Fluorescence imaging of lamellipodin-mediated biomolecular condensates on solid supported lipid bilayer membranes.

Biomolecular condensates play a major role in numerous cellular processes, including several that occur on the surface of lipid bilayer membranes. There is increasing evidence that cellular membrane trafficking phenomena, including the internalization of the plasma membrane through endocytosis, are mediated by multivalent protein-protein interactions that can lead to phase separation. We have recently found that proteins involved in the clathrin-independent endocytic pathway named Fast Endophilin Mediated Endocytosis can undergo liquid-liquid phase separation (LLPS) in solution and on lipid bilayer membranes. Here, the protein solution concentrations required for phase separation to be observed are significantly smaller compared to those required for phase separation in solution. LLPS is challenging to systematically characterize in cellular systems in general, and on biological membranes in particular. Model membrane approaches are more suitable for this purpose as they allow for precise control over the nature and amount of the components present in a mixture. Here we describe a method that enables the imaging of LLPS domain formation on solid supported lipid bilayers. These allow for facile imaging, provide long-term stability, and avoid clustering of vesicles and vesicle-attached features (such as buds and tethers) in the presence of multi-valent membrane interacting proteins.

Dynamin 1xA interacts with Endophilin A1 via its spliced long C-terminus for ultrafast endocytosis.

Dynamin 1 mediates fission of endocytic synaptic vesicles in the brain and has two major splice variants, Dyn1xA and Dyn1xB, which are nearly identical apart from the extended C-terminal region of Dyn1xA. Despite a similar set of binding partners, only Dyn1xA is enriched at endocytic zones and accelerates vesicle fission during ultrafast endocytosis. Here, we report that Dyn1xA achieves this localization by preferentially binding to Endophilin A1 through a newly defined binding site within its long C-terminal tail extension. Endophilin A1 binds this site at higher affinity than the previously reported site, and the affinity is determined by amino acids within the Dyn1xA tail but outside the binding site. This interaction is regulated by the phosphorylation state of two serine residues specific to the Dyn1xA variant. Dyn1xA and Endophilin A1 colocalize in patches near the active zone, and mutations disrupting Endophilin A binding to the long tail cause Dyn1xA mislocalization and stalled endocytic pits on the plasma membrane during ultrafast endocytosis. Together, these data suggest that the specificity for ultrafast endocytosis is defined by the phosphorylation-regulated interaction of Endophilin A1 with the C-terminal extension of Dyn1xA.

Autoantibodies against Endophilin A2 as a novel biomarker are beneficial to early diagnosis of breast cancer.

BACKGROUND: Due to the lack of early symptoms, breast cancer is frequently overlooked, leading to distant metastases and multi-organ lesions that directly threaten patients' lives. We have identified a novel tumor marker, antibodies to endophilin A2 (EA2), to improve early diagnosis of breast cancer. METHODS: Antibody levels of EA2 were analyzed in sera of patients with cancers of different origins and stages by indirect enzyme-linked immunosorbent assay (ELISA). Diagnostic accuracy and reference range were determined by the area under the receiver operating curve and distribution curve. The levels of EA2 antigen in sera were determined by sandwich ELISA. RESULTS: The levels of antibodies against EA2 were higher in sera of patients with breast cancer (P < 0.0001), liver cancer (P = 0.0005), gastric cancer (P = 0.0026), and colon cancer (P = 0.0349) than those in healthy controls, but not in patients with rectal cancer (P = 0.1151), leukemia (P = 0.7508), or lung cancer (P = 0.2247). The highest diagnostic value was for breast cancer, particularly in early cases (AUC = 0.8014) and those with distant metastases (AUC = 0.7885). The titers of EA2 antibodies in sera were correlated with levels of EA2 antigen in breast cancer patients. CONCLUSION: Antibodies to EA2 are novel blood biomarkers for early diagnosis of breast cancer that warrants further study in larger-scale cohort studies.

The CHD Protein Kismet Restricts the Synaptic Localization of Cell Adhesion Molecules at the Drosophila Neuromuscular Junction.

