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Down syndrome (DS) is the most common form of inherited intellectual disability caused by trisomy of chromosome 21, presenting with intellectual impairment, craniofacial abnormalities, cardiac defects, and gastrointestinal disorders. The Ts65Dn mouse model replicates many abnormalities of DS. We hypothesized that investigation of the cerebral cortex of fluoxetine-treated trisomic mice may provide proteomic signatures that identify therapeutic targets for DS. Subcellular fractionation of synaptosomes from cerebral cortices of age- and brain-area-matched samples from fluoxetine-treated vs. water-treated trisomic and euploid male mice were subjected to HPLC-tandem mass spectrometry. Analysis of the data revealed enrichment of trisomic risk genes that participate in regulation of synaptic vesicular traffic, pre-synaptic and post-synaptic development, and mitochondrial energy pathways during early brain development. Proteomic analysis of trisomic synaptic fractions revealed significant downregulation of proteins involved in synaptic vesicular traffic, including vesicular endocytosis (CLTA, CLTB, CLTC), synaptic assembly and maturation (EXOC1, EXOC3, EXOC8), anterograde axonal transport (EXOC1), neurotransmitter transport to PSD (SACM1L), endosomal-lysosomal acidification (ROGDI, DMXL2), and synaptic signaling (NRXN1, HIP1, ITSN1, YWHAG). Additionally, trisomic proteomes revealed upregulation of several trafficking proteins, involved in vesicular exocytosis (Rab5B), synapse elimination (UBE3A), scission of endocytosis (DBN1), transport of ER in dendritic spines (MYO5A), presynaptic activity-dependent bulk endocytosis (FMR1), and NMDA receptor activity (GRIN2A). Chronic fluoxetine treatment of Ts65Dn mice rescued synaptic vesicular abnormalities and prevented abnormal proteomic changes in adult Ts65Dn mice, pointing to therapeutic targets for potential treatment of DS. [ABSTRACT FROM AUTHOR]

CD36 is a type 2 cell surface scavenger receptor expressed in various tissues. In macrophages, CD36 recognizes oxidized low-density lipoprotein (ox-LDL), which promotes the formation of foam cells, the first step toward an atherosclerotic arterial lesion. CD36 possesses a variety of posttranslational modifications, among them N-glycosylation and O-GlcNAc modification. Some of the roles of these modifications on CD36 are known, such as N-linked glycosylation, which provides proper folding and trafficking to the plasma membrane in the human embryonic kidney. This study aimed to determine whether variations in the availability of UDP-GlcNAc could impact Rab-5-mediated endocytic trafficking and, therefore, the cellular localization of CD36. These preliminary results suggest that the availability of the substrate UDP-GlcNAc, modulated in response to treatment with Thiamet G (TMG), OSMI-1 (O-GlcNAcylation enzymes modulators) or Azaserine (HBP modulator), influences the localization of CD36 in J774 macrophages, and the endocytic trafficking as evidenced by the regulatory protein Rab-5, between the plasma membrane and the cytoplasm.

Studies on the pathogen–host interaction are crucial for the understanding of the mechanisms involved in the establishment, maintenance, and spread of infection. In recent years, our research group has observed that the P. brasiliensis species interact with integrin family receptors and increase the expression of α3 integrin in lung epithelial cells within 5 h of infection. Interestingly, α3 integrin levels were reduced by approximately 99% after 24 h of infection with P. brasiliensis compared to non-infected cells. In this work, we show that, during infection with this fungus, α3 integrin is increased in the late endosomes of A549 lung epithelial cells. We also observed that the inhibitor of the lysosomal activity bafilomycin A1 was able to inhibit the decrease in α3 integrin levels. In addition, the silencing of the charged multivesicular body protein 3 (CHMP3) inhibited the reduction in α3 integrin levels induced by P. brasiliensis in A549 cells. Thus, together, these results indicate that this fungus induces the degradation of α3 integrin in A549 lung epithelial cells by hijacking the host cell endolysosomal pathway. [ABSTRACT FROM AUTHOR]

