Extremely high spatiotemporal resolution microscopy for live cell imaging by single photon counting, noise elimination, and a novel restoration algorithm based on probability calculation.

Optical microscopy is essential for direct observation of dynamic phenomena in living cells. According to the classic optical theories, the images obtained through light microscopes are blurred for about half the wavelength of light, and therefore small structures below this "diffraction limit" were thought unresolvable by conventional optical microscopy. In reality, accurately obtained optical images contain complete information about the observed objects. Temporal resolution is also important for the observation of dynamic phenomena. A challenge exists here to overcome the trade-off between the time required for measurement and the accuracy of the measurement. The present paper describes a concrete methodology for reconstructing the structure of an observed object, based on the information contained in the image obtained by optical microscopy. It is realized by accurate single photon counting, complete noise elimination, and a novel restoration algorithm based on probability calculation. This method has been implemented in the Super-resolution Confocal Live Imaging Microscopy (SCLIM) we developed. The new system named SCLIM2M achieves unprecedented high spatiotemporal resolution. We have succeeded in capturing sub-diffraction-limit structures with millisecond-level dynamics of organelles and vesicles in living cells, which were never observed by conventional optical microscopy. Actual examples of the high-speed and high-resolution 4D observation of living cells are presented.

MARCH8 inhibits pseudorabies virus replication by trapping the viral cell-to-cell fusion complex in the trans-Golgi network.

The membrane-associated RING-CH 8 protein (MARCH8), a member of the E3 ubiquitin ligase family, has broad-spectrum antiviral activity. However, some viruses hijack MARCH8 to promote virus replication, highlighting its dual role in the viral lifecycle. Most studies on MARCH8 have focused on RNA viruses, leaving its role in DNA viruses largely unexplored. Pseudorabies virus (PRV) is a large DNA virus that poses a potential threat to humans. In this study, we found that MARCH8 inhibited PRV replication at the cell-to-cell fusion stage. Interestingly, our findings proved that MARCH8 blocks gB cleavage by recruiting furin but this activity does not inhibit viral infection in vitro. Furthermore, we confirmed that MARCH8 inhibits cell-to-cell fusion independent of its E3 ubiquitin ligase activity but dependent on the interaction with the cell-to-cell fusion complex (gB, gD, gH, and gL). Finally, we discovered that the distribution of the cell-to-cell fusion complex is significantly altered and trapped within the trans-Golgi network. Overall, our results indicate that human MARCH8 acts as a potent antiviral host factor against PRV via trapping the cell-to-cell fusion complex in the trans-Golgi network.

EYA protein complex is required for Wntless retrograde trafficking from endosomes to Golgi.

Retrograde transport of WLS (Wntless) from endosomes to trans-Golgi network (TGN) is required for efficient Wnt secretion during development. However, the molecular players connecting endosomes to TGN during WLS trafficking are limited. Here, we identified a role for Eyes Absent (EYA) proteins during retrograde trafficking of WLS to TGN in human cell lines. By using worm, fly, and zebrafish models, we found that the EYA-secretory carrier-associated membrane protein 3 (SCAMP3) axis is evolved in vertebrates. EYAs form a complex and interact with retromer on early endosomes. Retromer-bound EYA complex recruits SCAMP3 to endosomes, which is necessary for the fusion of WLS-containing endosomes to TGN. Loss of EYA complex or SCAMP3 leads to defective transport of WLS to TGN and failed Wnt secretion. EYA mutations found in patients with hearing loss form a dysfunctional EYA-retromer complex that fails to activate Wnt signaling. These findings identify the EYA complex as a component of retrograde trafficking of WLS from the endosome to TGN.

The lipid flippase ATP8A1 regulates the recruitment of ARF effectors to the trans-Golgi Network.

