Detecting abnormal cell behaviors from dry mass time series.

The prediction of pathological changes on single cell behaviour is a challenging task for deep learning models. Indeed, in self-supervised learning methods, no prior labels are used for the training and all of the information for event predictions are extracted from the data themselves. We present here a novel self-supervised learning model for the detection of anomalies in a given cell population, StArDusTS. Cells are monitored over time, and analysed to extract time-series of dry mass values. We assessed its performances on different cell lines, showing a precision of 96% in the automatic detection of anomalies. Additionally, anomaly detection was also associated with cell measurement errors inherent to the acquisition or analysis pipelines, leading to an improvement of the upstream methods for feature extraction. Our results pave the way to novel architectures for the continuous monitoring of cell cultures in applied research or bioproduction applications, and for the prediction of pathological cellular changes.

RAB6 and dynein drive post-Golgi apical transport to prevent neuronal progenitor delamination.

Radial glial (RG) cells are the neural stem cells of the developing neocortex. Apical RG (aRG) cells can delaminate to generate basal RG (bRG) cells, a cell type associated with human brain expansion. Here, we report that aRG delamination is regulated by the post-Golgi secretory pathway. Using in situ subcellular live imaging, we show that post-Golgi transport of RAB6+ vesicles occurs toward the minus ends of microtubules and depends on dynein. We demonstrate that the apical determinant Crumbs3 (CRB3) is also transported by dynein. Double knockout of RAB6A/A' and RAB6B impairs apical localization of CRB3 and induces a retraction of aRG cell apical process, leading to delamination and ectopic division. These defects are phenocopied by knockout of the dynein activator LIS1. Overall, our results identify a RAB6-dynein-LIS1 complex for Golgi to apical surface transport in aRG cells, and highlights the role of this pathway in the maintenance of neuroepithelial integrity.

Generating Rab6 Conditional Knockout Mice.

RAB6 GTPase is the most abundant Golgi-associated RAB protein and regulates several transport steps at the level of this organelle. Homozygous Rab6a knockout (k/o) is embryonic lethal in mouse. To study RAB6 function in cell lineages and tissues, we thus generated various conditional Rab6a knockout (k/o) mice using the Cre/lox system.

RAB6 GTPase regulates mammary secretory function by controlling the activation of STAT5.

The Golgi-associated RAB GTPases, RAB6A and RAB6A', regulate anterograde and retrograde transport pathways from and to the Golgi. In vitro, RAB6A/A' control several cellular functions including cell division, migration, adhesion and polarity. However, their role remains poorly described in vivo Here, we generated BlgCre; Rab6aF/F mice presenting a specific deletion of Rab6a in the mammary luminal secretory lineage during gestation and lactation. Rab6a loss severely impaired the differentiation, maturation and maintenance of the secretory tissue, compromising lactation. The mutant epithelium displayed a decreased activation of STAT5, a key regulator of the lactogenic process primarily governed by prolactin. Data obtained with a mammary epithelial cell line suggested that defective STAT5 activation might originate from a perturbed transport of the prolactin receptor, altering its membrane expression and signaling cascade. Despite the major functional defects observed upon Rab6a deletion, the polarized organization of the mammary epithelial bilayer was preserved. Altogether, our data reveal a crucial role for RAB6A/A' in the lactogenic function of the mammary gland and suggest that the trafficking pathways controlled by RAB6A/A' depend on cell-type specialization and tissue context.

RAB6 and microtubules restrict protein secretion to focal adhesions.

To ensure their homeostasis and sustain differentiated functions, cells continuously transport diverse cargos to various cell compartments and in particular to the cell surface. Secreted proteins are transported along intracellular routes from the endoplasmic reticulum through the Golgi complex before reaching the plasma membrane along microtubule tracks. Using a synchronized secretion assay, we report here that exocytosis does not occur randomly at the cell surface but on localized hotspots juxtaposed to focal adhesions. Although microtubules are involved, the RAB6-dependent machinery plays an essential role. We observed that, irrespective of the transported cargos, most post-Golgi carriers are positive for RAB6 and that its inactivation leads to a broad reduction of protein secretion. RAB6 may thus be a general regulator of post-Golgi secretion.