Simple Summary: In diabetic nephropathy, the injury of renal cells such as podocytes worsens the glomerular filtration process due to the cells' loss and detachment from the basal membrane. Intra- and intercellular communications in cells are partially mediated by the Rab3A/Rab27A system. We have observed that silencing the Rab3A/Rab27A system in podocytes experiencing glucose overload has a differential impact on differentiation, cytoskeleton organisation, apoptosis, CD63 distribution, and miRNA levels. Generally, when cells are subjected to glucose stress and the Rab system is silenced, we observed a reduction in the detrimental cell processes. These results suggest that the Rab3A/Rab27A system is a key participant in podocyte injury and vesicular traffic regulation in diabetic nephropathy. Diabetic nephropathy is a major complication in diabetic patients. Podocytes undergo loss and detachment from the basal membrane. Intra- and intercellular communication through exosomes are key processes for maintaining function, and the Rab3A/Rab27A system is an important counterpart. Previously, we observed significant changes in the Rab3A/Rab27A system in podocytes under glucose overload, demonstrating its important role in podocyte injury. We investigated the implication of silencing the Rab3A/Rab27A system in high glucose-treated podocytes and analysed the effect on differentiation, apoptosis, cytoskeletal organisation, vesicle distribution, and microRNA expression in cells and exosomes. For this, we subjected podocytes to high glucose and transfection through siRNAs, and we isolated extracellular vesicles and performed western blotting, transmission electron microscopy, RT-qPCR, immunofluorescence and flow cytometry assays. We found that silencing RAB3A and RAB27A generally leads to a decrease in podocyte differentiation and cytoskeleton organization and an increase in apoptosis. Moreover, CD63-positive vesicles experienced a pattern distribution change. Under high glucose, Rab3A/Rab27A silencing ameliorates some of these detrimental processes, suggesting a differential influence depending on the presence or absence of cellular stress. We also observed substantial expression changes in miRNAs that were relevant in diabetic nephropathy upon silencing and glucose treatment. Our findings highlight the Rab3A/Rab27A system as a key participant in podocyte injury and vesicular traffic regulation in diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Type I inositol polyphosphate-4-phosphatase (INPP4A) belongs to the group of phosphoinositide phosphatases controlling proliferation, apoptosis, and endosome function by hydrolyzing phosphatidylinositol 3,4-bisphosphate. INPP4A produces multiple transcripts encoding shorter and longer INPP4A isoforms with hydrophilic or hydrophobic C-terminus. Biallelic INPP4A truncating variants cause a spectrum of neurodevelopmental disorders ranging from moderate intellectual disability to postnatal microcephaly with developmental and epileptic encephalopathy and (ponto)cerebellar hypoplasia. We report a girl with the novel homozygous INPP4A variant NM_001134224.2:c.2840del/p.(Gly947Glufs*12) (isoform d). She presented with postnatal microcephaly, global developmental delay, visual impairment, myoclonic seizures, and pontocerebellar hypoplasia and died at the age of 27 months. The level of mutant INPP4A mRNAs in proband-derived leukocytes was comparable to controls suggesting production of C-terminally altered INPP4A isoforms. We transiently expressed eGFP-tagged INPP4A isoform a (NM_004027.3) wildtype and p.(Gly908Glufs*12) mutant [p.(Gly947Glufs*12) according to NM_001134224.2] as well as INPP4A isoform b (NM_001566.2) wildtype and p.(Asp915Alafs*2) mutant, previously reported in family members with moderate intellectual disability, in HeLa cells and determined their subcellular distributions. While INPP4A isoform a was preferentially found in perinuclear clusters co-localizing with the GTPase Rab5, isoform b showed a net-like distribution, possibly localizing near and/or on microtubules. Quantification of intracellular localization patterns of the two INPP4A mutants revealed significant differences compared with the respective wildtype and similarity with each other. Our data suggests an important non-redundant function of INPP4A isoforms with hydrophobic or hydrophilic C-terminus in the brain. [ABSTRACT FROM AUTHOR]

In mammalian retina, cholesterol excess is mainly metabolized to oxysterols by cytochromes P450 27A1 (CYP27A1) and 46A1 (CYP46A1) or removed on lipoprotein particles containing apolipoprotein E (APOE). In contrast, esterification by sterol-O-acyltransferase 1 (SOAT) plays only a minor role in this process. Accordingly, retinal cholesterol levels are unchanged in Soat1-/- mice but are increased in Cyp27a1-/-Cyp46a1-/- and Apoe-/- mice. Herein, we characterized Cyp27a1-/-Cyp46a1-/-Soat1-/- and Cyp27a1-/-Cyp46a1-/-Apoe-/- mice. In the former, retinal cholesterol levels, anatomical gross structure, and vasculature were normal, yet the electroretinographic responses were impaired. Conversely, in Cyp27a1-/-Cyp46a1-/-Apoe-/- mice, retinal cholesterol levels were increased while anatomical structure and vasculature were unaffected with only male mice showing a decrease in electroretinographic responses. Sterol profiling, qRT-PCR, proteomics, and transmission electron microscopy mapped potential compensatory mechanisms in the Cyp27a1-/-Cyp46a1-/-Soat1-/- and Cyp27a1-/-Cyp46a1-/-Apoe-/- retina. These included decreased cholesterol biosynthesis along with enhanced formation of intra- and extracellular vesicles, possibly a reserve mechanism for lowering retinal cholesterol. In addition, there was altered abundance of proteins in Cyp27a1-/-Cyp46a1-/-Soat1-/- mice that can affect photoreceptor function, survival, and retinal energy homeostasis (glucose and fatty acid metabolism). Therefore, the levels of retinal cholesterol do not seem to predict retinal abnormalities, and it is rather the network of compensatory mechanisms that appears to determine retinal phenotype.

Apolipoprotein E (APOE) is a component of lipid-transporting particles and a recognition ligand for receptors, which bind these particles. The APOE isoform ε2 is a risk factor for age-related macular degeneration; nevertheless, APOE absence in humans and mice does not significantly affect the retina. We found that retinal cholesterol biosynthesis and the levels of retinal cholesterol were increased in Apoe-/- mice, whereas cholesterol elimination by metabolism was decreased. No focal cholesterol deposits were observed in the Apoe-/- retina. Retinal proteomics identified the most abundant cholesterol-related proteins in WT mice and revealed that, of these cholesterol-related proteins, only APOA4 had increased expression in the Apoe-/- retina. In addition, there were changes in retinal abundance of proteins involved in proinflammatory and antiinflammatory responses, cellular cytoskeleton maintenance, vesicular traffic, and retinal iron homeostasis. The data obtained indicate that when APOE is absent, particles containing APOA1, APOA4, and APOJ still transport cholesterol in the intraretinal space, but these particles are not taken up by retinal cells. Therefore, cholesterol biosynthesis inside retinal cells increase, whereas metabolism to oxysterols decreases to prevent cells from cholesterol depletion. These and other compensatory changes underlie only a minor retinal phenotype in Apoe-/- mice.

