Physics, biology and the right chemistry

Joint studies that involve biologists and physicists are becoming more frequent and have contributed to the identification and understanding of physical parameters underlying key biological processes. Here, we illustrate the main findings resulting from a 10-year collaboration between a cell biologist and an experimental physicist, both interested in the mechanisms of intracellular transport and membrane dynamics in eukaryotic cells

Golgi localisation of GMAP210 requires two distinct cis-membrane binding mechanisms

<p>Abstract</p> <p>Background</p> <p>The Golgi apparatus in mammals appears as a ribbon made up of interconnected stacks of flattened cisternae that is positioned close to the centrosome in a microtubule-dependent manner. How this organisation is achieved and retained is not well understood. GMAP210 is a long coiled-coil cis-Golgi associated protein that plays a role in maintaining Golgi ribbon integrity and position and contributes to the formation of the primary cilium. An amphipathic alpha-helix able to bind liposomes <it>in vitro </it>has been recently identified at the first 38 amino acids of the protein (amphipathic lipid-packing sensor motif), and an ARF1-binding domain (Grip-related Arf-binding domain) was found at the C-terminus. To which type of membranes these two GMAP210 regions bind <it>in vivo </it>and how this contributes to GMAP210 localisation and function remains to be investigated.</p> <p>Results</p> <p>By using truncated as well as chimeric mutants and videomicroscopy we found that both the N-terminus and the C-terminus of GMAP210 are targeted to the cis-Golgi <it>in vivo</it>. The ALPS motif was identified as the N-terminal binding motif and appeared concentrated in the periphery of Golgi elements and between Golgi stacks. On the contrary, the C-terminal domain appeared uniformly distributed in the cis-cisternae of the Golgi apparatus. Strikingly, the two ends of the protein also behave differently in response to the drug Brefeldin A. The N-terminal domain redistributed to the endoplasmic reticulum (ER) exit sites, as does the full-length protein, whereas the C-terminal domain rapidly dissociated from the Golgi apparatus to the cytosol. Mutants comprising the full-length protein but lacking one of the terminal motifs also associated with the cis-Golgi with distribution patterns similar to those of the corresponding terminal end whereas a mutant consisting in fused N- and C-terminal ends exhibits identical localisation as the endogenous protein.</p> <p>Conclusion</p> <p>We conclude that the Golgi localisation of GMAP210 is the result of the combined action of the two N- and C-terminal domains that recognise different sub-regions of the cis-GA. Based on present and previous data, we propose a model in which GMAP210 would participate in homotypic fusion of cis-cisternae by anchoring the surface of cisternae via its C-terminus and projecting its distal N-terminus to bind the rims or to stabilise tubular structures connecting neighbouring cis-cisternae.</p

The late endocytic Rab39a GTPase regulates the interaction between multivesicular bodies and chlamydial inclusions.

Given their obligate intracellular lifestyle, Chlamydia trachomatis ensure their access to multiple host sources of essential lipids by interfering vesicular transport. These bacteria hijack Rab6-, Rab11- and Rab14-controlled trafficking pathways to acquire sphingomyelin from the Golgi apparatus. Another important source of sphingolipids, phospholipids and cholesterol are multivesicular bodies (MVBs). Despite their participation in chlamydial inclusion development and bacterial replication, the molecular mechanisms mediating MVBs-inclusion interaction remain unknown. In the present study, we demonstrate that Rab39a labels a subset of late endocytic vesicles -mainly MVBs- that move along microtubules. Moreover, Rab39a is actively recruited to chlamydial inclusions throughout the pathogen life cycle by a bacterial-driven process and depending on its GTP/GDP binding state. Interestingly, Rab39a participates in the delivery of MVB and host sphingolipids to maturing chlamydial inclusions thereby promoting inclusion growth and bacterial development. Altogether, our findings indicate that Rab39a favours chlamydial replication and infectivity. This is the first report showing a late endocytic Rab GTPase involved in chlamydial infection development.Fil: Gambarte Tudela, Julian Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Capmany, Anahi. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Romao, Maryse. Institute Curie; FranciaFil: Quintero, Cristian Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Miserey Lenkei, Stephanie. Institute Curie; FranciaFil: Raposo, Graça. Institute Curie; FranciaFil: Goud, Bruno. Institute Curie; FranciaFil: Damiani, Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentin

Synthesis and Characterization of an Epidermal Growth Factor Receptor selective Ru(II) Polypyridyl‐Nanobody Conjugate as a Photosensitizer for Photodynamic Therapy

International audienceThere is currently a surge for the development of novel photosensitizers (PSs) for photodynamic therapy (PDT) since those currently approved are not completely ideal. Among the tested compounds, we have previously investigated the use of Ru(II) polypyridyl complexes with a [Ru(bipy)2(dppz)]2+ and [Ru(phen)2(dppz)]2+ scaffold (bipy = 2,2'‐bipyridine; dppz = dipyrido[3,2‐a:2′,3′‐c]‐phenazine, phen = 1,10‐phenanthroline). These complexes selectively target DNA. However, since DNA is ubiquitous, it would be of great interest to increase the selectivity of our PDT PSs by linking them to a targeting vector in view of targeted PDT. Herein, we present the synthesis, characterization and in‐depth photophysical evaluation of a nanobody‐containing Ru(II) polypyridyl conjugate selective for the epidermal growth factor receptor (EGFR) in view of targeted PDT. Using ICP‐MS and confocal microscopy, we could demonstrate that our conjugate had a high selectivity for the EGFR receptor, which is a crucial oncological target as it is overexpressed and/or deregulated in a variety of solid tumors. However, contrary to expectations, this conjugate was found to not produce reactive oxygen species (ROS) in cancer cells and to be therefore not phototoxic

Characterization of single chain antibody targets through yeast two hybrid

<p>Abstract</p> <p>Background</p> <p>Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful <it>in vitro </it>selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain.</p> <p>Results</p> <p>We describe an approach that allows detailed characterization of a given antibody's target(s) using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID), efficiently narrowing the epitope-containing region.</p> <p>Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed.</p> <p>Conclusions</p> <p>Our method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise domain mapping for linear epitopes, confirmation of non-linear epitopes for conformational sensors, and detection of secondary binding partners. This approach may thus prove to be an elegant and rapid method for the target characterization of newly obtained scFv antibodies. It may be considered prior to any research application and particularly before any use of such recombinant antibodies in clinical medicine.</p

Patch-Based Markov Models for Event Detection in Fluorescence Bioimaging

International audienceThe study of protein dynamics is essential for understanding the multi-molecular complexes at subcellular levels. Fluorescent Protein (XFP)-tagging and time-lapse fluorescence microscopy enable to observe molecular dynamics and interactions in live cells, unraveling the live states of the matter. Original image analysis methods are then required to process challenging 2D or 3D image sequences. Recently, tracking methods that estimate the whole trajectories of moving objects have been successfully developed. In this paper, we address rather the detection of meaningful events in spatio-temporal fluorescence image sequences, such as apparent stable "stocking areas" involved in membrane transport. We propose an original patch-based Markov modeling to detect spatial irregularities in fluorescence images with low false alarm rates. This approach has been developed for real image sequences of cells expressing XFP-tagged Rab proteins, known to regulate membrane trafficking