A fluorogenic chemically induced dimerization technology for controlling, imaging and sensing protein proximity.

Molecular tools enabling the control and observation of the proximity of proteins are essential for studying the functional role of physical distance between two proteins. Here we present CATCHFIRE (chemically assisted tethering of chimera by fluorogenic-induced recognition), a chemically induced proximity technology with intrinsic fluorescence imaging and sensing capabilities. CATCHFIRE relies on genetic fusion to small dimerizing domains that interact upon addition of fluorogenic inducers of proximity that fluoresce upon formation of the ternary assembly, allowing real-time monitoring of the chemically induced proximity. CATCHFIRE is rapid and fully reversible and allows the control and tracking of protein localization, protein trafficking, organelle transport and cellular processes, opening new avenues for studying or controlling biological processes with high spatiotemporal resolution. Its fluorogenic nature allows the design of a new class of biosensors for the study of processes such as signal transduction and apoptosis.

Golgi maturation-dependent glycoenzyme recycling controls glycosphingolipid biosynthesis and cell growth via GOLPH3.

Glycosphingolipids are important components of the plasma membrane where they modulate the activities of membrane proteins including signalling receptors. Glycosphingolipid synthesis relies on competing reactions catalysed by Golgi-resident enzymes during the passage of substrates through the Golgi cisternae. The glycosphingolipid metabolic output is determined by the position and levels of the enzymes within the Golgi stack, but the mechanisms that coordinate the intra-Golgi localisation of the enzymes are poorly understood. Here, we show that a group of sequentially-acting enzymes operating at the branchpoint among glycosphingolipid synthetic pathways binds the Golgi-localised oncoprotein GOLPH3. GOLPH3 sorts these enzymes into vesicles for intra-Golgi retro-transport, acting as a component of the cisternal maturation mechanism. Through these effects, GOLPH3 controls the sub-Golgi localisation and the lysosomal degradation rate of specific enzymes. Increased GOLPH3 levels, as those observed in tumours, alter glycosphingolipid synthesis and plasma membrane composition thereby promoting mitogenic signalling and cell proliferation. These data have medical implications as they outline a novel oncogenic mechanism of action for GOLPH3 based on glycosphingolipid metabolism.

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.

Dendritic Cells Require TMEM176A/B Ion Channels for Optimal MHC Class II Antigen Presentation to Naive CD4+ T Cells.

Intracellular ion fluxes emerge as critical actors of immunoregulation but still remain poorly explored. In this study, we investigated the role of the redundant cation channels TMEM176A and TMEM176B (TMEM176A/B) in retinoic acid-related orphan receptor γt+ cells and conventional dendritic cells (DCs) using germline and conditional double knockout mice. Although Tmem176a/b appeared surprisingly dispensable for the protective function of Th17 and group 3 innate lymphoid cells in the intestinal mucosa, we found that they were required in conventional DCs for optimal Ag processing and presentation to CD4+ T cells. Using a real-time imaging method, we show that TMEM176A/B accumulate in dynamic post-Golgi vesicles preferentially linked to the late endolysosomal system and strongly colocalize with HLA-DM. Taken together, our results suggest that TMEM176A/B ion channels play a direct role in the MHC class II compartment of DCs for the fine regulation of Ag presentation and naive CD4+ T cell priming.

Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus.

Preclinical evidence depicts the capacity of redaporfin (Redp) to act as potent photosensitizer, causing direct antineoplastic effects as well as indirect immune-dependent destruction of malignant lesions. Here, we investigated the mechanisms through which photodynamic therapy (PDT) with redaporfin kills cancer cells. Subcellular localization and fractionation studies based on the physicochemical properties of redaporfin revealed its selective tropism for the endoplasmic reticulum (ER) and the Golgi apparatus (GA). When activated, redaporfin caused rapid reactive oxygen species-dependent perturbation of ER/GA compartments, coupled to ER stress and an inhibition of the GA-dependent secretory pathway. This led to a general inhibition of protein secretion by PDT-treated cancer cells. The ER/GA play a role upstream of mitochondria in the lethal signaling pathway triggered by redaporfin-based PDT Pharmacological perturbation of GA function or homeostasis reduces mitochondrial permeabilization. In contrast, removal of the pro-apoptotic multidomain proteins BAX and BAK or pretreatment with protease inhibitors reduced cell killing, yet left the GA perturbation unaffected. Altogether, these results point to the capacity of redaporfin to kill tumor cells via destroying ER/GA function.

The role of microtubules in secretory protein transport.