Initially discovered as the smallest component of the intraflagellar transport (IFT) system, the IFT20 protein has been found to be implicated in several unconventional mechanisms beyond its essential role in the assembly and maintenance of the primary cilium. IFT20 is now considered a key player not only in ciliogenesis but also in vesicular trafficking of membrane receptors and signaling proteins. Moreover, its ability to associate with a wide array of interacting partners in a cell-type specific manner has expanded the function of IFT20 to the regulation of intracellular degradative and secretory pathways. In this review, we will present an overview of the multifaceted role of IFT20 in both ciliated and non-ciliated cells. [ABSTRACT FROM AUTHOR]

Studies on the pathogen–host interaction are crucial for the understanding of the mechanisms involved in the establishment, maintenance, and spread of infection. In recent years, our research group has observed that the P. brasiliensis species interact with integrin family receptors and increase the expression of α3 integrin in lung epithelial cells within 5 h of infection. Interestingly, α3 integrin levels were reduced by approximately 99% after 24 h of infection with P. brasiliensis compared to non-infected cells. In this work, we show that, during infection with this fungus, α3 integrin is increased in the late endosomes of A549 lung epithelial cells. We also observed that the inhibitor of the lysosomal activity bafilomycin A1 was able to inhibit the decrease in α3 integrin levels. In addition, the silencing of the charged multivesicular body protein 3 (CHMP3) inhibited the reduction in α3 integrin levels induced by P. brasiliensis in A549 cells. Thus, together, these results indicate that this fungus induces the degradation of α3 integrin in A549 lung epithelial cells by hijacking the host cell endolysosomal pathway.

Endomembrane system compartments are significant elements in virtually all eukaryotic cells, supporting functions including protein synthesis, post‐translational modifications and protein/lipid targeting. In terms of membrane area the endoplasmic reticulum (ER) is the largest intracellular organelle, but the origins of proteins defining the organelle and the nature of lineage‐specific modifications remain poorly studied. To understand the evolution of factors mediating ER morphology and function we report a comparative genomics analysis of experimentally characterized ER‐associated proteins involved in maintaining ER structure. We find that reticulons, REEPs, atlastins, Ufe1p, Use1p, Dsl1p, TBC1D20, Yip3p and VAPs are highly conserved, suggesting an origin at least as early as the last eukaryotic common ancestor (LECA), although many of these proteins possess additional non‐ER functions in modern eukaryotes. Secondary losses are common in individual species and in certain lineages, for example lunapark is missing from the Stramenopiles and the Alveolata. Lineage‐specific innovations include protrudin, Caspr1, Arl6IP1, p180, NogoR, kinectin and CLIMP‐63, which are restricted to the Opisthokonta. Hence, much of the machinery required to build and maintain the ER predates the LECA, but alternative strategies for the maintenance and elaboration of ER shape and function are present in modern eukaryotes. Moreover, experimental investigations for ER maintenance factors in diverse eukaryotes are expected to uncover novel mechanisms. [ABSTRACT FROM AUTHOR]

Diabetic nephropathy is a major complication in diabetic patients. Podocytes undergo loss and detachment from the basal membrane. Intra- and intercellular communication through exosomes are key processes for maintaining function, and the Rab3A/Rab27A system is an important counterpart. Previously, we observed significant changes in the Rab3A/Rab27A system in podocytes under glucose overload, demonstrating its important role in podocyte injury. We investigated the implication of silencing the Rab3A/Rab27A system in high glucose-treated podocytes and analysed the effect on differentiation, apoptosis, cytoskeletal organisation, vesicle distribution, and microRNA expression in cells and exosomes. For this, we subjected podocytes to high glucose and transfection through siRNAs, and we isolated extracellular vesicles and performed western blotting, transmission electron microscopy, RT-qPCR, immunofluorescence and flow cytometry assays. We found that silencing RAB3A and RAB27A generally leads to a decrease in podocyte differentiation and cytoskeleton organization and an increase in apoptosis. Moreover, CD63-positive vesicles experienced a pattern distribution change. Under high glucose, Rab3A/Rab27A silencing ameliorates some of these detrimental processes, suggesting a differential influence depending on the presence or absence of cellular stress. We also observed substantial expression changes in miRNAs that were relevant in diabetic nephropathy upon silencing and glucose treatment. Our findings highlight the Rab3A/Rab27A system as a key participant in podocyte injury and vesicular traffic regulation in diabetic nephropathy.

CD36 is a type 2 cell surface scavenger receptor expressed in various tissues. In macrophages, CD36 recognizes oxidized low-density lipoprotein (ox-LDL), which promotes the formation of foam cells, the first step toward an atherosclerotic arterial lesion. CD36 possesses a variety of posttranslational modifications, among them N-glycosylation and O-GlcNAc modification. Some of the roles of these modifications on CD36 are known, such as N-linked glycosylation, which provides proper folding and trafficking to the plasma membrane in the human embryonic kidney. This study aimed to determine whether variations in the availability of UDP-GlcNAc could impact Rab-5-mediated endocytic trafficking and, therefore, the cellular localization of CD36. These preliminary results suggest that the availability of the substrate UDP-GlcNAc, modulated in response to treatment with Thiamet G (TMG), OSMI-1 (O-GlcNAcylation enzymes modulators) or Azaserine (HBP modulator), influences the localization of CD36 in J774 macrophages, and the endocytic trafficking as evidenced by the regulatory protein Rab-5, between the plasma membrane and the cytoplasm., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Loaeza-Reyes, Zenteno, Ramírez-Hernández, Salinas-Marin, Moreno-Rodríguez, Torres-Rosas, Argueta-Figueroa, Fernández-Rojas, Pina-Canseco, Acevedo-Mascarúa, Hernández-Antonio and Pérez-Cervera.)

Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß′ COPI coatomer subunits and demonstrate an accumulation of ‘coat-less’ vesicles that fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation.

The minor phospholipid, phosphatidylinositol 4-phosphate (PI4P), is emerging as a key regulator of lipid transfer in ER-membrane contact sites. Four different phosphatidylinositol 4-kinase (PI4K) enzymes generate PI4P in different membrane compartments supporting distinct cellular processes, many of which are crucial for the maintenance of cellular integrity but also hijacked by intracellular pathogens. While type III PI4Ks have been targeted by small molecular inhibitors, thus helping decipher their importance in cellular physiology, no inhibitors are available for the type II PI4Ks, which hinders investigations into their cellular functions. Here, we describe the identification of small molecular inhibitors of PI4K type II alpha (PI4K2A) by implementing a large scale small molecule high-throughput screening. A novel assay was developed that allows testing of selected inhibitors against PI4K2A in intact cells using a bioluminescence resonance energy transfer approach adapted to plate readers. The compounds disclosed here will pave the way to the optimization of PI4K2A inhibitors that can be used in cellular and animal studies to better understand the role of this enzyme in both normal and pathological states.

Messenger RNA (mRNA) localisation enables a high degree of spatiotemporal control on protein synthesis, which contributes to establishing the asymmetric protein distribution required to set up and maintain cellular polarity. As such, a tight control of mRNA localisation is essential for many biological processes during development and in adulthood, such as body axes determination in Drosophila melanogaster and synaptic plasticity in neurons. The mechanisms controlling how mRNAs are localised, including diffusion and entrapment, local degradation and directed active transport, are largely conserved across evolution and have been under investigation for decades in different biological models. In this review, we will discuss the standing of the field regarding directional mRNA transport in light of the recent discovery that RNA can hitchhike on cytoplasmic organelles, such as endolysosomes, and the impact of these transport modalities on our understanding of neuronal function during development, adulthood and in neurodegeneration. [ABSTRACT FROM AUTHOR]

Giardia intestinalis is a human parasite that causes a diarrheal disease in developing countries. G. intestinalis has a cytoskeleton (CSK) composed of microtubules and microfilaments, and the Giardia genome does not code for the canonical CSK-binding proteins described in other eukaryotic cells. To identify candidate actin and tubulin cross-linking proteins, we performed a BLAST analysis of the Giardia genome using a spectraplakins consensus sequence as a query. Based on the highest BLAST score, we selected a 259-kDa sequence designated as a cytoskeleton linker protein (CLP259). The sequence was cloned in three fragments and characterized by immunoprecipitation, confocal microscopy, and mass spectrometry (MS). CLP259 was located in the cytoplasm in the form of clusters of thick rods and colocalized with actin at numerous sites and with tubulin in the median body. Immunoprecipitation followed by mass spectrometry revealed that CLP259 interacts with structural proteins such as giardins, SALP-1, axonemal, and eight coiled-coils. The vesicular traffic proteins detected were Mu adaptin, Vacuolar ATP synthase subunit B, Bip, Sec61 alpha, NSF, AP complex subunit beta, and dynamin. These results indicate that CLP259 in trophozoites is a CSK linker protein for actin and tubulin and could act as a scaffold protein driving vesicular traffic. [ABSTRACT FROM AUTHOR]

Rab small GTPases regulate membrane traffic between distinct cellular compartments of all eukaryotes in a tempo‐spatially specific fashion. Rab small GTPases are also involved in the regulation of cytoskeleton and signalling. Membrane traffic and cytoskeletal regulation play pivotal role in the pathogenesis of Entamoeba histolytica, which is a protozoan parasite responsible for human amebiasis. E. histolytica is unique in that its genome encodes over 100 Rab proteins, containing multiple isotypes of conserved members (e.g., Rab7) and Entamoeba‐specific subgroups (e.g., RabA, B, and X). Among them, E. histolytica Rab7 is the most diversified group consisting of nine isotypes. While it was previously demonstrated that EhRab7A and EhRab7B are involved in lysosome and phagosome biogenesis, the individual roles of other Rab7 members and their coordination remain elusive. In this study, we characterised the third member of Rab7, Rab7D, to better understand the significance of the multiplicity of Rab7 isotypes in E. histolytica. Overexpression of EhRab7D caused reduction in phagocytosis of erythrocytes, trogocytosis (meaning nibbling or chewing of a portion) of live mammalian cells, and phagosome acidification and maturation. Conversely, transcriptional gene silencing of EhRab7D gene caused opposite phenotypes in phago/trogocytosis and phagosome maturation. Furthermore, EhRab7D gene silencing caused reduction in the attachment to and the motility on the collagen‐coated surface. Image analysis showed that EhRab7D was occasionally associated with lysosomes and prephagosomal vacuoles, but not with mature phagosomes and trogosomes. Finally, in silico prediction of structural organisation of EhRab7 isotypes identified unique amino acid changes on the effector binding surface of EhRab7D. Taken together, our data suggest that EhRab7D plays coordinated counteracting roles: a inhibitory role in phago/trogocytosis and lyso/phago/trogosome biogenesis, and an stimulatory role in adherence and motility, presumably via interaction with unique effectors. Finally, we propose the model in which three EhRab7 isotypes are sequentially involved in phago/trogocytosis. [ABSTRACT FROM AUTHOR]

