Early Endosomal Trafficking Research Articles

PDGFRα signaling regulates Srsf3 transcript binding to affect PI3K signaling and endosomal trafficking.

Signaling through the platelet-derived growth factor receptor alpha (PDGFRα) plays a critical role in craniofacial development, as mutations in PDGFRA are associated with cleft lip/palate in humans and Pdgfra mutant mouse models display varying degrees of facial clefting. Phosphatidylinositol 3-kinase (PI3K)/Akt is the primary effector of PDGFRα signaling during skeletal development in the mouse. We previously demonstrated that Akt phosphorylates the RNA-binding protein serine/arginine-rich splicing factor 3 (Srsf3) downstream of PI3K-mediated PDGFRα signaling in mouse embryonic palatal mesenchyme (MEPM) cells, leading to its nuclear translocation. We further showed that ablation of Srsf3 in the murine neural crest lineage results in severe midline facial clefting, due to defects in proliferation and survival of cranial neural crest cells, and widespread alternative RNA splicing (AS) changes. Here, we sought to determine the molecular mechanisms by which Srsf3 activity is regulated downstream of PDGFRα signaling to control AS of transcripts necessary for craniofacial development. We demonstrated via enhanced UV-crosslinking and immunoprecipitation (eCLIP) of MEPM cells that PDGF-AA stimulation leads to preferential binding of Srsf3 to exons and loss of binding to canonical Srsf3 CA-rich motifs. Through the analysis of complementary RNA-seq data, we showed that Srsf3 activity results in the preferential inclusion of exons with increased GC content and lower intron to exon length ratio. Moreover, we found that the subset of transcripts that are bound by Srsf3 and undergo AS upon PDGFRα signaling commonly encode regulators of PI3K signaling and early endosomal trafficking. Functional validation studies further confirmed that Srsf3 activity downstream of PDGFRα signaling leads to retention of the receptor in early endosomes and increases in downstream PI3K-mediated Akt signaling. Taken together, our findings reveal that growth factor-mediated phosphorylation of an RNA-binding protein underlies gene expression regulation necessary for mammalian craniofacial development.

Specific binding sites on Rhesus rotavirus capsid protein dictate the method of endocytosis inducing the murine model of biliary atresia.

Biliary atresia (BA) is the leading indication for pediatric liver transplantation. Rhesus rotavirus (RRV)-induced murine BA develops an obstructive cholangiopathy that mirrors the human disease. We have previously demonstrated the "SRL" motif on RRV's VP4 protein binds to heat shock cognate 70 protein (Hsc70) facilitating entry into cholangiocytes. In this study, we analyzed how binding to Hsc70 affects viral endocytosis, intracellular trafficking, and uniquely activates the signaling pathway that induces murine BA. Inhibition of clathrin- and dynamin-mediated endocytosis in cholangiocytes following infection demonstrated that blocking dynamin decreased the infectivity of RRV, whereas clathrin inhibition had no effect. Blocking early endosome trafficking resulted in decreased viral titers of RRV, whereas late endosome inhibition had no effect. After infection, TLR3 expression and p-NF-κB levels increased in cholangiocytes, leading to increased release of CXCL9 and CXCL10. Infected mice knocked out for TLR3 had decreased levels of CXCL9 and CXCL10, resulting in reduced NK cell numbers. Human patients with BA experienced an increase in CXCL10 levels, suggesting this as a possible pathway leading to biliary obstruction. Viruses that use Hsc70 for cell entry exploit a clathrin-independent pathway and traffic to the early recycling endosome uniquely activating NF-κB through TLR3, leading to the release of CXCL9 and CXCL10 and inducing NK cell recruitment. These results define how the "SRL" peptide found on RRV's VP4 protein modulates viral trafficking, inducing the host response leading to bile duct obstruction.NEW & NOTEWORTHY In this study, we have determined that the presence of the "SRL" peptide on RRV alters its method of endocytosis and intracellular trafficking through viral binding to heat shock cognate 70 protein. This initiates an inflammatory pathway that stimulates the release of cytokines associated with biliary damage and obstruction.

M6PR interacts with the HA2 subunit of influenza A virus to facilitate the fusion of viral and endosomal membranes.

