Nucleotide-free structures of KIF20A illuminate atypical mechanochemistry in this kinesin-6.

KIF20A is a critical kinesin for cell division and a promising anti-cancer drug target. The mechanisms underlying its cellular roles remain elusive. Interestingly, unusual coupling between the nucleotide- and microtubule-binding sites of this kinesin-6 has been reported, but little is known about how its divergent sequence leads to atypical motility properties. We present here the first high-resolution structure of its motor domain that delineates the highly unusual structural features of this motor, including a long L6 insertion that integrates into the core of the motor domain and that drastically affects allostery and ATPase activity. Together with the high-resolution cryo-electron microscopy microtubule-bound KIF20A structure that reveals the microtubule-binding interface, we dissect the peculiarities of the KIF20A sequence that influence its mechanochemistry, leading to low motility compared to other kinesins. Structural and functional insights from the KIF20A pre-power stroke conformation highlight the role of extended insertions in shaping the motor's mechanochemical cycle. Essential for force production and processivity is the length of the neck linker in kinesins. We highlight here the role of the sequence preceding the neck linker in controlling its backward docking and show that a neck linker four times longer than that in kinesin-1 is required for the activity of this motor.

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.

A comprehensive library of fluorescent constructs of SARS-CoV-2 proteins and their initial characterisation in different cell types.

BACKGROUND INFORMATION: Comprehensive libraries of plasmids for SARS-CoV-2 proteins with various tags (e.g., Strep, HA, Turbo) are now available. They enable the identification of numerous potential protein-protein interactions between the SARS-CoV-2 virus and host proteins. RESULTS: We present here a large library of SARS CoV-2 protein constructs fused with green and red fluorescent proteins and their initial characterisation in various human cell lines including lung epithelial cell models (A549, BEAS-2B), as well as in budding yeast. The localisation of a few SARS-CoV-2 proteins matches their proposed interactions with host proteins. These include the localisation of Nsp13 to the centrosome, Orf3a to late endosomes and Orf9b to mitochondria. CONCLUSIONS AND SIGNIFICANCE: This library should facilitate further cellular investigations, notably by imaging techniques.

Branched Actin Maintains Acetylated Microtubule Network in the Early Secretory Pathway.

In the early secretory pathway, the delivery of anterograde cargoes from the endoplasmic reticulum (ER) exit sites (ERES) to the Golgi apparatus is a multi-step transport process occurring via the ER-Golgi intermediate compartment (IC, also called ERGIC). While the role microtubules in ER-to-Golgi transport has been well established, how the actin cytoskeleton contributes to this process remains poorly understood. Here, we report that Arp2/3 inhibition affects the network of acetylated microtubules around the Golgi and induces the accumulation of unusually long RAB1/GM130-positive carriers around the centrosome. These long carriers are less prone to reach the Golgi apparatus, and arrival of anterograde cargoes to the Golgi is decreased upon Arp2/3 inhibition. Our data suggest that Arp2/3-dependent actin polymerization maintains a stable network of acetylated microtubules, which ensures efficient cargo trafficking at the late stage of ER to Golgi transport.

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.

Non-conventional Axonal Organelles Control TRPM8 Ion Channel Trafficking and Peripheral Cold Sensing.

TRPM8 is the main ion channel responsible for cold transduction in the somatosensory system. Nerve terminal availability of TRPM8 determines cold sensitivity, but how axonal secretory organelles control channel delivery remains poorly understood. Here we examine the distribution of TRPM8 and trafficking organelles in cold-sensitive peripheral axons and disrupt trafficking by targeting the ARF-GEF GBF1 pharmacologically or the small GTPase RAB6 by optogenetics. In axons of the sciatic nerve, inhibition of GBF1 interrupts TRPM8 trafficking and increases association with the trans-Golgi network, LAMP1, and Golgi satellites, which distribute profusely along the axonal shaft. Accordingly, both TRPM8-dependent ongoing activity and cold-evoked responses reversibly decline upon GBF1 inhibition in nerve endings of corneal cold thermoreceptors. Inhibition of RAB6, which also associates to Golgi satellites, decreases cold-induced responses in vivo. Our results support a non-conventional axonal trafficking mechanism controlling the availability of TRPM8 in axons and cold sensitivity in the peripheral nervous system.

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.

TBC1D8B Loss-of-Function Mutations Lead to X-Linked Nephrotic Syndrome via Defective Trafficking Pathways.