Apolipoprotein E (APOE) is a component of lipid-transporting particles and a recognition ligand for receptors, which bind these particles. The APOE isoform ε2 is a risk factor for age-related macular degeneration; nevertheless, APOE absence in humans and mice does not significantly affect the retina. We found that retinal cholesterol biosynthesis and the levels of retinal cholesterol were increased in Apoe−/− mice, whereas cholesterol elimination by metabolism was decreased. No focal cholesterol deposits were observed in the Apoe−/− retina. Retinal proteomics identified the most abundant cholesterol-related proteins in WT mice and revealed that, of these cholesterol-related proteins, only APOA4 had increased expression in the Apoe−/− retina. In addition, there were changes in retinal abundance of proteins involved in proinflammatory and antiinflammatory responses, cellular cytoskeleton maintenance, vesicular traffic, and retinal iron homeostasis. The data obtained indicate that when APOE is absent, particles containing APOA1, APOA4, and APOJ still transport cholesterol in the intraretinal space, but these particles are not taken up by retinal cells. Therefore, cholesterol biosynthesis inside retinal cells increase, whereas metabolism to oxysterols decreases to prevent cells from cholesterol depletion. These and other compensatory changes underlie only a minor retinal phenotype in Apoe−/− mice.

Rab proteins constitute the largest group of small GTPases and act as molecular switches in a wide variety of cellular processes, including proliferation, cytoskeleton assembly, and membrane trafficking in all eukaryotic cells. Rab21 has been reported in several eukaryotic cells, and our results suggest that in Entamoeba histolytica, Rab21 is involved in the vesicular traffic associated with the Golgi apparatus, where its function appears to be important to maintain the structure of this organelle. In addition, proteins such as Rab1A and Sec24, identified in this work associated with EhRab21, participate in the traffic of COPII vesicles from the endoplasmic reticulum to the Golgi apparatus and are necessary to maintain the latter's structure in human cells. In addition, EhRab21 probably affects the lysosome biogenesis, as indicated by an increase in the number of lysosomes as a result of the increase in EhRab21 activity. The participation of EhRab21 in the pathogenesis of amebiasis was verified on the amoebic liver abscess formation model using hamsters (Mesocricetus auratus), in which the overexpression of EhRab21Q64L (positive dominant mutant protein) decreased the number of liver abscesses formed. [ABSTRACT FROM AUTHOR]

Owing to the local enrichment of factors that influence its dynamics and organization, the actin cytoskeleton displays different shapes and functions within the same cell. In yeast cells, post-Golgi vesicles ride on long actin cables to the bud tip. The proteins Boi1 and Boi2 (Boi1/2) participate in tethering and docking these vesicles to the plasma membrane. Here, we show in Saccharomyces cerevisiae that Boi1/2 also recruit nucleation and elongation factors to form actin filaments at sites of exocytosis. Disrupting the connection between Boi1/2 and the nucleation factor Bud6 impairs filament formation, reduces the directed movement of the vesicles to the tip and shortens the vesicles' tethering time at the cortex. Transplanting Boi1 from the bud tip to the peroxisomal membrane partially redirects the actin cytoskeleton and the vesicular flow towards the peroxisome, and creates an alternative, rudimentary vesicle-docking zone. We conclude that Boi1/2, through interactions with Bud6 and Bni1, induce the formation of a cortical actin structure that receives and aligns incoming vesicles before fusion with the membrane. [ABSTRACT FROM AUTHOR]

The cell’s ability to communicate with the extracellular environment, with other cells, and with itself is a crucial feature of eukaryotic organisms. In the immune system, T lymphocytes assemble a specialized structure upon contact with antigen-presenting cells bearing a peptide-major histocompatibility complex ligand, known as the immunological synapse (IS). The IS has been extensively characterized as a signaling platform essential for T-cell activation. Moreover, emerging evidence identifies the IS as a device for vesicular traffic-mediated cell-to-cell communication as well as an active release site of soluble molecules. Here, we will review recent advances in the role of vesicular trafficking in IS assembly and focused secretion of microvesicles at the synaptic area in naïve T cells and discuss the role of the IS in transcellular communication.

Small GTPases of the Rab protein family control intracellular vesicular trafficking to allow their communication and maintenance. It is a common strategy for intracellular bacteria to exploit these pathways to shape their respective niches for survival. The subversion of Rabs for the generation of an intracellular environment favoring the pathogen has been described almost exclusively for intracellular bacteria that reside within bacterial containing vacuoles (BCVs). However, less is known about Rab subversion for bacteria that rupture the BCV to reach the host cytoplasm. Here, we provide recent examples of Rab targeting by both groups of intracellular bacteria with a special focus on Shigella, the causative agent of bacillary dysentery. Shigella recruits Rab11, the hallmark of the perinuclear recycling compartment to in situ formed macropinosomes at the entry foci via the bacterial effector IpgD. This leads to efficient BCV rupture and cytosolic escape. We discuss the concept of diverted recycling through host Rab GTPases that emerges as a novel pathogen strategy. [ABSTRACT FROM AUTHOR]