Influenza A virus (IAV) commandeers numerous host cellular factors for successful replication. However, very few host factors have been revealed to be involved in the fusion of viral envelope and late endosomal membranes. In this study, we identified cation-dependent mannose-6-phosphate receptor (M6PR) as a crucial host factor for the replication of IAV. We found that siRNA knockdown of M6PR expression significantly reduced the growth titers of different subtypes of IAV, and that the inhibitory effect of M6PR siRNA treatment on IAV growth was overcome by the complement of exogenously expressed M6PR. When A549 cells were treated with siRNA targeting M6PR, the nuclear accumulation of viral nucleoprotein (NP) was dramatically inhibited at early timepoints post-infection, indicating that M6PR engages in the early stage of the IAV replication cycle. By investigating the role of M6PR in the individual entry and post-entry steps of IAV replication, we found that the downregulation of M6PR expression had no effect on attachment, internalization, early endosome trafficking, or late endosome acidification. However, we found that M6PR expression was critical for the fusion of viral envelope and late endosomal membranes. Of note, M6PR interacted with the hemagglutinin (HA) protein of IAV, and further studies showed that the lumenal domain of M6PR and the ectodomain of HA2 mediated the interaction and directly promoted the fusion of the viral and late endosomal membranes, thereby facilitating IAV replication. Together, our findings highlight the importance of the M6PR-HA interaction in the fusion of viral and late endosomal membranes during IAV replication.

Transforming acidic coiled-coil containing protein 3 suppresses influenza A virus replication by impeding viral endosomal trafficking and nuclear import

Transforming acidic coiled-coil containing protein 3 (TACC3) is a motor spindle protein that plays an essential role in stabilization of the mitotic spindle. In this study, we show that the overexpression of TACC3 reduces the viral titers of multiple influenza A viruses (IAVs). In contrast, the downregulation of TACC3 increases IAVs propagation. Next, we map the target steps of TACC3 requirement to the early stages of viral replication. By confocal microscopy and nuclear plasma separation experiment, we reveal that overexpression of TACC3 results in a substantial decrease of IAV NP accumulation in the nuclei of infected cells. We further show that viral attachment and internalization are not affected by TACC3 overexpression and detect that the early and late endosomal trafficking of IAV in TACC3 overexpression cells is slower than negative control cells. These results suggest that TACC3 exerts an impaired effect on the endosomal trafficking and nuclear import of vRNP, thereby negatively regulating IAV replication. Moreover, the infection of different IAV subtypes decreases the expression level of TACC3 in turn. Consequently, we speculate that IAV ensures the generation of offspring virions by antagonizing the expression of inhibitory factor TACC3. Collectively, our results establish TACC3 as an important inhibitory factor for replication of the IAV, suggesting that TACC3 could be a potential target for the development of future antiviral compounds.

BAR domain is essential for early endosomal trafficking and dynamics in Ascochyta rabiei.

The online version contains supplementary material available at 10.1007/s13205-022-03451-5.

Central nervous system specific high molecular weight ALS2/alsin homophilic complex is enriched in mouse brain synaptosomes

ALS2/alsin, the causative gene product for a number of juvenile recessive motor neuron diseases, acts as a guanine nucleotide exchange factor (GEF) for Rab5, regulating early endosome trafficking and maturation. It has been demonstrated that ALS2 forms a tetramer, and this oligomerization is essential for its GEF activity and endosomal localization in established cancer cells. However, despite that ALS2 deficiency is implicated in neurological diseases, neither the subcellular distribution of ALS2 nor the form of its complex in the central nervous system (CNS) has been investigated. In this study, we showed that ALS2 in the brain was enriched both in synaptosomal and cytosolic fractions, while those in the liver were almost exclusively present in cytosolic fraction by differential centrifugation. Gel filtration chromatography revealed that cytosolic ALS2 prepared both from the brain and liver formed a tetramer. Remarkably, synaptosomal ALS2 existed as a high-molecular weight complex in addition to a tetramer. Such complex was also observed not only in embryonic brain but also several neuronal and glial cultures, but not in fibroblast-derived cell lines. Thus, the high-molecular weight ALS2 complex represents a unique form of ALS2-homophilic oligomers in the CNS, which may play a role in the maintenance of neural function.

