The Q loops of the human multidrug resistance transporter ABCB1 are necessary to couple drug binding to the ATP catalytic cycle.

For a primary active pump, such as the human ATP-binding-cassette (ABC) transporter ABCB1, coupling of drug-binding by the two transmembrane domains (TMDs) to the ATP catalytic cycle of the two nucleotide-binding domains (NBDs) is fundamental to the transport mechanism, but is poorly understood at the biochemical level. Structure data suggest that signals are transduced through intracellular loops of the TMDs that slot into grooves on the NBDs. At the base of these grooves is the Q loop. We therefore mutated the eponymous glutamine in one or both NBD Q loops and measured the effect on conformation and function by using a conformation-sensitive antibody (UIC2) and a fluorescent drug (Bodipy-verapamil), respectively. We showed that the double mutant is trapped in the inward-open state, which binds the drug, but cannot couple to the ATPase cycle. Our data also describe marked redundancy within the transport mechanism, because single-Q-loop mutants are functional for Bodipy-verapamil transport. This result allowed us to elucidate transduction pathways from twin drug-binding cavities to the Q loops using point mutations to favor one cavity over the other. Together, the data show that the Q loop is the central flexion point where the aspect of the drug-binding cavities is coupled to the ATP catalytic cycle.

Coxsackie adenovirus receptor (CAR) regulates integrin function through activation of p44/42 MAPK.

The coxsackie B virus and adenovirus receptor (CAR) is an attachment receptor for Adenovirus serotype 5 (Ad5) and in many cell types forms homodimers with neighbouring cells as part of a cell adhesion complex. CAR co-operates with cell surface integrin receptors to enable efficient viral entry, but little is known about the mechanism of crosstalk between these two receptor types. Here we show that overexpression of CAR in human epithelial cells leads to increased basal activation of p44/42 MAPK and this is required for efficient Ad5 infection. We demonstrate that CAR forms homodimers in cis and that this dimerisation is enhanced in the presence of Ad5 in a phospho-p44/42-dependent manner. CAR-induced p44/42 activation also leads to increased activation of beta1 and beta3 integrins. Analysis of CAR mutants demonstrates that the cyto domain of CAR is required for CAR-induced p44/42 activation, integrin activation and localisation to cell junctions. This data for the first time demonstrates that signalling downstream of CAR can have a dual effect on integrins and CAR itself in order to promote efficient viral binding to cell membranes.

Contribution of rare coding mutations in CD36 to type 2 diabetes and cardio-metabolic complications.

We sequenced coding regions of the cluster of differentiation 36 (CD36) gene in 184 French individuals of European ancestry presenting simultaneously with type 2 diabetes (T2D), arterial hypertension, dyslipidemia, and coronary heart disease. We identified rare missense mutations (p.Pro191Leu/rs143150225 and p.Ala252Val/rs147624636) in two heterozygous cases. The two CD36 mutation carriers had no family history of T2D and no clustering of cardio-metabolic complications. While the p.Pro191Leu mutation was found in 84 heterozygous carriers from five ethnic groups from the genome aggregation database (global frequency: 0.0297%, N = 141,321), only one European carrier of the p.Ala252Val mutation was identified (global frequency: 0.00040%, N = 125,523). The Pro191 and Ala252 amino acids were not conserved (74.8% and 68.9% across 131 animal species, respectively). In vitro experiments showed that the two CD36 mutant proteins are expressed and trafficked to the plasma membrane where they bind modified low-density-lipoprotein (LDL) cholesterol as normal. However, molecular modelling of the recent CD36 crystal structure showed that Pro191 was located at the exit/entrance gate of the lipid binding chamber and Ala252 was in line with the chamber. Overall, our data do not support a major contribution of CD36 rare coding mutations to T2D and its cardio-metabolic complications in the French population.

The use of fluorescence microscopy to visualise homotypic interactions of tomato spotted wilt virus nucleocapsid protein in living cells.

Fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) were employed to study homotypic protein-protein interactions in living cells. To this end, the nucleocapsid (N) protein of tomato spotted wilt virus (TSWV) was expressed as a fusion protein with either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP). Co-expression experiments of the two fusion proteins were carried out in baby hamster kidney (BHK21) cells. Both the wild type and the fusion proteins showed a peri-nuclear localisation pattern and were observed to form aggregates. In sensitised emission experiments, energy transfer was observed to take place from CFP to YFP when the two fluorophores were fused to TSWV N protein, indicating strongly homotypic interaction of the N proteins. This was confirmed by acceptor photobleaching studies as well as by FLIM experiments. All three methods showed interactions taking place, not only in the aggregates in the peri-nuclear region, but also throughout the cytoplasm. These experiments clearly demonstrated the potential of these fluorescence methods for studying the interactions of viral proteins in living cells.

