mediated regulation of actin cytoskeleton dynamics influences branching and nucleokinesis during neuronal locomotion. Altogether, our results reveal atypical roles of Kif21b on the actin cytoskeleton during migration of cortical projection neurons.

neuroscience↗

bioRxiv2022

Advancing Spectrally-Resolved Single Molecule Localization Microscopy using Deep Learning

Spectrally-Resolved Single Molecule Localization Microscopy (srSMLM) is a recent multidimensional technique enriching single molecule localization imaging by the simultaneous recording of single emitters spectra. As for SMLM, the localization precision is fundamentally limited by the number of photons collected per emitters. But srSMLM is more impacted because splitting the emission light from single emitters into a spatial and a spectral channel further reduces the number of photons available for each channel and impairs both spatial and spectral precision - or forces the sacrifice of one or the other. Here, we explored the potential of deep learning to overcome this limitation. We report srUnet - a Unet-based image processing that enhances the spectral and spatial signals and compensates for the signal loss inherent in recording the spectral component. We showed that localization and spectral precision of low-emitting species remain as good as those obtained with a high photons budget together with improving the fraction of localizations whose signal is both spatially and spectrally interpretable. srUnet is able to deal with spectral shift and its application to multicolour imaging in biological sample is straight-forward. srUnet advances spectrally resolved single molecule localization microscopy to achieve performance close to conventional SMLM without complicating its use.

biophysics↗

bioRxiv2022

The bacterial lectin LecA from P. aeruginosa alters membrane organization by dispersing ordered domains

The assembly and dynamic reorganization of plasma membrane nanodomains (also known as "lipid rafts") play key roles in host cell infection by human pathogens (e.g. viruses and bacteria). Viruses and bacteria can trigger the reorganization of lipid rafts which leads to membrane invaginations and downstream signaling that promote infection. Such reorganizations can be induced by interactions of bacterial or viral carbohydrate proteins (so-called lectins) with lipid raft glycosphingolipids (GSLs). Here, we studied the GSL globotriaosylceramide (Gb3) which is a key receptor involved in the cellular uptake of the gram-negative bacterium P. aeruginosa. The bacterial surface lectin LecA targets Gb3 and promotes bacterial invasion via the "lipid zipper" mechanism. However, the impact of LecA on the organization of membrane nanodomains is unknown yet. We mimicked of the plasma membrane using supported lipid bilayers (SLBs) that contained liquid-ordered (Lo, "raft-like", enriched in sphingolipids and GSLs) and liquid-disordered (Ld, "non-raft-like" enriched in DOPC) lipid domains. Upon interaction with LecA, the Lo domains in the SLBs reshaped and dispersed. Moreover, deformation of SLBs was observed as LecA formed membrane multilayers on SLBs surface. We further dissected this process to reveal the impact of Gb3 structure, bilayer composition and LecA valence on the Lo reorganization.

biophysics↗

bioRxiv2020

The two zinc fingers in the nucleocapsid domain of the HIV-1 Gag precursor are equivalent for the interaction with the genomic RNA in the cytoplasm, but not for the recruitment of the complexes at the plasma membrane

The HIV-1 Gag precursor specifically selects the unspliced viral genomic RNA (gRNA) from the bulk of cellular and spliced viral RNAs via its nucleocapsid (NC) domain and drives gRNA encapsidation at the plasma membrane (PM). To further identify the determinants governing the intracellular trafficking of Gag-gRNA complexes and their accumulation at the PM, we compared, in living and fixed cells, the interactions between gRNA and wild-type (WT) Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs), or a non-myristoylated version of Gag. Our data showed that the deletion of both ZFs simultaneously or the complete NC domain completely abolished intracytoplasmic Gag-gRNA interactions. Deletion of either ZF delayed the delivery of gRNA to the PM but did not prevent Gag-gRNA interactions in the cytoplasm, indicating that the two ZFs display redundant roles in this respect. However, ZF2 played a more prominent role than ZF1 in the accumulation of the ribonucleoprotein complexes at the PM. Finally, the myristate group which is mandatory for anchoring the complexes at the MP, was found to be dispensable for the association of Gag with the gRNA in the cytosol. STATEMENT of SIGNIFICANCEFormation of HIV-1 retroviral particles relies on specific interactions between the retroviral Gag precursor and the unspliced genomic RNA (gRNA). During the late phase of replication, Gag orchestrates the assembly of newly formed viruses at the plasma membrane (PM). It has been shown that the intracellular HIV-1 gRNA recognition is governed by the two-zinc finger (ZF) motifs of the nucleocapsid (NC) domain in Gag. Here we provided a clear picture of the role of ZFs in the cellular trafficking of Gag-gRNA complexes to the PM by showing that either ZF was sufficient to efficiently promote these interactions in the cytoplasm, while interestingly, ZF2 played a more prominent role in the relocation of these ribonucleoprotein complexes at the PM assembly sites.

biophysics↗

bioRxiv2018

Macromolecular organization of the pyoverdine biosynthetic pathway in Pseudomonas aeruginosa.

The biosynthesis of the pyoverdine siderophore in Pseudomonas aeruginosa inlvolves multiple enzymes including Non-Ribosomal Peptide Synthetases (NRPS). We used in vitro single-molecule tracking and FRET-FLIM (Forster resonance energy transfer measured by fluorescence lifetime microscopy) to explore the spatial partitioning of the ornithine hydroxylase PvdA and to characterize its interactions with NRPS. The statistical description of thousands of single PvdA traces in cells using jump distance distribution analysis showed PvdA was mostly diffusing bound to large complex in the cytoplasm with a small exchangeable trapped fraction diffusing slower. FRET-FLIM clearly showed PvdA are physically interacting with the four NRPS PvdJ, PvdI, PvdL and PvdD of the pyoverdine pathway in the cellular context. Our data provide evidence for strongly organized multi-enzymatic biosynthetic complexes named siderosomes responsible for the siderophore biosynthesis. PvdA binding mode is strikingly different according to the NRPS it is interacting with suggesting PvdA binding site have co-evolved with the enzymatic active sites of NRPS.

biophysics↗

bioRxiv2018

MemBright: a Family of Fluorescent Membrane Probes for Advanced Cellular Imaging and Neuroscience

The proper staining of the plasma membrane (PM) is critical in bioimaging as it delimits the cell. Herein, we developed MemBright: a family of six cyanine-based fluorescent turn-on PM probes that emit from orange to near-infrared when reaching the PM, and enable homogeneous and selective PM staining with excellent contrast in mono and two-photon microscopy. These probes are compatible with long-term live cell imaging and immunostaining. Moreover, MemBright label neurons in a brighter manner than surrounding cells allowing identification of neurons in acute brain tissue section and neuromuscular-junctions without any use of transfection or transgenic animals. At last, MemBright were used in super-resolution imaging to unravel the dendritic spines neck. 3D multicolor dSTORM in combination with immunostaining revealed en-passant synapse displaying endogenous glutamate receptors clustered at the axonal-dendritic contact site. MemBright probes thus constitute a universal toolkit for cell biology and neuroscience biomembrane imaging with a variety of microscopy techniques.

neuroscience↗