Defective RAB31-mediated megakaryocytic early endosomal trafficking of VWF, EGFR, and M6PR in RUNX1 deficiency

AbstractTranscription factor RUNX1 is a master regulator of hematopoiesis and megakaryopoiesis. RUNX1 haplodeficiency (RHD) is associated with thrombocytopenia and platelet granule deficiencies and dysfunction. Platelet profiling of our study patient with RHD showed decreased expression of RAB31, a small GTPase whose cell biology in megakaryocytes (MKs)/platelets is unknown. Platelet RAB31 messenger RNA was decreased in the index patient and in 2 additional patients with RHD. Promoter-reporter studies using phorbol 12-myristate 13-acetate–treated megakaryocytic human erythroleukemia cells revealed that RUNX1 regulates RAB31 via binding to its promoter. We investigated RUNX1 and RAB31 roles in endosomal dynamics using immunofluorescence staining for markers of early endosomes (EEs; early endosomal autoantigen 1) and late endosomes (CD63)/multivesicular bodies. Downregulation of RUNX1 or RAB31 (by small interfering RNA or CRISPR/Cas9) showed a striking enlargement of EEs, partially reversed by RAB31 reconstitution. This EE defect was observed in MKs differentiated from a patient-derived induced pluripotent stem cell line (RHD-iMKs). Studies using immunofluorescence staining showed that trafficking of 3 proteins with distinct roles (von Willebrand factor [VWF], a protein trafficked to α-granules; epidermal growth factor receptor; and mannose-6-phosphate) was impaired at the level of EE on downregulation of RAB31 or RUNX1. There was loss of plasma membrane VWF in RUNX1- and RAB31-deficient megakaryocytic human erythroleukemia cells and RHD-iMKs. These studies provide evidence that RAB31 is downregulated in RHD and regulates megakaryocytic vesicle trafficking of 3 major proteins with diverse biological roles. EE defect and impaired vesicle trafficking is a potential mechanism for the α-granule defects observed in RUNX1 deficiency.

Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice

Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3−/− mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3IRES-Cre/eR26-τGFP reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease.

Optimizing Long-Term Live Cell Imaging.

Live cell microscopy has become a common technique for exploring dynamic biological processes. When combined with fluorescent markers of cellular structures of interest, or fluorescent reporters of a biological activity of interest, live cell microscopy enables precise temporally and spatially resolved quantitation of the biological processes under investigation. However, because living cells are not normally exposed to light, live cell fluorescence imaging is significantly hindered by the effects of photodamage, which encompasses photobleaching of fluorophores and phototoxicity of the cells under observation. In this chapter, we outline several methods for optimizing and maintaining long-term imaging of live cells while simultaneously minimizing photodamage. This protocol demonstrates the intracellular trafficking of early and late endosomes following phagocytosis using both two and three dimensional imaging, but this protocol can easily be modified to image any biological process of interest in nearly any cell type.

Porcine Genome-Wide CRISPR Screen Identifies the Golgi Apparatus Complex Protein COG8 as a Pivotal Regulator of Influenza Virus Infection.

Avian and human influenza viruses bind to porcine sialic acid receptors to generate novel viruses that pose a potential pandemic threat to public health. Evidence suggests that the host factors regulating the influenza virus life cycle and viral reassortment are potential broad-spectrum antiviral drug targets, compared to the ineffective seasonal vaccines against highly pathogenic viruses, leading to drug resistance. After performing a genome-wide CRISPR-Cas9 screen targeting 13,735 genes in porcine cell lines, we identified several host factors critical for influenza virus infection-notably, a conserved oligomeric Golgi complex protein, COG8, which regulates viral protein transport and immune factor expression. Viral titers indicated that the loss of COG8 significantly enhanced cellular resistance to influenza (p < 0.005). Moreover, COG8 knockout reduced the colocalization between viral particles and early endosome marker (EEA1), indicating COG8's role in the early endosome trafficking events of the virus. COG8 deletion inhibited the retrograde transport from the endosome to the trans-Golgi network, thereby accumulating the influenza protein M2 in early endosomes. COG8 silencing enhanced the expression of immune-related genes, indicating COG8-mediated host immune responses affect virus replication. Our experiments have revealed COG8 as an essential factor in influenza virus infection.