Neutrophils respond nearly instantly to infection, rapidly deploying a potent enzymatic and chemical arsenal immediately upon entering an infected site. This capacity for rapid and potent responses is endowed by stores of antimicrobial proteins contained in readily mobilizable granules. These granules contain the proteins necessary to mediate the recruitment, chemotaxis, antimicrobial function and NET formation of neutrophils. Four granule types exist, and are sequentially deployed as neutrophils enter infected sites. Secretory vesicles are released first, enabling recruitment of neutrophils out of the blood. Next, specific and gelatinase granules are released to enable neutrophil migration and begin the formation of an antimicrobial environment. Finally, azurophilic granules release potent antimicrobial proteins at the site of infection and into phagosomes. The step-wise mobilization of these granules is regulated by calcium signaling, while specific trafficking regulators and membrane fusion complexes ensure the delivery of granules to the correct subcellular site. In this review, we describe neutrophil granules from their formation through to their deployment at the site of infection, focusing on recent developments in our understanding of the signaling pathways and vesicular trafficking mechanisms which mediate neutrophil degranulation. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) present internalized antigens to CD8 T cells through cross-presentation by major histocompatibility complex class I (MHC-I) molecules. While conventional cDC1 excel at cross-presentation, cDC2 can be licensed to cross-present during infection by signals from inflammatory receptors, most prominently Toll-like receptors (TLRs). At the core of the regulation of cross-presentation by TLRs is the control of subcellular MHC-I traffic. Within DCs, MHC-I are enriched within endosomal recycling compartments (ERC) and traffic to microbe-carrying phagosomes under the control of phagosome-compartmentalized TLR signals to favor CD8 T cell cross-priming to microbial antigens. Viral blockade of the transporter associated with antigen processing (TAP), known to inhibit the classic MHC-I presentation of cytoplasmic protein-derived peptides, depletes the ERC stores of MHC-I to simultaneously also block TLR-regulated cross-presentation. DCs counter this impairment in the two major pathways of MHC-I presentation to CD8 T cells by mobilizing noncanonical cross-presentation, which delivers MHC-I to phagosomes from a new location in the ER-Golgi intermediate compartment (ERGIC) where MHC-I abnormally accumulate upon TAP blockade. Noncanonical cross-presentation thus rescues MHC-I presentation and cross-primes TAP-independent CD8 T cells best-matched against target cells infected with immune evasive viruses. Because noncanonical cross-presentation relies on a phagosome delivery route of MHC-I that is not under TLR control, it risks potential cross-presentation of self-antigens during infection. Here I review these findings to illustrate how the subcellular route of MHC-I to phagosomes critically impacts the regulation of cross-presentation and the nature of the CD8 T cell response to infection and cancer. I highlight important and novel implications to CD8 T cell vaccines and immunotherapy. • Control of MHC-I traffic is at the heart of TLR regulation of cross-presentation. • ERC-to-phagosome MHC-I delivery is critical for phagocytic antigen cross-presentation. • TAP blockade inhibits classic MHC-presentation and TLR-regulated cross-presentation. • ERGIC-to-phagosome MHC-I delivery upon TAP blockade rescues MHC-I presentation. • DC-intrinsic noncanonical cross-presentation cross-primes TAP-independent CD8 T cells. [ABSTRACT FROM AUTHOR]

Ferritins are protein nanocages that accumulate inside their cavity thousands of oxidized iron atoms bound to oxygen and phosphates. Both characteristic types of eukaryotic ferritin subunits are present in secreted ferritins from insects, but here dimers between Ferritin 1 Heavy Chain Homolog (Fer1HCH) and Ferritin 2 Light Chain Homolog (Fer2LCH) are further stabilized by disulfide-bridge in the 24-subunit complex. We addressed ferritin assembly and iron loading in vivo using novel transgenic strains of Drosophila melanogaster. We concentrated on the intestine, where the ferritin induction process can be controlled experimentally by dietary iron manipulation. We showed that the expression pattern of Fer2LCH-Gal4 lines recapitulated iron-dependent endogenous expression of the ferritin subunits and used these lines to drive expression from UAS-mCherry-Fer2LCH transgenes. We found that the Gal4-mediated induction of mCherry-Fer2LCH subunits was too slow to effectively introduce them into newly formed ferritin complexes. Endogenous Fer2LCH and Fer1HCH assembled and stored excess dietary iron, instead. In contrast, when flies were genetically manipulated to co-express Fer2LCH and mCherry-Fer2LCH simultaneously, both subunits were incorporated with Fer1HCH in iron-loaded ferritin complexes. Our study provides fresh evidence that, in insects, ferritin assembly and iron loading in vivo are tightly regulated. [ABSTRACT FROM AUTHOR]

Biogenesis of the immune synapse at the interface between antigenpresenting cells and T cells assembles and organizes a large number of membrane proteins required for effective signaling through the T-cell receptor. We showed previously that the intraflagellar transport protein 20 (IFT20), a component of the intraflagellar transport system, controls polarized traffic during immune synapse assembly. To investigate the role of IFT20 in primary CD4+ T cells in vitro and in vivo, we generated mice bearing a conditional defect of IFT20 expression in T cells.We show that in the absence of IFT20, although cell spreading and the polarization of the centrosome were unaffected, T-cell receptor (TCR)-mediated signaling and recruitment of the signaling adaptor LAT (linker for activation of T cells) at the immune synapse were reduced. As a consequence, CD4+ T-cell activation and proliferation were also defective. In vivo, conditional IFT20-deficient mice failed to mount effective antigen-specific T-cell responses, and their T cells failed to induce colitis after adoptive transfer to Rag-/- mice. IFT20 is therefore required for the delivery of the intracellular pool of LAT to the immune synapse in naive primary T lymphocytes and for effective T-cell responses in vivo. [ABSTRACT FROM AUTHOR]