Formation of transport vesicles requires the coordinate activity of the coating machinery that selects cargo into the nascent vesicle and the membrane bending machinery that imparts curvature to the forming bud. Vesicle coating at the trans-Golgi Network (TGN) involves AP1, GGA2 and clathrin, which are recruited to membranes by activated ARF GTPases. The ARF activation at the TGN is mediated by the BIG1 and BIG2 guanine nucleotide exchange factors (GEFs). Membrane deformation at the TGN has been shown to be mediated by lipid flippases, including ATP8A1, that moves phospholipids from the inner to the outer leaflet of the TGN membrane. We probed a possible coupling between the coating and deformation machineries by testing for an interaction between BIG1, BIG2 and ATP8A1, and by assessing whether such an interaction may influence coating efficiency. Herein, we document that BIG1 and BIG2 co-localize with ATP8A1 in both, static and highly mobile TGN elements, and that BIG1 and BIG2 bind ATP8A1. We show that the interaction involves the catalytic Sec7 domain of the GEFs and the cytosolic C-terminal tail of ATP8A1. Moreover, we report that the expression of ATP8A1, but not ATP8A1 lacking the GEF-binding cytosolic tail, increases the generation of activated ARFs at the TGN and increases the selective recruitment of AP1, GGA2 and clathrin to TGN membranes. This occurs without increasing BIG1 or BIG2 levels at the TGN, suggesting that the binding of the ATP8A1 flippase tail to the Sec7 domain of BIG1/BIG2 increases their catalytic activity. Our results support a model in which a flippase component of the deformation machinery impacts the activity of the GEF component of the coating machinery.

Brefeldin A and M-COPA block the export of RTKs from the endoplasmic reticulum via simultaneous inactivation of ARF1, ARF4, and ARF5.

Normal receptor tyrosine kinases (RTKs) need to reach the plasma membrane (PM) for ligand-induced activation, whereas its cancer-causing mutants can be activated before reaching the PM in organelles, such as the Golgi/trans-Golgi network (TGN). Inhibitors of protein export from the endoplasmic reticulum (ER), such as brefeldin A (BFA) and 2-methylcoprophilinamide (M-COPA), can suppress the activation of mutant RTKs in cancer cells, suggesting that RTK mutants cannot initiate signaling in the ER. BFA and M-COPA block the function of ADP-ribosylation factors (ARFs) that play a crucial role in ER-Golgi protein trafficking. However, among ARF family proteins, the specific ARFs inhibited by BFA or M-COPA, that is, the ARFs involved in RTKs transport from the ER, remain unclear. In this study, we showed that M-COPA blocked the export of not only KIT but also PDGFRA/EGFR/MET RTKs from the ER. ER-retained RTKs could not fully transduce anti-apoptotic signals, thereby leading to cancer cell apoptosis. Moreover, a single knockdown of ARF1, ARF3, ARF4, ARF5, or ARF6 could not block ER export of RTKs, indicating that BFA/M-COPA treatment cannot be mimicked by the knockdown of only one ARF member. Interestingly, simultaneous transfection of ARF1, ARF4, and ARF5 siRNAs mirrored the effect of BFA/M-COPA treatment. Consistent with these results, in vitro pulldown assays showed that BFA/M-COPA blocked the function of ARF1, ARF4, and ARF5. Taken together, these results suggest that BFA/M-COPA targets at least ARF1, ARF4, and ARF5; in other words, RTKs require the simultaneous activation of ARF1, ARF4, and ARF5 for their ER export.

Therapeutic drug monitoring of vedolizumab therapy in inflammatory bowel disease.

