Diverse Fgfr1 signaling pathways and endocytic trafficking regulate mesoderm development.

The fibroblast growth factor (FGF) pathway is a conserved signaling pathway required for embryonic development. Activated FGF receptor 1 (FGFR1) drives multiple intracellular signaling cascade pathways, including ERK/MAPK and PI3K/AKT, collectively termed canonical signaling. However, unlike Fgfr1-null embryos, embryos containing hypomorphic mutations in Fgfr1 lacking the ability to activate canonical downstream signals are still able to develop to birth but exhibit severe defects in all mesodermal-derived tissues. The introduction of an additional signaling mutation further reduces the activity of Fgfr1, leading to earlier lethality, reduced somitogenesis, and more severe changes in transcriptional outputs. Genes involved in migration, ECM interaction, and phosphoinositol signaling were significantly downregulated, proteomic analysis identified changes in interactions with endocytic pathway components, and cells expressing mutant receptors show changes in endocytic trafficking. Together, we identified processes regulating early mesoderm development by mechanisms involving both canonical and noncanonical Fgfr1 pathways, including direct interaction with cell adhesion components and endocytic regulation.

Diverse Fgfr1 signaling pathways and endocytic trafficking regulate early mesoderm development.

The Fibroblast growth factor (FGF) pathway is a conserved signaling pathway required for embryonic development. Activated FGF receptor 1 (FGFR1) drives multiple intracellular signaling cascade pathways, including ERK/MAPK and PI3K/AKT, collectively termed canonical signaling. However, unlike Fgfr1 null embryos, embryos containing hypomorphic mutations in Fgfr1 lacking the ability to activate canonical downstream signals are still able to develop to birth, but exhibit severe defects in all mesodermal-derived tissues. The introduction of an additional signaling mutation further reduces the activity of Fgfr1, leading to earlier lethality, reduced somitogenesis, and more severe changes in transcriptional outputs. Genes involved in migration, ECM-interaction, and phosphoinositol signaling were significantly downregulated, proteomic analysis identified changes in interactions with endocytic pathway components, and cells expressing mutant receptors show changes in endocytic trafficking. Together, we identify processes regulating early mesoderm development by mechanisms involving both canonical and non-canonical Fgfr1 pathways, including direct interaction with cell adhesion components and endocytic regulation.

Insights into the role of derailed endocytic trafficking pathway in cancer: From the perspective of cancer hallmarks.

The endocytic trafficking pathway is a highly organized cellular program responsible for the regulation of membrane components and uptake of extracellular substances. Molecules internalized into the cell through endocytosis will be sorted for degradation or recycled back to membrane, which is determined by a series of sorting events. Many receptors, enzymes, and transporters on the membrane are strictly regulated by endocytic trafficking process, and thus the endocytic pathway has a profound effect on cellular homeostasis. However, the endocytic trafficking process is typically dysregulated in cancers, which leads to the aberrant retention of receptor tyrosine kinases and immunosuppressive molecules on cell membrane, the loss of adhesion protein, as well as excessive uptake of nutrients. Therefore, hijacking endocytic trafficking pathway is an important approach for tumor cells to obtain advantages of proliferation and invasion, and to evade immune attack. Here, we summarize how dysregulated endocytic trafficking process triggers tumorigenesis and progression from the perspective of several typical cancer hallmarks. The impact of endocytic trafficking pathway to cancer therapy efficacy is also discussed.

NeuM: A Neuron-Selective Probe Incorporates into Live Neuronal Membranes via Enhanced Clathrin-Mediated Endocytosis in Primary Neurons.