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture.

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Rab6-dependent retrograde traffic of LAT controls immune synapse formation and T cell activation.

The adapter molecule linker for activation of T cells (LAT) orchestrates the formation of signalosomes upon T cell receptor (TCR) stimulation. LAT is present in different intracellular pools and is dynamically recruited to the immune synapse upon stimulation. However, the intracellular traffic of LAT and its function in T lymphocyte activation are ill defined. We show herein that LAT, once internalized, transits through the Golgi-trans-Golgi network (TGN), where it is repolarized to the immune synapse. This retrograde transport of LAT depends on the small GTPase Rab6 and the target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) Syntaxin-16, two regulators of the endosome-to-Golgi/TGN retrograde transport. We also show in vitro in Syntaxin-16- or Rab6-silenced human cells and in vivo in CD4+ T lymphocytes of the Rab6 knockout mouse that this retrograde traffic controls TCR stimulation. These results establish that the retrograde traffic of LAT from the plasma membrane to the Golgi-TGN controls the polarized delivery of LAT at the immune synapse and T lymphocyte activation.

Coupling fission and exit of RAB6 vesicles at Golgi hotspots through kinesin-myosin interactions.

The actin and microtubule cytoskeletons play important roles in Golgi structure and function, but how they are connected remain poorly known. In this study, we investigated whether RAB6 GTPase, a Golgi-associated RAB involved in the regulation of several transport steps at the Golgi level, and two of its effectors, Myosin IIA and KIF20A participate in the coupling between actin and microtubule cytoskeleton. We have previously shown that RAB6-Myosin IIA interaction is critical for the fission of RAB6-positive transport carriers from Golgi/TGN membranes. Here we show that KIF20A is also involved in the fission process and serves to anchor RAB6 on Golgi/TGN membranes near microtubule nucleating sites. We provide evidence that the fission events occur at a limited number of hotspots sites. Our results suggest that coupling between actin and microtubule cytoskeletons driven by Myosin II and KIF20A ensures the spatial coordination between RAB6-positive vesicles fission from Golgi/TGN membranes and their exit along microtubules.

Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes.

Exocytic carriers convey neo-synthesized components from the Golgi apparatus to the cell surface. While the release and anterograde movement of Golgi-derived vesicles require the small GTPase RAB6, its effector ELKS promotes the targeting and docking of secretory vesicles to particular areas of the plasma membrane. Here, we show that specialized cell types exploit and divert the secretory pathway towards lysosome related organelles. In cultured melanocytes, the secretory route relies on RAB6 and ELKS to directly transport and dock Golgi-derived carriers to melanosomes. By delivering specific cargos, such as MART-1 and TYRP2/ DCT, the RAB6/ELKS-dependent secretory pathway controls the formation and maturation of melanosomes but also pigment synthesis. In addition, pigmentation defects are observed in RAB6 KO mice. Our data together reveal for the first time that the secretory pathway can be directed towards intracellular organelles of endosomal origin to ensure their biogenesis and function.

Persistent cell migration and adhesion rely on retrograde transport of ß(1) integrin.

Integrins have key functions in cell adhesion and migration. How integrins are dynamically relocalized to the leading edge in highly polarized migratory cells has remained unexplored. Here, we demonstrate that ß1 integrin (known as PAT-3 in Caenorhabditis elegans), but not ß3, is transported from the plasma membrane to the trans-Golgi network, to be resecreted in a polarized manner. This retrograde trafficking is restricted to the non-ligand-bound conformation of ß1 integrin. Retrograde trafficking inhibition abrogates several ß1-integrin-specific functions such as cell adhesion in early embryonic development of mice, and persistent cell migration in the developing posterior gonad arm of C. elegans. Our results establish a paradigm according to which retrograde trafficking, and not endosomal recycling, is the key driver for ß1 integrin function in highly polarized cells. These data more generally suggest that the retrograde route is used to relocalize plasma membrane machinery from previous sites of function to the leading edge of migratory cells.