Myeloid/Lymphoid Neoplasm with Eosinophilia and a Novel RUFY1-Pdgfrb Rearrangement

▪Myeloid/lymphoid neoplasms associated with eosinophilia (MLN-Eo) and tyrosine kinase fusion genes include rearrangements of PDGFRB with over 30 different partner genes, most commonly ETV6-PDGFRB. Rearrangements are evident by cytogenetic analysis and fluorescence in situ hybridization (FISH) break-apart probes. Herein, we describe a novel rearrangement partner for PDGFRB.A 25-year-old man with a medical history significant for gout and hypertension presented to medical care after several days of progressive dyspnea, chest pain, fatigue, epistaxis, and rash. Physical exam revealed palpable cervical, axillary, and inguinal lymphadenopathy and massive splenomegaly. By CT scan the spleen measured 25 cm and diffuse lymphadenopathy was identified in the chest, abdomen and pelvis with the largest lymph node mass measuring 8.9 x 2.5 cm. Transthoracic echocardiogram showed normal heart function and no evidence of myocardial injury. The patient subsequently developed respiratory failure requiring intubation and mechanical ventilation, shock requiring vasopressors, and renal failure requiring renal replacement therapy. Initial laboratory evaluation demonstrated a leukocytosis to 83.3 x 10 9/L with neutrophilic predominance with left shift and eosinophilia (10% of WBCs, absolute eosinophil count of 8.2 x 10 9/L); hemoglobin of 8.3 g/dL and hematocrit of 25%; and thrombocytopenia to 50 x 10 9/L. Ferritin was 660 ng/mL, lactate dehydrogenase 384 U/L, and uric acid 10.4 mg/dL. Secondary causes of eosinophilia including infection, immunodeficiency, atopy, medications, and rheumatologic disease were ruled out. The patient was positive for strongyloides IgG antibodies and received ivermectin.Core biopsy of a lymph node was non-diagnostic and could not repeated safely due to progressive severe thrombocytopenia. Skin biopsy demonstrated a perivascular leukocytoclasis, but no abnormal B or T cell population. Bone marrow aspirate and biopsy revealed a hypercellular marrow (90%) with mild trilineage dysplasia, granulocytic hyperplasia, moderate reticulin fibrosis, and slightly increased mast cells. Blasts were not increased and no hemophagocytic histiocytes were identified. Eosinophils accounted for 6.9% of myeloid cells. Flow cytometry did not reveal an abnormal B or T lymphoid, blast or myeloid population.Peripheral blood flow cytometry did not reveal an abnormal B or T-cell population and T-cell rearrangement studies showed an oligoclonal population. No clonal B cell population was identified.FISH, which identified a rearrangement of PDGFRB in 28% of cells, confirmed the diagnosis. Karyotype analysis revealed an abnormal male karyotype with a paracentric inversion of 5q with breakpoints at 5q32 and 5q35. FusionPlex® Solid Tumor Panel (Clinical Genomics Laboratory, University of Washington SOM) identified a novel fusion between RUFY1 and PDGFRB. RUFY1 is expressed in most tissues and encodes a protein that contains a RUN domain and a FYVE-type zinc finger domain and plays a role in early endosomal trafficking (Figure). We are currently demonstrating the fusion is oncogenic and sensitive to tyrosine kinase inhibition in Ba/F3 cells. The patient's remarkable clinical response, however, supports imatinib sensitivity and is in keeping with other published reports of MLN-Eo with PDGFRB rearrangements. Initial treatment included high dose steroids for 7 days and hydroxyurea briefly. Imatinib 400 mg daily was started, then decreased to 200 mg daily due to gastrointestinal toxicity. Follow-up is limited (8 weeks). However, the patient has recovered clinically. Six weeks after starting imatinib he had renal recovery and has achieved a complete hematologic response except for mild anemia due to resolving acute kidney injury. Splenomegaly and peripheral lymphadenopathy have resolved. PDGFRB FISH has declined to 9%.Inversions of chromosome 5 have very rarely been described in association with eosinophilia, but only one report has specifically identified a rearrangement of PDGFRB. This report described a novel rearrangement involving exons 12 to 23 of PDGFRB at 5q32 and the 5′ (UTR) of G3BP1 at 5q33.1. Notably, this specific rearrangement was undetectable by PDGFRB break-apart FISH. These reports and ours highlight the clinical relevance of NGS in identifying novel rearrangement partners. Furthermore, patient-specific ddPCR assays can be designed for sensitive monitoring. [Display omitted] DisclosuresOehler: BMS: Consultancy; OncLive: Honoraria; Pfizer: Research Funding; Takeda: Consultancy; Blueprint Medicines: Consultancy.