The development of a small-molecule probe designed to selectively target neurons would enhance the exploration of intricate neuronal structures and functions. Among such probes, NeuO stands out as the pioneer and has gained significant traction in the field of research. Nevertheless, neither the mechanism behind neuron-selectivity nor the cellular localization has been determined. Here, we introduce NeuM, a derivative of NeuO, designed to target neuronal cell membranes. Furthermore, we elucidate the mechanism behind the selective neuronal membrane trafficking that distinguishes neurons. In an aqueous buffer, NeuM autonomously assembles into micellar structures, leading to the quenching of its fluorescence (Φ=0.001). Upon exposure to neurons, NeuM micelles were selectively internalized into neuronal endosomes via clathrin-mediated endocytosis. Through the endocytic recycling pathway, NeuM micelles integrate into neuronal membrane, dispersing fluorescent NeuM molecules in the membrane (Φ=0.61). Molecular dynamics simulations demonstrated that NeuM, in comparison to NeuO, possesses optimal lipophilicity and molecular length, facilitating its stable incorporation into phospholipid layers. The stable integration of NeuM within neuronal membrane allows the prolonged monitoring of neurons, as well as the visualization of intricate neuronal structures.

Functional screening reveals Toxoplasma prenylated proteins required for endocytic trafficking and rhoptry protein sorting.

In the apicomplexans, endocytosed cargos (e.g., hemoglobin) are trafficked to a specialized organelle for digestion. This follows a unique endocytotic process at the micropore/cytostome in these parasites. However, the mechanism underlying endocytic trafficking remains elusive, due to the repurposing of classical endocytic proteins for the biogenesis of apical organelles. To resolve this issue, we have exploited the genetic tractability of the model apicomplexan Toxoplasma gondii, which ingests host cytosolic materials (e.g., green fluorescent protein[GFP]). We determined an association between protein prenylation and endocytic trafficking, and using an alkyne-labeled click chemistry approach, the prenylated proteome was characterized. Genome editing, using clustered regularly interspaced short palindromic repaet/CRISPR-associated nuclease 9 (CRISPR/Cas9), was efficiently utilized to generate genetically modified lines for the functional screening of 23 prenylated candidates. This identified four of these proteins that regulate the trafficking of endocytosed GFP vesicles. Among these proteins, Rab1B and YKT6.1 are highly conserved but are non-classical endocytic proteins in eukaryotes. Confocal imaging analysis showed that Rab1B and Ras are substantially localized to both the trans-Golgi network and the endosome-like compartments in the parasite. Conditional knockdown of Rab1B caused a rapid defect in secretory trafficking to the rhoptry bulb, suggesting a trafficking intersection role for the key regulator Rab1B. Further experiments confirmed a critical role for protein prenylation in regulating the stability/activity of these proteins (i.e., Rab1B and YKT6.1) in the parasite. Our findings define the molecular basis of endocytic trafficking and reveal a potential intersection function of Rab1B on membrane trafficking in T. gondii. This might extend to other related protists, including the malarial parasites. IMPORTANCE The protozoan Toxoplasma gondii establishes a permissive niche, in host cells, that allows parasites to acquire large molecules such as proteins. Numerous studies have demonstrated that the parasite repurposes the classical endocytic components for secretory sorting to the apical organelles, leaving the question of endocytic transport to the lysosome-like compartment unclear. Recent studies indicated that endocytic trafficking is likely to associate with protein prenylation in malarial parasites. This information promoted us to examine this association in the model apicomplexan T. gondii and to identify the key components of the prenylated proteome that are involved. By exploiting the genetic tractability of T. gondii and a host GFP acquisition assay, we reveal four non-classical endocytic proteins that regulate the transport of endocytosed cargos (e.g., GFP) in T. gondii. Thus, we extend the principle that protein prenylation regulates endocytic trafficking and elucidate the process of non-classical endocytosis in T. gondii and potentially in other related protists.

Sphingosine Kinases Promote Ebola Virus Infection and Can Be Targeted to Inhibit Filoviruses, Coronaviruses, and Arenaviruses Using Late Endocytic Trafficking to Enter Cells.

