register interest

Dr Sergi Padilla-Parra

Research Area: Microbiology
Technology Exchange: In vivo imaging, Microscopy (Confocal), Microscopy (Video) and Protein interaction
Scientific Themes: Immunology & Infectious Disease
Keywords: Quantitative Fluorescence Microscopy, Single Virus Particle Tracking, FRET by FLIM, FCS and FCCS and Endocytosis
Web Links:

Methodological Quantification of the Spatio-Temporal Regulation of cell-factors controlling Endocytic Routes during Virus Entry

The study of entry mechanisms of enveloped viruses and how they are targeted to different endocytic routes, from internalization to trafficking and fusion is crucial for many reasons: first, virus-cell interactions constitutes an area that is not completely explored and novel strategies to develop drugs and vaccines capable of blocking infection are needed, also viruses are interesting means to deliver drugs and macromolecules within the cell and finally, they are important tools to better understand new mechanisms and concepts related to virus entry (i.e. endocytosis) in structural and molecular biology.

We propose a unique combination of approaches based on quantitative fluorescence microscopy among others; to unveil the spatial and temporal regulation of the host-cell factors implied in endocytic viral entry. We will produce a quantitative microscopy-based framework that will allow to systematically studying how different viruses enter the cell via different routes of endocytosis, and also to discover how different endocytic pathways are regulated in the presence/absence of pathogens.

Name Department Institution Country
Professor Gregory B Melikyan Emory University United States
Professor Kohji Takei Okayama University Japan
Professor Christian Siebold Structural Biology Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Professor A. Radu Aricescu Structural Biology Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Professor E. Yvonne Jones FRS FMedSci Structural Biology Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Dr Marc Tramier IGDR-University of Rennes 1 France
Prof Christian Eggeling (RDM) Investigative Medicine Division Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Jakobsdottir GM, Iliopoulou M, Nolan R, Alvarez L, Compton AA, Padilla-Parra S. 2017. On the Whereabouts of HIV-1 Cellular Entry and Its Fusion Ports Trends in Molecular Medicine, | Show Abstract | Read more

© 2017 Elsevier Ltd. 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. Recent studies of HIV-1 infection have described the role of dynamin-2 (DNM2) in stabilization of the fusion pore in the plasma membrane of CD4 + T cells, and also shown that DNM2 in these cells might not be involved in HIV-1 virion endocytosis.The role of endocytosis during cell-cell HIV-1 transmission is controversial: some reports state that HIV-1 might mature and fuse within endosomal compartments in the target cell, while others state that endocytosis might not lead to productive infection. In this scenario, the virus would be recycled back towards the plasma membrane and fuse. This is important because endosomal delivery of the virus could serve to evade neutralization by circulating HIV-1-specific antibodies.Recent studies have highlighted the importance of the role played by host cells during HIV-1 entry. Indeed, inhibition of HIV-1 entry can be modulated by innate immunity, and by restriction factors such as IFITM and SERINC.

Nolan R, Alvarez LAJ, Elegheert J, Iliopoulou M, Jakobsdottir GM, Rodriguez-Muñoz M, Aricescu AR, Padilla-Parra S. 2017. nandb—number and brightness in R with a novel automatic detrending algorithm Bioinformatics, | Read more

Herbomel G, Hatte G, Roul J, Padilla-Parra S, Tassan JP, Tramier M. 2017. Actomyosin-generated tension on cadherin is similar between dividing and non-dividing epithelial cells in early Xenopus laevis embryos. Sci Rep, 7 pp. 45058. | Show Abstract | Read more

Epithelia represent a unique situation where polarized cells must maintain sufficiently strong cell-cell contacts to guarantee the epithelial integrity indispensable for barrier functions. Nevertheless, epithelia must also keep sufficient plasticity which is crucial during development and morphogenesis. Adherens junctions and mechanical forces produced by the actomyosin cytoskeleton are major players for epithelial integrity maintenance and plasticity regulations. To understand how the epithelium is able to meet such a challenge, it is indispensable to determine how cellular junctions and mechanical forces acting at adherens junctions are regulated. Here, we investigate the tensile forces acting on adherens junctions via cadherin during cell division in the Xenopus embryos epithelium. Using the recently developed E-cadherin FRET tension sensor and a fastFLIM prototype microscope, we were able to measure mechanical forces applied on cadherin at cell-cell junctions. We have shown that the Xenopus epithelium is under tension, approximately 3 pN which remains stable, indicating that tensile forces acting on cadherin at the adherens junction are at equilibrium. Unexpectedly, mechanical tension across cadherin was similar between dividing and non-dividing epithelial cells.

Russell RA, Chojnacki J, Jones DM, Johnson E, Do T, Eggeling C, Padilla-Parra S, Sattentau QJ. 2017. Astrocytes Resist HIV-1 Fusion but Engulf Infected Macrophage Material. Cell Rep, 18 (6), pp. 1473-1483. | Show Abstract | Read more

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.

Demeautis C, Sipieter F, Roul J, Chapuis C, Padilla-Parra S, Riquet FB, Tramier M. 2017. Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM. Sci Rep, 7 pp. 41026. | Show Abstract | Read more

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.

Jones DM, Alvarez LA, Nolan R, Ferriz M, Sainz Urruela R, Massana-Muñoz X, Novak-Kotzer H, Dustin ML, Padilla-Parra S. 2017. Dynamin-2 Stabilizes the HIV-1 Fusion Pore with a Low Oligomeric State. Cell Rep, 18 (2), pp. 443-453. | Show Abstract | Read more

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.

