Integrating single cell transcriptomics and volume electron microscopy confirms the presence of pancreatic acinar-like cells in sea urchins

The identity and function of a given cell type relies on the differential expression of gene batteries that promote diverse phenotypes and functional specificities. Therefore, the identification of the molecular and morphological fingerprints of cell types across taxa is essential for untangling their evolution. Here we use a multidisciplinary approach to identify the molecular and morphological features of an exocrine, pancreas-like cell type harbored within the sea urchin larval gut. Using single cell transcriptomics, we identify various cell populations with a pancreatic-like molecular fingerprint that are enriched within the S. purpuratus larva digestive tract. Among these, in the region where they reside, the midgut/stomach domain, we find that populations of exocrine pancreas-like cells have a unique regulatory wiring distinct from the rest the of the cell types of the same region. Furthermore, Serial Block-face scanning Electron Microscopy (SBEM) of the exocrine cells shows that this reported molecular diversity is associated to distinct morphological features that reflect the physiological and functional properties of this cell type. Therefore, we propose that these sea urchin exocrine cells are homologous to the well-known mammalian pancreatic acinar cells and thus we trace the origin of this particular cell type to the time of deuterostome diversification. Overall, our approach allows a thorough characterization of a complex cell type and shows how both the transcriptomic and morphological information contribute to disentangling the evolution of cell types and organs such as the pancreatic cells and pancreas. Keywords: SBEM; acinar cells; evolution of cell types; morphology; pancreas; scRNAseq; sea urchin

Bioelectrochemistry by fluorescent cyclic voltammetry

Understanding the factors influencing the ET characteristics of redox proteins confined at an electrochemical interface is of fundamental importance from both pure (fundamental science) and applied (biosensory) perspectives. This thesis reports on progress made in the emerging field of coupled electrochemical characterization and optical imaging in moving the analysis of redox-active films to molecular scales. More specifically the combination of cyclic voltammetry and wide-field Total Internal Reflection (TIRF) microscopy, here named ‘Fluorescent Cyclic Voltammetry’ (FCV), was applied to monitoring the response of surface-confined redox active proteins at submonolayer concentrations.The combined submicrometre spatial resolution and photon capture efficiency of an inverted TIRF configuration enabled the redox reactions of localized populations of proteins to be directly imaged at scales down to a few hundreds of molecules. This represents a 6-9 orders of magnitude enhancement in sensitivity with respect to classical current signals observed in bioelectrochemical analysis. Importantly, measurements of redox potentials at this scale could be achieved from both natural and artificially designed bioelectrochemical fluorescent switches and shed fundamental light on the thermodynamic and kinetic dispersion within a population of surface confined metalloproteins.The first three chapters of this thesis provide an overview of the relevant literature and a theoretical background to both the rapidly expanding fields of electroactive monolayers bioelectrochemistry and TIRF imaging.The initial design and construction of a robust electrochemically and optically addressable fluorescent switch, crucial to the applicability of FCV is reported in chapter 5. The generation of optically transparent, and chemically modifiable electrode surfaces suitable for FCV are also described.Chapter 6 describes the response of the surface confined azurin-based switch. Analysis of the spatially-resolved redox reaction of zeptomole samples in various conditions enables the mapping of thermodynamic dispersion across the sampled areas.In chapter 7 the newly developed FCV detection method was extended to investigate more complex bioelectrochemical systems containing multiple electron transferring redox centres and responding optically at different wavelengths. This approach provides a platform for spectral resolution of different electrochemical processes on the same sample.Finally in chapter 8 an electrochemical procedure is proposed for investigating the kinetic response of redox proteins using a fundamentally new methodology based on interfacial capacitance. In using variations in the surface chemistry to tune the rate of electron transfer, the approach was shown to be a robust and facile means of characterising redox active films in considerably more detail than possible through standard electrochemical methodologies. Ultimately, it can be applied to probe dispersion within protein populations and represents a powerful means of analysing molecular films more generally.</p