Steroid-resistant nephrotic syndrome (SRNS) is characterized by high-range proteinuria and most often focal and segmental glomerulosclerosis (FSGS). Identification of mutations in genes causing SRNS has improved our understanding of disease mechanisms and highlighted defects in the podocyte, a highly specialized glomerular epithelial cell, as major factors in disease pathogenesis. By exome sequencing, we identified missense mutations in TBC1D8B in two families with an X-linked early-onset SRNS with FSGS. TBC1D8B is an uncharacterized Rab-GTPase-activating protein likely involved in endocytic and recycling pathways. Immunofluorescence studies revealed TBC1D8B presence in human glomeruli, and affected individual podocytes displayed architectural changes associated with migration defects commonly found in FSGS. In zebrafish we demonstrated that both knockdown and knockout of the unique TBC1D8B ortholog-induced proteinuria and that this phenotype was rescued by human TBC1D8B mRNA injection, but not by either of the two mutated mRNAs. We also showed an interaction between TBC1D8B and Rab11b, a key protein in vesicular recycling in cells. Interestingly, both internalization and recycling processes were dramatically decreased in affected individuals' podocytes and fibroblasts, confirming the crucial role of TBC1D8B in the cellular recycling processes, probably as a Rab11b GTPase-activating protein. Altogether, these results confirmed that pathogenic variations in TBC1D8B are involved in X-linked podocytopathy and points to alterations in recycling processes as a mechanism of SRNS.

Ezrin enrichment on curved membranes requires a specific conformation or interaction with a curvature-sensitive partner.

One challenge in cell biology is to decipher the biophysical mechanisms governing protein enrichment on curved membranes and the resulting membrane deformation. The ERM protein ezrin is abundant and associated with cellular membranes that are flat, positively or negatively curved. Using in vitro and cell biology approaches, we assess mechanisms of ezrin's enrichment on curved membranes. We evidence that wild-type ezrin (ezrinWT) and its phosphomimetic mutant T567D (ezrinTD) do not deform membranes but self-assemble anti-parallelly, zipping adjacent membranes. EzrinTD's specific conformation reduces intermolecular interactions, allows binding to actin filaments, which reduces membrane tethering, and promotes ezrin binding to positively-curved membranes. While neither ezrinTD nor ezrinWT senses negative curvature alone, we demonstrate that interacting with curvature-sensing I-BAR-domain proteins facilitates ezrin enrichment in negatively-curved membrane protrusions. Overall, our work demonstrates that ezrin can tether membranes, or be targeted to curved membranes, depending on conformations and interactions with actin and curvature-sensing binding partners.

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.

Small RAB GTPases Regulate Multiple Steps of Mitosis.

GTPases of the RAB family are key regulators of multiple steps of membrane trafficking. Several members of the RAB GTPase family have been implicated in mitotic progression. In this review, we will first focus on the function of endosome-associated RAB GTPases reported in early steps of mitosis, spindle pole maturation, and during cytokinesis. Second, we will discuss the role of Golgi-associated RAB GTPases at the metaphase/anaphase transition and during cytokinesis.

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.

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

Given their obligate intracellular lifestyle, Chlamydia trachomatis ensure that they have access to multiple host sources of essential lipids by interfering with vesicular transport. These bacteria hijack Rab6-, Rab11- and Rab14-controlled trafficking pathways to acquire sphingomyelin from the Golgi complex. 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 the interaction between MVBs and chlamydial inclusions 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 that depends on the Rab39a GTP- or GDP-binding state. Interestingly, Rab39a participates in the delivery of MVBs and host sphingolipids to maturing chlamydial inclusions, thereby promoting inclusion growth and bacterial development. Taken together, our findings indicate that Rab39a favours chlamydial replication and infectivity. This is the first report showing that a late endocytic Rab GTPase is involved in chlamydial infection development.

Analysis of the interactions between Rab GTPases and class V myosins.

Myosins are actin-based motor proteins that are involved in a wide variety of cellular processes such as membrane transport, muscle contraction, and cell division. Humans have over 40 myosins that can be placed into 18 classes, the malfunctioning of a number of which can lead to disease. There are three members of the human class V myosin family, myosins Va, Vb, and Vc. People lacking functional myosin Va suffer from a rare autosomal recessive disease called Griscelli's Syndrome type I (GS1) that is characterized by severe neurological defects and partial albinism. Mutations in the myosin Vb gene lead to an epithelial disorder called microvillus inclusion disease (MVID) that is often fatal in infants. The class V myosins have been implicated in the transport of diverse cargoes such as melanosomes in pigment cells, synaptic vesicles in neurons, RNA transcripts in a variety of cell types, and organelles such as the endoplasmic reticulum. The Rab GTPases play a critical role in recruiting class V myosins to their cargo. We recently published a study in which we used the yeast two-hybrid system to systematically test myosin Va for its ability to interact with each member of the human Rab GTPase family. We present here a detailed description of this yeast two-hybrid "living chip" assay. Furthermore, we present a protocol for validating positive interactions obtained from this screen by coimmunoprecipitation.