The appropriate expression and localization of cell surface cell adhesion molecules must be tightly regulated for optimal synaptic growth and function. How neuronal plasma membrane proteins, including cell adhesion molecules, cycle between early endosomes and the plasma membrane is poorly understood. Here we show that the Drosophila homolog of the chromatin remodeling enzymes CHD7 and CHD8, Kismet, represses the synaptic levels of several cell adhesion molecules. Neuroligins 1 and 3 and the integrins αPS2 and ßPS are increased at kismet mutant synapses but Kismet only directly regulates transcription of neuroligin 2. Kismet may therefore regulate synaptic CAMs indirectly by activating transcription of gene products that promote intracellular vesicle trafficking including endophilin B (endoB) and/or rab11. Knock down of EndoB in all tissues or neurons increases synaptic FasII while knock down of EndoB in kis mutants does not produce an additive increase in FasII. In contrast, neuronal expression of Rab11, which is deficient in kis mutants, leads to a further increase in synaptic FasII in kis mutants. These data support the hypothesis that Kis influences the synaptic localization of FasII by promoting intracellular vesicle trafficking through the early endosome.

Endophilin A2 controls touch and mechanical allodynia via kinesin-mediated Piezo2 trafficking.

BACKGROUND: Tactile and mechanical pain are crucial to our interaction with the environment, yet the underpinning molecular mechanism is still elusive. Endophilin A2 (EndoA2) is an evolutionarily conserved protein that is documented in the endocytosis pathway. However, the role of EndoA2 in the regulation of mechanical sensitivity and its underlying mechanisms are currently unclear. METHODS: Male and female C57BL/6 mice (8-12 weeks) and male cynomolgus monkeys (7-10 years old) were used in our experiments. Nerve injury-, inflammatory-, and chemotherapy-induced pathological pain models were established for this study. Behavioral tests of touch, mechanical pain, heat pain, and cold pain were performed in mice and nonhuman primates. Western blotting, immunostaining, co-immunoprecipitation, proximity ligation and patch-clamp recordings were performed to gain insight into the mechanisms. RESULTS: The results showed that EndoA2 was primarily distributed in neurofilament-200-positive (NF200+) medium-to-large diameter dorsal root ganglion (DRG) neurons of mice and humans. Loss of EndoA2 in mouse NF200+ DRG neurons selectively impaired the tactile and mechanical allodynia. Furthermore, EndoA2 interacted with the mechanically sensitive ion channel Piezo2 and promoted the membrane trafficking of Piezo2 in DRG neurons. Moreover, as an adaptor protein, EndoA2 also bound to kinesin family member 5B (KIF5B), which was involved in the EndoA2-mediated membrane trafficking process of Piezo2. Loss of EndoA2 in mouse DRG neurons damaged Piezo2-mediated rapidly adapting mechanically activated currents, and re-expression of EndoA2 rescued the MA currents. In addition, interference with EndoA2 also suppressed touch sensitivity and mechanical hypersensitivity in nonhuman primates. CONCLUSIONS: Our data reveal that the KIF5B/EndoA2/Piezo2 complex is essential for Piezo2 trafficking and for sustaining transmission of touch and mechanical hypersensitivity signals. EndoA2 regulates touch and mechanical allodynia via kinesin-mediated Piezo2 trafficking in sensory neurons. Our findings identify a potential new target for the treatment of mechanical pain.

Alveolar-capillary endocytosis and trafficking in acute lung injury and acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is associated with high morbidity and mortality but lacks specific therapeutic options. Diverse endocytic processes play a key role in all phases of acute lung injury (ALI), including the initial insult, development of respiratory failure due to alveolar flooding, as a consequence of altered alveolar-capillary barrier function, as well as in the resolution or deleterious remodeling after injury. In particular, clathrin-, caveolae-, endophilin- and glycosylphosphatidyl inositol-anchored protein-mediated endocytosis, as well as, macropinocytosis and phagocytosis have been implicated in the setting of acute lung damage. This manuscript reviews our current understanding of these endocytic pathways and subsequent intracellular trafficking in various phases of ALI, and also aims to identify potential therapeutic targets for patients with ARDS.

Biology of endophilin and it's role in disease.

