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.

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.

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.

Tongluo Huatan capsule improves cognitive function by regulating the endocytosis of N-methyl-D-aspartic acid receptors mediated by clathrin in a rat model of vascular dementia.

OBJECTIVE: To explore the neuroprotective mechanisms of Tongluo Huatan capsule (THC) in a rat model of vascular dementia (VD). METHODS: A rat model of VD was established by repeated clamping of bilateral common carotid arteries with the intraperitoneal injection of sodium nitroprusside solution. VD rats were administered THC, memantine hydrochloride, or distilled water daily for 14 d after operation. Learning and memory abilities were assessed using the step-down passive avoidance test, novel object recognition (NOR) test, and Morris water maze (MWM) test. Pathological changes in the hippocampus were observed through hematoxylin and eosin and Nissl staining. The expression levels of clathrin, RAB5B, and N-methyl-D-aspartic acid receptor 1 (NMDAR1) were measured by immunohistochemistry staining, real-time quantitative polymerase chain reaction and Western blot. RESULTS: Rats in VD group showed impaired learning and memory abilities (step-down passive avoidance, NOR, and MWM) and abnormalities in neuronal morphology (light microscopy) in the hippocampus. The mRNA or protein expression levels of clathrin and RAB5B were decreased, and NMDAR1 was increased in hippocampal tissues (P < 0.05). Administration of THC promoted the learning and memory abilities and the morphological structure of hippocampal neurons in VD rats. Besides, THC enhanced mRNA or protein expression levels of clathrin and RAB5B, and decreased NMDAR1 (P < 0.05). CONCLUSION: THC may improve cognitive functions by regulating the endocytosis of NMDA receptors mediated by clathrin.

Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission.

G protein-coupled receptors (GPCRs) are considered to function primarily at the plasma membrane, where they interact with extracellular ligands and couple to G proteins that transmit intracellular signals. Consequently, therapeutic drugs are designed to target GPCRs at the plasma membrane. Activated GPCRs undergo clathrin-dependent endocytosis. Whether GPCRs in endosomes control pathophysiological processes in vivo and are therapeutic targets remains uncertain. We investigated the contribution of endosomal signaling of the calcitonin receptor-like receptor (CLR) to pain transmission. Calcitonin gene-related peptide (CGRP) stimulated CLR endocytosis and activated protein kinase C (PKC) in the cytosol and extracellular signal regulated kinase (ERK) in the cytosol and nucleus. Inhibitors of clathrin and dynamin prevented CLR endocytosis and activation of cytosolic PKC and nuclear ERK, which derive from endosomal CLR. A cholestanol-conjugated antagonist, CGRP8-37, accumulated in CLR-containing endosomes and selectively inhibited CLR signaling in endosomes. CGRP caused sustained excitation of neurons in slices of rat spinal cord. Inhibitors of dynamin, ERK, and PKC suppressed persistent neuronal excitation. CGRP8-37-cholestanol, but not unconjugated CGRP8-37, prevented sustained neuronal excitation. When injected intrathecally to mice, CGRP8-37-cholestanol inhibited nociceptive responses to intraplantar injection of capsaicin, formalin, or complete Freund's adjuvant more effectively than unconjugated CGRP8-37 Our results show that CLR signals from endosomes to control pain transmission and identify CLR in endosomes as a therapeutic target for pain. Thus, GPCRs function not only at the plasma membrane but also in endosomes to control complex processes in vivo. Endosomal GPCRs are a drug target that deserve further attention.

Unconventional EGF-induced ERK1/2-mediated Kv1.3 endocytosis.

The potassium channel Kv1.3 plays roles in immunity, neuronal development and sensory discrimination. Regulation of Kv1.3 by kinase signaling has been studied. In this context, EGF binds to specific receptors (EGFR) and triggers tyrosine kinase-dependent signaling, which down-regulates Kv1.3 currents. We show that Kv1.3 undergoes EGF-dependent endocytosis. This EGF-mediated mechanism is relevant because is involved in adult neural stem cell fate determination. We demonstrated that changes in Kv1.3 subcellular distribution upon EGFR activation were due to Kv1.3 clathrin-dependent endocytosis, which targets the Kv1.3 channels to the lysosomal degradative pathway. Interestingly, our results further revealed that relevant tyrosines and other interacting motifs, such as PDZ and SH3 domains, were not involved in the EGF-dependent Kv1.3 internalization. However, a new, and yet undescribed mechanism, of ERK1/2-mediated threonine phosphorylation is crucial for the EGF-mediated Kv1.3 endocytosis. Our results demonstrate that EGF triggers the down-regulation of Kv1.3 activity and its expression at the cell surface, which is important for the development and migration of adult neural progenitors.

Buckwheat trypsin inhibitor enters Hep G2 cells by clathrin-dependent endocytosis.

Recombinant buckwheat trypsin inhibitor (rBTI) was studied to evaluate if it could enter cancer cells and to determine the mechanism. Fluorescein isothiocyanate-labelled buckwheat trypsin inhibitor (FITC-BTI) entered Hep G2 cells in a concentration-dependent manner. FITC-BTI colocalised with labelled transferrin (Tf) in the punctate structure, implying that rBTI enters Hep G2 cells by clathrin-dependent endocytosis. Incubation of Hep G2 cells with different chemical inhibitors abolished diffuse, but not punctate fluorescence, thus indicating that membrane potential plays a critical role in this process. Impairment of clathrin-mediated endocytosis by RNAi with clathrin heavy chain greatly reduced or completely abolished both diffuse and punctate fluorescence, further supporting a theory of a single route of endocytosis. Consistent with our working hypothesis, Hep G2 cells which were arrested in the M phase did not show any vesicular or diffuse FITC-BTI. We conclude from these results that both endocytosis and membrane potential are required for rBTI entry into Hep G2 cells.

Controlled elimination of clathrin heavy-chain expression in DT40 lymphocytes.

We exploited the high rate of homologous recombination shown by the chicken B cell line DT40 to inactivate the endogenous alleles for clathrin heavy chain and replace them with human clathrin complementary DNA under the control of a tetracycline-regulatable promoter. Clathrin repression perturbed the activities of Akt-mediated and mitogen-activated protein kinase-mediated signaling pathways and induced apoptosis; this finding suggests that in DT40 cells clathrin helps to maintain the integrity of antiapoptotic survival pathways. We also describe a variant cell line in which these signaling pathways were unaffected by clathrin down-regulation. This variant cell line did not undergo apoptosis in the absence of clathrin and was used to examine the effects of clathrin depletion on membrane-trafficking pathways. Receptor-mediated and fluid-phase endocytosis were both substantially inhibited, and transferrin-receptor recycling was modestly inhibited. Surprisingly, clathrin removal did not affect the morphology or biochemical composition of lysosomes.