Wills QF, Mellado-Gomez E, Nolan R, Warner D, Sharma E, Broxholme J, Wright B, Lockstone H, James W, Lynch M et al. 2017. The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq. BMC Genomics, 18 (1), pp. 53. | Show Abstract | Read more

BACKGROUND: 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. RESULTS: We introduce the programmable Polaris™ microfluidic lab-on-chip for single-cell sequencing, which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1, such as an altered oxidative stress response, have a large paracrine signaling component. Furthermore, we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation, both associated with the oxidative stress response and altered proteostasis. Interestingly, SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'), with no known co-regulation. CONCLUSION: As single-cell methods continue to mature, so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture, live-cell imaging, and single-cell sequencing, we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages, for example, react to inflammation and form treatment resistant HIV reservoirs.

Kong Y, Janssen BJ, Malinauskas T, Vangoor VR, Coles CH, Kaufmann R, Ni T, Gilbert RJ, Padilla-Parra S, Pasterkamp RJ, Jones EY. 2016. Structural Basis for Plexin Activation and Regulation. Neuron, 91 (3), pp. 548-560. | Show Abstract | Read more

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.

Zhao Y, Ren J, Harlos K, Jones DM, Zeltina A, Bowden TA, Padilla-Parra S, Fry EE, Stuart DI. 2016. Toremifene interacts with and destabilizes the Ebola virus glycoprotein. Nature, 535 (7610), pp. 169-172. | Show Abstract | Read more

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.

Padilla-Parra S, Dustin ML. 2016. Actin Dynamics and HIV-1 Entry. Trends Mol Med, 22 (5), pp. 354-356. | Show Abstract | Read more

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.

Jones DM, Padilla-Parra S. 2016. The β-Lactamase Assay: Harnessing a FRET Biosensor to Analyse Viral Fusion Mechanisms. Sensors (Basel), 16 (7), pp. 950-950. | Show Abstract | Read more

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.

Jones DM, Padilla-Parra S. 2015. Imaging real-time HIV-1 virion fusion with FRET-based biosensors. Sci Rep, 5 (1), pp. 13449. | Show Abstract | Read more

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.

Healey EG, Bishop B, Elegheert J, Bell CH, Padilla-Parra S, Siebold C. 2015. Repulsive guidance molecule is a structural bridge between neogenin and bone morphogenetic protein. Nat Struct Mol Biol, 22 (6), pp. 458-465. | Show Abstract | Read more

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.

Padilla-Parra S, Audugé N, Tramier M, Coppey-Moisan M. 2015. Time-domain fluorescence lifetime imaging microscopy: a quantitative method to follow transient protein-protein interactions in living cells. Cold Spring Harb Protoc, 2015 (6), pp. 508-521. | Show Abstract | Read more

Quantitative analysis in Förster resonance energy transfer (FRET) imaging studies of protein-protein interactions within live cells is still a challenging issue. Many cellular biology applications aim at the determination of the space and time variations of the relative amount of interacting fluorescently tagged proteins occurring in cells. This relevant quantitative parameter can be, at least partially, obtained at a pixel-level resolution by using fluorescence lifetime imaging microscopy (FLIM). Indeed, fluorescence decay analysis of a two-component system (FRET and no FRET donor species), leads to the intrinsic FRET efficiency value (E) and the fraction of the donor-tagged protein that undergoes FRET (fD). To simultaneously obtain fD and E values from a two-exponential fit, data must be acquired with a high number of photons, so that the statistics are robust enough to reduce fitting ambiguities. This is a time-consuming procedure. However, when fast-FLIM acquisitions are used to monitor dynamic changes in protein-protein interactions at high spatial and temporal resolutions in living cells, photon statistics and time resolution are limited. In this case, fitting procedures are unreliable, even for single lifetime donors. We introduce the concept of a minimal fraction of donor molecules involved in FRET (mfD), obtained from the mathematical minimization of fD. Here, we discuss different FLIM techniques and the compromises that must be made between precision and time invested in acquiring FLIM measurements. We show that mfD constitutes an interesting quantitative parameter for fast FLIM because it gives quantitative information about transient interactions in live cells.

Padilla-Parra S, Marin M, Kondo N, Melikyan GB. 2014. Pinpointing retrovirus entry sites in cells expressing alternatively spliced receptor isoforms by single virus imaging. Retrovirology, 11 (1), pp. 47. | Show Abstract | Read more

BACKGROUND: 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. RESULTS: Here, we visualized single avian retrovirus co-trafficking with markers for early (Rab5) and late (Rab7) endosomes, acidification of endosomal lumen and the resulting viral fusion measured by the viral content release into the cytoplasm. Virus-carrying vesicles either merged with the existing Rab5-positive early endosomes or slowly accumulated Rab5. The Rab5 recruitment to virus-carrying endosomes correlated with acidification of their lumen. Viral fusion occurred either in early (Rab5-positive) or intermediate (Rab5- and Rab7-positive) compartments. Interestingly, different isoforms of the cognate receptor directed virus entry from distinct endosomes. In cells expressing the transmembrane receptor, viruses preferentially entered and fused with slowly maturing early endosomes prior to accumulation of Rab7. By comparison, in cells expressing the GPI-anchored receptor, viruses entered both slowly and quickly maturing endosomes and fused with early (Rab5-positive) and intermediate (Rab5- and Rab7-positive) compartments. CONCLUSIONS: Since the rate of low pH-triggered fusion was independent of the receptor isoform, we concluded that the sites of virus entry are determined by the kinetic competition between endosome maturation and viral fusion. Our findings demonstrate the ability of this retrovirus to enter cells via alternative endocytic pathways and establish infection by releasing its content from distinct endosomal compartments.