Microtubules are part of the dynamic cytoskeleton network and composed of tubulin dimers. They are the main tracks used in cells to organize organelle positioning and trafficking of cargos. In this Review, we compile recent findings on the involvement of microtubules in anterograde protein transport. First, we highlight the importance of microtubules in organelle positioning. Second, we discuss the involvement of microtubules within different trafficking steps, in particular between the endoplasmic reticulum and the Golgi complex, traffic through the Golgi complex itself and in post-Golgi processes. A large number of studies have assessed the involvement of microtubules in transport of cargo from the Golgi complex to the cell surface. We focus here on the role of kinesin motor proteins and protein interactions in post-Golgi transport, as well as the impact of tubulin post-translational modifications. Last, in light of recent findings, we highlight the role microtubules have in exocytosis, the final step of secretory protein transport, occurring close to focal adhesions.

Microtubule-independent secretion requires functional maturation of Golgi elements.

The Golgi complex is responsible for processing and sorting of secretory cargos. Microtubules are known to accelerate the transport of proteins from the endoplasmic reticulum (ER) to the Golgi complex and from the Golgi to the plasma membrane. However, whether post-Golgi transport strictly requires microtubules is still unclear. Using the retention using selective hooks (RUSH) system to synchronize the trafficking of cargos, we show that anterograde transport of tumor necrosis factor (TNF) is strongly reduced without microtubules. We show that two populations of Golgi elements co-exist in these cells. A centrally located and giantin-positive Golgi complex that sustains trafficking, and newly formed peripheral Golgi mini-stacks that accumulate cargos in cells without microtubules. Using a genome-edited GFP-giantin cell line, we observe that the trafficking-competent Golgi population corresponds to the pre-existing population that was present before removal of microtubules. All Golgi elements support trafficking after long-term depletion of microtubules and after relocation of Golgi proteins to the ER after treatment with Brefeldin A. Our results demonstrate that functional maturation of Golgi elements is needed to ensure post-Golgi trafficking, and that microtubule-driven post-Golgi transport is not strictly required.

Dymeclin deficiency causes postnatal microcephaly, hypomyelination and reticulum-to-Golgi trafficking defects in mice and humans.

Dymeclin is a Golgi-associated protein whose deficiency causes Dyggve-Melchior-Clausen syndrome (DMC, MIM #223800), a rare recessively inherited spondyloepimetaphyseal dysplasia consistently associated with postnatal microcephaly and intellectual disability. While the skeletal phenotype of DMC patients has been extensively described, very little is known about their cerebral anomalies, which result in brain growth defects and cognitive dysfunction. We used Dymeclin-deficient mice to determine the cause of microcephaly and to identify defective mechanisms at the cellular level. Brain weight and volume were reduced in all mutant mice from postnatal day 5 onward. Mutant mice displayed a narrowing of the frontal cortex, although cortical layers were normally organized. Interestingly, the corpus callosum was markedly thinner, a characteristic we also identified in DMC patients. Consistent with this, the myelin sheath was thinner, less compact and not properly rolled, while the number of mature oligodendrocytes and their ability to produce myelin basic protein were significantly decreased. Finally, cortical neurons from mutant mice and primary fibroblasts from DMC patients displayed substantially delayed endoplasmic reticulum to Golgi trafficking, which could be fully rescued upon Dymeclin re-expression. These findings indicate that Dymeclin is crucial for proper myelination and anterograde neuronal trafficking, two processes that are highly active during postnatal brain maturation.

Improving the Solubility of Artificial Ligands of Streptavidin to Enable More Practical Reversible Switching of Protein Localization in Cells.

Chemical inducers that can control target-protein localization in living cells are powerful tools to investigate dynamic biological systems. We recently reported the retention using selective hook or "RUSH" system for reversible localization change of proteins of interest by addition/washout of small-molecule artificial ligands of streptavidin (ALiS). However, the utility of previously developed ALiS was restricted by limited solubility in water. Here, we overcame this problem by X-ray crystal structure-guided design of a more soluble ALiS derivative (ALiS-3), which retains sufficient streptavidin-binding affinity for use in the RUSH system. The ALiS-3-streptavidin interaction was characterized in detail. ALiS-3 is a convenient and effective tool for dynamic control of α-mannosidase II localization between ER and Golgi in living cells.

Artificial Ligands of Streptavidin (ALiS): Discovery, Characterization, and Application for Reversible Control of Intracellular Protein Transport.

Artificial ligands of streptavidin (ALiS) with association constants of ∼10(6) M(-1) were discovered by high-throughput screening of our chemical library, and their binding characteristics, including X-ray crystal structure of the streptavidin complex, were determined. Unlike biotin and its derivatives, ALiS exhibits fast dissociation kinetics and excellent cell permeability. The streptavidin-ALiS system provides a novel, practical compound-dependent methodology for repeated reversible cycling of protein localization between intracellular organella.

Synchronization of secretory protein traffic in populations of cells.

