Beyond its preferential niche: Brucella abortus RNA down-modulates the IFN-γ-induced MHC-I expression in epithelial and endothelial cells.

Brucella abortus (Ba) is a pathogen that survives inside macrophages. Despite being its preferential niche, Ba infects other cells, as shown by the multiple signs and symptoms humans present. This pathogen can evade our immune system. Ba displays a mechanism of down-modulating MHC-I on monocytes/macrophages in the presence of IFN-γ (when Th1 response is triggered) without altering the total expression of MHC-I. The retained MHC-I proteins are located within the Golgi Apparatus (GA). The RNA of Ba is one of the PAMPs that trigger this phenomenon. However, we acknowledged whether this event could be triggered in other cells relevant during Ba infection. Here, we demonstrate that Ba RNA reduced the surface expression of MHC-I induced by IFN-γ in the human bronchial epithelium (Calu-6), the human alveolar epithelium (A-549) and the endothelial microvasculature (HMEC) cell lines. In Calu-6 and HMEC cells, Ba RNA induces the retention of MHC-I in the GA. This phenomenon was not observed in A-549 cells. We then evaluated the effect of Ba RNA on the secretion of IL-8, IL-6 and MCP-1, key cytokines in Ba infection. Contrary to our expectations, HMEC, Calu-6 and A-549 cells treated with Ba RNA had higher IL-8 and IL-6 levels compared to untreated cells. In addition, we showed that Ba RNA down-modulates the MHC-I surface expression induced by IFN-γ on human monocytes/macrophages via the pathway of the Epidermal Growth Factor Receptor (EGFR). So, cells were stimulated with an EGFR ligand-blocking antibody (Cetuximab) and Ba RNA. Neutralization of the EGFR to some extent reversed the down-modulation of MHC-I mediated by Ba RNA in HMEC and A-549 cells. In conclusion, this is the first study exploring a central immune evasion strategy, such as the downregulation of MHC-I surface expression, beyond monocytes and could shed light on how it persists effectively within the host, enduring unseen and escaping CD8+ T cell surveillance.

GOLPH3 Participates in Mitochondrial Fission and Is Necessary to Sustain Bioenergetic Function in MDA-MB-231 Breast Cancer Cells.

In this study, we investigated the inter-organelle communication between the Golgi apparatus (GA) and mitochondria. Previous observations suggest that GA-derived vesicles containing phosphatidylinositol 4-phosphate (PI(4)P) play a role in mitochondrial fission, colocalizing with DRP1, a key protein in this process. However, the functions of these vesicles and potentially associated proteins remain unknown. GOLPH3, a PI(4)P-interacting GA protein, is elevated in various types of solid tumors, including breast cancer, yet its precise role is unclear. Interestingly, GOLPH3 levels influence mitochondrial mass by affecting cardiolipin synthesis, an exclusive mitochondrial lipid. However, the mechanism by which GOLPH3 influences mitochondria is not fully understood. Our live-cell imaging analysis showed GFP-GOLPH3 associating with PI(4)P vesicles colocalizing with YFP-DRP1 at mitochondrial fission sites. We tested the functional significance of these observations with GOLPH3 knockout in MDA-MB-231 cells of breast cancer, resulting in a fragmented mitochondrial network and reduced bioenergetic function, including decreased mitochondrial ATP production, mitochondrial membrane potential, and oxygen consumption. Our findings suggest a potential negative regulatory role for GOLPH3 in mitochondrial fission, impacting mitochondrial function and providing insights into GA-mitochondria communication.

Memories of heat: autophagy and Golgi recovery.

Fluctuations in temperature severely impact crop yield and trigger various plant response mechanisms. In a recent study, Zhou et al. discovered a non-canonical role of autophagy in mediating Golgi apparatus restoration after short-term heat stress (HS). Their results further suggest a critical, yet previously unknown, mechanism of autophagy-related (ATG)-8 in Golgi reassembly after HS.

The ubiquitin E3 ligase TRIM10 promotes STING aggregation and activation in the Golgi apparatus.

