calibration-free-vitro-quantification-protein-homo-oligomerization

Calibration-free <em>In Vitro</em> Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

University of Oxford

Biocruces Health Research Centre

Basque Foundation for Science

The majority of viruses enter host cells via endocytosis. Current knowledge of viral entry pathways is largely based upon infectivity measurements following genetic and/or pharmacological interventions that disrupt vesicular trafficking and maturation. Imaging of single virus entry in living cells provides a powerful means to delineate viral trafficking pathways and entry sites under physiological conditions.

Pinpointing retrovirus entry sites in cells expressing alternatively spliced receptor isoforms by single virus imaging.

The integral membrane protein LIMP-2 has been a paradigm for mannose 6-phosphate receptor (MPR) independent lysosomal targeting, binding to β-glucocerebrosidase (β-GCase) and directing it to the lysosome, before dissociating in the late-endosomal/lysosomal compartments. Here we report structural results illuminating how LIMP-2 binds and releases β-GCase according to changes in pH, via a histidine trigger, and suggesting that LIMP-2 localizes the ceramide portion of the substrate adjacent to the β-GCase catalytic site. Remarkably, we find that LIMP-2 bears P-Man9GlcNAc2 covalently attached to residue N325, and that it binds MPR, via mannose 6-phosphate, with a similar affinity to that observed between LIMP-2 and β-GCase. The binding sites for β-GCase and the MPR are functionally separate, so that a stable ternary complex can be formed. By fluorescence lifetime imaging microscopy, we also demonstrate that LIMP-2 interacts with MPR in living cells. These results revise the accepted view of LIMP-2-β-GCase lysosomal targeting. 

Lysosome sorting of β-glucocerebrosidase by LIMP-2 is targeted by the mannose 6-phosphate receptor.

Repulsive guidance molecules (RGMs) control crucial processes including cell motility, adhesion, immune-cell regulation and systemic iron metabolism. RGMs signal via the neogenin (NEO1) and the bone morphogenetic protein (BMP) pathways. Here, we report crystal structures of the N-terminal domains of all human RGM family members in complex with the BMP ligand BMP2, revealing a new protein fold and a conserved BMP-binding mode. Our structural and functional data suggest a pH-linked mechanism for RGM-activated BMP signaling and offer a rationale for RGM mutations causing juvenile hemochromatosis. We also determined the crystal structure of the ternary BMP2-RGM-NEO1 complex, which, along with solution scattering and live-cell super-resolution fluorescence microscopy, indicates BMP-induced clustering of the RGM-NEO1 complex. Our results show how RGM acts as the central hub that links BMP and NEO1 and physically connects these fundamental signaling pathways. 

Repulsive guidance molecule is a structural bridge between neogenin and bone morphogenetic protein.

We have produced a novel, simple and rapid method utilising genetically encodable FRET-based biosensors to permit the detection of HIV-1 virion fusion in living cells. These biosensors show high sensitivity both spatially and temporally, and allow the real-time recovery of HIV-1 fusion kinetics in both single cells and cell populations simultaneously. 

Imaging real-time HIV-1 virion fusion with FRET-based biosensors.

Cortical F-actin plays important roles during HIV-1 infection. A recent paper in Cell highlights the involvement of F-actin in enhancing dendritic cell (DC)-mediated HIV-1 infection of T cells. Such a critical mechanism of HIV-1 trans-enhancement between human DCs and T cells had not been analyzed in molecular detail before this work.

Actin Dynamics and HIV-1 Entry.

The β-lactamase (BlaM) assay was first revealed in 1998 and was demonstrated to be a robust Förster resonance energy transfer (FRET)-based reporter system that was compatible with a range of commonly-used cell lines. Today, the BlaM assay is available commercially as a kit and can be utilised readily and inexpensively for an array of experimental procedures that require a fluorescence-based readout. One frequent application of the BlaM assay is the measurement of viral fusion-the moment at which the genetic material harboured within virus particles is released into the cytosol following successful entry. The flexibility of the system permits evaluation of not only total fusion levels, but also the kinetics of fusion. However, significant variation exists in the scientific literature regarding the methodology by which the assay is applied to viral fusion analysis, making comparison between results difficult. In this review we draw attention to the disparity of these methodologies and examine the advantages and disadvantages of each approach. Successful strategies shown to render viruses compatible with BlaM-based analyses are also discussed. 