Nitric oxide may regulate focal adhesion turnover and cell migration in MDA-MB-231 breast cancer cells by modulating early endosome trafficking

Abstract Cell migration is an essential process for wound healing, metastasis and inflammation. Focal adhesions (FA) are local regions of plasma membrane consisting of multiprotein complexes providing adhesive contact between the cell and the extracellular matrix (ECM). FA turnover regulates different signalling pathways implicated in various cellular responses (e.g. cell migration). Endocytosis, specifically the dynamin and clathrin pathways, is known to regulate cell migration by modulating FA dynamics. In this study, we investigated whether NO activity regulates cell migration, FA dynamics and early endosome trafficking in MDA-MB-231 cells. The assessment of cell migration showed a slowing down of cell migration and an increased duration of FA turnover in cells treated with inhibitors of NO synthase (NOS) such as L-NAME or 1400W. In addition, these treatments were found to exhibit no effect on transferrin and dextran uptake mediated by endocytosis and micropinocytosis, respectively. The number of early endosome antigen 1 (EEA1)-positive endosomes was reduced while their sizes were found to increase in cells treated with L-NAME or 1400W. In contrast, these inhibitors did not affect the number nor the size of Rab5-positive endosomes. Furthermore, we demonstrated that EEA1, endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) were colocalised. Using the biotin switch assay followed by western blot, we showed that early endosome proteins such as APPL1, EEA1, Rab5 were found to be S-nitrosylated. These results were further supported by the sequence analysis performed with the GPS-SNO algorithm which predicted the S-nitrosylation of these endosomal proteins. Taken together, our findings suggest that NO might be involved in cell migration and FA turnover through early endosome trafficking in MDA-MB-231 cells. Running title: Nitric oxide in MDA-MB-231 breast cancer cells

Modulation of fungal virulence through CRZ1 regulated F-BAR-dependent actin remodeling and endocytosis in chickpea infecting phytopathogen Ascochyta rabiei.

Polarized hyphal growth of filamentous pathogenic fungi is an essential event for host penetration and colonization. The long-range early endosomal trafficking during hyphal growth is crucial for nutrient uptake, sensing of host-specific cues, and regulation of effector production. Bin1/Amphiphysin/Rvs167 (BAR) domain-containing proteins mediate fundamental cellular processes, including membrane remodeling and endocytosis. Here, we identified a F-BAR domain protein (ArF-BAR) in the necrotrophic fungus Ascochyta rabiei and demonstrate its involvement in endosome-dependent fungal virulence on the host plant Cicer arietinum. We show that ArF-BAR regulates endocytosis at the hyphal tip, localizes to the early endosomes, and is involved in actin dynamics. Functional studies involving gene knockout and complementation experiments reveal that ArF-BAR is necessary for virulence. The loss-of-function of ArF-BAR gene results in delayed formation of apical septum in fungal cells near growing hyphal tip that is crucial for host penetration, and impaired secretion of a candidate effector having secretory signal peptide for translocation across the endoplasmic reticulum membrane. The mRNA transcripts of ArF-BAR were induced in response to oxidative stress and infection. We also show that ArF-BAR is able to tubulate synthetic liposomes, suggesting the functional role of F-BAR domain in membrane tubule formation in vivo. Further, our studies identified a stress-induced transcription factor, ArCRZ1 (Calcineurin-responsive zinc finger 1), as key transcriptional regulator of ArF-BAR expression. We propose a model in which ArCRZ1 functions upstream of ArF-BAR to regulate A. rabiei virulence through a mechanism that involves endocytosis, effector secretion, and actin cytoskeleton regulation.

Porcine Circovirus Type 3 Enters Into PK15 Cells Through Clathrin- and Dynamin-2-Mediated Endocytosis in a Rab5/Rab7 and pH-Dependent Fashion.