Padilla-Parra S, Audugé N, Coppey-Moisan M, Tramier M. 2014. Quantitative Study of protein-protein interactions in live cell by dual-color fluorescence correlation spectroscopy Methods in Molecular Biology, 1076 pp. 683-698. | Show Abstract | Read more

Dual-color FCS is a powerful method to monitor protein-protein interactions in living cells. The main idea is based on the cross-correlation analysis of temporal fluorescence intensity fluctuations of two fluorescent proteins to obtain their co-diffusion and relative concentration. But, when performing these experiments, the spectral overlap in the emission of the two colors produces an artifact that corrupts the cross-correlation data: spectral bleed-through. We have shown that problems with cross talk are overcome with Fluorescence Lifetime Correlation Spectroscopy (FLCS). FLCS applied to dual-color cross-correlation, utilizing for example eGFP and mCherry fluorescent proteins, allows the determination of protein-protein interactions in living cells without the need of spectral bleed-through calibration. Here, we present in detail how this methodology can be implemented using a commercial setup (Microtime from PicoQuant, SP8 SMD from Leica or any conventional confocal with PicoQuant TCSPC module, and also with a Becker and Hickl TCSPC module). The dual-color FLCS experimental procedure where the different laser intensities do not have to be controlled during the experiment constitutes a very powerful technique to quantitatively study protein interactions in live samples. © 2014 Springer Science+Business Media, LLC.

Zhao Y, Ren J, Padilla-Parra S, Fry EE, Stuart DI. 2014. Lysosome sorting of β-glucocerebrosidase by LIMP-2 is targeted by the mannose 6-phosphate receptor. Nat Commun, 5 pp. 4321. | Show Abstract | Read more

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.

Padilla-Parra S, Marin M, Gahlaut N, Suter R, Kondo N, Melikyan GB. 2013. Fusion of mature HIV-1 particles leads to complete release of a gag-GFP-based content marker and raises the intraviral pH. PLoS One, 8 (8), pp. e71002. | Show Abstract | Read more

By imaging the release of a GFP-based viral content marker produced upon virus maturation, we have previously found that HIV-1 fuses with endosomes. In contrast, fusion at the cell surface did not progress beyond a lipid mixing stage (hemifusion). However, recent evidence suggesting that free GFP can be trapped within the mature HIV-1 capsid raises concerns that this content marker may not be released immediately after the formation of a fusion pore. To determine whether a significant portion of GFP is trapped in the mature capsid, we first permeabilized the viral membrane with saponin. The overwhelming majority of pseudoviruses fully released GFP while the remaining particles exhibited partial loss or no loss of content. The extent of GFP release correlated with HIV-1 maturation, implying that incomplete Gag processing, but not GFP entrapment by mature capsids, causes partial content release. Next, we designed a complementary assay for visualizing pore formation by monitoring the intraviral pH with an additional pH-sensitive fluorescent marker. The loss of GFP through saponin-mediated pores was associated with a concomitant increase in the intraviral pH due to equilibration with the pH of an external buffer. We next imaged single HIV-cell fusion and found that these events were manifested in a highly correlated loss of content and increase in the intraviral pH, as it equilibrated with the cytosolic pH. Fused or saponin-permeabilized pseudoviruses that partially lost GFP did not release the remaining content marker under conditions expected to promote the capsid dissociation. We were thus unable to detect significant entrapment of GFP by the mature HIV-1 capsid. Together, our results validate the use of the GFP-based content marker for imaging single virus fusion and inferring the sites of HIV-1 entry.

Nolan R, Padilla-Parra S. 2013. Easier unit tests and better examples with exampletestr and covr Wellcome Open Research, 2 pp. 31-31. | Read more

Leray A, Padilla-Parra S, Roul J, Héliot L, Tramier M. 2013. Spatio-Temporal Quantification of FRET in living cells by fast time-domain FLIM: a comparative study of non-fitting methods [corrected]. PLoS One, 8 (7), pp. e69335. | Show Abstract | Read more

Förster Resonance Energy Transfer (FRET) measured with Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique to investigate spatio-temporal regulation of protein-protein interactions in living cells. When using standard fitting methods to analyze time domain FLIM, the correct estimation of the FRET parameters requires a high number of photons and therefore long acquisition times which are incompatible with the observation of dynamic protein-protein interactions. Recently, non-fitting strategies have been developed for the analysis of FLIM images: the polar plot or "phasor" and the minimal fraction of interacting donor mfD . We propose here a novel non-fitting strategy based on the calculation of moments. We then compare the performance of these three methods when shortening the acquisition time: either by reducing the number of counted photons N or the number of temporal channels Nch , which is particularly adapted for the original fast-FLIM prototype presented in this work that employs the time gated approach. Based on theoretical calculations, Monte Carlo simulations and experimental data, we determine the domain of validity of each method. We thus demonstrate that the polar approach remains accurate for a large range of conditions (low N, Nch or small fractions of interacting donor fD ). The validity domain of the moments method is more restricted (not applicable when fD <0.25 or when Nch  = 4) but it is more precise than the polar approach. We also demonstrate that the mfD is robust in all conditions and it is the most precise strategy; although it does not strictly provide the fraction of interacting donor. We show using the fast-FLIM prototype (with an acquisition rate up to 1 Hz) that these non-fitting strategies are very powerful for on-line analysis on a standard computer and thus for quantifying automatically the spatio-temporal activation of Rac-GTPase in living cells by FRET.

