Role of the dynamin-related protein 2 family and SH3P2 in clathrin-mediated endocytosis in Arabidopsis thaliana.

Clathrin-mediated endocytosis (CME) is vital for the regulation of plant growth and development through controlling plasma membrane protein composition and cargo uptake. CME relies on the precise recruitment of regulators for vesicle maturation and release. Homologues of components of mammalian vesicle scission are strong candidates to be part of the scission machinery in plants, but the precise roles of these proteins in this process are not fully understood. Here, we characterised the roles of the plant dynamin-related protein 2 (DRP2) family (hereafter DRP2s) and SH3-domain containing protein 2 (SH3P2), the plant homologue to recruiters of dynamins, such as endophilin and amphiphysin, in CME by combining high-resolution imaging of endocytic events in vivo and characterisation of the purified proteins in vitro. Although DRP2s and SH3P2 arrive similarly late during CME and physically interact, genetic analysis of the sh3p123 triple mutant and complementation assays with non-SH3P2-interacting DRP2 variants suggest that SH3P2 does not directly recruit DRP2s to the site of endocytosis. These observations imply that, despite the presence of many well-conserved endocytic components, plants have acquired a distinct mechanism for CME.

Chitosan/dsRNA polyplex nanoparticles advance environmental RNA interference efficiency through activating clathrin-dependent endocytosis.

Chitosan, as a promising gene nanocarrier for enhancing RNA interference (RNAi) efficiency, displays tremendous application prospects in addressing dsRNA delivery concerns. However, the molecular mechanism of chitosan/dsRNA polyplex nanoparticles (PNs) for advancing dsRNA delivery efficiency remains largely unknown. Here, chitosan/dsRNA PNs were prepared by an electrostatic attraction method. The results showed that the chitosan/dsRNA PNs significantly advance stability, and cellular uptake efficiency of dsRNA, and RNAi efficiency. RNA-Seq and qPCR assays further revealed that chitosan/dsRNA PNs upregulated the key clathrin heavy chain (CHC) gene for activating clathrin-dependent endocytosis (CDE) pathway. Additionally, inhibition of CDE hindered the robust RNAi responses of chitosan/dsRNA PNs using an inhibitor (chlorpromazine) and an RNAi-of-RNAi strategy. Ultimately, microscale thermophoresis assay confirmed that chitosan/dsRNA PNs directly bound to CHC protein, which was a core component in CDE, to advance RNAi efficiency. To our knowledge, our findings firstly illuminate the molecular mechanism how chitosan nanoparticles-based RNAi deliver dsRNA for enhancing RNAi efficiency. Above mechanism will advance the extensive utilization of nanocarrier-based RNAi in pest management and gene delivery.

An Alternative Mechanism of Subcellular Iron Uptake Deficiency in Cardiomyocytes.

BACKGROUND: Systemic defects in intestinal iron absorption, circulation, and retention cause iron deficiency in 50% of patients with heart failure. Defective subcellular iron uptake mechanisms that are independent of systemic absorption are incompletely understood. The main intracellular route for iron uptake in cardiomyocytes is clathrin-mediated endocytosis. METHODS: We investigated subcellular iron uptake mechanisms in patient-derived and CRISPR/Cas-edited induced pluripotent stem cell-derived cardiomyocytes as well as patient-derived heart tissue. We used an integrated platform of DIA-MA (mass spectrometry data-independent acquisition)-based proteomics and signaling pathway interrogation. We employed a genetic induced pluripotent stem cell model of 2 inherited mutations (TnT [troponin T]-R141W and TPM1 [tropomyosin 1]-L185F) that lead to dilated cardiomyopathy (DCM), a frequent cause of heart failure, to study the underlying molecular dysfunctions of DCM mutations. RESULTS: We identified a druggable molecular pathomechanism of impaired subcellular iron deficiency that is independent of systemic iron metabolism. Clathrin-mediated endocytosis defects as well as impaired endosome distribution and cargo transfer were identified as a basis for subcellular iron deficiency in DCM-induced pluripotent stem cell-derived cardiomyocytes. The clathrin-mediated endocytosis defects were also confirmed in the hearts of patients with DCM with end-stage heart failure. Correction of the TPM1-L185F mutation in DCM patient-derived induced pluripotent stem cells, treatment with a peptide, Rho activator II, or iron supplementation rescued the molecular disease pathway and recovered contractility. Phenocopying the effects of the TPM1-L185F mutation into WT induced pluripotent stem cell-derived cardiomyocytes could be ameliorated by iron supplementation. CONCLUSIONS: Our findings suggest that impaired endocytosis and cargo transport resulting in subcellular iron deficiency could be a relevant pathomechanism for patients with DCM carrying inherited mutations. Insight into this molecular mechanism may contribute to the development of treatment strategies and risk management in heart failure.

Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5ß1.

Reticular adhesions (RAs) consist of integrin αvß5 and harbor flat clathrin lattices (FCLs), long-lasting structures with similar molecular composition as clathrin-mediated endocytosis (CME) carriers. Why FCLs and RAs colocalize is not known. Here, we show that RAs are assembled at FCLs in a process controlled by fibronectin (FN) and its receptor, integrin α5ß1. We observed that cells on FN-rich matrices displayed fewer FCLs and RAs. CME machinery inhibition abolished RAs and live-cell imaging showed that RA establishment requires FCL coassembly. The inhibitory activity of FN was mediated by the activation of integrin α5ß1 at Tensin1-positive fibrillar adhesions. Conventionally, endocytosis disassembles cellular adhesions by internalizing their components. Our results present a novel paradigm in the relationship between these two processes by showing that endocytic proteins can actively function in the assembly of cell adhesions. Furthermore, we show this novel adhesion assembly mechanism is coupled to cell migration via unique crosstalk between cell-matrix adhesions.

Conserved NIMA kinases regulate multiple steps of endocytic trafficking.

Human NIMA-related kinases have primarily been studied for their roles in cell cycle progression (NEK1/2/6/7/9), checkpoint-DNA-damage control (NEK1/2/4/5/10/11), and ciliogenesis (NEK1/4/8). We previously showed that Caenorhabditis elegans NEKL-2 (NEK8/9 homolog) and NEKL-3 (NEK6/7 homolog) regulate apical clathrin-mediated endocytosis (CME) in the worm epidermis and are essential for molting. Here we show that NEKL-2 and NEKL-3 also have distinct roles in controlling endosome function and morphology. Specifically, loss of NEKL-2 led to enlarged early endosomes with long tubular extensions but showed minimal effects on other compartments. In contrast, NEKL-3 depletion caused pronounced defects in early, late, and recycling endosomes. Consistently, NEKL-2 was strongly localized to early endosomes, whereas NEKL-3 was localized to multiple endosomal compartments. Loss of NEKLs also led to variable defects in the recycling of two resident cargoes of the trans-Golgi network (TGN), MIG-14/Wntless and TGN-38/TGN38, which were missorted to lysosomes after NEKL depletion. In addition, defects were observed in the uptake of clathrin-dependent (SMA-6/Type I BMP receptor) and independent cargoes (DAF-4/Type II BMP receptor) from the basolateral surface of epidermal cells after NEKL-2 or NEKL-3 depletion. Complementary studies in human cell lines further showed that siRNA knockdown of the NEKL-3 orthologs NEK6 and NEK7 led to missorting of the mannose 6-phosphate receptor from endosomes. Moreover, in multiple human cell types, depletion of NEK6 or NEK7 disrupted both early and recycling endosomal compartments, including the presence of excess tubulation within recycling endosomes, a defect also observed after NEKL-3 depletion in worms. Thus, NIMA family kinases carry out multiple functions during endocytosis in both worms and humans, consistent with our previous observation that human NEKL-3 orthologs can rescue molting and trafficking defects in C. elegans nekl-3 mutants. Our findings suggest that trafficking defects could underlie some of the proposed roles for NEK kinases in human disease.

Clathrin-dependent endocytosis plays a critical role in larval and pupal development, and female oocyte production in the red flour beetle (Tribolium castaneum).