Porcine circovirus type 3 (PCV3) invades multiple tissues and organs of pigs of different ages and are widely spread throughout pig farms, emerging as an important viral pathogen that can potentially damage the pig industry worldwide. Since PCV3 is a newly discovered virus, many aspects of its life cycle remain unknown. Porcine kidney epithelial cells are important host targets for PCV3. Here, we used systematic approaches to dissect the molecular mechanisms underlying the cell entry and intracellular trafficking of PCV3 in PK15 cells, a cell line of porcine kidney epithelial origin. A large number of PCV3 viral particles were found to colocalize with clathrin but not caveolin-1 after entry, and PCV3 infection was significantly decreased when treated with chlorpromazine, dynasore, knockdown of clathrin heavy chain expression via RNA interference, or overexpression of a dominant-negative mutant of EPS15 in PCV3-infected cells. After internalization, the viral particles were further observed to colocalize with Rab5 and Rab7, and knockdown of both expression by RNA interference significantly inhibited PCV3 replication. We also found that PCV3 infection was impeded by ammonium chloride treatment, which indicated the requirement of an acidic environment for viral entry. Taken together, our findings demonstrate that PCV3 enters PK15 cells through a clathrin- and dynamin-2-mediated endocytic pathway, which requires early and late endosomal trafficking, as well as an acidic environment, providing an insightful theoretical basis for further understanding the PCV3 life cycle and its pathogenesis.

Early Endosomal Trafficking Component BEN2/VPS45 Plays a Crucial Role in Internal Tissues in Regulating Root Growth and Meristem Size in Arabidopsis.

Polar auxin transport is involved in multiple aspects of plant development, including root growth, lateral root branching, embryogenesis, and vasculature development. PIN-FORMED (PIN) auxin efflux proteins exhibit asymmetric distribution at the plasma membrane (PM) and collectively play pivotal roles in generating local auxin accumulation, which underlies various auxin-dependent developmental processes. In previous research, it has been revealed that endosomal trafficking components BEN1/BIG5 (ARF GEF) and BEN2/VPS45 (Sec1/Munc 18 protein) function in intracellular trafficking of PIN proteins in Arabidopsis. Mutations in both BEN1 and BEN2 resulted in defects in polar PIN localization, auxin response gradients, and in root architecture. In this study, we have attempted to gain insight into the developmental roles of these trafficking components. We showed that while genetic or pharmacological disturbances of auxin distribution reduced dividing cells in the root tips and resulted in reduced root growth, the same manipulations had only moderate impact on ben1; ben2 double mutants. In addition, we established transgenic lines in which BEN2/VPS45 is expressed under control of tissue-specific promoters and demonstrated that BEN2/VPS45 regulates the intracellular traffic of PIN proteins in cell-autonomous manner, at least in stele and epidermal cells. Furthermore, BEN2/VPS45 rescued the root architecture defects when expressed in internal tissues of ben1; ben2 double mutants. These results corroborate the roles of the endosomal trafficking component BEN2/VPS45 in regulation of auxin-dependent developmental processes, and suggest that BEN2/VPS45 is required for sustainable root growth, most likely through regulation of tip-ward auxin transport through the internal tissues of root.

Long-distance early endosome motility in Aspergillus fumigatus promotes normal hyphal growth behaviors in controlled microenvironments but is dispensable for virulence.

In filamentous fungi, early endosomes are continuously trafficked to, and from, the growing hyphal tip by microtubule-based motor proteins, serving as platforms for the long-distance transport of diverse cargos including mRNA, signaling molecules, and other organelles which hitchhike on them. While the cellular machinery for early endosome motility in filamentous fungi is fairly well characterized, the broader physiological significance of this process remains less well understood. We set out to determine the importance of long-distance early endosome trafficking in Aspergillus fumigatus, an opportunistic human pathogenic fungus that can cause devastating pulmonary infections in immunocompromised individuals. We first characterized normal early endosome motile behavior in A. fumigatus, then generated a mutant in which early endosome motility is severely perturbed through targeted deletion of the gene encoding for FtsA, one of a complex of proteins that links early endosomes to their motor proteins. Using a microfluidics-based approach we show that contact-induced hyphal branching behaviors are impaired in ΔftsA mutants, but that FtsA-mediated early endosome motility is dispensable for virulence in an invertebrate infection model. Overall, our study provides new insight into early endosome motility in an important human pathogenic fungus.