Entry of enveloped viruses in host cells requires the fusion of viral and host cell membranes, a process that is facilitated by viral fusion proteins protruding from the viral envelope. These viral fusion proteins need to be triggered by host factors, and for some viruses, this event occurs inside endosomes and/or lysosomes. Consequently, these 'late-penetrating viruses' must be internalized and delivered to entry-conducive intracellular vesicles. Because endocytosis and vesicular trafficking are tightly regulated cellular processes, late-penetrating viruses also depend on specific host proteins for efficient delivery to the site of fusion, suggesting that these could be targeted for antiviral therapy. In this study, we investigated a role for sphingosine kinases (SKs) in viral entry and found that chemical inhibition of sphingosine kinase 1 (SK1) and/or SK2 and knockdown of SK1/2 inhibited entry of Ebola virus (EBOV) into host cells. Mechanistically, inhibition of SK1/2 prevented EBOV from reaching late-endosomes and lysosomes that contain the EBOV receptor, Niemann Pick C1 (NPC1). Furthermore, we present evidence that suggests that the trafficking defect caused by SK1/2 inhibition occurs independently of sphingosine-1-phosphate (S1P) signaling through cell-surface S1P receptors. Lastly, we found that chemical inhibition of SK1/2 prevents entry of other late-penetrating viruses, including arenaviruses and coronaviruses, and inhibits infection by replication-competent EBOV and SARS-CoV-2 in Huh7.5 cells. In sum, our results highlight an important role played by SK1/2 in endocytic trafficking, which can be targeted to inhibit entry of late-penetrating viruses and could serve as a starting point for the development of broad-spectrum antiviral therapeutics.

The Drosophila AWP1 ortholog Doctor No regulates JAK/STAT signaling for left-right asymmetry in the gut by promoting receptor endocytosis.

Many organs of Drosophila show stereotypical left-right (LR) asymmetry; however, the underlying mechanisms remain elusive. Here, we have identified an evolutionarily conserved ubiquitin-binding protein, AWP1/Doctor No (Drn), as a factor required for LR asymmetry in the embryonic anterior gut. We found that drn is essential in the circular visceral muscle cells of the midgut for JAK/STAT signaling, which contributes to the first known cue for anterior gut lateralization via LR asymmetric nuclear rearrangement. Embryos homozygous for drn and lacking its maternal contribution showed phenotypes similar to those with depleted JAK/STAT signaling, suggesting that Drn is a general component of JAK/STAT signaling. Absence of Drn resulted in specific accumulation of Domeless (Dome), the receptor for ligands in the JAK/STAT signaling pathway, in intracellular compartments, including ubiquitylated cargos. Dome colocalized with Drn in wild-type Drosophila. These results suggest that Drn is required for the endocytic trafficking of Dome, which is a crucial step for activation of JAK/STAT signaling and the subsequent degradation of Dome. The roles of AWP1/Drn in activating JAK/STAT signaling and in LR asymmetric development may be conserved in various organisms.

RhoB as a tumor suppressor: It's all about localization.

The small GTPase RhoB is distinguished from other Rho proteins by its unique subcellular localization in endosomes, multivesicular bodies, and nucleus. Despite high sequence homology with RhoA and RhoC, RhoB is mainly associated with tumor suppressive function, while RhoA and RhoC support oncogenic transformation in most malignancies. RhoB regulates the endocytic trafficking of signaling molecules and cytoskeleton remodeling, thereby controlling growth, apoptosis, stress response, immune function, and cell motility in various contexts. Some of these functions may be ascribed to RhoB's unique subcellular localization to endocytic compartments. Here we describe the pleiotropic roles of RhoB in cancer suppression in the context of its subcellular localization, and we discuss possible therapeutic avenues to pursue and highlight priorities for future research.

Proteome-Wide Analysis Reveals TFEB Targets for Establishment of a Prognostic Signature to Predict Clinical Outcomes of Colorectal Cancer.