BACKGROUND: Clathrin-dependent endocytosis is a vesicular transport process by which cells take macromolecules from the extracellular space to the intracellular space. It plays important roles in various cellular functions, but its biological significance in insect development and reproduction has not been well studied. RESULTS: We characterized and functionally analyzed four major clathrin-dependent endocytic pathway genes (TcChc, TcAP50, TcVhaSFD, TcRab7) in Tribolium castaneum. RNA interference (RNAi) by injecting double-stranded RNA (dsRNA) targeting each gene at three doses (50, 100, or 200 ng per insect) in 20-day-old larvae led to 100% larval mortality. When the expressions of TcChc, TcVhaSFD, and TcRab7 were suppressed by injecting their respective dsRNAs at each dose in 1-day-old pupae, the adults that emerged from the dsRNA-injected pupae were deformed, with the absence of wing development. The deformed adults died within 2 days after eclosion. When the expression of TcAP50 was suppressed by injecting its dsRNA into 1-day-old pupae, although no apparent deformed adults were observed, all the adults died within 35 days after eclosion. In addition, when the expressions of TcChc and TcVhaSFD were suppressed by injecting their respective dsRNAs at a reduced dose (10 ng per insect) in 5-day-old pupae, the ovarian development and oocyte production in the resultant females were completely inhibited. CONCLUSION: Our results indicate that clathrin-dependent endocytosis is essential for insect development and reproduction. The results from this study can help researchers identify potential molecular targets for developing novel strategies for insect pest management. © 2023 Society of Chemical Industry.

A universal method to analyze cellular internalization mechanisms via endocytosis without non-specific cross-effects.

Endocytosis is an essential biological process for nutrient absorption and intercellular communication; it can also be used to accelerate the cellular internalization of drug delivery carriers. Clarifying the cellular uptake mechanisms of unidentified endogenous and exogenous molecules and designing new effective drug delivery systems require an accurate, specific endocytosis analysis methodology. Therefore, we developed a method to specifically evaluate cellular internalization via three main endocytic pathways: clathrin- and caveolae-mediated endocytosis, and macropinocytosis. We first revealed that most known endocytosis inhibitors had no specific inhibitory effect or were cytotoxic. Second, we successfully established an alternative method using small interfering RNA to knock down dynamin-2 and caveolin-1, which are necessary for clathrin- and caveolae-mediated endocytosis, in HeLa cells. Third, we established another method to specifically analyze macropinocytosis using rottlerin on A431 cells. Finally, we validated the proposed methods by testing the cellular internalization of a biological molecule (insulin) and carriers (nanoparticles and cell-penetrating peptides). Through this study, we established versatile methods to precisely and specifically evaluate endocytosis of newly developed biopharmaceuticals or drug delivery systems.

The endocytic TPLATE complex internalizes ubiquitinated plasma membrane cargo.

Endocytosis controls the perception of stimuli by modulating protein abundance at the plasma membrane. In plants, clathrin-mediated endocytosis is the most prominent internalization pathway and relies on two multimeric adaptor complexes, the AP-2 and the TPLATE complex (TPC). Ubiquitination is a well-established modification triggering endocytosis of cargo proteins, but how this modification is recognized to initiate the endocytic event remains elusive. Here we show that TASH3, one of the large subunits of TPC, recognizes ubiquitinated cargo at the plasma membrane via its SH3 domain-containing appendage. TASH3 lacking this evolutionary specific appendage modification allows TPC formation but the plants show severely reduced endocytic densities, which correlates with reduced endocytic flux. Moreover, comparative plasma membrane proteomics identified differential accumulation of multiple ubiquitinated cargo proteins for which we confirm altered trafficking. Our findings position TPC as a key player for ubiquitinated cargo internalization, allowing future identification of target proteins under specific stress conditions.

Transcriptomic-Guided Phosphonate Utilization Analysis Unveils Evidence of Clathrin-Mediated Endocytosis and Phospholipid Synthesis in the Model Diatom, Phaeodactylum tricornutum.