BACKGROUND: Therapeutic drug monitoring is effective for optimizing anti-tumor necrosis factor therapies in inflammatory bowel disease, but for vedolizumab, a gut-selective leucocyte migration inhibitor, data are scarce. METHODS: Observational cohort study including 116 bio-experienced inflammatory bowel disease patients treated with vedolizumab for active luminal disease. Biobanked trough blood samples (n = 676) covering 96% of patients were analyzed using a drug-binding immunofluorometric assay. Steroid-free treatment outcomes were classified by clinical disease activity indices and objective findings, primarily endoscopy. RESULTS: Patients with clinical remission to vedolizumab induction therapy (37%) had significantly higher trough levels than those without at weeks 6 (mean 34.1 vs 28.0 µg/mL, P = 0.03) and 10 (34.8 vs 27.5 µg/mL, P = 0.01). Optimal thresholds for discrimination were 32.4 µg/mL (AUCROC 0.66, P = 0.04) and 23.5 (AUCROC 0.67, P = 0.01), respectively. This positive association persisted during maintenance phase with 11.9 µg/mL (AUCROC 0.69, P < 0.01) associated with clinical remission (37%) and 15.3 (AUCROC 0.74, P < 0.001) for objective remission (46%). Stratification by temporal evolution of treatment effects revealed higher induction and maintenance vedolizumab levels in persistent and slow responders as compared to secondary or persistent failures. Pharmacokinetics was influenced by rare formation of anti-vedolizumab antibodies (2%), and to a lesser extent gender and albumin during induction, but not disease severity, concomitant steroids, or thiopurine metabolites. Switching to subcutaneous administrations resulted in 2.3-fold increase in steady-state trough levels. CONCLUSION: Our study supports maintaining adequate drug exposure being essential for sustained positive outcomes of vedolizumab and emphasizes individualized, therapeutic drug monitoring-based treatment regimens. Controlled trials and pharmacokinetic modeling are, however, needed.

Arabidopsis HAPLESS13/AP-1µ is critical for pollen sac formation and tapetal function.

The production of excess and viable pollen grains is critical for reproductive success of flowering plants. Pollen grains are produced within anthers, the male reproductive organ whose development involves precisely controlled cell differentiation, division, and intercellular communication. In Arabidopsis thaliana, specification of an archesporial cell (AC) at four corners of a developing anther, followed by programmed cell divisions, generates four pollen sacs, walled by four cell layers among which the tapetum is in close contact with developing microspores. Tapetum secretes callose-dissolving enzymes to release microspores at early stages and undergoes programmed cell death (PCD) to deliver nutrients and signals for microspore development at later stages. Except for transcription factors, plasma membrane (PM)-associated and secretory peptides have also been demonstrated to mediate anther development. Adaptor protein complexes (AP) recruit both cargos and coat proteins during vesicle trafficking. Arabidopsis AP-1µ/HAPLESS13 (HAP13) is a core component of AP-1 for protein sorting at the trans-Golgi network/early endosomes (TGN/EE). We report here that Arabidopsis HAP13 is critical for pollen sac formation and for sporophytic control of pollen production. Functional loss of HAP13 causes a reduction in pollen sac number. It also results in the dysfunction of tapetum such that secretory function of tapetum at early stages and PCD of tapetum at later stages are both compromised. We further show that the expression of SPL, the polar distribution of auxin maximum, as well as the asymmetric distribution of PIN1 are interfered in hap13 anthers, which in combination may lead to male sterility in hap13.

Intracellularly delivered human lactoferrin functions as an activator of Na+/H+ exchanger 7.

Here, we report that human lactoferrin (hLF), known for its anticancer properties, induced intracellular activation of the Na+/H+ exchanger (NHE) 7 in human lung cancer PC-9 cells. Compared to non-fused hLF, the fusion of human serum albumin (HSA) with hLF (hLF-HSA) facilitated its internalization into PC-9 cells in a caveolae-mediated manner, thereby exhibiting enhanced anti-proliferative effects. Although hLF alone did not exhibit any discernible effects, hLF-HSA resulted in organelle alkalization as detected using an acidotropic pH indicator. hLF-HSA-induced elevation of organelle pH and inhibition of cancer growth were abolished by NHE7 siRNA. hLF-HSA upregulated NHE7. Thus, upon cellular uptake, hLF-HSA triggers proton leakage through the upregulation of NHE7. This process led to organelle alkalization, probably in the trans-Golgi network (TGN) as suggested by the localization of NHE7 in PC-9 cells, thereby suppressing lung cancer cell growth. Forcing the cellular uptake of hLF alone using a caveolae-mediated endocytosis activator led to an increase in organelle pH. Furthermore, cell entry of hLF also activated proton-loading NHE7, leading to organelle acidification in the pancreatic cancer cell line MIA PaCa-2. Therefore, the intracellularly delivered hLF functions as an activator of NHE7.