Recycling endosome tubule morphogenesis from sorting endosomes requires the kinesin motor KIF13A.

Early endosomes consist of vacuolar sorting and tubular recycling domains that segregate components fated for degradation in lysosomes or reuse by recycling to the plasma membrane or Golgi. The tubular transport intermediates that constitute recycling endosomes function in cell polarity, migration, and cytokinesis. Endosomal tubulation and fission require both actin and intact microtubules, but although factors that stabilize recycling endosomal tubules have been identified, those required for tubule generation from vacuolar sorting endosomes (SEs) remain unknown. We show that the microtubule motor KIF13A associates with recycling endosome tubules and controls their morphogenesis. Interfering with KIF13A function impairs the formation of endosomal tubules from SEs with consequent defects in endosome homeostasis and cargo recycling. Moreover, KIF13A interacts and cooperates with RAB11 to generate endosomal tubules. Our data illustrate how a microtubule motor couples early endosome morphogenesis to its motility and function.

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.

Studying mitochondria and microtubule localization and dynamics in standardized cell shapes.

Mammalian cells show a large diversity in shape and are both shape-changing and mobile when cultured on conventional uniform substrates. The use of micropatterning techniques limits the number of variable parameters, by imposing shape and standardized adhesive areas on the cells, which facilitates analysis. By changing size or shape of the micropattern, for example, forcing a polar axis on the cell, it is possible to study how these parameters impact organelle organization, distribution, and dynamics inside the cell. To study the mitochondrial network, which is composed of dynamic tubular organelles dependent on the microtubule cytoskeleton for its distribution, it is important to be able to distinguish between distinct mitochondria. Here, we present a practical method with which we spread the cells on large patterns created with deep UV technique, which not only makes the cells uniform in size and shape as well as immobile, and therefore easier to compare and analyze, but also expands the mitochondrial network and allows for an easier tracking of appropriately labeled individual mitochondria.

A new vesicular scaffolding complex mediates the G-protein-coupled 5-HT1A receptor targeting to neuronal dendrites.

Although essential for their neuronal function, the molecular mechanisms underlying the dendritic targeting of serotonin G-protein-coupled receptors are poorly understood. Here, we characterized a Yif1B-dependent vesicular scaffolding complex mediating the intracellular traffic of the rat 5-HT(1A) receptor (5-HT(1A)R) toward dendrites. By combining directed mutagenesis, GST-pull down, and surface plasmon resonance, we identified a tribasic motif in the C-tail of the 5-HT(1A)R on which Yif1B binds directly with high affinity (K(D) ≈ 37 nM). Moreover, we identified Yip1A, Rab6, and Kif5B as new partners of the 5-HT(1A)R/Yif1B complex, and showed that their expression in neurons is also crucial for the dendritic targeting of the 5-HT(1A)R. Live videomicroscopy revealed that 5-HT(1A)R, Yif1B, Yip1A, and Rab6 traffic in vesicles exiting the soma toward the dendritic tree, and also exhibit bidirectional motions, sustaining their role in 5-HT(1A)R dendritic targeting. Hence, we propose a new trafficking pathway model in which Yif1B is the scaffold protein recruiting the 5-HT(1A)R in a complex including Yip1A and Rab6, with Kif5B and dynein as two opposite molecular motors coordinating the traffic of vesicles along dendritic microtubules. This targeting pathway opens new insights for G-protein-coupled receptors trafficking in neurons.

KLHL3 mutations cause familial hyperkalemic hypertension by impairing ion transport in the distal nephron.

Familial hyperkalemic hypertension (FHHt) is a Mendelian form of arterial hypertension that is partially explained by mutations in WNK1 and WNK4 that lead to increased activity of the Na(+)-Cl(-) cotransporter (NCC) in the distal nephron. Using combined linkage analysis and whole-exome sequencing in two families, we identified KLHL3 as a third gene responsible for FHHt. Direct sequencing of 43 other affected individuals revealed 11 additional missense mutations that were associated with heterogeneous phenotypes and diverse modes of inheritance. Polymorphisms at KLHL3 were not associated with blood pressure. The KLHL3 protein belongs to the BTB-BACK-kelch family of actin-binding proteins that recruit substrates for Cullin3-based ubiquitin ligase complexes. KLHL3 is coexpressed with NCC and downregulates NCC expression at the cell surface. Our study establishes a role for KLHL3 as a new member of the complex signaling pathway regulating ion homeostasis in the distal nephron and indirectly blood pressure.