Phenotypic characterisation of RAB6A knockout mouse embryonic fibroblasts.

BACKGROUND INFORMATION: Rab6 is one of the most conserved Rab GTPaes throughout evolution and the most abundant Rab protein associated with the Golgi complex. The two ubiquitous Rab isoforms, Rab6A and Rab6A', that are generated by alternative splicing of the RAB6A gene, regulate several transport steps at the Golgi level, including retrograde transport between endosomes and Golgi, anterograde transport between Golgi and the plasma membrane, and intra-Golgi and Golgi to endoplasmic reticulum transport. RESULTS: We have generated mice with a conditional null allele of RAB6A. Mice homozygous for the RAB6A null allele died at an early stage of embryonic development. Mouse embryonic fibroblasts (MEFs) were isolated from RAB6A(loxP/loxP) Rosa26-CreERT2 and incubated with 4-hydroxy tamoxifen, resulting in the efficient depletion of Rab6A and Rab6A'. We show that Rab6 depletion affects cell growth, alters Golgi morphology and decreases the Golgi-associated levels of some known Rab6 effectors such as Bicaudal-D and myosin II. We also show that Rab6 depletion protects MEFs against ricin toxin and delays VSV-G secretion. CONCLUSIONS: Our study shows that RAB6 is an essential gene required for normal embryonic development. We confirm in MEF cells most of the functions previously attributed to the two ubiquitous Rab6 isoforms.

Background fluorescence estimation and vesicle segmentation in live cell imaging with conditional random fields.

Image analysis applied to fluorescence live cell microscopy has become a key tool in molecular biology since it enables to characterize biological processes in space and time at the subcellular level. In fluorescence microscopy imaging, the moving tagged structures of interest, such as vesicles, appear as bright spots over a static or nonstatic background. In this paper, we consider the problem of vesicle segmentation and time-varying background estimation at the cellular scale. The main idea is to formulate the joint segmentation-estimation problem in the general conditional random field framework. Furthermore, segmentation of vesicles and background estimation are alternatively performed by energy minimization using a min cut-max flow algorithm. The proposed approach relies on a detection measure computed from intensity contrasts between neighboring blocks in fluorescence microscopy images. This approach permits analysis of either 2D + time or 3D + time data. We demonstrate the performance of the so-called C-CRAFT through an experimental comparison with the state-of-the-art methods in fluorescence video-microscopy. We also use this method to characterize the spatial and temporal distribution of Rab6 transport carriers at the cell periphery for two different specific adhesion geometries.

Fast high-resolution 3D total internal reflection fluorescence microscopy by incidence angle scanning and azimuthal averaging.

Total internal reflection fluorescence microscopy (TIRFM) is the method of choice to visualize a variety of cellular processes in particular events localized near the plasma membrane of live adherent cells. This imaging technique not relying on particular fluorescent probes provides a high sectioning capability. It is, however, restricted to a single plane. We present here a method based on a versatile design enabling fast multiwavelength azimuthal averaging and incidence angles scanning to computationally reconstruct 3D images sequences. We achieve unprecedented 50-nm axial resolution over a range of 800 nm above the coverslip. We apply this imaging modality to obtain structural and dynamical information about 3D actin architectures. We also temporally decipher distinct Rab11a-dependent exocytosis events in 3D at a rate of seven stacks per second.

Mechanical role of actin dynamics in the rheology of the Golgi complex and in Golgi-associated trafficking events.

BACKGROUND: In vitro studies have shown that physical parameters, such as membrane curvature, tension, and composition, influence the budding and fission of transport intermediates. Endocytosis in living cells also appears to be regulated by the mechanical load experienced by the plasma membrane. In contrast, how these parameters affect intracellular membrane trafficking in living cells is not known. To address this question, we investigate here the impact of a mechanical stress on the organization of the Golgi complex and on the formation of transport intermediates from the Golgi complex. RESULTS: Using confocal microscopy, we visualize the deformation of Rab6-positive Golgi membranes applied by an internalized microsphere trapped in optical tweezers and simultaneously measure the corresponding forces. Our results show that the force necessary to deform Golgi membranes drops when actin dynamics is altered and correlates with myosin II activity. We also show that the applied stress has a long-range effect on Golgi membranes, perturbs the dynamics of Golgi-associated actin, and induces a sharp decrease in the formation of Rab6-positive vesicles from the Golgi complex as well as tubulation of Golgi membranes. CONCLUSIONS: We suggest that acto-myosin contractility strongly contributes to the local rigidity of the Golgi complex and regulates the mechanics of the Golgi complex to control intracellular membrane trafficking.

