Elaine Del Nery

Bicaudal-D regulates COPI-independent Golgi-ER transport by recruiting the dynein-dynactin motor complex

The small GTPase Rab6a is involved in the regulation of membrane traffic from the Golgi apparatus towards the endoplasmic reticulum (ER) in a coat complex coatomer protein I (COPI)-independent pathway. Here, we used a yeast two-hybrid approach to identify binding partners of Rab6a. In particular, we identified the dynein–dynactin-binding protein Bicaudal-D1 (BICD1), one of the two mammalian homologues of Drosophila Bicaudal-D. BICD1 and BICD2 colocalize with Rab6a on the trans-Golgi network (TGN) and on cytoplasmic vesicles, and associate with Golgi membranes in a Rab6-dependent manner. Overexpression of BICD1 enhances the recruitment of dynein–dynactin to Rab6a-containing vesicles. Conversely, overexpression of the carboxy-terminal domain of BICD, which can interact with Rab6a but not with cytoplasmic dynein, inhibits microtubule minus-end-directed movement of green fluorescent protein (GFP)–Rab6a vesicles and induces an accumulation of Rab6a and COPI-independent ER cargo in peripheral structures. These data suggest that coordinated action between Rab6a, BICD and the dynein–dynactin complex controls COPI-independent Golgi–ER transport.

Rab6A and Rab6A' GTPases play non-overlapping roles in membrane trafficking.

The closely related Rab6 isoforms, Rab6A and Rab6A′, have been shown to regulate vesicular trafficking within the Golgi and post‐Golgi compartments, but studies using dominant active or negative mutant suggested conflicting models. Here, we report that reduction in the expression of Rab6 isoform using specific small interfering RNA reveals noticeable differences in the Rab6A and Rab6A′ biological functions. Surprisingly, Rab6A seems to be largely dispensable in membrane trafficking events, whereas knocking down the expression of Rab6A′ hampers the intracellular transport of the retrograde cargo marker, the Shiga Toxin B‐subunit along the endocytic pathway, and causes defects in Golgi‐ associated protein recycling through the endoplasmic reticulum. We also showed that Rab6A′ is required for cell cycle progression through mitosis and identify Ile62 as a key residue for uncoupling Rab6A′ functions in mitosis and retrograde trafficking. Thus, our work shows that Rab6A and Rab6A′ perform different functions within the cell and suggests a novel role for Rab6A′ as the major Rab6 isoform regulating previously described Rab6‐dependent transport pathways.

Screening out irrelevant cell-based models of disease.

The common and persistent failures to translate promising preclinical drug candidates into clinical success highlight the limited effectiveness of disease models currently used in drug discovery. An apparent reluctance to explore and adopt alternative cell- and tissue-based model systems, coupled with a detachment from clinical practice during assay validation, contributes to ineffective translational research. To help address these issues and stimulate debate, here we propose a set of principles to facilitate the definition and development of disease-relevant assays, and we discuss new opportunities for exploiting the latest advances in cell-based assay technologies in drug discovery, including induced pluripotent stem cells, three-dimensional (3D) co-culture and organ-on-a-chip systems, complemented by advances in single-cell imaging and gene editing technologies. Funding to support precompetitive, multidisciplinary collaborations to develop novel preclinical models and cell-based screening technologies could have a key role in improving their clinical relevance, and ultimately increase clinical success rates.

Distinct roles of RalA and RalB in the progression of cytokinesis are supported by distinct RalGEFs

The Ras family G‐proteins RalA and RalB make critical non‐overlapping contributions to the generation of a tumorigenic regulatory network, supporting bypass of the normal restraints on both cell proliferation and survival. The Sec6/8 complex, or exocyst, has emerged as a principal direct effector complex for Ral GTPases. Here, we show that RalA and RalB support mitotic progression through mobilization of the exocyst for two spatially and kinetically distinct steps of cytokinesis. RalA is required to tether the exocyst to the cytokinetic furrow in early cytokinesis. RalB is then required for recruitment of the exocyst to the midbody of this bridge to drive abscission and completion of cytokinesis. The collaborative action of RalA and RalB is specified by discrete subcellular compartmentalization and unique pairs of RalGEF proteins that provide inputs from both Ras‐family protein‐dependent and protein‐independent regulatory cues. This suggests that Ral GTPases integrate diverse upstream signals to choreograph multiple roles for the exocyst in mitotic progression.

Cleavage of Toll-like receptor 3 by cathepsins B and H is essential for signaling

Toll-like receptor (TLR) 3 is an endosomal TLR that mediates immune responses against viral infections upon activation by its ligand double-stranded RNA, a replication intermediate of most viruses. TLR3 is expressed widely in the body and activates both the innate and adaptive immune systems. However, little is known about how TLR3 intracellular trafficking and maturation are regulated. Here we show that newly synthesized endogenous TLR3 is transported through the ER and Golgi apparatus to endosomes, where it is rapidly cleaved. TLR3 protein expression is up-regulated by its own ligand, leading to the accumulation of its cleaved form. In agreement with its proposed role as a transporter, UNC93B1 expression is required for TLR3 cleavage and signaling. Furthermore, TLR3 signaling and cleavage are sensitive to cathepsin inhibition. Cleavage occurs between aa 252 and 346, and results in a functional receptor that signals upon activation. A truncated form of TLR3 lacking the N-terminal 345 aa also signals from acidic compartments in response to ligand activation. Screening of the human cathepsin family by RNA interference identified cathepsins B and H as key mediators of TLR3 processing. Taken together, our data indicate that TLR3 proteolytic processing is essential for its function, and suggest a mechanism of tight control of TLR3 signaling and thus immunity.