Leray A, Padilla-Parra S, Roul J, Héliot L, Tramier M. 2013. Correction: 827Spatio-Temporal Quantification of FRET in Living Cells by Fast Time-Domain FLIM: A Comparative Study of Non-Fitting Methods. PLoS One, 8 (8), | Show Abstract | Read more

[This corrects the article on p. e69335 in vol. 8.].

Padilla-Parra S, Matos PM, Kondo N, Marin M, Santos NC, Melikyan GB. 2012. Quantitative imaging of endosome acidification and single retrovirus fusion with distinct pools of early endosomes. Proc Natl Acad Sci U S A, 109 (43), pp. 17627-17632. | Show Abstract | Read more

Diverse enveloped viruses enter host cells through endocytosis and fuse with endosomal membranes upon encountering acidic pH. Currently, the pH dynamics in virus-carrying endosomes and the relationship between acidification and viral fusion are poorly characterized. Here, we examined the entry of avian retrovirus that requires two sequential stimuli--binding to a cognate receptor and low pH--to undergo fusion. A genetically encoded sensor incorporated into the viral membrane was used to measure the pH in virus-carrying endosomes. Acid-induced virus fusion was visualized as the release of a fluorescent viral content marker into the cytosol. The pH values in early acidic endosomes transporting the virus ranged from 5.6 to 6.5 but were relatively stable over time for a given vesicle. Analysis of viral motility and luminal pH showed that cells expressing the transmembrane isoform of the receptor (TVA950) preferentially sorted the virus into slowly trafficking, less acidic endosomes. In contrast, viruses internalized by cells expressing the GPI-anchored isoform (TVA800) were uniformly distributed between stationary and mobile compartments. We found that the lag times between acidification and fusion were significantly shorter and fusion pores were larger in dynamic endosomes than in more stationary compartments. Despite the same average pH within mobile compartments of cells expressing alternative receptor isoforms, TVA950 supported faster fusion than TVA800 receptor. Collectively, our results suggest that fusion steps downstream of the low-pH trigger are modulated by properties of intracellular compartments harboring the virus.

Demirkhanyan LH, Marin M, Padilla-Parra S, Zhan C, Miyauchi K, Jean-Baptiste M, Novitskiy G, Lu W, Melikyan GB. 2012. Multifaceted mechanisms of HIV-1 entry inhibition by human α-defensin. J Biol Chem, 287 (34), pp. 28821-28838. | Show Abstract | Read more

The human neutrophil peptide 1 (HNP-1) is known to block the human immunodeficiency virus type 1 (HIV-1) infection, but the mechanism of inhibition is poorly understood. We examined the effect of HNP-1 on HIV-1 entry and fusion and found that, surprisingly, this α-defensin inhibited multiple steps of virus entry, including: (i) Env binding to CD4 and coreceptors; (ii) refolding of Env into the final 6-helix bundle structure; and (iii) productive HIV-1 uptake but not internalization of endocytic markers. Despite its lectin-like properties, HNP-1 could bind to Env, CD4, and other host proteins in a glycan- and serum-independent manner, whereas the fusion inhibitory activity was greatly attenuated in the presence of human or bovine serum. This demonstrates that binding of α-defensin to molecules involved in HIV-1 fusion is necessary but not sufficient for blocking the virus entry. We therefore propose that oligomeric forms of defensin, which may be disrupted by serum, contribute to the anti-HIV-1 activity perhaps through cross-linking virus and/or host glycoproteins. This notion is supported by the ability of HNP-1 to reduce the mobile fraction of CD4 and coreceptors in the plasma membrane and to precipitate a core subdomain of Env in solution. The ability of HNP-1 to block HIV-1 uptake without interfering with constitutive endocytosis suggests a novel mechanism for broad activity against this and other viruses that enter cells through endocytic pathways.

Padilla-Parra S, Tramier M. 2012. FRET microscopy in the living cell: different approaches, strengths and weaknesses. Bioessays, 34 (5), pp. 369-376. | Show Abstract | Read more

New imaging methodologies in quantitative fluorescence microscopy, such as Förster resonance energy transfer (FRET), have been developed in the last few years and are beginning to be extensively applied to biological problems. FRET is employed for the detection and quantification of protein interactions, and of biochemical activities. Herein, we review the different methods to measure FRET in microscopy, and more importantly, their strengths and weaknesses. In our opinion, fluorescence lifetime imaging microscopy (FLIM) is advantageous for detecting inter-molecular interactions quantitatively, the intensity ratio approach representing a valid and straightforward option for detecting intra-molecular FRET. Promising approaches in single molecule techniques and data analysis for quantitative and fast spatio-temporal protein-protein interaction studies open new avenues for FRET in biological research.