Dephosphorylation of transcription factor EB (TFEB) at Ser142 and Ser138 determines its nuclear localization and transcriptional activity. The link between TFEB-associated genes and colorectal cancer (CRC) progression and prognosis remains unclear. To systematically identify the targets of TFEB, we performed data-independent acquisition (DIA)-based quantitative proteomics to compare global protein changes in wild-type (WT) DLD1 cells and TFEBWT- or TFEBS142A/S138A (activated status)-expressing DLD1 cells. A total of 6048 proteins were identified and quantified in three independent experiments. The differentially expressed proteins in TFEBS142A/S138A versus TFEBWT and TFEBWT versus control groups were compared, and 60 proteins were identified as products of TFEB transcriptional regulation. These proteins were significantly associated with vesicular endocytic trafficking, the HIF-1 signaling pathway, and metabolic processes. Furthermore, we generated a TFEB-associated gene signature using a univariate and LASSO Cox regression model to screen robust prognostic markers. An eight-gene signature (PLSCR3, SERPINA1, ATP6V1C2, TIMP1, SORT1, MAP2, KDM4B, and DDAH2) was identified. According to the signature, patients were assigned to high-risk and low-risk groups. Higher risk scores meant worse overall survival and higher epithelial-mesenchymal transition (EMT) scores. Additionally, as per the clinicopathological parameters and gene signature, a nomogram was constructed that was utilized to enhance the quantification capacity in risk assessment for individual patients. This research shows that TFEB directly mediates network effects in CRC, and the identified TFEB gene signature-based model may provide important information for the clinical judgment of prognosis.

Listeria InlB Expedites Vacuole Escape and Intracellular Proliferation by Promoting Rab7 Recruitment via Vps34.

Rapid phagosomal escape mediated by listeriolysin O (LLO) is a prerequisite for Listeria monocytogenes intracellular replication and pathogenesis. Escape takes place within minutes after internalization from vacuoles that are negative to the early endosomal Rab5 GTPase and positive to the late endosomal Rab7. Using mutant analysis, we found that the listerial invasin InlB was required for optimal intracellular proliferation of L. monocytogenes. Starting from this observation, we determined in HeLa cells that InlB promotes early phagosomal escape and efficient Rab7 acquisition by the Listeria-containing vacuole (LCV). Recruitment of the class III phosphoinositide 3-kinase (PI3K) Vps34 to the LCV and accumulation of its lipid product, phosphatidylinositol 3-phosphate (PI3P), two key endosomal maturation mediators, were also dependent on InlB. Small interfering RNA (siRNA) knockdown experiments showed that Vps34 was required for Rab7 recruitment and early (LLO-mediated) escape and supported InlB-dependent intracellular proliferation. Together, our data indicate that InlB accelerates LCV conversion into an escape-favorable Rab7 late phagosome via subversion of class III PI3K/Vps34 signaling. Our findings uncover a new function for the InlB invasin in Listeria pathogenesis as an intracellular proliferation-promoting virulence factor. IMPORTANCE Avoidance of lysosomal killing by manipulation of the endosomal compartment is a virulence mechanism assumed to be largely restricted to intravacuolar intracellular pathogens. Our findings are important because they show that cytosolic pathogens like L. monocytogenes, which rapidly escape the phagosome after internalization, can also extensively subvert endocytic trafficking as part of their survival strategy. They also clarify that, instead of delaying phagosome maturation (to allow time for LLO-dependent disruption, as currently thought), via InlB L. monocytogenes appears to facilitate the rapid conversion of the phagocytic vacuole into an escape-conducive late phagosome. Our data highlight the multifunctionality of bacterial virulence factors. At the cell surface, the InlB invasin induces receptor-mediated phagocytosis via class I PI3K activation, whereas after internalization it exploits class III PI3K (Vsp34) to promote intracellular survival. Systematically elucidating the mechanisms by which Listeria interferes with PI3K signaling all along the endocytic pathway may lead to novel anti-infective therapies.

Kinesin-1 Regulates Endocytic Trafficking of Classical Swine Fever Virus along Acetylated Microtubules.