A role for the Rab6A′ GTPase in the inactivation of the Mad2‐spindle checkpoint

The two isoforms of the Rab6 GTPase, Rab6A and Rab6A′, regulate a retrograde transport route connecting early endosomes and the endoplasmic reticulum via the Golgi complex in interphasic cells. Here we report that when Rab6A′ function is altered cells are unable to progress normally through mitosis. Such cells are blocked in metaphase, despite displaying a normal Golgi fragmentation and with the Mad2‐spindle checkpoint activated. Furthermore, the Rab6 effector p150Glued, a subunit of the dynein/dynactin complex, remains associated with some kinetochores. A similar phenotype was observed when GAPCenA, a GTPase‐activating protein of Rab6, was depleted from cells. Our results suggest that Rab6A′ likely regulates the dynamics of the dynein/dynactin complex at the kinetochores and consequently the inactivation of the Mad2‐spindle checkpoint. Rab6A′, through its interaction with p150Glued and GAPCenA, may thus participate in a pathway involved in the metaphase/anaphase transition.

A Novel Organelle Map Framework for High-Content Cell Morphology Analysis in High Throughput

A screening procedure was developed that takes advantage of the cellular normalization by micropatterning and a novel quantitative organelle mapping approach that allows unbiased and automated cell morphology comparison using black-box statistical testing. Micropatterns of extracellular matrix proteins force cells to adopt a reproducible shape and distribution of intracellular compartments avoiding strong cell-to-cell variation that is a major limitation of classical culture conditions. To detect changes in cell morphology induced by compound treatment, fluorescently labeled intracellular structures from several tens of micropatterned cells were transformed into probabilistic density maps. Then, the similarity or difference between two given density maps was quantified using statistical testing that evaluates differences directly from the data without additional analysis or any subjective decision. The versatility of this organelle mapping approach for different magnifications and its performance for different cell shapes has been assessed. Density-based analysis detected changes in cell morphology due to compound treatment in a small-scale proof-of-principle screen demonstrating its compatibility with high-throughput screening. This novel tool for high-content and high-throughput cellular phenotyping can potentially be used for a wide range of applications from drug screening to careful characterization of cellular processes.

A Probabilistic Model for Cell Population Phenotyping Using HCS Data

High Content Screening (HCS) platforms allow screening living cells under a wide range of experimental conditions and give access to a whole panel of cellular responses to a specific treatment. The outcome is a series of cell population images. Within these images, the heterogeneity of cellular response to the same treatment leads to a whole range of observed values for the recorded cellular features. Consequently, it is difficult to compare and interpret experiments. Moreover, the definition of phenotypic classes at a cell population level remains an open question, although this would ease experiments analyses. In the present work, we tackle these two questions. The input of the method is a series of cell population images for which segmentation and cellular phenotype classification has already been performed. We propose a probabilistic model to represent and later compare cell populations. The model is able to fully exploit the HCS-specific information: “dependence structure of population descriptors” and “within-population variability”. The experiments we carried out illustrate how our model accounts for this specific information, as well as the fact that the model benefits from considering them. We underline that these features allow richer HCS data analysis than simpler methods based on single cellular feature values averaged over each well. We validate an HCS data analysis method based on control experiments. It accounts for HCS specificities that were not taken into account by previous methods but have a sound biological meaning. Biological validation of previously unknown outputs of the method constitutes a future line of work.

MYO1C facilitates arrival at the Golgi apparatus through stabilization of branched actin

We aim at the identification of myosin motor proteins that control trafficking at the Golgi apparatus. In addition to the known Golgi-associated myosins MYO6, MYO18A and MYH9 (myosin IIA), we identify MYO1C as a novel player at the Golgi. We demonstrate that depletion of MYO1C induces Golgi apparatus fragmentation and decompaction. MYO1C accumulates at dynamic structures around the Golgi apparatus that colocalize with Golgi-associated actin dots. Interestingly, MYO1C depletion leads to loss of cellular F-actin, and Golgi apparatus decompaction is also observed after the inhibition or loss of the Arp2/3 complex. We show that the functional consequences of MYO1C depletion is a delay in the arrival of incoming transport carriers, both from the anterograde and retrograde routes. We propose that MYO1C stabilizes branched actin at the Golgi apparatus that facilitates the arrival of incoming transport at the Golgi.

Regulation of eIF4F complex by the peptidyl prolyl isomerase FKBP7 in taxane-resistant prostate cancer

Targeted therapies that exploit the signaling pathways involved in prostate cancer are required to overcome chemoresistance and improve treatment outcomes for men. Molecular chaperones play a key role in the regulation of protein homeostasis and are potential targets to alleviate chemoresistance. Using image-based high content siRNA functional screening based on a gene expression signature, we identified FKBP7, a molecular chaperone overexpressed in docetaxel-resistant and in cabazitaxel-resistant prostate cancer cells. FKBP7 was upregulated in human prostate cancers and correlated with the recurrence in patients receiving Docetaxel. FKBP7 silencing showed that FKBP7 is required to maintain the growth of chemoresistant cell lines and of chemoresistant tumors in mice. Mass spectrometry analysis revealed that FKBP7 interacts with the eIF4G component of the eIF4F translation initiation complex to mediate survival of chemoresistant cells. Using small molecule inhibitors of eIF4A, the RNA helicase component of eIF4F, we were able to overcome docetaxel and cabazitaxel resistance.