Padilla-Parra S, Marin M, Melikian GB. 2012. Imaging Single Virus Entry and Fusion in Live Cells Microscopy and Microanalysis, 18 (S2), pp. 1968-1969. | Read more

Padilla-Parra S, Marin M, Kondo N, Melikyan GB. 2012. Synchronized retrovirus fusion in cells expressing alternative receptor isoforms releases the viral core into distinct sub-cellular compartments. PLoS Pathog, 8 (5), pp. e1002694. | Show Abstract | Read more

Disparate enveloped viruses initiate infection by fusing with endosomes. However, the highly diverse and dynamic nature of endosomes impairs mechanistic studies of fusion and identification of sub-cellular sites supporting the nucleocapsid release. We took advantage of the extreme stability of avian retrovirus-receptor complexes at neutral pH and of acid-dependence of virus-endosome fusion to isolate the latter step from preceding asynchronous internalization/trafficking steps. Viruses were trapped within endosomes in the presence of NH₄Cl. Removal of NH₄Cl resulted in a quick and uniform acidification of all subcellular compartments, thereby initiating synchronous viral fusion. Single virus imaging demonstrated that fusion was initiated within seconds after acidification and often culminated in the release of the viral core from an endosome. Comparative studies of cells expressing either the transmembrane or GPI-anchored receptor isoform revealed that the transmembrane receptor delivered the virus to more fusion-permissive compartments. Thus the identity of endosomal compartments, in addition to their acidity, appears to modulate viral fusion. A more striking manifestation of the virus delivery to distinct compartments in the presence of NH₄Cl was the viral core release into the cytosol of cells expressing the transmembrane receptor and into endosomes of cells expressing the GPI-anchored isoform. In the latter cells, the newly released cores exhibited restricted mobility and were exposed to a more acidic environment than the cytoplasm. These cores appear to enter into the cytosol after an additional slow temperature-dependent step. We conclude that the NH₄Cl block traps the virus within intralumenal vesicles of late endosomes in cells expressing the GPI-anchored receptor. Viruses surrounded by more than one endosomal membrane release their core into the cytoplasm in two steps--fusion with an intralumenal vesicle followed by a yet unknown temperature-dependent step that liberates the core from late endosomes.

Padilla-Parra S, Audugé N, Coppey-Moisan M, Tramier M. 2011. Dual-color fluorescence lifetime correlation spectroscopy to quantify protein-protein interactions in live cell. Microsc Res Tech, 74 (8), pp. 788-793. | Show Abstract | Read more

Dual-color fluorescence correlation spectroscopy is an interesting method to quantify protein interaction in living cells. But, when performing these experiments, one must compensate for a known spectral bleed through artifact that corrupts cross-correlation data. In this article, problems with crosstalk were overcome with an approach based on fluorescence lifetime correlation spectroscopy (FLCS). We show that FLCS applied to dual-color EGFP and mCherry cross-correlation allows the determination of protein-protein interactions in living cells without the need of spectral bleed through calibration. The methodology was validated by using EGFP-mCherry tandem in comparison with coexpressed EGFP and mCherry in live cell. The dual-color FLCS experimental procedure where the different laser intensities do not have to be controlled during experiment is really very helpful to study quantitatively protein interactions in live sample.

Padilla-Parra S, Auduge N, Coppey-Moisan M, Tramier M. 2011. Non fitting based FRET–FLIM analysis approaches applied to quantify protein–protein interactions in live cells Biophysical Reviews, 3 (2), pp. 63-70. | Show Abstract | Read more

New imaging methodologies in quantitative fluorescence microscopy and nanoscopy have been developed in the last few years and are beginning to be extensively applied to biological problems, such as the localization and quantification of protein interactions. Fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM) is currently employed not only in biophysics or chemistry but also in bio-medicine, thanks to new advancements in technology and also new developments in data treatment. FRET-FLIM can be a very useful tool to ascertain protein interactions occurring in single living cells. In this review, we stress the importance of increasing the acquisition speed when working in vivo employing Time-Domain FLIM. The development of the new mathematical-based non-fitting methods allows the determining of the fraction of interacting donor without the requirement of high count statistics, and thus allows the performing of high speed acquisitions in FRET-FLIM to still be quantitative. © 2011 International Union for Pure and Applied Biophysics (IUPAB) and Springer.

Padilla-Parra S, Auduge N, Coppey-Moisan M, Tramier M. 2011. Non fitting based FRET-FLIM analysis approaches applied to quantify protein-protein interactions in live cells Biophysical Reviews, 3 (2), pp. 63-70. | Show Abstract | Read more

New imaging methodologies in quantitative fluorescence microscopy and nanoscopy have been developed in the last few years and are beginning to be extensively applied to biological problems, such as the localization and quantification of protein interactions. Fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM) is currently employed not only in biophysics or chemistry but also in bio-medicine, thanks to new advancements in technology and also new developments in data treatment. FRET-FLIM can be a very useful tool to ascertain protein interactions occurring in single living cells. In this review, we stress the importance of increasing the acquisition speed when working in vivo employing Time-Domain FLIM. The development of the new mathematical-based non-fitting methods allows the determining of the fraction of interacting donor without the requirement of high count statistics, and thus allows the performing of high speed acquisitions in FRET-FLIM to still be quantitative. © 2011 International Union for Pure and Applied Biophysics (IUPAB) and Springer.