STING is an endoplasmic reticulum-resident protein regulating innate immunity. After binding with cyclic guanosine monophosphate-AMP (cGAMP), STING translocates from the endoplasmic reticulum (ER) to the Golgi apparatus to stimulate TBK1 and IRF3 activation, leading to expression of type I interferon. However, the exact mechanism concerning STING activation remains largely enigmatic. Here, we identify tripartite motif 10 (TRIM10) as a positive regulator of STING signaling. TRIM10-deficient macrophages exhibit reduced type I interferon production upon double-stranded DNA (dsDNA) or cGAMP stimulation and decreased resistance to herpes simplex virus 1 (HSV-1) infection. Additionally, TRIM10-deficient mice are more susceptible to HSV-1 infection and exhibit faster melanoma growth. Mechanistically, TRIM10 associates with STING and catalyzes K27- and K29-linked polyubiquitination of STING at K289 and K370, which promotes STING trafficking from the ER to the Golgi apparatus, formation of STING aggregates, and recruitment of TBK1 to STING, ultimately enhancing the STING-dependent type I interferon response. Our study defines TRIM10 as a critical activator in cGAS-STING-mediated antiviral and antitumor immunity.

A second chance at yeast early endosomes.

Post-endocytic recycling in yeast has been posited to transit solely through the Golgi, raising the possibility that yeast lack early endosomes. In this issue, Laidlaw and colleagues (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202109137) describe a yeast endosomal recycling pathway that gives proteins a second chance to return to the plasma membrane.

Golgi Phosphoprotein 3 Regulates the Physical Association of Glycolipid Glycosyltransferases.

Glycolipid glycosylation is an intricate process that mainly takes place in the Golgi by the complex interplay between glycosyltransferases. Several features such as the organization, stoichiometry and composition of these complexes may modify their sorting properties, sub-Golgi localization, enzymatic activity and in consequence, the pattern of glycosylation at the plasma membrane. In spite of the advance in our comprehension about physiological and pathological cellular states of glycosylation, the molecular basis underlying the metabolism of glycolipids and the players involved in this process remain not fully understood. In the present work, using biochemical and fluorescence microscopy approaches, we demonstrate the existence of a physical association between two ganglioside glycosyltransferases, namely, ST3Gal-II (GD1a synthase) and ß3GalT-IV (GM1 synthase) with Golgi phosphoprotein 3 (GOLPH3) in mammalian cultured cells. After GOLPH3 knockdown, the localization of both enzymes was not affected, but the fomation of ST3Gal-II/ß3GalT-IV complex was compromised and glycolipid expression pattern changed. Our results suggest a novel control mechanism of glycolipid expression through the regulation of the physical association between glycolipid glycosyltransferases mediated by GOLPH3.

Clathrin adaptor AP-1-mediated Golgi export of amyloid precursor protein is crucial for the production of neurotoxic amyloid fragments.

One of the hallmarks of Alzheimer's disease is the accumulation of toxic amyloid-ß (Aß) peptides in extracellular plaques. The direct precursor of Aß is the carboxyl-terminal fragment ß (or C99) of the amyloid precursor protein (APP). C99 is detected at elevated levels in Alzheimer's disease brains, and its intracellular accumulation has been linked to early neurotoxicity independently of Aß. Despite this, the causes of increased C99 levels are poorly understood. Here, we demonstrate that APP interacts with the clathrin vesicle adaptor AP-1 (adaptor protein 1), and we map the interaction sites on both proteins. Using quantitative kinetic trafficking assays, established cell lines and primary neurons, we also show that this interaction is required for the transport of APP from the trans-Golgi network to endosomes. In addition, disrupting AP-1-mediated transport of APP alters APP processing and degradation, ultimately leading to increased C99 production and Aß release. Our results indicate that AP-1 regulates the subcellular distribution of APP, altering its processing into neurotoxic fragments.

BARS Influences Neuronal Development by Regulation of Post-Golgi Trafficking.