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is an important and highly infectious pig disease worldwide. Kinesin-1, a molecular motor responsible for transporting cargo along the microtubule, has been demonstrated to be involved in the infections of diverse viruses. However, the role of kinesin-1 in the CSFV life cycle remains unknown. Here, we first found that Kif5B played a positive role in CSFV entry by knockdown or overexpression of Kif5B. Subsequently, we showed that Kif5B was associated with the endosomal and lysosomal trafficking of CSFV in the early stage of CSFV infection, which was reflected by the colocalization of Kif5B and Rab7, Rab11, or Lamp1. Interestingly, trichostatin A (TSA) treatment promoted CSFV proliferation, suggesting that microtubule acetylation facilitated CSFV endocytosis. The results of chemical inhibitors and RNA interference showed that Rac1 and Cdc42 induced microtubule acetylation after CSFV infection. Furthermore, confocal microscopy revealed that cooperation between Kif5B and dynein help CSFV particles move in both directions along microtubules. Collectively, our study shed light on the role of kinesin motor Kif5B in CSFV endocytic trafficking, indicating the dynein/kinesin-mediated bidirectional CSFV movement. The elucidation of this study provides the foundation for developing CSFV antiviral drugs. IMPORTANCE The minus end-directed cytoplasmic dynein and the plus end-directed kinesin-1 are the molecular motors that transport cargo on microtubules in intracellular trafficking, which plays a notable role in the life cycles of diverse viruses. Our previous studies have reported that the CSFV entry host cell is dependent on the microtubule-based motor dynein. However, little is known about the involvement of kinesin-1 in CSFV infection. Here, we revealed the critical role of kinesin-1 that regulated the viral endocytosis along acetylated microtubules induced by Cdc42 and Rac1 after CSFV entry. Mechanistically, once CSFV transported by dynein met an obstacle, it recruited kinesin-1 to move in reverse to the anchor position. This study extends the theoretical basis of intracellular transport of CSFV and provides a potential target for the control and treatment of CSFV infection.

The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins.

The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as viroporins. Here, we show that neither SARS-CoV-2 nor SARS-CoV-1 Orf3a form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a positively charged aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a's ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.

Mechanistic investigation into selective cytotoxic activities of gold nanoparticles functionalized with epidermal growth factor variants.

Epidermal growth factor (EGF) gains unique selective cytotoxicity against cancer cells upon conjugation with gold nanoparticles (GNPs). We have previously developed several lysine-free EGF mutants for favorable interactions between the nanoparticle conjugates with EGF receptor (EGFR) and found one mutant (SR: K28S/K48R) showing stronger anticancer activities. However, the exact mechanisms for the selective cytotoxicity enhancement in the SR mutant remained unsolved. In this study, we analyzed how the nanoparticle conjugates of EGF variants interacted differently with A431 cancer cells, in terms of receptor binding, activation, and trafficking. Our results indicate that the essential feature of the SR-GNP conjugates in the cytotoxicity enhancement is their preferential activation of the clathrin-independent endocytosis pathway. It is suggested that we should focus on not only ligand-receptor binding affinity but also the selectivity of the receptor endocytic route to optimize the anticancer effects in this modality.

Imbalance of NRG1-ERBB2/3 signalling underlies altered myelination in Charcot-Marie-Tooth disease 4H.

Charcot-Marie-Tooth (CMT) disease is one of the most common inherited neurological disorders, affecting either axons from the motor and/or sensory neurons or Schwann cells of the peripheral nervous system (PNS) and caused by more than 100 genes. We previously identified mutations in FGD4 as responsible for CMT4H, an autosomal recessive demyelinating form of CMT disease. FGD4 encodes FRABIN, a GDP/GTP nucleotide exchange factor, particularly for the small GTPase Cdc42. Remarkably, nerves from patients with CMT4H display excessive redundant myelin figures called outfoldings that arise from focal hypermyelination, suggesting that FRABIN could play a role in the control of PNS myelination. To gain insights into the role of FGD4/FRABIN in Schwann cell myelination, we generated a knockout mouse model (Fgd4SC-/-), with conditional ablation of Fgd4 in Schwann cells. We show that the specific deletion of FRABIN in Schwann cells leads to aberrant myelination in vitro, in dorsal root ganglia neuron/Schwann cell co-cultures, as well as in vivo, in distal sciatic nerves from Fgd4SC-/- mice. We observed that those myelination defects are related to an upregulation of some interactors of the NRG1 type III/ERBB2/3 signalling pathway, which is known to ensure a proper level of myelination in the PNS. Based on a yeast two-hybrid screen, we identified SNX3 as a new partner of FRABIN, which is involved in the regulation of endocytic trafficking. Interestingly, we showed that the loss of FRABIN impairs endocytic trafficking, which may contribute to the defective NRG1 type III/ERBB2/3 signalling and myelination. Using RNA-Seq, in vitro, we identified new potential effectors of the deregulated pathways, such as ERBIN, RAB11FIP2 and MAF, thereby providing cues to understand how FRABIN contributes to proper ERBB2 trafficking or even myelin membrane addition through cholesterol synthesis. Finally, we showed that the re-establishment of proper levels of the NRG1 type III/ERBB2/3 pathway using niacin treatment reduces myelin outfoldings in nerves of CMT4H mice. Overall, our work reveals a new role of FRABIN in the regulation of NRG1 type III/ERBB2/3 NRG1signalling and myelination and opens future therapeutic strategies based on the modulation of the NRG1 type III/ERBB2/3 pathway to reduce CMT4H pathology and more generally other demyelinating types of CMT disease.