The β-Lactamase Assay: Harnessing a FRET Biosensor to Analyse Viral Fusion Mechanisms.

Ebola viruses (EBOVs) are responsible for repeated outbreaks of fatal infections, including the recent deadly epidemic in West Africa. There are currently no approved therapeutic drugs or vaccines for the disease. EBOV has a membrane envelope decorated by trimers of a glycoprotein (GP, cleaved by furin to form GP1 and GP2 subunits), which is solely responsible for host cell attachment, endosomal entry and membrane fusion. GP is thus a primary target for the development of antiviral drugs. Here we report the first, to our knowledge, unliganded structure of EBOV GP, and high-resolution complexes of GP with the anticancer drug toremifene and the painkiller ibuprofen. The high-resolution apo structure gives a more complete and accurate picture of the molecule, and allows conformational changes introduced by antibody and receptor binding to be deciphered. Unexpectedly, both toremifene and ibuprofen bind in a cavity between the attachment (GP1) and fusion (GP2) subunits at the entrance to a large tunnel that links with equivalent tunnels from the other monomers of the trimer at the three-fold axis. Protein–drug interactions with both GP1 and GP2 are predominately hydrophobic. Residues lining the binding site are highly conserved among filoviruses except Marburg virus (MARV), suggesting that MARV may not bind these drugs. Thermal shift assays show up to a 14 °C decrease in the protein melting temperature after toremifene binding, while ibuprofen has only a marginal effect and is a less potent inhibitor. These results suggest that inhibitor binding destabilizes GP and triggers premature release of GP2, thereby preventing fusion between the viral and endosome membranes. Thus, these complex structures reveal the mechanism of inhibition and may guide the development of more powerful anti-EBOV drugs.

Toremifene interacts with and destabilizes the Ebola virus glycoprotein.

Class A plexins (PlxnAs) act as semaphorin receptors and control diverse aspects of nervous system development and plasticity, ranging from axon guidance and neuron migration to synaptic organization. PlxnA signaling requires cytoplasmic domain dimerization, but extracellular regulation and activation mechanisms remain unclear. Here we present crystal structures of PlxnA (PlxnA1, PlxnA2, and PlxnA4) full ectodomains. Domains 1-9 form a ring-like conformation from which the C-terminal domain 10 points away. All our PlxnA ectodomain structures show autoinhibitory, intermolecular "head-to-stalk" (domain 1 to domain 4-5) interactions, which are confirmed by biophysical assays, live cell fluorescence microscopy, and cell-based and neuronal growth cone collapse assays. This work reveals a 2-fold role of the PlxnA ectodomains: imposing a pre-signaling autoinhibitory separation for the cytoplasmic domains via intermolecular head-to-stalk interactions and supporting dimerization-based PlxnA activation upon ligand binding. More generally, our data identify a novel molecular mechanism for preventing premature activation of axon guidance receptors. 

Structural Basis for Plexin Activation and Regulation.

Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity, despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages.

The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq.

One of the key research areas surrounding HIV-1 concerns the regulation of the fusion event that occurs between the virus particle and the host cell during entry. Even if it is universally accepted that the large GTPase dynamin-2 is important during HIV-1 entry, its exact role during the first steps of HIV-1 infection is not well characterized. Here, we have utilized a multidisciplinary approach to study the DNM2 role during fusion of HIV-1 in primary resting CD4 T and TZM-bl cells. We have combined advanced light microscopy and functional cell-based assays to experimentally assess the role of dynamin-2 during these processes. Overall, our data suggest that dynamin-2, as a tetramer, might help to establish hemi-fusion and stabilizes the pore during HIV-1 fusion.

Dynamin-2 Stabilizes the HIV-1 Fusion Pore with a Low Oligomeric State.