TGN-020 Alleviate Inflammation and Apoptosis After Cerebral Ischemia-Reperfusion Injury in Mice Through Glymphatic and ERK1/2 Signaling Pathway.

Post-stroke acute inhibition of aquaporin 4 (AQP4) is known to exacerbate inflammation and apoptosis, yet the underlying mechanisms are not fully understood. The objective of this study was to investigate the specific mechanism of inflammation and apoptosis following cerebral ischemia-reperfusion (I/R) injury using the AQP4-specific inhibitor, N-(1,3,4-thiadiazol-2-yl) pyridine-3-carboxamide dihydrochloride (TGN-020). Ischemic stroke was induced in mice using the middle cerebral artery occlusion (MCAO) model. The C57/BL6 mice were randomly divided into three groups as follows: sham operation, I/R 48 h, and TGN-020 + I/R 48 h treatment. All mice were subjected to a series of procedures. These procedures encompassed 2,3,5-triphenyltetrazolium chloride (TTC) staining, neurological scoring, fluorescence tracing, western blotting, immunofluorescence staining, and RNA sequencing (RNA-seq). The glymphatic function in the cortex surrounding cerebral infarction was determined using tracer, glial fibrillary acid protein (GFAP), AQP4 co-staining, and beta-amyloid precursor protein (APP) staining; differential genes were detected using RNA-seq. The influence of TGN-020 on the extracellular signal-regulated kinase 1/2 (ERK) 1/2 pathway was confirmed using the ERK1/2 pathway agonists Ro 67-7467. Additionally, we examined the expression of inflammation associated with microglia and astrocytes after TGN-020 and Ro 67-7467 treatment. Compared with I/R group, TGN-020 alleviated glymphatic dysfunction by inhibiting astrocyte proliferation and reducing tracer accumulation in the peri-infarct area. RNA-seq showed that the differentially expressed genes were mainly involved in the activation of astrocytes and microglia and in the ERK1/2 pathway. Western blot and immunofluorescence further verified the expression of associated inflammation. The inflammation and cell apoptosis induced by I/R are mitigated by TGN-020. This mitigation occurs through the improvement of glymphatic function and the inhibition of the ERK1/2 pathway.

Erythrocyte membrane-coated nanocarriers modified by TGN for Alzheimer's disease.

Alzheimer's disease (AD) is an aging-related neurodegenerative disease, and the main pathological feature was ß-amyloid protein (Aß) deposition. Recently, bioactive materials-based drug delivery system has been widely investigated for the treatment of AD. In this study, we developed a red blood cells (RBC) membrane-coated polycaprolactone (PCL) nanoparticles (NPs) loading with a therapeutic agent for AD, curcumin (Cur). A functional peptide TGNYKALHPHN (TGN) was conjugated to the surface of membrane for blood-brain barrier (BBB) transport (TGN-RBC-NPs-Cur). TGN peptide can be recognized by receptors on the BBB and has great potential for brain transport. To confirm the targeted delivery of Cur to the brain, a cell co-culturing immortalized human cerebral microvascular endothelial cells and human brain astrocytes glioblastoma (hCMEC/D3 and U-118MG) in vitro model was established. As a result, the BBB transporting ratio of TGN-RBC-NPs-FITC was 29.64% at 12 h which was approximately eight-fold than RBC-NPs-FITC. The improvement of drug accumulation in the AD lesion was confirmed by the NPs modified with the BBB-penetrating peptide in the fluorescence imaging and quantitative analysis with UPLC-MS/MS in vivo. The neuroprotective effects were evaluated with new object recognition behavioral test, in vitro AD cell model, dendritic spine stain, GFAP and IBA1 immunofluorescence stain. The spatial learning and memory abilities of the AD model mice treated with TGN-RBC-NPs-Cur were obviously enhanced compared with the AD control mice and were also better than Cur at the same dosage. These results were consistent with the values of protection index of rat adrenal pheochromocytoma cells (PC12 cells) treated by Aß25-35. TGN-RBC-NPs-Cur increased the dendritic segments densities and restrained activation of microglia and astrocytes of AD mice, as well as reversed cognitive function of AD mice. All of the results demonstrated TGN-RBC-NPs-Cur a promising therapeutic strategy for delaying the progression of AD by designing biomimetic nanosystems to deliver drugs into the brain.