The CryoCapsule: simplifying correlative light to electron microscopy.

Correlating complementary multiple scale images of the same object is a straightforward means to decipher biological processes. Light microscopy and electron microscopy are the most commonly used imaging techniques, yet despite their complementarity, the experimental procedures available to correlate them are technically complex. We designed and manufactured a new device adapted to many biological specimens, the CryoCapsule, that simplifies the multiple sample preparation steps, which at present separate live cell fluorescence imaging from contextual high-resolution electron microscopy, thus opening new strategies for full correlative light to electron microscopy. We tested the biological application of this highly optimized tool on three different specimens: the in vitro Xenopus laevis mitotic spindle, melanoma cells over-expressing YFP-langerin sequestered in organized membranous subcellular organelles and a pigmented melanocytic cell in which the endosomal system was labeled with internalized fluorescent transferrin.

C11ORF24 is a novel type I membrane protein that cycles between the Golgi apparatus and the plasma membrane in Rab6-positive vesicles.

The Golgi apparatus is an intracellular compartment necessary for post-translational modification, sorting and transport of proteins. It plays a key role in mitotic entry through the Golgi mitotic checkpoint. In order to identify new proteins involved in the Golgi mitotic checkpoint, we combine the results of a knockdown screen for mitotic phenotypes and a localization screen. Using this approach, we identify a new Golgi protein C11ORF24 (NP_071733.1). We show that C11ORF24 has a signal peptide at the N-terminus and a transmembrane domain in the C-terminal region. C11ORF24 is localized on the Golgi apparatus and on the trans-Golgi network. A large part of the protein is present in the lumen of the Golgi apparatus whereas only a short tail extends into the cytosol. This cytosolic tail is well conserved in evolution. By FRAP experiments we show that the dynamics of C11ORF24 in the Golgi membrane are coherent with the presence of a transmembrane domain in the protein. C11ORF24 is not only present on the Golgi apparatus but also cycles to the plasma membrane via endosomes in a pH sensitive manner. Moreover, via video-microscopy studies we show that C11ORF24 is found on transport intermediates and is colocalized with the small GTPase RAB6, a GTPase involved in anterograde transport from the Golgi to the plasma membrane. Knocking down C11ORF24 does not lead to a mitotic phenotype or an intracellular transport defect in our hands. All together, these data suggest that C11ORF24 is present on the Golgi apparatus, transported to the plasma membrane and cycles back through the endosomes by way of RAB6 positive carriers.

Birbeck granule-like "organized smooth endoplasmic reticulum" resulting from the expression of a cytoplasmic YFP-tagged langerin.

Langerin is required for the biogenesis of Birbeck granules (BGs), the characteristic organelles of Langerhans cells. We previously used a Langerin-YFP fusion protein having a C-terminal luminal YFP tag to dynamically decipher the molecular and cellular processes which accompany the traffic of Langerin. In order to elucidate the interactions of Langerin with its trafficking effectors and their structural impact on the biogenesis of BGs, we generated a YFP-Langerin chimera with an N-terminal, cytosolic YFP tag. This latter fusion protein induced the formation of YFP-positive large puncta. Live cell imaging coupled to a fluorescence recovery after photobleaching approach showed that this coalescence of proteins in newly formed compartments was static. In contrast, the YFP-positive structures present in the pericentriolar region of cells expressing Langerin-YFP chimera, displayed fluorescent recovery characteristics compatible with active membrane exchanges. Using correlative light-electron microscopy we showed that the coalescent structures represented highly organized stacks of membranes with a pentalaminar architecture typical of BGs. Continuities between these organelles and the rough endoplasmic reticulum allowed us to identify the stacks of membranes as a form of "Organized Smooth Endoplasmic Reticulum" (OSER), with distinct molecular and physiological properties. The involvement of homotypic interactions between cytoplasmic YFP molecules was demonstrated using an A206K variant of YFP, which restored most of the Langerin traffic and BG characteristics observed in Langerhans cells. Mutation of the carbohydrate recognition domain also blocked the formation of OSER. Hence, a "double-lock" mechanism governs the behavior of YFP-Langerin, where asymmetric homodimerization of the YFP tag and homotypic interactions between the lectin domains of Langerin molecules participate in its retention and the subsequent formation of BG-like OSER. These observations confirm that BG-like structures appear wherever Langerin accumulates and confirm that membrane trafficking effectors dictate their physiology and, illustrate the importance of molecular interactions in the architecture of intracellular membranes.