Yamada H, Padilla-Parra S, Park SJ, Itoh T, Chaineau M, Monaldi I, Cremona O, Benfenati F, De Camilli P, Coppey-Moisan M et al. 2009. Dynamic interaction of amphiphysin with N-WASP regulates actin assembly. J Biol Chem, 284 (49), pp. 34244-34256. | Show Abstract | Read more

Amphiphysin 1, an endocytic adaptor concentrated at synapses that couples clathrin-mediated endocytosis to dynamin-dependent fission, was also shown to have a regulatory role in actin dynamics. Here, we report that amphiphysin 1 interacts with N-WASP and stimulates N-WASP- and Arp2/3-dependent actin polymerization. Both the Src homology 3 and the N-BAR domains are required for this stimulation. Acidic liposome-triggered, N-WASP-dependent actin polymerization is strongly impaired in brain cytosol of amphiphysin 1 knock-out mice. FRET-FLIM analysis of Sertoli cells, where endogenously expressed amphiphysin 1 co-localizes with N-WASP in peripheral ruffles, confirmed the association between the two proteins in vivo. This association undergoes regulation and is enhanced by stimulating phosphatidylserine receptors on the cell surface with phosphatidylserine-containing liposomes that trigger ruffle formation. These results indicate that actin regulation is a key function of amphiphysin 1 and that such function cooperates with the endocytic adaptor role and membrane shaping/curvature sensing properties of the protein during the endocytic reaction.

Padilla-Parra S, Audugé N, Lalucque H, Mevel JC, Coppey-Moisan M, Tramier M. 2009. Quantitative comparison of different fluorescent protein couples for fast FRET-FLIM acquisition. Biophys J, 97 (8), pp. 2368-2376. | Show Abstract | Read more

The fluorescent-protein based fluorescence resonance energy transfer (FRET) approach is a powerful method for quantifying protein-protein interactions in living cells, especially when combined with fluorescence lifetime imaging microscopy (FLIM). To compare the performance of different FRET couples for FRET-FLIM experiments, we first tested enhanced green fluorescent protein (EGFP) linked to different red acceptors (mRFP1-EGFP, mStrawberry-EGFP, HaloTag (TMR)-EGFP, and mCherry-EGFP). We obtained a fraction of donor engaged in FRET (f(D)) that was far from the ideal case of one, using different mathematical models assuming a double species model (i.e., discrete double exponential fixing the donor lifetime and double exponential stretched for the FRET lifetime). We show that the relatively low f(D) percentages obtained with these models may be due to spectroscopic heterogeneity of the acceptor population, which is partially caused by different maturation rates for the donor and the acceptor. In an attempt to improve the amount of donor protein engaged in FRET, we tested mTFP1 as a donor coupled to mOrange and EYFP, respectively. mTFP1 turned out to be at least as good as EGFP for donor FRET-FLIM experiments because 1), its lifetime remained constant during light-induced fluorescent changes; 2), its fluorescence decay profile was best fitted with a single exponential model; and 3), no photoconversion was detected. The f(D) value when combined with EYFP as an acceptor was the highest of all tandems tested (0.7). Moreover, in the context of fast acquisitions, we obtained a minimal f(D) (mf(D)) for mTFP1-EYFP that was almost two times greater than that for mCherry-EGFP (0.65 vs. 0.35). Finally, we compared EGFP and mTFP1 in a biological situation in which the fusion proteins were highly immobile, and EGFP and mTFP1 were linked to the histone H4 (EGFP-H4 and mTFP1-H4) in fast FLIM acquisitions. In this particular case, the fluorescence intensity was more stable for EGFP-H4 than for mTFP1-H4. Nevertheless, we show that mTFP1/EYFP stands alone as the best FRET-FLIM couple in terms of f(D) analysis.

Padilla-Parra S, Audugé N, Coppey-Moisan M, Tramier M. 2008. Quantitative FRET analysis by fast acquisition time domain FLIM at high spatial resolution in living cells. Biophys J, 95 (6), pp. 2976-2988. | Show Abstract | Read more

Quantitative analysis in Förster resonance energy transfer (FRET) experiments in live cells for protein interaction studies is still a challenging issue. In a two-component system (FRET and no FRET donor species), fitting of fluorescence lifetime imaging microscopy (FLIM) data gives the fraction of donor molecules involved in FRET (f(D)) and the intrinsic transfer efficiency. But when fast FLIM acquisitions are used to monitor dynamic changes in protein-protein interactions at high spatial and temporal resolutions in living cells, photon statistics and time resolution are limited. In this case, fitting procedures are not reliable, even for single lifetime donors. We introduce the new concept of a minimal fraction of donor molecules involved in FRET (mf(D)), coming from the mathematical minimization of f(D). We find particular advantage in the use of mf(D) because it can be obtained without fitting procedures and it is derived directly from FLIM data. mf(D) constitutes an interesting quantitative parameter for live cell studies because it is related to the minimal relative concentration of interacting proteins. For multi-lifetime donors, the process of fitting complex fluorescence decays to find at least four reliable lifetimes is a near impossible task. Here, mf(D) extension for multi-lifetime donors is the only quantitative determinant. We applied this methodology for imaging the interaction between the bromodomains of TAF(II250) and acetylated histones H4 in living cells at high resolution. We show the existence of discrete acetylated chromatin domains where the minimal fraction of bromodomain interacting with acetylated H4 oscillates from 0.26 to 0.36 and whose size is smaller than half of one micron cube. We demonstrate that mf(D) by itself is a useful tool to investigate quantitatively protein interactions in live cells, especially when using fast FRET-FLIM acquisition times.