Sorting secretory proteins.

A receptor protein called TGN46 has an important role in sorting secretory proteins into vesicles going to different destinations inside cells.

Copper-independent lysosomal localisation of the Wilson disease protein ATP7B.

In hepatocytes, the Wilson disease protein ATP7B resides on the trans-Golgi network (TGN) and traffics to peripheral lysosomes to export excess intracellular copper through lysosomal exocytosis. We found that in basal copper or even upon copper chelation, a significant amount of ATP7B persists in the endolysosomal compartment of hepatocytes but not in non-hepatic cells. These ATP7B-harbouring lysosomes lie in close proximity of ~10 nm to the TGN. ATP7B constitutively distributes itself between the sub-domain of the TGN with a lower pH and the TGN-proximal lysosomal compartments. The presence of ATP7B on TGN-lysosome colocalising sites upon Golgi disruption suggested a possible exchange of ATP7B directly between the TGN and its proximal lysosomes. Manipulating lysosomal positioning significantly alters the localisation of ATP7B in the cell. Contrary to previous understanding, we found that upon copper chelation in a copper-replete hepatocyte, ATP7B is not retrieved back to TGN from peripheral lysosomes; rather, ATP7B recycles to these TGN-proximal lysosomes to initiate the next cycle of copper transport. We report a hitherto unknown copper-independent lysosomal localisation of ATP7B and the importance of TGN-proximal lysosomes but not TGN as the terminal acceptor organelle of ATP7B in its retrograde pathway.

Golgi retention and oncogenic KIT signaling via PLCγ2-PKD2-PI4KIIIß activation in gastrointestinal stromal tumor cells.

Most gastrointestinal stromal tumors (GISTs) develop due to gain-of-function mutations in the tyrosine kinase gene, KIT. We recently showed that mutant KIT mislocalizes to the Golgi area and initiates uncontrolled signaling. However, the molecular mechanisms underlying its Golgi retention remain unknown. Here, we show that protein kinase D2 (PKD2) is activated by the mutant, which causes Golgi retention of KIT. In PKD2-inhibited cells, KIT migrates from the Golgi region to lysosomes and subsequently undergoes degradation. Importantly, delocalized KIT cannot trigger downstream activation. In the Golgi/trans-Golgi network (TGN), KIT activates the PKD2-phosphatidylinositol 4-kinase IIIß (PKD2-PI4KIIIß) pathway through phospholipase Cγ2 (PLCγ2) to generate a PI4P-rich membrane domain, where the AP1-GGA1 complex is aberrantly recruited. Disruption of any factors in this cascade results in the release of KIT from the Golgi/TGN. Our findings show the molecular mechanisms underlying KIT mislocalization and provide evidence for a strategy for inhibition of oncogenic signaling.

TGN020 application against aquaporin 4 improved multiple sclerosis by inhibiting astrocytes, microglia, and NLRP3 inflammasome in a cuprizone mouse model.