Neurons are highly polarized cells requiring precise regulation of trafficking and targeting of membrane proteins to generate and maintain different and specialized compartments, such as axons and dendrites. Disruption of the Golgi apparatus (GA) secretory pathway in developing neurons alters axon/dendritic formation. Therefore, detailed knowledge of the mechanisms underlying vesicles exiting from the GA is crucial for understanding neuronal polarity. In this study, we analyzed the role of Brefeldin A-Ribosylated Substrate (CtBP1-S/BARS), a member of the C-terminal-binding protein family, in the regulation of neuronal morphological polarization and the exit of membrane proteins from the Trans Golgi Network. Here, we show that BARS is expressed during neuronal development in vitro and that RNAi suppression of BARS inhibits axonal and dendritic elongation in hippocampal neuronal cultures as well as largely perturbed neuronal migration and multipolar-to-bipolar transition during cortical development in situ. In addition, using plasma membrane (PM) proteins fused to GFP and engineered with reversible aggregation domains, we observed that expression of fission dominant-negative BARS delays the exit of dendritic and axonal membrane protein-containing carriers from the GA. Taken together, these data provide the first set of evidence suggesting a role for BARS in neuronal development by regulating post-Golgi membrane trafficking.

A gold revision of the Golgi Dynamics (GOLD) domain structure and associated cell functionalities.

The classical secretory pathway is the key membrane-based delivery system in eukaryotic cells. Several families of proteins involved in the secretory pathway, with functionalities going from cargo sorting receptors to the maintenance and dynamics of secretory organelles, share soluble globular domains predicted to mediate protein-protein interactions. One of them is the 'Golgi Dynamics' (GOLD) domain, named after its strong association with the Golgi apparatus. There are many GOLD-containing protein families, such as the transmembrane emp24 domain-containing proteins (TMED/p24 family), animal SEC14-like proteins, human Golgi resident protein ACBD3, a splice variant of TICAM2 called TRAM with GOLD domain, and FYCO1. Here, we critically review the state-of-the-art knowledge of the structures and functions of the main representatives of GOLD-containing proteins in vertebrates. We provide the first unified description of the GOLD domain structure across different families since the first high-resolution structure was determined. With a brand-new update on the definition of the GOLD domain, we also discuss how its tertiary structure fits the ß-sandwich-like fold map and give exciting new directions for forthcoming studies.

The dendritic ERGIC: Microtubule and actin cytoskeletons participate in stop-and-go movement of mobile carriers between stable structures.

The endoplasmic reticulum (ER)-to-Golgi intermediate compartment (ERGIC) is a membranous organelle that mediates protein transport between the ER and the Golgi apparatus. In neurons, clusters of these vesiculotubular structures are situated throughout the cell in proximity to the ER, passing cargo to the cis-Golgi cisternae, located mainly in the perinuclear region. Although ERGIC markers have been identified in neurons, the distribution and dynamics of neuronal ERGIC structures have not been characterized yet. Here, we show that long-distance ERGIC transport occurs via an intermittent mechanism in dendrites, with mobile elements moving between stationary structures. Slow and fast live-cell imaging have captured stable ERGIC structures remaining in place over long periods of time, as well as mobile ERGIC structures advancing very short distances along dendrites. These short distances have been consistent with the lengths between the stationary ERGIC structures. Kymography revealed ERGIC elements that moved intermittently, emerging from and fusing with stationary ERGIC structures. Interestingly, this movement apparently depends not only on the integrity of the microtubule cytoskeleton, as previously reported, but on the actin cytoskeleton as well. Our results indicate that the dendritic ERGIC has a dual nature, with both stationary and mobile structures. The neural ERGIC network transports proteins via a stop-and-go movement in which both the microtubule and the actin cytoskeletons participate.

Rab1b-GBF1-ARF1 Secretory Pathway Axis Is Required for Birnavirus Replication.

Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide. We previously demonstrated that IBDV hijacks the endocytic pathway for establishing the viral replication complexes on endosomes associated with the Golgi complex (GC). Here, we report that IBDV reorganizes the GC to localize the endosome-associated replication complexes without affecting its secretory functionality. By analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b for viral replication. Rab1b comprises a key regulator of GC transport and we demonstrate that transfecting the negative mutant Rab1b N121I or knocking down Rab1b expression by RNA interference significantly reduces the yield of infectious viral progeny. Furthermore, we showed that the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), which activates the small GTPase ADP ribosylation factor 1 (ARF1), is required for IBDV replication, since inhibiting its activity by treatment with brefeldin A (BFA) or golgicide A (GCA) significantly reduces the yield of infectious viral progeny. Finally, we show that ARF1 dominant negative mutant T31N overexpression hampered IBDV infection. Taken together, these results demonstrate that IBDV requires the function of the Rab1b-GBF1-ARF1 axis to promote its replication, making a substantial contribution to the field of birnavirus-host cell interactions. IMPORTANCE Birnaviruses are unconventional members of the dsRNA viruses, with the lack of a transcriptionally active core being the main differential feature. This structural trait, among others that resemble those of the plus single-stranded (+ssRNA) viruses features, suggests that birnaviruses might follow a different replication program from that conducted by prototypical dsRNA members and the hypothesis that birnaviruses could be evolutionary links between +ssRNA and dsRNA viruses has been argued. Here, we present original data showing that IBDV-induced GC reorganization and the cross talk between IBDV and the Rab1b-GBF1-ARF1 mediate the intracellular trafficking pathway. The replication of several +ssRNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. Thus, our findings make a substantial contribution to the field of birnavirus-host cell interactions and provide further evidence supporting the proposed evolutionary connection role of birnaviruses, an aspect which we consider especially relevant for researchers working in the virology field.

Structural and thermodynamic analyses of human TMED1 (p24γ1) Golgi dynamics.

The transmembrane emp24 domain-containing (TMED) proteins, also called p24 proteins, are members of a family of sorting receptors present in all representatives of the Eukarya and abundantly present in all subcompartments of the early secretory pathway, namely the endoplasmic reticulum (ER), the Golgi, and the intermediate compartment. Although essential during the bidirectional transport between the ER and the Golgi, there is still a lack of information regarding the TMED's structure across different subfamilies. Besides, although the presence of a TMED homo-oligomerization was suggested previously based on crystallographic contacts observed for the isolated Golgi Dynamics (GOLD) domain, no further analyses of its presence in solution were done. Here, we describe the first high-resolution structure of a TMED1 GOLD representative and its biophysical characterization in solution. The crystal structure showed a dimer formation that is also present in solution in a salt-dependent manner, suggesting that the GOLD domain can form homodimers in solution even in the absence of the TMED1 coiled-coil region. A molecular dynamics description of the dimer stabilization, with a phylogenetic analysis of the residues important for the oligomerization and a model for the orientation towards the lipid membrane, are also presented.

Myristoylation and its effects on the human Golgi Reassembly and Stacking Protein 55.

GRASP55 is a myristoylated protein localized in the medial/trans-Golgi faces and involved in the Golgi structure maintenance and the regulation of unconventional secretion pathways. It is believed that GRASP55 achieves its main functionalities in the Golgi organization by acting as a tethering factor. When bound to the lipid bilayer, its orientation relative to the membrane surface is restricted to determine its proper trans-oligomerization. Despite the paramount role of myristoylation in GRASP function, the impact of such protein modification on the membrane-anchoring properties and the structural organization of GRASP remains elusive. Here, an optimized protocol for the myristoylation in E. coli of the membrane-anchoring domain of GRASP55 is presented. The biophysical properties of the myristoylated/non-myristoylated GRASP55 GRASP domain were characterized in a membrane-mimicking micellar environment. Although myristoylation did not cause any impact on the protein's secondary structure, according to our circular dichroism data, it had a significant impact on the protein's thermal stability and solubility. Electrophoresis of negatively charged liposomes incubated with the two GRASP55 constructions showed different electrophoretic mobility for the myristoylated anchored protein only, thus demonstrating that myristoylation is essential for the biological membrane anchoring. Molecular dynamics simulations were used to further explore the anchoring process in determining the restricted orientation of GRASPs in the membrane.

zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways.