Endophilin is an evolutionarily conserved family of protein that involves in a range of intracellular membrane dynamics. This family consists of five isoforms, which are distributed in various tissues. Recent studies have shown that Endophilin regulates diseases pathogenesis, including neurodegenerative diseases, tumors, cardiovascular diseases, and autoimmune diseases. In vivo, it regulates different biological functions such as vesicle endocytosis, mitochondrial morphological changes, apoptosis and autophagosome formation. Functional studies confirmed the role of Endophilin in development and progression of these diseases. In this study, we have comprehensively discussed the complex function of Endophilin and how the family contributes to diseases development. It is hoped that this study will provide new ideas for targeting Endophilin in diseases.

Dynamin 1xA interacts with Endophilin A1 via its spliced long C-terminus for ultrafast endocytosis.

Dynamin 1 (Dyn1) has two major splice variants, xA and xB, with unique C-terminal extensions of 20 and 7 amino acids, respectively. Of these, only Dyn1xA is enriched at endocytic zones and accelerates vesicle fission during ultrafast endocytosis. Here, we report that the long tail variant, Dyn1xA, achieves this localization by preferentially binding to Endophilin A through a newly defined Class II binding site overlapping with its extension, at a site spanning the splice boundary. Endophilin binds this site at higher affinity than the previously reported site, and this affinity is determined by amino acids outside the binding sites acting as long distance elements within the xA tail. Their interaction is regulated by the phosphorylation state of two serine residues specific to the xA variant. Dyn1xA and Endophilin colocalize in patches near the active zone of synapses. Mutations selectively disrupting Endophilin binding to the long extension cause Dyn1xA mislocalization along axons. In these mutants, endocytic pits are stalled on the plasma membrane during ultrafast endocytosis. These data suggest that the specificity for ultrafast endocytosis is defined by the phospho-regulated interaction of Endophilin A through a newly identified site of Dyn1xA's long tail.

The role of EndophilinA1 in chronic unpredicted mild stress-induced depression model mice.

BACKGROUND: Depression is a common mental disease, accompanied by anxiety and persistent depression. Endophilin A1 (EPA1) is a brain-specific protein enriched in synaptic terminals that is primarily expressed in the central nervous system. It has been reported that EPA1 is involved in neurotransmitter release, which indicates that the protein may be involved in depression. However, it is unclear whether EPA1 is implicated in the development of depression. METHODS: The mice depression model was established by chronic unpredicted mild stress (CUMS). Depression-like behaviors were detected by sucrose preference test (SPT), forced swim test (FST), tail-suspension test (TST) and open-field test (OFT). Neuronal histopathology was applied by hematoxylin and eosin stain (H&E), and Nissl stain. EPA1, NLRP1 inflammatory complexes, NADPH oxidase2 (NOX2), synaptic-related protein expression of the mice were tested by western blot. Immunofluorescence was applied to detect the expression of EPA1 and ROS in mice hippocampus. EPA1 knockdown was performed by an adeno-associated virus (AAV) vector containing EPA1-shRNA-EGFP infusion. RESULT: CUMS exposure induced depressive-like behaviors and increased the expression of EPA1 in the hippocampus. Knockdown hippocampal EPA1 ameliorated CUMS-induced depressive-like behaviors, decreased calcium (Ca2+) overload, decreased ROS generation and NOX2 expression, inhibited NLRP1 inflammasome-driven neuroinflammation, and restored the levels of BDNF, PSD95, GAP-43, SYN, and MAP-2 in the hippocampus. CONCLUSION: EPA1 contributes to CUMS induced depressive-like behaviors and the mechanism may be related to NLRP1 inflammasome-driven inflammatory response, regulating calcium ion homeostasis and ROS generation, and alleviating synaptic function damage. This indicated that EPA1 may participate in the occurrence and development of depression.

The SH3 binding site in front of the WH1 domain contributes to the membrane binding of the BAR domain protein endophilin A2.