Bioelectrochemistry by fluorescent cyclic voltammetry

Understanding the factors influencing the ET characteristics of redox proteins confined at an electrochemical interface is of fundamental importance from both pure (fundamental science) and applied (biosensory) perspectives. This thesis reports on progress made in the emerging field of coupled electrochemical characterization and optical imaging in moving the analysis of redox-active films to molecular scales. More specifically the combination of cyclic voltammetry and wide-field Total Internal Reflection (TIRF) microscopy, here named ‘Fluorescent Cyclic Voltammetry’ (FCV), was applied to monitoring the response of surface-confined redox active proteins at submonolayer concentrations. The combined submicrometre spatial resolution and photon capture efficiency of an inverted TIRF configuration enabled the redox reactions of localized populations of proteins to be directly imaged at scales down to a few hundreds of molecules. This represents a 6-9 orders of magnitude enhancement in sensitivity with respect to classical current signals observed in bioelectrochemical analysis. Importantly, measurements of redox potentials at this scale could be achieved from both natural and artificially designed bioelectrochemical fluorescent switches and shed fundamental light on the thermodynamic and kinetic dispersion within a population of surface confined metalloproteins. The first three chapters of this thesis provide an overview of the relevant literature and a theoretical background to both the rapidly expanding fields of electroactive monolayers bioelectrochemistry and TIRF imaging. The initial design and construction of a robust electrochemically and optically addressable fluorescent switch, crucial to the applicability of FCV is reported in chapter 5. The generation of optically transparent, and chemically modifiable electrode surfaces suitable for FCV are also described. Chapter 6 describes the response of the surface confined azurin-based switch. Analysis of the spatially-resolved redox reaction of zeptomole samples in various conditions enables the mapping of thermodynamic dispersion across the sampled areas. In chapter 7 the newly developed FCV detection method was extended to investigate more complex bioelectrochemical systems containing multiple electron transferring redox centres and responding optically at different wavelengths. This approach provides a platform for spectral resolution of different electrochemical processes on the same sample. Finally in chapter 8 an electrochemical procedure is proposed for investigating the kinetic response of redox proteins using a fundamentally new methodology based on interfacial capacitance. In using variations in the surface chemistry to tune the rate of electron transfer, the approach was shown to be a robust and facile means of characterising redox active films in considerably more detail than possible through standard electrochemical methodologies. Ultimately, it can be applied to probe dispersion within protein populations and represents a powerful means of analysing molecular films more generally.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Capacitance Spectroscopy: A Versatile Approach To Resolving the Redox Density of States and Kinetics in Redox-Active Self-Assembled Monolayers

Redox active self-assembled monolayers inherently possess both electrochemically addressable and polarizable components. The latter will contribute, with additional parasitic terms, to the <i>iR</i> drop effects within any form of electronic analysis, potentially distorting results. A capacitive analysis of such interfaces (Electroactive Monolayer Capacitance Spectroscopy), presented here, enables a clean mapping of both the thermodynamic and kinetic faradaic characteristics in a single experimental run, with parasitic nonfaradaic contributions (polarization and resistance terms) both spectrally resolved and cleanly removed. The methodology enables a rapid and undistorted quantification of accessible redox site density of states (reported directly by redox capacitance), molecular surface coverage, electron transfer kinetics, and reorganization energies with comparatively little experimental effort. Exemplified here with electroactive copper protein and ferrocene films the approach is equally applicable to any redox active interface

Identification of host dependency factors involved in SARS-CoV-2 replication organelle formation through proteomics and ultrastructural analysis.