Phosphonates are important components of marine organic phosphorus, but their bioavailability and catabolism by eukaryotic phytoplankton remain enigmatic. Here, diatom Phaeodactylum tricornutum was used to investigate the bioavailability of phosphonates and describe the underlying molecular mechanism. The results showed that 2-aminoethylphosphonic acid (2-AEP) can be utilized as an alternative phosphorus source. Comparative transcriptomics revealed that the utilization of 2-AEP comprised 2 steps, including molecular uptake through clathrin-mediated endocytosis and incorporation into the membrane phospholipids in the form of diacylglyceryl-2-AEP (DAG-2-AEP). In the global ocean, we found the prevalence and dynamic expression pattern of key genes that are responsible for vesicle formation (CLTC, AP-2) and DAG-AEP synthesis (PCYT2, EPT1) in diatom assemblages. This study elucidates a distinctive mechanism of phosphonate utilization by diatoms, and discusses the ecological implications. IMPORTANCE Phosphonates contribute ~25% of total dissolved organic phosphorus in the ocean, and are found to be important for marine phosphorus biogeochemical cycle. As a type of biogenic phosphonate produced by microorganisms, 2-aminoethylphosphonic acid (2-AEP) widely exists in the ocean. It is well known that 2-AEP can be cleaved and utilized by prokaryotes, but its ability to support the growth of eukaryotic phytoplankton remains unclear. Our research identified the bioavailability of 2-AEP for the diatom Phaeodactylum tricornutum, and proposed a distinctive metabolic pathway of 2-AEP utilization. Different from the enzymatic hydrolysis of phosphonates, the results suggested that P. tricornutum utilizes 2-AEP by incorporating it into phospholipid instead of cleaving the C-P bond. Moreover, the ubiquitous distribution of associated representative gene transcripts in the environmental assemblages and the higher gene transcript abundance in the cold regions were observed, which suggests the possible environmental adaption of 2-AEP utilization by diatoms.

Cationic lipopolymeric nanoplexes containing the CRISPR/Cas9 ribonucleoprotein for genome surgery.

sgRNA/Cas9 ribonucleoproteins (RNPs) provide a site-specific robust gene-editing approach avoiding the mutagenesis and unwanted off-target effects. However, the high molecular weight (∼165 kDa), hydrophilicity and net supranegative charge (∼-20 mV) hinder the intracellular delivery of these RNPs. In the present study, we have prepared cationic RNPs lipopolymeric nanoplexes that showed a size of 117.3 ± 7.64 nm with +6.17 ± 1.04 mV zeta potential and >90% entrapment efficiency of RNPs. Further, these RNPs lipopolymeric nanoplexes showed good complexation efficiency and were found to be stable for 12 h with fetal bovine serum. These RNPs lipopolymeric nanoplexes did not induce any significant cytotoxicity in HEK293T cells, and were efficiently uptaken via a clathrin-mediated pathway with optimal transfection efficiency and nuclear localization after 48 h. Further, HEK293T cells having the mGFP insert were used as a cell line model for gene editing, wherein the loss of the mGFP signal was observed as a function of gene editing after transfection with mGFP targeting RNPs lipopolymeric nanoplexes. Further, the T7 endonuclease and TIDE assay data showed a decent gene editing efficiency. Additionally, the lipopolymeric nanoplexes were able to transfect muscle cells in vivo, when injected intra-muscularly. Collectively, this study explored the potential of cationic lipopolymeric nanoplexes for delivering gene-editing endonucleases.

Eps15/Pan1p is a master regulator of the late stages of the endocytic pathway.

Endocytosis is a multistep process involving the sequential recruitment and action of numerous proteins. This process can be divided into two phases: an early phase, in which sites of endocytosis are formed, and a late phase in which clathrin-coated vesicles are formed and internalized into the cytosol, but how these phases link to each other remains unclear. In this study, we demonstrate that anchoring the yeast Eps15-like protein Pan1p to the peroxisome triggers most of the events occurring during the late phase at the peroxisome. At this ectopic location, Pan1p recruits most proteins that function in the late phases-including actin nucleation promoting factors-and then initiates actin polymerization. Pan1p also recruited Prk1 kinase and actin depolymerizing factors, thereby triggering disassembly immediately after actin assembly and inducing dissociation of endocytic proteins from the peroxisome. These observations suggest that Pan1p is a key regulator for initiating, processing, and completing the late phase of endocytosis.

Ubiquitin-specific protease TRE17/USP6 promotes tumor cell invasion through the regulation of glycoprotein CD147 intracellular trafficking.