Jakobsdottir GM, Iliopoulou M, Nolan R, Alvarez L, Compton AA, Padilla-Parra S. 2017. On the Whereabouts of HIV-1 Cellular Entry and Its Fusion Ports Trends in Molecular Medicine, | Show Abstract | Read more

© 2017 Elsevier Ltd. 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. Recent studies of HIV-1 infection have described the role of dynamin-2 (DNM2) in stabilization of the fusion pore in the plasma membrane of CD4 + T cells, and also shown that DNM2 in these cells might not be involved in HIV-1 virion endocytosis.The role of endocytosis during cell-cell HIV-1 transmission is controversial: some reports state that HIV-1 might mature and fuse within endosomal compartments in the target cell, while others state that endocytosis might not lead to productive infection. In this scenario, the virus would be recycled back towards the plasma membrane and fuse. This is important because endosomal delivery of the virus could serve to evade neutralization by circulating HIV-1-specific antibodies.Recent studies have highlighted the importance of the role played by host cells during HIV-1 entry. Indeed, inhibition of HIV-1 entry can be modulated by innate immunity, and by restriction factors such as IFITM and SERINC.

Nolan R, Alvarez LAJ, Elegheert J, Iliopoulou M, Jakobsdottir GM, Rodriguez-Muñoz M, Aricescu AR, Padilla-Parra S. 2017. nandb—number and brightness in R with a novel automatic detrending algorithm Bioinformatics, | Read more

Russell RA, Chojnacki J, Jones DM, Johnson E, Do T, Eggeling C, Padilla-Parra S, Sattentau QJ. 2017. Astrocytes Resist HIV-1 Fusion but Engulf Infected Macrophage Material. Cell Rep, 18 (6), pp. 1473-1483. | Show Abstract | Read more

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.

Jones DM, Alvarez LA, Nolan R, Ferriz M, Sainz Urruela R, Massana-Muñoz X, Novak-Kotzer H, Dustin ML, Padilla-Parra S. 2017. Dynamin-2 Stabilizes the HIV-1 Fusion Pore with a Low Oligomeric State. Cell Rep, 18 (2), pp. 443-453. | Show Abstract | Read more

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.

Padilla-Parra S, Dustin ML. 2016. Actin Dynamics and HIV-1 Entry. Trends Mol Med, 22 (5), pp. 354-356. | Show Abstract | Read more

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.

Jones DM, Padilla-Parra S. 2015. Imaging real-time HIV-1 virion fusion with FRET-based biosensors. Sci Rep, 5 (1), pp. 13449. | Show Abstract | Read more

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.

Padilla-Parra S, Marin M, Kondo N, Melikyan GB. 2014. Pinpointing retrovirus entry sites in cells expressing alternatively spliced receptor isoforms by single virus imaging. Retrovirology, 11 (1), pp. 47. | Show Abstract | Read more

BACKGROUND: 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. RESULTS: Here, we visualized single avian retrovirus co-trafficking with markers for early (Rab5) and late (Rab7) endosomes, acidification of endosomal lumen and the resulting viral fusion measured by the viral content release into the cytoplasm. Virus-carrying vesicles either merged with the existing Rab5-positive early endosomes or slowly accumulated Rab5. The Rab5 recruitment to virus-carrying endosomes correlated with acidification of their lumen. Viral fusion occurred either in early (Rab5-positive) or intermediate (Rab5- and Rab7-positive) compartments. Interestingly, different isoforms of the cognate receptor directed virus entry from distinct endosomes. In cells expressing the transmembrane receptor, viruses preferentially entered and fused with slowly maturing early endosomes prior to accumulation of Rab7. By comparison, in cells expressing the GPI-anchored receptor, viruses entered both slowly and quickly maturing endosomes and fused with early (Rab5-positive) and intermediate (Rab5- and Rab7-positive) compartments. CONCLUSIONS: Since the rate of low pH-triggered fusion was independent of the receptor isoform, we concluded that the sites of virus entry are determined by the kinetic competition between endosome maturation and viral fusion. Our findings demonstrate the ability of this retrovirus to enter cells via alternative endocytic pathways and establish infection by releasing its content from distinct endosomal compartments.

Zhao Y, Ren J, Padilla-Parra S, Fry EE, Stuart DI. 2014. Lysosome sorting of β-glucocerebrosidase by LIMP-2 is targeted by the mannose 6-phosphate receptor. Nat Commun, 5 pp. 4321. | Show Abstract | Read more

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.

Padilla-Parra S, Marin M, Gahlaut N, Suter R, Kondo N, Melikyan GB. 2013. Fusion of mature HIV-1 particles leads to complete release of a gag-GFP-based content marker and raises the intraviral pH. PLoS One, 8 (8), pp. e71002. | Show Abstract | Read more