The Bin-Amphiphysin-Rvs (BAR) domain of endophilin binds to the cell membrane and shapes it into a tubular shape for endocytosis. Endophilin has a Src-homology 3 (SH3) domain at their C-terminal. The SH3 domain interacts with the proline-rich motif (PRM) that is found in proteins such as neural Wiskott-Aldrich syndrome protein (N-WASP). Here, we re-examined the binding sites of the SH3 domain of endophilin in N-WASP by machine learning-based prediction and identified the previously unrecognized binding site. In addition to the well-recognized PRM at the central proline-rich region, we found a PRM in front of the N-terminal WASP homology 1 (WH1) domain of N-WASP (NtPRM) as a binding site of the endophilin SH3 domain. Furthermore, the diameter of the membrane tubules in the presence of NtPRM mutant was narrower and wider than that in the presence of N-WASP and in its absence, respectively. Importantly, the NtPRM of N-WASP was involved in the membrane localization of endophilin A2 in cells. Therefore, the NtPRM contributes to the binding of endophilin to N-WASP in membrane remodeling.

Synaptotagmin-11 Inhibits Synaptic Vesicle Endocytosis via Endophilin A1.

Synaptic vesicle (SV) endocytosis is a critical and well-regulated process for the maintenance of neurotransmission. We previously reported that synaptotagmin-11 (Syt11), an essential non-Ca2+-binding Syt associated with brain diseases, inhibits neuronal endocytosis (Wang et al., 2016). Here, we found that Syt11 deficiency caused accelerated SV endocytosis and vesicle recycling under sustained stimulation and led to the abnormal membrane partition of synaptic proteins in mouse hippocampal boutons of either sex. Furthermore, our study revealed that Syt11 has direct but Ca2+-independent binding with endophilin A1 (EndoA1), a membrane curvature sensor and endocytic protein recruiter, with high affinity. EndoA1-knockdown significantly reversed Syt11-KO phenotype, identifying EndoA1 as a main inhibitory target of Syt11 during SV endocytosis. The N-terminus of EndoA1 and the C2B domain of Syt11 were responsible for this interaction. A peptide (amino acids 314-336) derived from the Syt11 C2B efficiently blocked Syt11-EndoA1 binding both in vitro and in vivo Application of this peptide inhibited SV endocytosis in WT hippocampal neurons but not in EndoA1-knockdown neurons. Moreover, intracellular application of this peptide in mouse calyx of Held terminals of either sex effectively hampered both fast and slow SV endocytosis at physiological temperature. We thus propose that Syt11 ensures the precision of protein retrieval during SV endocytosis by inhibiting EndoA1 function at neuronal terminals.SIGNIFICANCE STATEMENT Endocytosis is a key stage of synaptic vesicle (SV) recycling. SV endocytosis retrieves vesicular membrane and protein components precisely to support sustained neurotransmission. However, the molecular mechanisms underlying the regulation of SV endocytosis remain elusive. Here, we reported that Syt11-KO accelerated SV endocytosis and impaired membrane partition of synaptic proteins. EndoA1 was identified as a main inhibitory target of Syt11 during SV endocytosis. Our study reveals a novel inhibitory mechanism of SV endocytosis in preventing hyperactivation of endocytosis, potentially safeguarding the recycling of synaptic proteins during sustained neurotransmission.

The Role of Endophilin A1 in Lipopolysaccharide-Induced Parkinson's Disease Model Mice.

BACKGROUND: Endophilin A1 (EPA1) is encoded by the SH3GL2 gene, and SH3GL2 was designated as a Parkinson's disease (PD) risk locus by genome-wide association analysis, suggesting that EPA1 may be involved in the occurrence and development of PD. OBJECTIVE: To investigate the role of EPA1 in lipopolysaccharide (LPS)-induced PD model mice. METHODS: The mice PD model was prepared by injecting LPS into the substantia nigra (SN), and the changes in the behavioral data of mice in each group were observed. The damage of dopaminergic neurons, activation of microglia, and reactive oxygen species (ROS) generation were detected by immunofluorescence method; calcium ion concentration was detected by calcium content detection kit; EPA1 and inflammation and its related indicators were detected by western blot method. EPA1 knockdown was performed by an adeno-associated virus vector containing EPA1-shRNA-eGFP infusion. RESULTS: LPS-induced PD model mice developed behavioral dysfunction, SN dopaminergic nerve damage, significantly increased calcium ion, calpain 1, and ROS production, activated NLRP1 inflammasome and promoted pro-inflammatory cell release, and SN EPA1 knockdown improves behavioral disorders, alleviates dopaminergic neuron damage, reduces calcium, calpain 1, ROS generation, and blocks NLRP1 inflammasome-driven inflammatory responses. CONCLUSION: The expression of EPA1 in the SN of LPS-induced PD model mice was increased, and it played a role in promoting the occurrence and development of PD. EPA1 knockdown inhibited the NLRP1 inflammasome activation, decreased the release of inflammatory factors and ROS generation, and alleviated dopaminergic neuron damage. This indicated that EPA1 may participating in the occurrence and development of PD.