To dissect secretory traffic, we developed the retention using selective hooks (RUSH) system. RUSH is a two-state assay based on the reversible interaction of a hook protein fused to core streptavidin and stably anchored in the donor compartment with a reporter protein of interest fused to streptavidin-binding peptide (SBP). Biotin addition causes a synchronous release of the reporter from the hook. Using the RUSH system, we analyzed different transport characteristics of various Golgi and plasma membrane reporters at physiological temperature in living cells. Using dual-color simultaneous live-cell imaging of two cargos, we observed intra- and post-Golgi segregation of cargo traffic, consistent with observation in other systems. We show preliminarily that the RUSH system is usable for automated screening. The system should help increase the understanding of the mechanisms of trafficking and enable screens for molecules that perturb pathological protein transport.

Lysosomal membrane proteins LAMP1 and LIMP2 are segregated in the Golgi apparatus independently of their clathrin adaptor binding motif.

To reach the lysosome, lysosomal membrane proteins (LMPs) are translocated in the endoplasmic reticulum after synthesis and then transported to the Golgi apparatus. The existence of a direct transport from the Golgi apparatus to the endosomes but also of an indirect route through the plasma membrane has been described. Clathrin adaptor binding motifs contained in the cytosolic tail of LMPs have been described as key players in their intracellular trafficking. Here we used the RUSH assay to synchronize the biosynthetic transport of multiple LMPs. After exiting the Golgi apparatus, RUSH-synchronized LAMP1 was addressed to the cell surface both after overexpression or at endogenous level. Its YXXΦ motif was not involved in the transport from the Golgi apparatus to the plasma membrane but in its endocytosis. LAMP1 and LIMP2 were sorted from each other after reaching the Golgi apparatus. LIMP2 was incorporated in punctate structures for export from the Golgi apparatus from which LAMP1 is excluded. LIMP2-containing post-Golgi transport intermediates did not rely neither on its adaptor binding signal nor on its C-terminal cytoplasmic domain.

The many routes of Golgi-dependent trafficking.

The Golgi apparatus lies at the center of intracellular trafficking, controlling secretory and retrograde routes. Its role is essential both to ensure housekeeping cellular functions and to sustain highly differentiated ones. While Golgi dynamics and function have been studied by many groups for several decades now, and intensely for more than 40 years, the mechanisms of Golgi-dependent secretion, how anterograde and retrograde intra-Golgi transports are controlled, as well as the regulation of the Golgi stacked structure and function are some of the many questions that are still open. We will review here the main characteristics of intra-Golgi trafficking discuss the different models that were built to explain the diversity of intra-Golgi routes and its dynamics and propose an integrative model.

Transfer learning for versatile and training free high content screening analyses.

High content screening (HCS) is a technology that automates cell biology experiments at large scale. A High Content Screen produces a high amount of microscopy images of cells under many conditions and requires that a dedicated image and data analysis workflow be designed for each assay to select hits. This heavy data analytic step remains challenging and has been recognized as one of the burdens hindering the adoption of HCS. In this work we propose a solution to hit selection by using transfer learning without additional training. A pretrained residual network is employed to encode each image of a screen into a discriminant representation. The deep features obtained are then corrected to account for well plate bias and misalignment. We then propose two training-free pipelines dedicated to the two main categories of HCS for compound selection: with or without positive control. When a positive control is available, it is used alongside the negative control to compute a linear discriminant axis, thus building a classifier without training. Once all samples are projected onto this axis, the conditions that best reproduce the positive control can be selected. When no positive control is available, the Mahalanobis distance is computed from each sample to the negative control distribution. The latter provides a metric to identify the conditions that alter the negative control's cell phenotype. This metric is subsequently used to categorize hits through a clustering step. Given the lack of available ground truth in HCS, we provide a qualitative comparison of the results obtained using this approach with results obtained with handcrafted image analysis features for compounds and siRNA screens with or without control. Our results suggests that the fully automated and generic pipeline we propose offers a good alternative to handcrafted dedicated image analysis approaches. Furthermore, we demonstrate that this solution select conditions of interest that had not been identified using the primary dedicated analysis. Altogether, this approach provides a fully automated, reproducible, versatile and comprehensive alternative analysis solution for HCS encompassing compound-based or downregulation screens, with or without positive controls, without the need for training or cell detection, or the development of a dedicated image analysis workflow.

Identification of a family of effectors secreted by the type III secretion system that are conserved in pathogenic Chlamydiae.