The PI3K lipid kinases are involved in signal transduction and intracellular vesicular traffic, endowing these enzymes with multiple cellular functions and important roles in normal physiology and disease. In this mini-review, we aim to distill from the vast PI3K literature the key relevant concepts for successful targeting of this pathway in disease. Of the eight isoforms of PI3K, the class I PI3Ks have been implicated in the aetiology and maintenance of various diseases, most prominently cancer, overgrowth syndromes, inflammation and autoimmunity, with emerging potential roles in metabolic and cardiovascular disorders. The development of class I PI3K inhibitors, mainly for use in cancer and inflammation, is a very active area of drug development. In 2014, an inhibitor of the p110δ isoform of PI3K was approved for the treatment of specific human B cell malignancies. The key therapeutic indications of targeting each class I PI3K isoform are summarized and discussed. [ABSTRACT FROM AUTHOR]

We show here that the G protein‐coupled receptor (GPCR) sphingosine 1‐phosphate receptor 1 (S1PR1) activated by its natural lipid ligand S1P or the synthetic agonist FTY720P is rapidly internalized by the clathrin‐dependent endocytic pathway. Depletion of activated S1PR1 from the cell surface relies on clathrin, its endocytic adaptor AP‐2 dynamin and the non‐visual βarrestin 1 and βarrestin 2, suggesting that activated S1PR1 is downregulated from the cell surface using the canonical GPCR entry route. The sphingosine 1‐phosphate receptor 1 (S1PR1) is one of five G protein‐coupled receptors activated by the lipid sphingosine 1‐phosphate (S1P). Stimulation of S1PR1 by binding S1P or the synthetic agonist FTY720P results in rapid desensitization, associated in part with depletion of receptor from the cell surface. We report here combining spinning disc confocal fluorescence microscopy and flow cytometry to show that rapid internalization of activated S1PR1 relies on a functional clathrin‐mediated endocytic pathway. Uptake of activated S1PR1 was strongly inhibited in cells disrupted in their clathrin‐mediated endocytosis by depleting clathrin or AP‐2 or by treating cells with dynasore‐OH. The uptake of activated S1P1R was strongly inhibited in cells lacking both β‐arrestin 1 and β‐arrestin 2, indicating that activated S1PR1 follows the canonical route of endocytosis for G‐protein coupled receptor's (GPCR)'s. [ABSTRACT FROM AUTHOR]

Cross-presentation is the culmination of complex subcellular processes that allow the processing of exogenous proteins and the presentation of resultant peptides on major histocompatibility class I (MHC-I) molecules to CD8 T cells. Dendritic cells (DCs) are a cell type that uniquely specializes in cross-presentation, mainly in the context of viral or non-viral infection and cancer. DCs have an extensive network of endovesicular pathways that orchestrate the biogenesis of an ideal cross-presentation compartment where processed antigen, MHC-I molecules, and the MHC-I peptide loading machinery all meet. As a central conveyor of information to CD8 T cells, cross-presentation allows cross-priming of T cells which carry out robust adaptive immune responses for tumor and viral clearance. Cross-presentation can be canonical or noncanonical depending on the functional status of the transporter associated with antigen processing (TAP), which in turn influences the vesicular route of MHC-I delivery to internalized antigen and the cross-presented repertoire of peptides. Because TAP is a central node in MHC-I presentation, it is targeted by immune evasive viruses and cancers. Thus, understanding the differences between canonical and noncanonical cross-presentation may inform new therapeutic avenues against cancer and infectious disease. Defects in cross-presentation on a cellular and genetic level lead to immune-related disease progression, recurrent infection, and cancer progression. In this chapter, we review the process of cross-presentation beginning with the DC subsets that conduct cross-presentation, the signals that regulate cross-presentation, the vesicular trafficking pathways that orchestrate cross-presentation, the modes of cross-presentation, and ending with disease contexts where cross-presentation plays a role., (Copyright © 2023. Published by Elsevier Inc.)

Acrosome is a specialized organelle in spermatozoids necessary for fertilization of oocyte. Acrosome is formed, according to various theories, either from vesicles of the Golgi apparatus or from endosomes and lysosomes. Hepatocyte growth factor regulated tyrosine kinase substrate (Hrs) is a multidomain protein component of ESCRT-0 complex, that participates in sorting of proteins absorbed during endocytosis. It has been shown that, in mammals, Hgs protein (Hrs homologue) possesses the ability to bind the acrosome, but whether Drosophila Hrs has a similar capacity has not been determined. We found that two forms of Hrs protein are expressed in Drosophila testes: a long form, Hrs-B, and a truncated form, Hrs-A. The later lacks a VHS domain and a portion of FYVE domain, both directly involved in Hrs anchoring to endosomes. We also determined that, unlike mammal Hgs, Drosophila Hrs-B isoform and an almost identical truncated form, Hrs8 ([290-760]), are localized not to acrosome, but to spermatocyte fusomes. This localization requires a portion of the protein molecule located between amino acid residues 383 and 472, which presumably correspond to NF2- and/or Stam-binding domains. In situ hybridization for hrs mRNA showed that the gene is expressed during early spermatogenesis. This is consistent with our data that the Hrs protein binds to spermatocyte fusomes and is absent during later spermatogenesis stages. In addition, we have shown that Hrs is not involved in regulation or implementation of cytokinesis in spermatocytes. Finally, despite the absence of Hrs on Drosophila acrosomes, we detected endosome markers Rab4, Rab7, and Rab11 on this organelle. Thus, our data support the endosomal hypothesis of acrosome biogenesis. [ABSTRACT FROM AUTHOR]