By imaging the release of a GFP-based viral content marker produced upon virus maturation, we have previously found that HIV-1 fuses with endosomes. In contrast, fusion at the cell surface did not progress beyond a lipid mixing stage (hemifusion). However, recent evidence suggesting that free GFP can be trapped within the mature HIV-1 capsid raises concerns that this content marker may not be released immediately after the formation of a fusion pore. To determine whether a significant portion of GFP is trapped in the mature capsid, we first permeabilized the viral membrane with saponin. The overwhelming majority of pseudoviruses fully released GFP while the remaining particles exhibited partial loss or no loss of content. The extent of GFP release correlated with HIV-1 maturation, implying that incomplete Gag processing, but not GFP entrapment by mature capsids, causes partial content release. Next, we designed a complementary assay for visualizing pore formation by monitoring the intraviral pH with an additional pH-sensitive fluorescent marker. The loss of GFP through saponin-mediated pores was associated with a concomitant increase in the intraviral pH due to equilibration with the pH of an external buffer. We next imaged single HIV-cell fusion and found that these events were manifested in a highly correlated loss of content and increase in the intraviral pH, as it equilibrated with the cytosolic pH. Fused or saponin-permeabilized pseudoviruses that partially lost GFP did not release the remaining content marker under conditions expected to promote the capsid dissociation. We were thus unable to detect significant entrapment of GFP by the mature HIV-1 capsid. Together, our results validate the use of the GFP-based content marker for imaging single virus fusion and inferring the sites of HIV-1 entry.

Padilla-Parra S, Matos PM, Kondo N, Marin M, Santos NC, Melikyan GB. 2012. Quantitative imaging of endosome acidification and single retrovirus fusion with distinct pools of early endosomes. Proc Natl Acad Sci U S A, 109 (43), pp. 17627-17632. | Show Abstract | Read more

Diverse enveloped viruses enter host cells through endocytosis and fuse with endosomal membranes upon encountering acidic pH. Currently, the pH dynamics in virus-carrying endosomes and the relationship between acidification and viral fusion are poorly characterized. Here, we examined the entry of avian retrovirus that requires two sequential stimuli--binding to a cognate receptor and low pH--to undergo fusion. A genetically encoded sensor incorporated into the viral membrane was used to measure the pH in virus-carrying endosomes. Acid-induced virus fusion was visualized as the release of a fluorescent viral content marker into the cytosol. The pH values in early acidic endosomes transporting the virus ranged from 5.6 to 6.5 but were relatively stable over time for a given vesicle. Analysis of viral motility and luminal pH showed that cells expressing the transmembrane isoform of the receptor (TVA950) preferentially sorted the virus into slowly trafficking, less acidic endosomes. In contrast, viruses internalized by cells expressing the GPI-anchored isoform (TVA800) were uniformly distributed between stationary and mobile compartments. We found that the lag times between acidification and fusion were significantly shorter and fusion pores were larger in dynamic endosomes than in more stationary compartments. Despite the same average pH within mobile compartments of cells expressing alternative receptor isoforms, TVA950 supported faster fusion than TVA800 receptor. Collectively, our results suggest that fusion steps downstream of the low-pH trigger are modulated by properties of intracellular compartments harboring the virus.

Demirkhanyan LH, Marin M, Padilla-Parra S, Zhan C, Miyauchi K, Jean-Baptiste M, Novitskiy G, Lu W, Melikyan GB. 2012. Multifaceted mechanisms of HIV-1 entry inhibition by human α-defensin. J Biol Chem, 287 (34), pp. 28821-28838. | Show Abstract | Read more

The human neutrophil peptide 1 (HNP-1) is known to block the human immunodeficiency virus type 1 (HIV-1) infection, but the mechanism of inhibition is poorly understood. We examined the effect of HNP-1 on HIV-1 entry and fusion and found that, surprisingly, this α-defensin inhibited multiple steps of virus entry, including: (i) Env binding to CD4 and coreceptors; (ii) refolding of Env into the final 6-helix bundle structure; and (iii) productive HIV-1 uptake but not internalization of endocytic markers. Despite its lectin-like properties, HNP-1 could bind to Env, CD4, and other host proteins in a glycan- and serum-independent manner, whereas the fusion inhibitory activity was greatly attenuated in the presence of human or bovine serum. This demonstrates that binding of α-defensin to molecules involved in HIV-1 fusion is necessary but not sufficient for blocking the virus entry. We therefore propose that oligomeric forms of defensin, which may be disrupted by serum, contribute to the anti-HIV-1 activity perhaps through cross-linking virus and/or host glycoproteins. This notion is supported by the ability of HNP-1 to reduce the mobile fraction of CD4 and coreceptors in the plasma membrane and to precipitate a core subdomain of Env in solution. The ability of HNP-1 to block HIV-1 uptake without interfering with constitutive endocytosis suggests a novel mechanism for broad activity against this and other viruses that enter cells through endocytic pathways.

Padilla-Parra S, Tramier M. 2012. FRET microscopy in the living cell: different approaches, strengths and weaknesses. Bioessays, 34 (5), pp. 369-376. | Show Abstract | Read more

New imaging methodologies in quantitative fluorescence microscopy, such as Förster resonance energy transfer (FRET), have been developed in the last few years and are beginning to be extensively applied to biological problems. FRET is employed for the detection and quantification of protein interactions, and of biochemical activities. Herein, we review the different methods to measure FRET in microscopy, and more importantly, their strengths and weaknesses. In our opinion, fluorescence lifetime imaging microscopy (FLIM) is advantageous for detecting inter-molecular interactions quantitatively, the intensity ratio approach representing a valid and straightforward option for detecting intra-molecular FRET. Promising approaches in single molecule techniques and data analysis for quantitative and fast spatio-temporal protein-protein interaction studies open new avenues for FRET in biological research.

1187

Thank you for registering your interest

We were unable to record your request to register for interest in future opportunities. Please try again and if problems persist contact us at webteam@ndm.ox.ac.uk