Here we introduce zapalog-mediated endoplasmic reticulum trap (zapERtrap), which allows one to use light to precisely trigger forward trafficking of diverse integral membrane proteins from internal secretory organelles to the cell surface with single cell and subcellular spatial resolution. To demonstrate its utility, we use zapERtrap in neurons to dissect where synaptic proteins emerge at the cell surface when processed through central (cell body) or remote (dendrites) secretory pathways. We reveal rapid and direct long-range trafficking of centrally processed proteins deep into the dendritic arbor to synaptic sites. Select proteins were also trafficked to the plasma membrane of the axon initial segment, revealing a novel surface trafficking hotspot. Proteins locally processed through dendritic secretory networks were widely dispersed before surface insertion, challenging assumptions for precise trafficking at remote sites. These experiments provide new insights into compartmentalized secretory trafficking and showcase the tunability and spatiotemporal control of zapERtrap, which will have broad applications for regulating cell signaling and function.

Imaging the electrical activity of organelles in living cells.

Eukaryotic cells are complex systems compartmentalized in membrane-bound organelles. Visualization of organellar electrical activity in living cells requires both a suitable reporter and non-invasive imaging at high spatiotemporal resolution. Here we present hVoSorg, an optical method to monitor changes in the membrane potential of subcellular membranes. This method takes advantage of a FRET pair consisting of a membrane-bound voltage-insensitive fluorescent donor and a non-fluorescent voltage-dependent acceptor that rapidly moves across the membrane in response to changes in polarity. Compared to the currently available techniques, hVoSorg has advantages including simple and precise subcellular targeting, the ability to record from individual organelles, and the potential for optical multiplexing of organellar activity.

From cartoons to quantitative models in Golgi transport.

BACKGROUND: Cell biology is evolving to become a more formal and quantitative science. In particular, several mathematical models have been proposed to address Golgi self-organisation and protein and lipid transport. However, most scientific articles about the Golgi apparatus are still using static cartoons that miss the dynamism of this organelle. RESULTS: In this report, we show that schematic drawings of Golgi trafficking can be easily translated into an agent-based model using the Repast platform. The simulations generate an active interplay among cisternae and vesicles rendering quantitative predictions about Golgi stability and transport of soluble and membrane-associated cargoes. The models can incorporate complex networks of molecular interactions and chemical reactions by association with COPASI, a software that handles ordinary differential equations. CONCLUSIONS: The strategy described provides a simple, flexible and multiscale support to analyse Golgi transport. The simulations can be used to address issues directly linked to the mechanism of transport or as a way to incorporate the complexity of trafficking to other cellular processes that occur in dynamic organelles. SIGNIFICANCE: We show that the rules implicitly present in most schematic representations of intracellular trafficking can be used to build dynamic models with quantitative outputs that can be compared with experimental results.

Host Retromer Protein Sorting Nexin 2 Interacts with Human Respiratory Syncytial Virus Structural Proteins and is Required for Efficient Viral Production.