LEAP: a novel LC3C-dependent pathway connects autophagy, endocytic trafficking and signaling.

Macroautophagy/autophagy acts to promote homeostasis and is increasingly understood to selectively target cargo for degradation. The LC3-family of proteins mediate diverse yet distinct cargo recruitment to phagophores. However, what underlies specificity for cargo engagement among LC3 proteins is poorly understood. Using an unbiased protein interaction screen of LC3B and LC3C, we uncover a novel LC3C-endocytic-associated-pathway (LEAP) that recruits selective plasma membrane (PM) cargo to phagophores. We show LC3C but not LC3B localizes to peripheral endosomes and engages proteins that traffic between the PM, endosomes and autophagosomes. We establish that endocytic LC3C binds cargo internalized from the PM, including MET receptor tyrosine kinase and TFRC (transferrin receptor), and targets them toward autophagic degradation. These findings identify LEAP as an unexpected LC3C-dependent pathway, providing new understanding of selective coupling of PM signaling and autophagic degradation with important implications in cancer and other disease states.

Juvenile mucopolysaccharidosis plus disease caused by a missense mutation in VPS33A.

A rare and fatal disease resembling mucopolysaccharidosis in infants, is caused by impaired intracellular endocytic trafficking due to deficiency of core components of the intracellular membrane-tethering protein complexes, HOPS, and CORVET. Whole exome sequencing identified a novel VPS33A mutation in a patient suffering from a variant form of mucopolysaccharidosis. Electron and confocal microscopy, immunoblotting, and glycosphingolipid trafficking experiments were undertaken to investigate the effects of the mutant VPS33A in patient-derived skin fibroblasts. We describe an attenuated juvenile form of VPS33A-related syndrome-mucopolysaccharidosis plus in a man who is homozygous for a hitherto unknown missense mutation (NM_022916.4: c.599 G>C; NP_075067.2:p. Arg200Pro) in a conserved region of the VPS33A gene. Urinary glycosaminoglycan (GAG) analysis revealed increased heparan, dermatan sulphates, and hyaluronic acid. We showed decreased abundance of VPS33A in patient derived fibroblasts and provided evidence that the p.Arg200Pro mutation leads to destablization of the protein and proteasomal degradation. As in the infantile form of mucopolysaccharidosis plus, the endocytic compartment in the fibroblasts also expanded-a phenomenon accompanied by increased endolysosomal acidification and impaired intracellular glycosphingolipid trafficking. Experimental treatment of the patient's cultured fibroblasts with the proteasome inhibitor, bortezomib, or exposure to an inhibitor of glucosylceramide synthesis, eliglustat, improved glycosphingolipid trafficking. To our knowledge this is the first report of an attenuated juvenile form of VPS33A insufficiency characterized by appreciable residual endosomal-lysosomal trafficking and a milder mucopolysaccharidosis plus than the disease in infants. Our findings expand the proof of concept of redeploying clinically approved drugs for therapeutic exploitation in patients with juvenile as well as infantile forms of mucopolysaccharidosis plus disease.

Regulation of oxidative stress-induced autophagy by ATG9A ubiquitination.