The phospho-regulated amphiphysin/endophilin interaction is required for synaptic vesicle endocytosis.

The multidomain adaptor protein amphiphysin-1 (Amph1) is an important coordinator of clathrin-mediated endocytosis in non-neuronal cells and synaptic vesicle (SV) endocytosis at central nerve terminals. Amph1 contains a lipid-binding N-BAR (Bin/Amphiphysin/Rvs) domain, central proline-rich (PRD) and clathrin/AP2 (CLAP) domains, and a C-terminal SH3 domain. Amph1 interacts with both lipids and proteins, with all of these interactions required for SV endocytosis, with the exception of the Amph1 PRD. The Amph1 PRD associates with the endocytosis protein endophilin A1, however, the role of this interaction in SV endocytosis has not been investigated. In this study, we set out to determine whether the Amph1 PRD and its interaction with endophilin A1 was essential for efficient SV endocytosis at typical small central synapses. To achieve this, domain-specific interactions of Amph1 were validated using in vitro GST pull-down assays, with the role of these interactions in SV endocytosis determined in molecular replacement experiments in primary neuronal culture. Using this approach, we confirmed important roles for CLAP and SH3 domain interactions of Amph1 in the control of SV endocytosis. Importantly, we identified the interaction site for endophilin A1 within the Amph1 PRD and exploited specific binding mutants to reveal a key role for this interaction in SV endocytosis. Finally, we determined that the formation of the Amph1-endophilin A1 complex is dependent on the phosphorylation status of Amph1-S293 within the PRD and that the phosphorylation status of this residue is essential for efficient SV regeneration. This work, therefore, reveals a key role for the dephosphorylation-dependent Amph1-endophilin A1 interaction in efficient SV endocytosis.

Identifying mRNAs Residing in Myelinating Oligodendrocyte Processes as a Basis for Understanding Internode Autonomy.

In elaborating and maintaining myelin sheaths on multiple axons/segments, oligodendrocytes distribute translation of some proteins, including myelin basic protein (MBP), to sites of myelin sheath assembly, or MSAS. As mRNAs located at these sites are selectively trapped in myelin vesicles during tissue homogenization, we performed a screen to identify some of these mRNAs. To confirm locations, we used real-time quantitative polymerase chain reaction (RT-qPCR), to measure mRNA levels in myelin (M) and 'non-myelin' pellet (P) fractions, and found that five (LPAR1, TRP53INP2, TRAK2, TPPP, and SH3GL3) of thirteen mRNAs were highly enriched in myelin (M/P), suggesting residences in MSAS. Because expression by other cell-types will increase p-values, some MSAS mRNAs might be missed. To identify non-oligodendrocyte expression, we turned to several on-line resources. Although neurons express TRP53INP2, TRAK2 and TPPP mRNAs, these expressions did not invalidate recognitions as MSAS mRNAs. However, neuronal expression likely prevented recognition of KIF1A and MAPK8IP1 mRNAs as MSAS residents and ependymal cell expression likely prevented APOD mRNA assignment to MSAS. Complementary in situ hybridization (ISH) is recommended to confirm residences of mRNAs in MSAS. As both proteins and lipids are synthesized in MSAS, understanding myelination should not only include efforts to identify proteins synthesized in MSAS, but also the lipids.