Disordered expression and distribution of plasma membrane proteins at the cell surface leads to diverse malignant phenotypes in tumors, including cell invasion. The ubiquitin-specific protease TRE17/USP6, an oncogene identified in Ewing sarcoma, is highly expressed in several cancers and locally aggressive tumor-like lesions. We have previously demonstrated that TRE17 regulates the trafficking of plasma membrane proteins that enter cells via clathrin-independent endocytosis (CIE); TRE17 prevents CIE cargo proteins from being targeted to lysosomes for degradation by deubiquitylating them. However, functional insights into the effects of TRE17-mediated CIE cargo trafficking on cell invasion remain unknown. Here, we show that increased expression of TRE17 enhances invasiveness of the human sarcoma cell line HT-1080 by elevating the cell surface levels of the membrane glycoprotein CD147, which plays a central role in tumor progression. We demonstrate overexpression of TRE17 decreases ubiquitylated CD147, which is accompanied by suppression of CD147 transport to lysosomes, resulting in the stabilization and increase of cell surface-localized CD147. On the other hand, we show knockdown of TRE17 decreases cell surface CD147, which is coupled with reduced production of matrix metalloproteinases, the enzymes responsible for extracellular matrix degradation. Furthermore, we demonstrate that inhibition of CD147 by a specific inhibitor alleviated the TRE17-promoted tumor cell invasion. We therefore propose a model for the pathogenesis of TRE17-driven tumors in which TRE17 increases CD147 at the cell surface by preventing its lysosomal degradation, which in turn enhances matrix metalloproteinase synthesis and matrix degradation, thereby promoting tumor cell invasion.

Low complexity RGG-motif containing proteins Scd6 and Psp2 act as suppressors of clathrin heavy chain deficiency.

Clathrin, made up of the heavy- and light-chains, constitutes one of the most abundant proteins involved in intracellular protein trafficking and endocytosis. YPR129W, which encodes RGG-motif containing translation repressor was identified as a part of the multi-gene construct (SCD6) that suppressed clathrin deficiency. However, the contribution of YPR129W alone in suppressing clathrin deficiency has not been documented. This study identifies YPR129W as a necessary and sufficient gene in a multi-gene construct SCD6 that suppresses clathrin deficiency. Importantly, we also identify cytoplasmic RGG-motif protein encoding gene PSP2 as another novel suppressor of clathrin deficiency. Detailed domain analysis of the two suppressors reveals that the RGG-motif of both Scd6 and Psp2 is important for suppressing clathrin deficiency. Interestingly, the endocytosis function of clathrin heavy chain assayed by internalization of GFP-Snc1 and α-factor secretion activity are not complemented by either Scd6 or Psp2. We further observe that inhibition of TORC1 compromises the suppression activity of both SCD6 and PSP2 to different extent, suggesting that two suppressors are differentially regulated. Scd6 granules increased based on its RGG-motif upon Chc1 depletion. Strikingly, Psp2 overexpression increased the abundance of ubiquitin-conjugated proteins in Chc1 depleted cells in its RGG-motif dependent manner and also decreased the accumulation of GFP-Atg8 foci. Overall based on our results using SCD6 and PSP2, we identify a novel role of RGG-motif containing proteins in suppressing clathrin deficiency. Since both the suppressors are RNA-binding proteins, this study opens an exciting avenue for exploring the connection between clathrin function and post-transcriptional gene control processes.

AP1/2ß-mediated exocytosis of tapetum-specific transporters is required for pollen development in Arabidopsis thaliana.