Pi4KIIα Regulates Unconditioned Stimulus-Retrieval-Induced Fear Memory Reconsolidation through Endosomal Trafficking of AMPA Receptors.

SummaryTargeting memory reconsolidation is an effective intervention for treating posttraumatic stress disorder (PTSD). Disrupting unconditioned stimulus (US)-retrieval-induced fear memory reconsolidation has become an effective therapeutic approach to attenuate fear memory, but the underlying molecular mechanisms remain unknown. Here, we report that US-retrieval-dependent increase in phosphatidylinositol 4-kinase IIα (Pi4KIIα) promotes early endosomal trafficking of AMPA receptors, leading to the enhancement of synaptic efficacy in basolateral amygdala (BLA) neurons. The inhibition of Pi4KIIα by an inhibitor or short hairpin RNA impaired contextual fear memory reconsolidation. This disruptive effect persisted for at least 2 weeks, which was restored by Pi4KIIα overexpression with TAT-Pi4KIIα. Furthermore, the blockade of early endosomal trafficking following US retrieval reduced synaptosomal membrane GluA1 levels and decreased subsequent fear expression. These data demonstrate that Pi4KIIα in the BLA is crucial for US-retrieval-induced fear memory reconsolidation, the inhibition of which might be an effective therapeutic strategy for treating PTSD.

1715. Modulation of RAB5A early endosome trafficking in response to Kras mediated macropinocytic fluxes in pancreatic cancer cells

1715. Modulation of RAB5A early endosome trafficking in response to Kras mediated macropinocytic fluxes in pancreatic cancer cells

Innate Immune Signals Induce Anterograde Endosome Transport Promoting MHC Class I Cross-Presentation

Both cross-presentation of antigens by dendritic cells, a key pathway triggering Tcell immunity and immune tolerance, and survival of several pathogens residing in intracellular vacuoles are intimately linked to delayed maturation of vesicles containing internalized antigens and microbes. However, how early endosome or phagosome identity is maintained is incompletely understood. We show that Toll-like receptor 4 (TLR4) and Fc receptor ligation induces interaction of the GTPase Rab14 with the kinesin KIF16b mediating plus-end-directed microtubule transport of endosomes. As a result, Rab14 recruitment to phagosomes delays their maturation and killing of an internalized pathogen. Enhancing anterograde transport by overexpressing Rab14, promoting the GTP-bound Rab14 state, or inhibiting retrograde transport upregulates cross-presentation. Conversely, reducing Rab14 expression, destabilizing Rab14 endosomes, and inhibiting anterograde microtubule transport by Kif16b knockdown compromise cross-presentation. Therefore, regulation of early endosome trafficking by innate immune signals is a critical parameter in cross-presentation by dendritic cells.

Alteration of endosomal trafficking is associated with early-onset parkinsonism caused by SYNJ1 mutations

Recently, a new form of autosomal recessive early-onset parkinsonism (PARK20), due to mutations in the gene encoding the phosphoinositide phosphatase, Synaptojanin 1 (Synj1), has been reported. Several genes responsible for hereditary forms of Parkinson’s disease are implicated in distinct steps of the endolysosomal pathway. However, the nature and the degree of endocytic membrane trafficking impairment in early-onset parkinsonism remains elusive. Here, we show that depletion of Synj1 causes drastic alterations of early endosomes, which become enlarged and more numerous, while it does not affect the morphology of late endosomes both in non-neuronal and neuronal cells. Moreover, Synj1 loss impairs the recycling of transferrin, while it does not alter the trafficking of the epidermal growth factor receptor. The ectopic expression of Synj1 restores the functions of early endosomes, and rescues these trafficking defects in depleted cells. Importantly, the same alterations of early endosomal compartments and trafficking defects occur in fibroblasts of PARK20 patients. Our data indicate that Synj1 plays a crucial role in regulating the homeostasis and functions of early endosomal compartments in different cell types, and highlight defective cellular pathways in PARK20. In addition, they strengthen the link between endosomal trafficking and Parkinson’s disease.