Endophilin-A/SH3GL2 calcium switch for synaptic autophagy induction is impaired by a Parkinson's risk variant.

At the synapse, proteins are reused several times during neuronal activity, causing a decline in protein function over time. Although emerging evidence supports a role of autophagy in synaptic function, the precise molecular mechanisms linking neuronal activity, autophagy and synaptic dysfunction are vastly unknown. We show how extracellular calcium influx in the pre-synaptic terminal constitutes the initial stimulus for autophagosome formation in response to neuronal activity. This mechanism likely acts to rapidly support synaptic homeostasis and protein quality control when intense neuronal activity challenges the synaptic proteome. We identified a residue in the flexible region of EndoA (Endophilin A) that dictates calcium-dependent EndoA mobility from the plasma membrane to the cytosol, where this protein interacts with autophagic membranes to promote autophagosome formation. We discovered that a novel Parkinson's disease-risk mutation in SH3GL2 (SH3 domain containing GRB2 like 2, endophilin A1) disrupts the calcium sensing of SH3GL2, leading to an immobile protein that cannot respond to calcium influx and therefore disrupting autophagy induction at synapses. Our work shows how neuronal activity is connected with autophagy to maintain synaptic homeostasis and survival.

N-BAR and F-BAR proteins-endophilin-A3 and PSTPIP1-control clathrin-independent endocytosis of L1CAM.

Recent advances in the field demonstrate the high diversity and complexity of endocytic pathways. In the current study, we focus on the endocytosis of L1CAM. This glycoprotein plays a major role in the development of the nervous system, and is involved in cancer development and is associated with metastases and poor prognosis. Two L1CAM isoforms are subject to endocytosis: isoform 1, described as a clathrin-mediated cargo; isoform 2, whose endocytosis has never been studied. Deciphering the molecular machinery of isoform 2 internalisation should contribute to a better understanding of its pathophysiological role. First, we demonstrated in our cellular context that both isoforms of L1CAM are mainly a clathrin-independent cargo, which was not expected for isoform 1. Second, the mechanism of L1CAM endocytosis is specifically mediated by the N-BAR domain protein endophilin-A3. Third, we discovered PSTPIP1, an F-BAR domain protein, as a novel actor in this endocytic process. Finally, we identified galectins as endocytic partners and negative regulators of L1CAM endocytosis. In summary, the interplay of the BAR proteins endophilin-A3 and PSTPIP1, and galectins fine tune the clathrin-independent endocytosis of L1CAM.

lncR26319/miR-2834/EndophilinA axis regulates oogenesis of the silkworm, Bombyx mori.

Oocyte maturation is critical for insect reproduction. Vitellogenesis, the timely production and uptake of vitellogenin (Vg), is crucial for female fecundity. Vg is synthesized in fat body and absorbed by the oocytes through endocytosis during insect oogenesis. In the silkworm, Bombyx mori, we discovered that a nucleus-enriched long-noncoding RNA (lncRNA) lncR26319 regulates Endophilin A (EndoA) - a member of the endophilin family of endocytic proteins - through competitive binding to miR-2834. The lncR26319-miR-2834-EndoA axis was required for Vg endocytosis in the silkworm; loss of EndoA or overexpression of miR-2834 significantly reduced egg numbers in virgin moths. In addition, accumulation of miR-2834 resulted in pupal and adult deformation and reduced fecundity in females. The expression of Vg, 30-kDa (30K) protein, and egg-specific protein (Esp) decreased after knockdown of EndoA or overexpression of miR-2834, while knockdown of miR-2834 had an opposite effect on the expression of Vg, 30K protein gene, and Esp. These results suggest that the lncR26319-miR-2834-EndoA axis contributes to the endocytic activity in the Vg uptake and leads to the normal progression of oogenesis in the silkworm. Thus, miR-2834 and EndoA are crucial for female reproduction and could be potential targets for new pest management strategies in lepidopterans.

Endophilin A2 protects against renal fibrosis by targeting TGF-ß/Smad signaling.