Remodeling of the cellular endomembrane system by viruses allows for efficient and coordinated replication of the viral genome in distinct subcellular compartments termed replication organelles. As a critical step in the viral life cycle, replication organelle formation is an attractive target for therapeutic intervention, but factors central to this process are only partially understood. In this study, we corroborate that two viral proteins, nsp3 and nsp4, are the major drivers of membrane remodeling in SARS-CoV-2 infection. We further report a number of host cell factors interacting with these viral proteins and supporting the viral replication cycle, some of them by contributing to the formation of the SARS-CoV-2 replication organelle

Targeted volume correlative light and electron microscopy of an environmental marine microorganism

International audienceABSTRACT Photosynthetic microalgae are responsible for an important fraction of CO2 fixation and O2 production on Earth. Three-dimensional (3D) ultrastructural characterization of these organisms in their natural environment can contribute to a deeper understanding of their cell biology. However, the low throughput of volume electron microscopy (vEM) methods along with the complexity and heterogeneity of environmental samples pose great technical challenges. In the present study, we used a workflow based on a specific electron microscopy sample preparation method compatible with both light and vEM imaging in order to target one cell among a complex natural community. This method revealed the 3D subcellular landscape of a photosynthetic dinoflagellate, which we identified as Ensiculifera tyrrhenica, with quantitative characterization of multiple organelles. We show that this cell contains a single convoluted chloroplast and show the arrangement of the flagellar apparatus with its associated photosensitive elements. Moreover, we observed partial chromatin unfolding, potentially associated with transcription activity in these organisms, in which chromosomes are permanently condensed. Together with providing insights in dinoflagellate biology, this proof-of-principle study illustrates an efficient tool for the targeted ultrastructural analysis of environmental microorganisms in heterogeneous mixes

Zika virus prM protein contains cholesterol binding motifs required for virus entry and assembly

For successful infection of host cells and virion production, enveloped viruses, including Zika virus (ZIKV), extensively rely on cellular lipids. However, how virus protein–lipid interactions contribute to the viral life cycle remains unclear. Here, we employ a chemo-proteomics approach with a bifunctional cholesterol probe and show that cholesterol is closely associated with the ZIKV structural protein prM. Bioinformatic analyses, reverse genetics alongside with photoaffinity labeling assays, and atomistic molecular dynamics simulations identified two functional cholesterol binding motifs within the prM transmembrane domain. Loss of prM–cholesterol association has a bipartite effect reducing ZIKV entry and leading to assembly defects. We propose a model in which membrane-resident M facilitates cholesterol-supported lipid exchange during endosomal entry and, together with cholesterol, creates a platform promoting virion assembly. In summary, we identify a bifunctional role of prM in the ZIKV life cycle by mediating viral entry and virus assembly in a cholesterol-dependent manner.Peer reviewe

Zika virus prM protein contains cholesterol binding motifs required for virus entry and assembly - Molecular Dynamics Simulation Dataset