The signals that orchestrate the process of T cell activation are coordinated at the specialized interface that forms upon contact with an antigen presenting cell displaying a specific MHC-associated peptide ligand, known as the immune synapse. The central role of vesicular traffic in the assembly of the immune synapse has emerged only in recent years with the finding that sustained T-cell receptor ( TCR) signaling involves delivery of TCR/ CD3 complexes from an intracellular pool associated with recycling endosomes. A number of receptors as well as membrane-associated signaling mediators have since been demonstrated to exploit this process to localize to the immune synapse. Here, we will review our current understanding of the mechanisms responsible for TCR recycling, with a focus on the intraflagellar transport system, a multimolecular complex that is responsible for the assembly and function of the primary cilium which we have recently implicated in polarized endosome recycling to the immune synapse. [ABSTRACT FROM AUTHOR]

Humans and yeast possess alkaline ceramidases located in the early secretory pathway. Single deletions of the highly homologous yeast alkaline ceramidases YPC1 and YDC1 have very little genetic interactions or phenotypes. Here, we performed chemical-genetic screens to find deletions/conditions that would alter the growth of ypc1∆ydc1∆ double mutants. These screens were essentially negative, demonstrating that ceramidase activity is not required for cell growth even under genetic stresses. A previously reported protein targeting defect of ypc1∆ could not be reproduced and reported abnormalities in sphingolipid biosynthesis detected by metabolic labeling do not alter the mass spectrometric lipid profile of ypc1∆ydc1∆ cells. Ceramides of ypc1∆ydc1∆ remained normal even in presence of aureobasidin A, an inhibitor of inositolphosphorylceramide synthase. Moreover, in caloric restriction conditions Ypc1p reduces chronological life span. A novel finding is that, when working backwards as a ceramide synthase in vivo, Ypc1p prefers C24 and C26 fatty acids as substrates, whereas it prefers C16:0, when solubilized in detergent and working in vitro. Therefore, its physiological activity may not only concern the minor ceramides containing C14 and C16. Intriguingly, so far the sole discernable benefit of conserving YPC1 for yeast resides with its ability to convey relative resistance toward H2O2. [ABSTRACT FROM AUTHOR]

Apicomplexans are obligate intracellular parasites that invade the host cell in an active process that relies on unique secretory organelles (micronemes, rhoptries and dense granules) localized at the apical tip of these highly polarized eukaryotes. In order for the contents of these specialized organelles to reach their final destination, these proteins are sorted post-Golgi and it has been speculated that they pass through endosomal-like compartments ( ELCs), where they undergo maturation. Here, we characterize a Toxoplasma gondii homologue of Syntaxin 6 ( TgStx6), a well-established marker for the early endosomes and trans Golgi network ( TGN) in diverse eukaryotes. Indeed, TgStx6 appears to have a role in the retrograde transport between ELCs, the TGN and the Golgi, because overexpression of TgStx6 results in the development of abnormally shaped parasites with expanded ELCs, a fragmented Golgi and a defect in inner membrane complex maturation. Interestingly, other organelles such as the micronemes, rhoptries and the apicoplast are not affected, establishing the TGN as a major sorting compartment where several transport pathways intersect. It therefore appears that Toxoplasma has retained a plant-like secretory pathway. [ABSTRACT FROM AUTHOR]

Cross-presentation involves the presentation of peptides derived from internalized cargo on major histocompatibility complex class I molecules by dendritic cells, a process critical for tolerance and immunity. Detailed studies of the pathways mediating cross-presentation have revealed that this process takes place in a specialized subcellular compartment with a unique set of proteins. In this review, we focus on the recently appreciated role for intracellular vesicular traffic, which serves to equip compartments such as endosomes and phagosomes with the necessary apparatus for conducting the various steps of cross-presentation. We also consider how these pathways may integrate with inflammatory signals particularly from pattern recognition receptors that detect the presence of microbial components during infection. We discuss the consequences of such signals on initiating cross-presentation to stimulate adaptive CD8T cell responses. [ABSTRACT FROM AUTHOR]

Abstract: Dictyostelium cells are genetically haploid and therefore easily analyzed for mutant phenotypes. In the past, many tools and molecular markers have been developed for a quantitative and qualitative analysis of the endocytic pathway in these amoebae. This review outlines parallels and discrepancies between mutants in Dictyostelium, the corresponding mammalian cells and the symptoms of human patients affected by lysosomal and trafficking defects. Situations where knowledge from Dictyostelium may potentially help understand human disease and vice versa are also addressed. [Copyright &y& Elsevier]

Abstract: Entamoeba invadens is a reptilian enteric protozoan parasite closely related to the human pathogen Entamoeba histolytica and a good model organism of encystation. To understand the molecular mechanism of vesicular trafficking involved in the encystation of Entamoeba, we examined the conservation of Rab small GTPases between the two species. E. invadens has over 100 Rab genes, similar to E. histolytica. Most of the Rab subfamilies are conserved between the two species, while a number of species-specific Rabs are also present. We annotated all E. invadens Rabs according to the previous nomenclature [Saito-Nakano, Y., Loftus, B.J., Hall, N., Nozaki, T., 2005. The diversity of Rab GTPases in Entamoeba histolytica. Experimental Parasitology 110, 244–252]. Comparative genomic analysis suggested that the fundamental vesicular traffic machinery is well conserved, while there are species-specific protein transport mechanisms. We also reviewed the function of Rabs in Entamoeba, and proposed the use of the annotation of E. invadens Rab genes to understand the ubiquitous importance of Rab-mediated membrane trafficking during important biological processes including differentiation in Entamoeba. [Copyright &y& Elsevier]