Chlamydiae are Gram-negative, obligate intracellular pathogens that replicate within a membrane-bounded compartment termed an inclusion. Throughout their development, they actively modify the eukaryotic environment. The type III secretion (TTS) system is the main process by which the bacteria translocate effector proteins into the inclusion membrane and the host cell cytoplasm. Here we describe a family of type III secreted effectors that are present in all pathogenic chlamydiae and absent in the environment-related species. It is defined by a common domain of unknown function, DUF582, that is present in four or five proteins in each Chlamydiaceae species. We show that the amino-terminal extremity of DUF582 proteins functions as a TTS signal. DUF582 proteins from C. trachomatis CT620, CT621, and CT711 are expressed at the middle and late phases of the infectious cycle. Immunolocalization further revealed that CT620 and CT621 are secreted into the host cell cytoplasm, as well as within the lumen of the inclusion, where they do not associate with bacterial markers. Finally, we show that DUF582 proteins are present in nuclei of infected cells, suggesting that members of the DUF582 family of effector proteins may target nuclear cell functions. The expansion of this family of proteins in pathogenic chlamydiae and their conservation among the different species suggest that they play important roles in the infectious cycle.

Retention Using Selective Hooks-Synchronized Secretion to Measure Local Exocytosis.

Proteins destined to be exposed to the extracellular space enter the secretory pathway at the level of the endoplasmic reticulum. Proteins are then transported to the Golgi apparatus and addressed to their destination compartment, such as the plasma membrane for exocytic cargos. Exocytosis constitutes the last step of the anterograde transport of secretory cargos. Exocytic vesicles fuse with the plasma membrane, releasing soluble proteins to the extracellular milieu and transmembrane proteins to the plasma membrane. In order to monitor local exocytosis of cargos, we describe in this chapter how to perform synchronization of the anterograde transport of an exocytic cargo of interest using the retention using selective hooks (RUSH) assay in combination with selective protein immobilization (SPI). SPI is based on the coating of coverslips with anti-green fluorescent protein (GFP) antibodies, which capture the GFP-tagged RUSH cargos once exposed to the cell surface after its release by the addition of biotin.

Rab7-harboring vesicles are carriers of the transferrin receptor through the biosynthetic secretory pathway.

The biosynthetic secretory pathway is particularly challenging to investigate as it is underrepresented compared to the abundance of the other intracellular trafficking routes. Here, we combined the retention using selective hook (RUSH) to a CRISPR-Cas9 gene editing approach (eRUSH) and identified Rab7-harboring vesicles as an important intermediate compartment of the Golgi-to-plasma membrane transport of neosynthesized transferrin receptor (TfR). These vesicles did not exhibit degradative properties and were not associated to Rab6A-harboring vesicles. Rab7A was transiently associated to neosynthetic TfR-containing post-Golgi vesicles but dissociated before fusion with the plasma membrane. Together, our study reveals a role for Rab7 in the biosynthetic secretory pathway of the TfR, highlighting the diversity of the secretory vesicles' nature.

Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9.

Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type.

Production of reactive oxygen species is turned on and rapidly shut down in epithelial cells infected with Chlamydia trachomatis.

Reactive oxygen species (ROS) are many-faceted compounds involved in cell defense against pathogens, as well as in cell signaling. Their involvement in the response to infection in epithelial cells remains poorly documented. Here, we investigated the production of ROS during infection with Chlamydia trachomatis, a strict intracellular pathogen, in HeLa cells. C. trachomatis induced a transient increase in the ROS level within a few hours, followed by a return to basal level 9 hours after infection. At this time point, the host enzyme dedicated to ROS production, NADPH oxidase, could no longer be activated by external stimuli, such as interleukin-1beta. In addition, Rac, a regulatory subunit of the NADPH oxidase complex, was relocated to the membrane of the compartment in which the bacteria develop, the inclusion, while other subunits were not. Altogether, these results indicate that C. trachomatis infection elicits the production of ROS and that the bacteria rapidly target the activity of NADPH oxidase to shut it down. Prevention of ROS production at the onset of the bacterial developmental cycle might delay the host response to infection.

Distinct anterograde trafficking pathways of BACE1 and amyloid precursor protein from the TGN and the regulation of amyloid-ß production.

Processing of amyloid precursor protein (APP) by the ß-secretase BACE1 is the initial step of the amyloidogenic pathway to generate amyloid-ß (Aß). Although newly synthesized BACE1 and APP are transported along the secretory pathway, it is not known whether BACE1 and APP share the same post-Golgi trafficking pathways or are partitioned into different transport routes. Here we demonstrate that BACE1 exits the Golgi in HeLa cells and primary neurons by a pathway distinct from the trafficking pathway for APP. By using the Retention Using Selective Hooks system, we show that BACE1 is transported from the trans-Golgi network to the plasma membrane in an AP-1- and Arf1/4-dependent manner. Subsequently, BACE1 is endocytosed to early and recycling endosomes. Perturbation of BACE1 post-Golgi trafficking results in an increase in BACE1 cleavage of APP and increased production of both Aß40 and Aß42. These findings reveal that Golgi exit of BACE1 and APP in primary neurons is tightly regulated, resulting in their segregation along different transport routes, which limits APP processing.