&lt;p&gt;The molecular dynamics (MD) simulation dataset. The contents:&lt;/p&gt; &lt;ul&gt; &lt;li&gt;&lt;strong&gt;5ire_BIOMT_expanded.pdb&lt;/strong&gt;: The complete biological assembly of the&nbsp;cryo-EM structure of Zika Virus (PDB ID:5IRE)&nbsp;&lt;/li&gt; &lt;li&gt;&lt;strong&gt;5ire_Mprotein_BIOMT_expanded.pdb&lt;/strong&gt;:&nbsp;The M proteins extracted from the complete biological assembly of the&nbsp;cryo-EM structure of Zika Virus (PDB ID:5IRE).&nbsp; The biological assembly shows the dimeric organization of M proteins.&lt;/li&gt; &lt;li&gt;&lt;strong&gt;0chol.zip, 10chol.zip, 20chol.zip, and 30chol.zip&lt;/strong&gt; contain&nbsp;simulation input and output files for the simulated membrane compositions: 0:100, 10:90, 20:80, 30:70 (mol%:mol%) Cholesterol:POPC, respectively.&nbsp; &lt;ul&gt; &lt;li&gt;In each zip file, there are 5 directories: &lt;strong&gt;wt,&nbsp;R253L+F257A,&nbsp;R253L+F257S, K275L+Y278A,&nbsp;K275L+Y278S&lt;/strong&gt;&nbsp;corresponding to each simulated&nbsp;M protein dimer variant: wild type, CARC 2-A, CARC 2-S, CARC 3-A, and CARC 3-S.&nbsp;In each directory, there are the following files: &lt;ul&gt; &lt;li&gt;&lt;strong&gt;toppar&lt;/strong&gt;: This directory contains all force field topologies and parameters&lt;/li&gt; &lt;li&gt;&lt;strong&gt;topol.top&lt;/strong&gt;: GROMACS&nbsp;topology (top) file&lt;/li&gt; &lt;li&gt;&lt;strong&gt;index.ndx&lt;/strong&gt;: GROMACS index (ndx) file&lt;/li&gt; &lt;li&gt;&lt;strong&gt;prod.mdp&lt;/strong&gt;: GROMACS MD&nbsp;parameters (mdp)&nbsp; file&lt;/li&gt; &lt;li&gt;&lt;strong&gt;0, 1, 2, 3, 4, 5, 6, 7, 8, 9&lt;/strong&gt;: These directories contain the simulation&nbsp;inputs and outputs for each simulation&nbsp;repeat. In each of these directories, there are the following files:&nbsp; &lt;ul&gt; &lt;li&gt;&lt;strong&gt;t0.pdb&lt;/strong&gt;: The pdb file of the starting&nbsp;coordinates&lt;/li&gt; &lt;li&gt;&lt;strong&gt;prod0.tpr&lt;/strong&gt;: GROMACS binary run input (tpr) file&nbsp;&lt;/li&gt; &lt;li&gt;&lt;strong&gt;prod0.edr&lt;/strong&gt;: GROMACS energy (edr) file&lt;/li&gt; &lt;li&gt;&lt;strong&gt;prod0.gro&lt;/strong&gt;: GROMACS output coordinates and velocities after&nbsp;1 microsecond of simulation&lt;/li&gt; &lt;li&gt;&lt;strong&gt;prod0.cpt&lt;/strong&gt;: GROMACS checkpoint file&nbsp;after 1 microsecond of simulation&lt;/li&gt; &lt;li&gt;&lt;strong&gt;noW.pdb&lt;/strong&gt;: The pdb file of the starting&nbsp;coordinates with all water molecules removed&lt;/li&gt; &lt;li&gt;&lt;strong&gt;noW.xtc&lt;/strong&gt;:&nbsp; GROMACS compressed trajectory (xtc)&nbsp;file with all water molecules removed&lt;/li&gt; &lt;/ul&gt; &lt;/li&gt; &lt;/ul&gt; &lt;/li&gt; &lt;/ul&gt; &lt;/li&gt; &lt;/ul&gt

MOSPD2 is an endoplasmic reticulum–lipid droplet tether functioning in LD homeostasis

International audienceMembrane contact sites between organelles are organized by protein bridges. Among the components of these contacts, the VAP family comprises ER–anchored proteins, such as MOSPD2, that function as major ER–organelle tethers. MOSPD2 distinguishes itself from the other members of the VAP family by the presence of a CRAL-TRIO domain. In this study, we show that MOSPD2 forms ER–lipid droplet (LD) contacts, thanks to its CRAL-TRIO domain. MOSPD2 ensures the attachment of the ER to LDs through a direct protein–membrane interaction. The attachment mechanism involves an amphipathic helix that has an affinity for lipid packing defects present at the surface of LDs. Remarkably, the absence of MOSPD2 markedly disturbs the assembly of lipid droplets. These data show that MOSPD2, in addition to being a general ER receptor for inter-organelle contacts, possesses an additional tethering activity and is specifically implicated in the biology of LDs via its CRAL-TRIO domain