AP-1 and AP-2 adaptor protein (AP) complexes mediate clathrin-dependent trafficking at the trans-Golgi network (TGN) and the plasma membrane, respectively. Whereas AP-1 is required for trafficking to plasma membrane and vacuoles, AP-2 mediates endocytosis. These AP complexes consist of four subunits (adaptins): two large subunits (ß1 and γ for AP-1 and ß2 and α for AP-2), a medium subunit µ, and a small subunit σ. In general, adaptins are unique to each AP complex, with the exception of ß subunits that are shared by AP-1 and AP-2 in some invertebrates. Here, we show that the two putative Arabidopsis thaliana AP1/2ß adaptins co-assemble with both AP-1 and AP-2 subunits and regulate exocytosis and endocytosis in root cells, consistent with their dual localization at the TGN and plasma membrane. Deletion of both ß adaptins is lethal in plants. We identified a critical role of ß adaptins in pollen wall formation and reproduction, involving the regulation of membrane trafficking in the tapetum and pollen germination. In tapetal cells, ß adaptins localize almost exclusively to the TGN and mediate exocytosis of the plasma membrane transporters such as ATP-binding cassette (ABC)G9 and ABCG16. This study highlights the essential role of AP1/2ß adaptins in plants and their specialized roles in specific cell types.

Overexpression of Ribosomal Protein S6 Kinase A4 (RPS6KA4) Predicts a Poor Prognosis in Hepatocellular Carcinoma Patients: A Study Based on TCGA Samples.

AIM: This study aims to comprehensively analyse the Ribosomal Protein S6 Kinase A4 (RPS6KA4) and determine the prognostic value for hepatocellular carcinoma (HCC). BACKGROUND: Liver cancer is a common type of tumor worldwide, and HCC accounts for about 75 to 85% of all primary liver cancer cases. The Ribosomal S6 protein kinases (RSK) family plays an important regulatory role in cell growth, movement, survival, and proliferation. METHODS: We collected the expression and clinicopathological features of RPS6KA4 in The Cancer Genome Atlas (TCGA) cohort and evaluated the prognostic value of RPS6KA4 in HCC. Gene Ontology (GO)/ Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) were performed to determine the enrichment pathways of RPS6KA4. Correlation between RPS6KA4 expression and immune infiltration was analyzed. Protein-protein interaction (PPI) network analysis was performed to screen hub genes. RESULTS: RPS6KA4 overexpression is statistically significant in HCC relative to normal tissues (P < 0.001). Increased expression of RPS6KA4 is associated with higher T stage (p=0.021), pathological stage (p=0.006), α-fetoprotein (AFP) value (p=0.026), and vascular invasion (p=0.023) of HCC. Overexpression of RPS6KA4 predicted worse overall survival (OS, P=0.002), disease-specific survival (DSS, P=0.012), and progress-free interval (PFI, P=0.031) for HCC. Univariate/multivariate Cox regression analysis confirmed that RPS6KA4 was an independent risk factor for HCC (P=0.002 in univariate analysis; P=0.014 in multivariate analysis). GO/KEGG analysis and GSEA analysis suggest that RPS6KA4 plays a precancer role in HCC through epigenetics, cell adhesion, tumor-driven GTPase pathways, infection-related carcinogenesis, and adaptive immunity. Immune infiltration analysis confirmed the strong negative relationship between RPS6KA4 and B cells, CD4+ T cells, macrophages, neutrophils, as well as dendritic cells. Protein-protein interactions (PPI) analysis and hub gene identification revealed the cancer-promoting effects of RPS6KA4 related to RSKs, AP-2, clathrin, and MAPK/ ERK pathways. CONCLUSION: RPS6KA4 is a potentially valuable molecule for understanding HCC tumorigenesis. Increased RPS6KA4 might be a promising prognostic factor for low HCC survival.

Structural similarity-based prediction of host factors associated with SARS-CoV-2 infection and pathogenesis.

The current pandemic resulted from SARS-CoV-2 still remains as the major public health concern globally. The precise mechanism of viral pathogenesis is not fully understood, which remains a major hurdle for medical intervention. Here we generated an interactome profile of protein-protein interactions based on host and viral protein structural similarities information. Further computational biological study combined with Gene enrichment analysis predicted key enriched pathways associated with viral pathogenesis. The results show that axon guidance, membrane trafficking, vesicle-mediated transport, apoptosis, clathrin-mediated endocytosis, Vpu mediated degradation of CD4 T cell, and interferon-gamma signaling are key events associated in SARS-CoV-2 life cycle. Further, degree centrality analysis reveals that IRF1/9/7, TP53, and CASP3, UBA52, and UBC are vital proteins for IFN-γ-mediated signaling, apoptosis, and proteasomal degradation of CD4, respectively. We crafted chronological events of the virus life cycle. The SARS-CoV-2 enters through clathrin-mediated endocytosis, and the genome is trafficked to the early endosomes in a RAB5-dependent manner. It is predicted to replicate in a double-membrane vesicle (DMV) composed of the endoplasmic reticulum, autophagosome, and ERAD machinery. The SARS-CoV-2 down-regulates host translational machinery by interacting with protein kinase R, PKR-like endoplasmic reticulum kinase, and heme-regulated inhibitor and can phosphorylate eIF2a. The virion assembly occurs in the ER-Golgi intermediate compartment (ERGIC) organized by the spike and matrix protein. Collectively, we have established a spatial link between viral entry, RNA synthesis, assembly, pathogenesis, and their associated diverse host factors, those could pave the way for therapeutic intervention.