Human respiratory syncytial virus (HRSV) envelope glycoproteins traffic to assembly sites through the secretory pathway, while nonglycosylated proteins M and N are present in HRSV inclusion bodies but must reach the plasma membrane, where HRSV assembly happens. Little is known about how nonglycosylated HRSV proteins reach assembly sites. Here, we show that HRSV M and N proteins partially colocalize with the Golgi marker giantin, and the glycosylated F and nonglycosylated N proteins are closely located in the trans-Golgi, suggesting their interaction in that compartment. Brefeldin A compromised the trafficking of HRSV F and N proteins and inclusion body sizes, indicating that the Golgi is important for both glycosylated and nonglycosylated HRSV protein traffic. HRSV N and M proteins colocalized and interacted with sorting nexin 2 (SNX2), a retromer component that shapes endosomes in tubular structures. Glycosylated F and nonglycosylated N HRSV proteins are detected in SNX2-laden aggregates with intracellular filaments projecting from their outer surfaces, and VPS26, another retromer component, was also found in inclusion bodies and filament-shaped structures. Similar to SNX2, TGN46 also colocalized with HRSV M and N proteins in filamentous structures at the plasma membrane. Cell fractionation showed enrichment of SNX2 in fractions containing HRSV M and N proteins. Silencing of SNX1 and 2 was associated with reduction in viral proteins, HRSV inclusion body size, syncytium formation, and progeny production. The results indicate that HRSV structural proteins M and N are in the secretory pathway, and SNX2 plays an important role in the traffic of HRSV structural proteins toward assembly sites.IMPORTANCE The present study contributes new knowledge to understand HRSV assembly by providing evidence that nonglycosylated structural proteins M and N interact with elements of the secretory pathway, shedding light on their intracellular traffic. To the best of our knowledge, the present contribution is important given the scarcity of studies about the traffic of HRSV nonglycosylated proteins, especially by pointing to the involvement of SNX2, a retromer component, in the HRSV assembly process.

Human Golgi phosphoprotein 3 is an effector of RAB1A and RAB1B.

Golgi phosphoprotein 3 (GOLPH3) is a peripheral membrane protein localized at the trans-Golgi network that is also distributed in a large cytosolic pool. GOLPH3 has been involved in several post-Golgi protein trafficking events, but its precise function at the molecular level is not well understood. GOLPH3 is also considered the first oncoprotein of the Golgi apparatus, with important roles in several types of cancer. Yet, it is unknown how GOLPH3 is regulated to achieve its contribution in the mechanisms that lead to tumorigenesis. Binding of GOLPH3 to Golgi membranes depends on its interaction to phosphatidylinositol-4-phosphate. However, an early finding showed that GTP promotes the binding of GOLPH3 to Golgi membranes and vesicles. Nevertheless, it remains largely unknown whether this response is consequence of the function of GTP-dependent regulatory factors, such as proteins of the RAB family of small GTPases. Interestingly, in Drosophila melanogaster the ortholog of GOLPH3 interacts with- and behaves as effector of the ortholog of RAB1. However, there is no experimental evidence implicating GOLPH3 as a possible RAB1 effector in mammalian cells. Here, we show that human GOLPH3 interacted directly with either RAB1A or RAB1B, the two isoforms of RAB1 in humans. The interaction was nucleotide dependent and it was favored with GTP-locked active state variants of these GTPases, indicating that human GOLPH3 is a bona fide effector of RAB1A and RAB1B. Moreover, the expression in cultured cells of the GTP-locked variants resulted in less distribution of GOLPH3 in the Golgi apparatus, suggesting an intriguing model of GOLPH3 regulation.

Biophysical characterization of intrinsically disordered human Golgi matrix protein GRASP65.

Golgi Reassembly and Stacking Proteins (GRASPs), including GRASP65/GRASP55, were firstly found as stacking factors of Golgi cisternae. Their involvement in other processes, such as unconventional protein secretion (UPS), have been demonstrated, suggesting GRASPs act as interaction hubs. However, structural details governing GRASP functions are not understood thoroughly. Here, we explored the structural features of human cis-Golgi GRASP65 in aqueous solution and compared them with those from trans-Golgi GRASP55. Besides their distinct Golgi localization, GRASP65/55 also seem to be selectively recruited to mitosis-related events or to UPS. Despite preserving the monomeric form in solution seen for GRASP55, as inferred from our SEC-MALS and DLS data, GRASP65 exhibited higher intrinsic disorder and susceptibility to denaturant than GRASP55 (disorder prediction, urea denaturation and circular dichroism data). Moreover, spectroscopic and microscopic studies showed for GRASP65 the same temperature-dependent amorphous aggregation and time-dependent amyloid fibrillation at 37 °C seen for GRASP55. In the latter case, however, GRASP65 presented a lower aggregation rate than GRASP55. The present and previous data evidenced that intrinsic disorder and formation of higher-order oligomers, such as amyloid fibrils, are common features within GRASP family potentially impacting the protein's participation in cell processes.

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.