Renal fibrosis underlies all forms of end-stage kidney disease. Endophilin A2 (EndoA2) plays a role in nephrotic syndrome; however, its effect on renal fibrosis remains unknown. Here, we demonstrate that EndoA2 protects against kidney interstitial fibrosis via the transforming growth factor-ß (TGF-ß)/Smad signaling pathway. Mouse kidneys with fibrosis or kidney biopsy specimens from patients with fibrotic nephropathy had lower levels of EndoA2 protein expression than that in kidneys without fibrosis. In vivo overexpression of EndoA2 with the endophilin A2 transgene (EndoA2Tg ) notably prevented renal fibrosis, decreased the protein expression of profibrotic molecules, suppressed tubular injury, and reduced apoptotic tubular cells in the obstructed kidney cortex of mice with unilateral ureteral obstruction (UUO). In vivo and in vitro overexpression of EndoA2 markedly inhibited UUO- or TGF-ß1-induced phosphorylation of Smad2/3 and tubular epithelial cells dedifferentiation. Furthermore, EndoA2 was co-immunoprecipitated with the type II TGF-ß receptor (TßRII), thus inhibiting the binding of the type I TGF-ß receptor (TßRI) to TßRII. These findings indicate that EndoA2 mitigates renal fibrosis, at least partially, via modulating the TGF-ß/Smad signaling. Targeting EndoA2 may be a new potential therapeutic strategy for treatment of renal fibrosis.

Rac1, the actin cytoskeleton and microtubules are key players in clathrin-independent endophilin-A3-mediated endocytosis.

Endocytic mechanisms actively regulate plasma membrane composition and sustain fundamental cellular functions. Recently, we identified a clathrin-independent endocytic (CIE) modality mediated by the BAR domain protein endophilin-A3 (endoA3, encoded by SH3GL3), which controls the cell surface homeostasis of the tumor marker CD166 (also known as ALCAM). Deciphering the molecular machinery of endoA3-dependent CIE should therefore contribute to a better understanding of its pathophysiological role, which remains so far unknown. Here, we investigate the role of actin, Rho GTPases and microtubules, which are major players in CIE processes, in this mechanism. We show that the actin cytoskeleton is dynamically associated with endoA3- and CD166-positive endocytic carriers, and that its perturbation strongly inhibits the process of CD166 uptake. We also reveal that the Rho GTPase Rac1, but not Cdc42, is a master regulator of this endocytic route. Finally, we provide evidence that microtubules and kinesin molecular motors are required to potentiate endoA3-dependent endocytosis. Of note, our study also highlights potential compensation phenomena between endoA3-dependent CIE and macropinocytosis. Altogether, our data deepen our understanding of this CIE modality and further differentiate it from other unconventional endocytic mechanisms. This article has an associated First Person interview with the first author of the paper.

17ß-Estradiol nongenomically induces vasodilation is enhanced by promoting phosphorylation of endophilin A2.

ObjectiveA previous study found that the tyrosine phosphorylation of endophilin A2 (Endo II) was responsible for increase surface expression of MT1-MMP and ECM degradation; however, there is little information about whether Endo II could influence membrane estrogen receptors (mERs) and its functions.Materials and methodsIn the present study, Human umbilical vein endothelial cells (HUVECs) were treated with E2, PPT, DPN, ICI 182780, Endo siRNA or negative control siRNA, and the biological behavior of the treated cells was observed. The mice were randomly divided into AAV-control-shRNA + Ach, AAV-Endo II-shRNA + Ach, AAV-control-shRNA + E2, AAV-Endo II-shRNA + E2 groups and the thoracic aorta were isolated, cut into 2-mm rings, then the wall tension was detected.ResultsWe found that 17ß-Estradiol (E2) enhanced mERα protein level, which was further increased after knocking down Endo II, the mechanism maybe involved in E2-induced tyrosine phosphorylation of Endo II. In addition, we also observed that Endo II blocked the activation of Akt, ERK1/2 and eNOS signaling in HUVECs treated with E2. E2 induced vasodilation was significantly increased by silencing of Endo II expression.ConclusionOur study provided a sound basis to selective modulate Endo II for E2's nongenomic pathway, which can be benefit for cardiovascular system.