Boron uptake in rice is regulated post-translationally via a clathrin-independent pathway.

Uptake of boron (B) in rice (Oryza sativa) is mediated by the Low silicon rice 1 (OsLsi1) channel, belonging to the NOD26-like intrinsic protein III subgroup, and the efflux transporter B transporter 1 (OsBOR1). However, it is unknown how these transporters cooperate for B uptake and how they are regulated in response to B fluctuations. Here, we examined the response of these two transporters to environmental B changes at the transcriptional and posttranslational level. OsBOR1 showed polar localization at the proximal side of both the exodermis and endodermis of mature root region, forming an efficient uptake system with OsLsi1 polarly localized at the distal side of the same cell layers. Expression of OsBOR1 and OsLsi1 was unaffected by B deficiency and excess. However, although OsLsi1 protein did not respond to high B at the protein level, OsBOR1 was degraded in response to high B within hours, which was accompanied with a significant decrease of total B uptake. The high B-induced degradation of OsBOR1 was inhibited in the presence of MG-132, a proteasome inhibitor, without disturbance of the polar localization. In contrast, neither the high B-induced degradation of OsBOR1 nor its polarity was affected by induced expression of dominant-negative mutated dynamin-related protein 1A (OsDRP1AK47A) or knockout of the mu subunit (AP2M) of adaptor protein-2 complex, suggesting that clathrin-mediated endocytosis is not involved in OsBOR1 degradation and polar localization. These results indicate that, in contrast to Arabidopsis thaliana, rice has a distinct regulatory mechanism for B uptake through clathrin-independent degradation of OsBOR1 in response to high B.

Both Clathrin-Mediated and Membrane Microdomain-Associated Endocytosis Contribute to the Cellular Adaptation to Hyperosmotic Stress in Arabidopsis.

As sessile organisms, plants must directly deal with an often complex and adverse environment in which hyperosmotic stress is one of the most serious abiotic factors, challenging cellular physiology and integrity. The plasma membrane (PM) is the hydrophobic barrier between the inside and outside environments of cells and is considered a central compartment in cellular adaptation to diverse stress conditions through dynamic PM remodeling. Endocytosis is a powerful method for rapid remodeling of the PM. In animal cells, different endocytic pathways are activated in response to osmotic stress, while only a few reports are related to the endocytosis response pathway and involve a mechanism in plant cells upon hyperosmotic stress. In this study, using different endocytosis inhibitors, the microdomain-specific dye di-4-ANEPPDHQ, variable-angle total internal reflection fluorescence microscopy (VA-TIRFM), and confocal microscopy, we discovered that internalized Clathrin Light Chain-Green Fluorescent Protein (CLC-GFP) increased under hyperosmotic conditions, accompanied by decreased fluorescence intensity of CLC-GFP at the PM. CLC-GFP tended to have higher diffusion coefficients and a fraction of CLC-GFP molecules underwent slower diffusion upon hyperosmotic stress. Meanwhile, an increased motion range of CLC-GFP was found under hyperosmotic treatment compared with the control. In addition, the order of the PM decreased, but the order of the endosome increased when cells were in hyperosmotic conditions. Hence, our results demonstrated that clathrin-mediated endocytosis and membrane microdomain-associated endocytosis both participate in the adaptation to hyperosmotic stress. These findings will help to further understand the role and the regulatory mechanism involved in plant endocytosis in helping plants adapt to osmotic stress.