In multiple sclerosis (MS), activation of the astrocytes and microglia induces a cascading inflammatory response. Overexpression of the aquaporin 4 (AQP4) in the glia is a trigger for this reaction. This study aimed to block AQP4 by injecting TGN020 to alleviate the symptoms of MS. Total of 30 male mice were randomly divided into control (intact), cuprizone model of MS (fed with 0.2% cuprizone for 35 days), and TGN020-treated (received daily intraperitoneal injections of 200 mg/kg TGN020 with cuprizone intake) groups. Astrogliosis, M1-M2 microglia polarization, NLRP3 inflammasome activation, and demyelination were investigated in the corpus callosum by immunohistochemistry, real-time PCR, western blot, and luxol fast blue staining. The Rotarod test was performed for a behavior assessment. AQP4 inhibition caused a significant decrease in the expression of the astrocyte-specific marker, GFAP. It also changed the microglia polarization from M1 to M2 indicated by a significant downregulation of iNOS, CD86, MHC-ІІ, and upregulation of arginase1, CD206, and TREM-2. In addition, western blot data showed a significant decrease in the NLRP3, caspase1, and IL-1b proteins in the treatment group, which indicated inflammasome inactivation. The molecular changes following the TGN020 injection resulted in remyelination and motor recovery enhancement in the treatment group. In conclusion, the results draw the attention to the role of AQP4 in the cuprizone model of MS.

The yeast endocytic early/sorting compartment exists as an independent sub-compartment within the trans-Golgi network.

Although budding yeast has been extensively used as a model organism for studying organelle functions and intracellular vesicle trafficking, whether it possesses an independent endocytic early/sorting compartment that sorts endocytic cargos to the endo-lysosomal pathway or the recycling pathway has long been unclear. The structure and properties of the endocytic early/sorting compartment differ significantly between organisms; in plant cells, the trans-Golgi network (TGN) serves this role, whereas in mammalian cells a separate intracellular structure performs this function. The yeast syntaxin homolog Tlg2p, widely localizing to the TGN and endosomal compartments, is presumed to act as a Q-SNARE for endocytic vesicles, but which compartment is the direct target for endocytic vesicles remained unanswered. Here we demonstrate by high-speed and high-resolution 4D imaging of fluorescently labeled endocytic cargos that the Tlg2p-residing compartment within the TGN functions as the early/sorting compartment. After arriving here, endocytic cargos are recycled to the plasma membrane or transported to the yeast Rab5-residing endosomal compartment through the pathway requiring the clathrin adaptors GGAs. Interestingly, Gga2p predominantly localizes at the Tlg2p-residing compartment, and the deletion of GGAs has little effect on another TGN region where Sec7p is present but suppresses dynamics of the Tlg2-residing early/sorting compartment, indicating that the Tlg2p- and Sec7p-residing regions are discrete entities in the mutant. Thus, the Tlg2p-residing region seems to serve as an early/sorting compartment and function independently of the Sec7p-residing region within the TGN.

Distinct functional domains of the epsin-related Ent5p, a cargo adaptor for the SNARE Tlg2p in transport between endosomes and Golgi.

The epsin-related adaptor proteins Ent3p and Ent5p participate in budding of clathrin coated vesicles in transport between trans-Golgi network and endosomes in yeast. Transport of the arginine permease Can1p was analyzed, which recycles between plasma membrane and endosomes and can be targeted to the vacuole for degradation. ent3∆ cells accumulate Can1p-GFP in endosomes. Can1p-GFP is transported faster to the vacuole upon induction of degradation in ent5∆ cells than in wild type cells. The C-terminal domain of Ent5p was sufficient to restore recycling of the secretory SNARE GFP-Snc1p between plasma membrane and TGN in ent3∆ ent5∆ cells. The SNARE Tlg2p was identified as interaction partner of the Ent5p ENTH domain by in vitro binding assays and the interaction site on Ent5p was mapped. Tlg2p functions in transport from early endosomes to the trans-Golgi network and in homotypic fusion of these organelles. Tlg2p is partially shifted to denser fractions in sucrose density gradients of organelles from ent5∆ cells while distribution of Kex2p is unaffected demonstrating that Ent5p acts as cargo adaptor for Tlg2p in vivo. Taken together we show that Ent3p and Ent5p have different roles in transport and function as cargo adaptors for distinct SNAREs.