High levels of reactive oxygen species (ROS) result in oxidative stress, which damages cells and leads to the development of many diseases. Macroautophagy/autophagy plays an important role in protecting cells from diverse stress stimuli including oxidative stress. However, the molecular mechanisms of autophagy activation in response to oxidative stress remain largely unclear. In this study, we showed that TRAF6 mediates oxidative stress-induced ATG9A ubiquitination at two C-terminal lysine residues (K581 and K838). ATG9A ubiquitination promotes its association with BECN1, BECN1-PIK3C3/VPS34-UVRAG complex assembly and PIK3C3/VPS34 activation, thereby activating autophagy and endocytic trafficking. We also identified TNFAIP3/A20 as a negative regulator of oxidative-induced autophagy by counteracting TRAF6-mediated ATG9A ubiquitination. Moreover, ATG9A depletion attenuates LPS-induced autophagy and causes aberrant TLR4 signaling and inflammatory responses. Our findings revealed a critical role of ATG9A ubiquitination in oxidative stress-induced autophagy, endocytic trafficking and innate immunity.

Endocytic trafficking induces lateral root founder cell specification in Arabidopsis thaliana in a process distinct from the auxin-induced pathway.

Plants can modify their body structure, such as their root architecture, post-embryonically. For example, Arabidopsis thaliana can develop lateral roots as part of an endogenous program or in response to biotic and abiotic stimuli. Root pericycle cells are specified to become lateral root founder cells, initiating lateral root organogenesis. We used the endocytic trafficking inducer Sortin2 to examine the role of endomembrane trafficking in lateral root founder cell specification. Our results indicate that Sortin2 stimulation turns on a de novo program of lateral root primordium formation that is distinct from the endogenous program driven by auxin. In this distinctive mechanism, extracellular calcium uptake and endocytic trafficking toward the vacuole are required for lateral root founder cell specification upstream of the auxin module led by AUX/IAA28. The auxin-dependent TIR1/AFB F-boxes and auxin polar transport are dispensable for the endocytic trafficking-dependent lateral root founder cell specification; however, a different set of F-box proteins and a functional SCF complex are required. The endocytic trafficking could constitute a convenient strategy for organogenesis in response to environmental conditions.

Beta-amyloid pore linked to controlled calcium influx into the cell: A new paradigm for Alzheimer's Disease.

Despite tremendous worldwide efforts, clinical trials assessing Alzheimer's disease (AD)-related therapeutics have been relentlessly unsuccessful. Hence, there is an urgent need to challenge old hypotheses with novel paradigms. An emerging concept is that the amyloid-beta (Aß) peptide, which was until recently deemed a major player in the cause of AD, may instead modulate synaptic plasticity and protect against excitotoxicity. The link between Aß-mediated synaptic plasticity and Aß trafficking is central for understanding AD pathogenesis and remains a perplexing relationship. The crossover between Aß pathological and physiological roles is subtle and remains controversial. Based on existing literature, as a signaling molecule, Aß is proposed to modulate its own turnover and synaptic plasticity through what is currently believed to be the cause of AD: the transient formation of pore-like oligomers. A change of perspective regarding how Aß pores exert a protective function will unavoidably revolutionize the entire field of anti-amyloid drug development.

SARS-CoV-2 spike protein harnesses SNX27-mediated endocytic recycling pathway.

SARS-CoV-2 is an enveloped positive-sense RNA virus that depends on host factors for all stages of its life. Membrane receptor ACE2 is a well-established factor for SARS-CoV-2 docking. In addition to ACE2, whole-genome genetic screens have identified additional proteins, such as endosomal trafficking regulators SNX27 and retromer, as key host factors required for SARS-CoV-2 infection. However, it is poorly understood how SARS-CoV-2 utilize host endocytic transport pathways to produce productive infection. Here, we report that SNX27 interacts with the SARS-CoV-2 spike (S) protein to facilitate S protein surface expression. Interestingly, S protein binds to the PDZ domain of SNX27, although it does not contain a PDZ-binding motif (PDZbm). Either abrogation of the SNX27 PDZ domain or S protein "MTSC" motif, which is critical for SNX27 binding, decreases surface expression of S protein and viral production. Collectively, our study highlights a novel approach utilized by SARS-CoV-2 to facilitate virion trafficking to establish virus infection.