CAR modulates E-cadherin dynamics in the presence of adenovirus type 5.

Adenovirus (Ad) serotype 5 (Ad5) fiber competitively binds to the coxsackievirus and Ad receptor (CAR) to attach Ad5 to target cells and also disrupts cell junctions and facilitates virus escape at a late stage in Ad5 infection. Here we demonstrate that paracellular permeability in MCF7 and CAR overexpressing MCF7 (FLCARMCF7) cells is increased within minutes following the addition of Ad5 to cells. This is brought about, at least in part, by altering the molecular dynamics of E-cadherin, a key component of the cell-cell adhesion complex. We also demonstrate that the increase in E-cadherin mobility is constitutively altered by the presence of CAR at FLCARMCF7 cell junctions. As increased paracellular permeability was observed early after the addition of Ad5 to cells, we postulate that this may represent a mechanism by which Ad5 could disrupt cell junctions to facilitate further access to its cell receptors.

The DIADEM data sets: representative light microscopy images of neuronal morphology to advance automation of digital reconstructions.

The comprehensive characterization of neuronal morphology requires tracing extensive axonal and dendritic arbors imaged with light microscopy into digital reconstructions. Considerable effort is ongoing to automate this greatly labor-intensive and currently rate-determining process. Experimental data in the form of manually traced digital reconstructions and corresponding image stacks play a vital role in developing increasingly more powerful reconstruction algorithms. The DIADEM challenge (short for DIgital reconstruction of Axonal and DEndritic Morphology) successfully stimulated progress in this area by utilizing six data set collections from different animal species, brain regions, neuron types, and visualization methods. The original research projects that provided these data are representative of the diverse scientific questions addressed in this field. At the same time, these data provide a benchmark for the types of demands automated software must meet to achieve the quality of manual reconstructions while minimizing human involvement. The DIADEM data underwent extensive curation, including quality control, metadata annotation, and format standardization, to focus the challenge on the most substantial technical obstacles. This data set package is now freely released ( http://diademchallenge.org ) to train, test, and aid development of automated reconstruction algorithms.

Tomato spotted wilt virus Gc and N proteins interact in vivo.

Tomato spotted wilt virus (TSWV) virions consist of a nucleocapsid core surrounded by a membrane containing glycoproteins Gn and Gc. To unravel the protein interactions involved in the membrane acquisition of RNPs, TSWV nucleocapsid protein (N), Gn and Gc were expressed and analyzed in BHK21 cells. Upon coexpression of Gn, Gc and N, a partial colocalization of N with both glycoproteins was observed in the Golgi region. In contrast, upon coexpression of Gc and N in the absence of Gn, both proteins colocalized to a distinct non-Golgi perinuclear region. Using FLIM and FRET, interaction was demonstrated between N and Gc, but not between N and Gn, and was only observed in the region where both proteins accumulated. The genuine character of N-Gc interaction was confirmed by its presence in purified virus and RNP preparations. The results are discussed in view of TSWV particle assembly taking place at the Golgi complex.

The cytoplasmic domain of tomato spotted wilt virus Gn glycoprotein is required for Golgi localisation and interaction with Gc.

Envelopment of tomato spotted wilt virus nucleocapsids occurs at the Golgi stacks of infected cells. This is also the place where the two membrane glycoproteins Gn and Gc accumulate upon coexpression. The required Golgi retention signal has previously been demonstrated to reside within Gn. Using a series of truncated Gn proteins, the Golgi retention signal was mapped to a stretch of 10 amino acids on this protein's cytoplasmic tail, 20 residues downstream the transmembrane domain. Studies on the intracellular distribution of chimeric Gc proteins in which the cytoplasmic tail and/or transmembrane domain were exchanged by those from Gn, demonstrated the additional requirement of the Gn transmembrane domain for Golgi targeting. Truncated Gn constructs lacking the C-terminal 20 amino acids but still localising to the Golgi were no longer able to redirect Gc, suggesting the requirement of this domain for interaction with Gc.