Protein sorting from endosomes to the TGN.

Retrograde transport from endosomes to the trans-Golgi network is essential for recycling of protein and lipid cargoes to counterbalance anterograde membrane traffic. Protein cargo subjected to retrograde traffic include lysosomal acid-hydrolase receptors, SNARE proteins, processing enzymes, nutrient transporters, a variety of other transmembrane proteins, and some extracellular non-host proteins such as viral, plant, and bacterial toxins. Efficient delivery of these protein cargo molecules depends on sorting machineries selectively recognizing and concentrating them for their directed retrograde transport from endosomal compartments. In this review, we outline the different retrograde transport pathways governed by various sorting machineries involved in endosome-to-TGN transport. In addition, we discuss how this transport route can be analyzed experimentally.

Live Cell Imaging of Yeast Golgi Dynamics.

Fluorescence imaging of live cells allows for the observation of dynamic processes inside cells in real time. Here we describe a strategy to image clathrin-coated vesicle dynamics in a single focal plane at the trans-Golgi network of the yeast Saccharomyces cerevisiae. This method can be readily adapted for live cell imaging of a diverse set of dynamic processes within cells.

Cab45 deficiency leads to the mistargeting of progranulin and prosaposin and aberrant lysosomal positioning.

The trans-Golgi Network (TGN) sorts molecular "addresses" and sends newly synthesized proteins to their destination via vesicular transport carriers. Despite the functional significance of packaging processes at the TGN, the sorting of soluble proteins remains poorly understood. Recent research has shown that the Golgi resident protein Cab45 is a significant regulator of secretory cargo sorting at the TGN. Cab45 oligomerizes upon transient Ca2+ influx, recruits soluble cargo molecules (clients), and packs them in sphingomyelin-rich transport carriers. However, the identity of client molecules packed into Cab45 vesicles is scarce. Therefore, we used a precise and highly efficient secretome analysis technology called hiSPECs. Intriguingly, we observed that Cab45 deficient cells manifest hypersecretion of lysosomal hydrolases. Specifically, Cab45 deficient cells secrete the unprocessed precursors of prosaposin (PSAP) and progranulin (PGRN). In addition, lysosomes in these cells show an aberrant perinuclear accumulation suggesting a new role of Cab45 in lysosomal positioning. This work uncovers a yet unknown function of Cab45 in regulating lysosomal function.

Plant elicitor peptide induces endocytosis of plasma membrane proteins in Arabidopsis.

In plants, the regulation of plasma membrane (PM) dynamics through endocytosis plays a crucial role in responding to external environmental cues and defending against pathogens. The Arabidopsis plant elicitor peptides (Peps), originating from precursor proteins called PROPEPs, have been implicated in various aspects of plant immunity. This study delves into the signaling pathway of Peps, particularly Pep1, and its effect on PM protein internalization. Using PIN2 and BRI1 as PM markers, we demonstrated that Pep1 stimulates the endocytosis of these PM-localized proteins through clathrin-mediated endocytosis (CME). CLC2 and CLC3, two light chains of clathrin, are vital for Pep1-induced PIN2-GFP and BRI1-GFP internalization.The internalized PIN2 and BRI1 are subsequently transported to the vacuole via the trans-Golgi network/early endosome (TGN/EE) and prevacuolar compartment (PVC) pathways. Intriguingly, salicylic acid (SA) negatively regulates the effect of Pep1 on PM endocytosis. This study sheds light on a previously unknown signaling pathway by which danger peptides like Pep1 influence PM dynamics, contributing to a deeper understanding of the function of plant elicitor peptide.