Monitoring of different signalling enzymes in a single assay using multiplex biosensing provides a multidimensional workspace to elucidate biological processes, signalling pathway crosstalk, and determine precise sequence of events at the single living cell level. In this study, we interrogate the complexity in cAMP/PKA-MAPK/ERK1&2 crosstalk by using multi-parameter biosensing experiments to correlate biochemical activities simultaneously in time and space. Using a single excitation wavelength dual colour FLIM method we are able to detect fluorescence lifetime images of two donors to simultaneously measure PKA and ERK1&2 kinase activities in the same cellular localization by using FRET biosensors. To this end, we excite two FRET donors mTFP1 and LSSmOrange with a 440 nm wavelength and we alleviate spectral bleed-through associated limitations with the very dim-fluorescent acceptor ShadowG for mTFP1 and the red-shifted mKate2 for LSSmOrange. The simultaneous recording of PKA and ERK1&2 kinase activities reveals concomitant EGF-mediated activations of both kinases in HeLa cells. Under these conditions the subsequent Forskolin-induced cAMP release reverses the transient increase of EGF-mediated ERK1&2 kinase activity while reinforcing PKA activation. Here we propose a validated methodology for multiparametric kinase biosensing in living cells using FRET-FLIM.

Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM.

HIV-1 disseminates to diverse tissues and establishes long-lived viral reservoirs. These reservoirs include the CNS, in which macrophage-lineage cells, and as suggested by many studies, astrocytes, may be infected. Here, we have investigated astrocyte infection by HIV-1. We confirm that astrocytes trap and internalize HIV-1 particles for subsequent release but find no evidence that these particles infect the cell. Astrocyte infection was not observed by cell-free or cell-to-cell routes using diverse approaches, including luciferase and GFP reporter viruses, fixed and live-cell fusion assays, multispectral flow cytometry, and super-resolution imaging. By contrast, we observed intimate interactions between HIV-1-infected macrophages and astrocytes leading to signals that might be mistaken for astrocyte infection using less stringent approaches. These results have implications for HIV-1 infection of the CNS, viral reservoir formation, and antiretroviral therapy.

Astrocytes Resist HIV-1 Fusion but Engulf Infected Macrophage Material.

In spite of the utility of unit tests, most R package developers do not write them.  makes it easier to  writing unit tests by creating shells/skeletons of unit tests based on the examples in the user's package documentation. When completed, these unit tests test whether said examples run  . By combining the functionality of  with that of , having ensured that their examples adequately demonstrate the features of their package, the developer can have much of the work of constructing a comprehensive set of unit tests done for them.

-An easy start to unit testing R packages.

HIV-1 disseminates to diverse tissues through different cell types and establishes long-lived reservoirs. The exact cellular compartment where fusion occurs differs depending on the cell type and mode of viral transmission. This implies that HIV-1 may modulate a number of common host cell factors in different cell types. In this review, we evaluate recent advances on the host cell factors that play an important role in HIV-1 entry and fusion. New insights from restriction factors inhibiting virus-cell fusion in vitro may contribute to the development of future therapeutic interventions. Collectively, novel findings underline the need for potent, host-directed therapies that disrupt the earliest stages of the virus life cycle and preclude the emergence of resistant viral variants.

On the Whereabouts of HIV-1 Cellular Entry and Its Fusion Ports.

An R package for performing number and brightness image analysis, with the implementation of a novel automatic detrending algorithm.

nandb-number and brightness in R with a novel automatic detrending algorithm.

The possibility to detect and quantify protein-protein interactions with good spatial and temporal resolutions in live cells is crucial in biology. Number and brightness is a powerful approach to detect both protein aggregation/desegregation dynamics and stoichiometry in live cells. Importantly, this technique can be applied in commercial set ups: both camera based and laser scanning microscopes. It provides pixel-by-pixel information on protein oligomeric states. If performed with two colours, the technique can retrieve the stoichiometry of the reaction under study. In this review, we discuss the strengths and weaknesses of the technique, stressing which are the correct acquisition parameters for a given microscope, the main challenges in analysis, and the limitations of the technique.

Sergi Padilla-Parra

Cellular Imaging Group,
Wellcome Centre Human Genetics,
Division of Structural Biology,
Cellular Imaging Group, Wellcome Centre Human Genetics,
Division of Structural Biology, Wellcome Centre Human Genetics

University of Oxford

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Sergi Padilla-Parra

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Cellular Imaging Group,Wellcome Centre Human Genetics,Division of Structural Biology,Cellular Imaging Group, Wellcome Centre Human Genetics,Division of Structural Biology, Wellcome Centre Human Genetics

University of Oxford

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Cellular Imaging Group,
Wellcome Centre Human Genetics,
Division of Structural Biology,
Cellular Imaging Group, Wellcome Centre Human Genetics,
Division of Structural Biology, Wellcome Centre Human Genetics