A Rab11A/myosin Vb/Rab11-FIP2 complex frames two late recycling steps of langerin from the ERC to the plasma membrane.

A large body of knowledge relating to the constitution of Rab GTPase/Rab effector complexes and their impact on both membrane domain organization and overall membrane trafficking has been built up in recent years. However in the context of the live cell there are still many questions that remain to be answered, such as where and when these complexes assemble and where they perform their primary function(s). We describe here the dynamic processes that take place in the final steps of the Rab11A dependent recycling pathway, in the context of the membrane platform constituted by Myosin Vb, Rab11A, and Rab11-FIP2. We first confirm that a series of previously reported observations obtained during the study of a number of trafficking cargoes also apply to langerin. Langerin is a cargo molecule that traffics through Rab11A-positive membrane domains of the endosomal recycling pathway. In order to explore the relative dynamics of this set of partners, we make extensive use of a combinatory approach of Live-FRET, fast FRAP video, fast confocal and TIRF microscopy modalities. Our data show that the Myosin Vb/Rab11A/Rab11-FIP2 platform is spatially involved in the regulation of langerin trafficking at two distinct sites within live cells, first at the sorting site in the endosomal recycling compartment (ERC) where transport vesicles are formed, and subsequently, in a strict time-defined order, at the very late stage of docking/tethering and fusion of these langerin recycling vesicles to the plasma membrane.

Glycan-modifying bacteria-derived soluble factors from Bacteroides thetaiotaomicron and Lactobacillus casei inhibit rotavirus infection in human intestinal cells.

Rotaviruses attach to intestinal cells in a process that requires glycan recognition. Some bacteria from the gut microflora have been shown to modify cell-surface glycans. In this study, human intestinal cultured cells were incubated with bacteria-derived soluble factors and infected with rotavirus. Results show that only bacterial soluble factors that increase cell-surface galactose namely, those of Bacteroides thetaiotaomicron and Lactobacillus casei were able to efficiently block rotavirus infections. Increasing cell-surface galactose using galactosyltransferase resulted in a similar blockage of rotavirus infections. These results indicate that manipulation of cell-surface intestinal glycans by bacterial soluble factors can prevent rotavirus infection in a species-specific manner, and should now be considered a potential therapeutic approach against rotavirus infection.

Probabilistic density maps to study global endomembrane organization.

We developed a computational imaging approach that describes the three-dimensional spatial organization of endomembranes from micromanipulation-normalized mammalian cells with probabilistic density maps. Applied to several well-known marker proteins, this approach revealed the average steady-state organization of early endosomes, multivesicular bodies or lysosomes, endoplasmic reticulum exit sites, the Golgi apparatus and Golgi-derived transport carriers in crossbow-shaped cells. The steady-state organization of each tested endomembranous population was well-defined, unique and in some cases depended on the cellular adhesion geometry. Density maps of all endomembrane populations became stable when pooling several tens of cells only and were reproducible in independent experiments, allowing construction of a standardized cell model. We detected subtle changes in steady-state organization induced by disruption of the cellular cytoskeleton, with statistical significance observed for just 20 cells. Thus, combining micropatterning with construction of endomembrane density maps allows the systematic study of intracellular trafficking determinants.