Plasma Membrane Localization Research Articles

Neuropilin-1 interacts with VE-cadherin and TGFBR2 to stabilize adherens junctions and prevent activation of endothelium under flow.

Linear and disturbed flow differentially regulate gene expression, with disturbed flow priming endothelial cells (ECs) for a proinflammatory, atheroprone expression profile and phenotype. Here, we investigated the role of the transmembrane protein neuropilin-1 (NRP1) in ECs exposed to flow using cultured ECs, mice with an endothelium-specific knockout of NRP1, and a mouse model of atherosclerosis. We demonstrated that NRP1 was a constituent of adherens junctions that interacted with VE-cadherin and promoted its association with p120 catenin, stabilizing adherens junctions and inducing cytoskeletal remodeling in alignment with the direction of flow. We also showed that NRP1 interacted with transforming growth factor-β (TGF-β) receptor II (TGFBR2) and reduced the plasma membrane localization of TGFBR2 and TGF-β signaling. NRP1 knockdown increased the abundance of proinflammatory cytokines and adhesion molecules, resulting in increased leukocyte rolling and atherosclerotic plaque size. These findings describe a role for NRP1 in promoting endothelial function and reveal a mechanism by which NRP1 reduction in ECs may contribute to vascular disease by modulating adherens junction signaling and promoting TGF-β signaling and inflammation.

Salicylic acid attenuates brassinosteroid signaling via protein de-S-acylation.

Brassinosteroids (BRs) are important plant hormones involved in many aspects of development. Here, we show that BRASSINOSTEROID SIGNALING KINASEs (BSKs), key components of the BR pathway, are precisely controlled via de-S-acylation mediated by the defense hormone salicylic acid (SA). Most Arabidopsis BSK members are substrates of S-acylation, a reversible protein lipidation that is essential for their membrane localization and physiological function. We establish that SA interferes with the plasma membrane localization and function of BSKs by decreasing their S-acylation levels, identifying ABAPT11 (ALPHA/BETA HYDROLASE DOMAIN-CONTAINING PROTEIN 17-LIKE ACYL PROTEIN THIOESTERASE 11) as an enzyme whose expression is quickly induced by SA. ABAPT11 de-S-acylates most BSK family members, thus integrating BR and SA signaling for the control of plant development. In summary, we show that BSK-mediated BR signaling is regulated by SA-induced protein de-S-acylation, which improves our understanding of the function of protein modifications in plant hormone cross talk.

Gene silencing of endothelial von Willebrand factor reduces the susceptibility of human endothelial cells to SARS-CoV-2 infection.

Mechanisms underlying vascular endothelial susceptibility to infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not fully understood. Emerging evidence indicates that patients lacking von Willebrand factor (vWF), an endothelial hallmark, are less severely affected by SARS-CoV-2 infection, yet the precise role of endothelial vWF in modulating coronavirus entry into endothelial cells is unknown. In the present study, we demonstrated that effective gene silencing by short interfering RNA (siRNA) for vWF expression in resting human umbilical vein endothelial cells (HUVECs) significantly reduced by 56% the cellular levels of SARS-CoV-2 genomic RNA. Similar reduction in intracellular SARS-CoV-2 genomic RNA levels was observed in non-activated HUVECs treated with siRNA targeting angiotensin-converting enzyme 2 (ACE2), the cellular gateway to coronavirus. By integrating quantitative information from real-time PCR and high-resolution confocal imaging, we demonstrated that ACE2 gene expression and its plasma membrane localization in HUVECs were both markedly reduced after treatment with siRNA anti-vWF or anti-ACE2. Conversely, siRNA anti-ACE2 did not reduce endothelial vWF gene expression and protein levels. Finally, SARS-CoV-2 infection of viable HUVECs was enhanced by overexpression of vWF, which increased ACE2 levels. Of note, we found a similar increase in interferon-β mRNA levels following transfection with untargeted, anti-vWF or anti-ACE2 siRNA and pcDNA3.1-WT-VWF. We envision that siRNA targeting endothelial vWF will protect against productive endothelial infection by SARS-CoV-2 through downregulation of ACE2 expression and might serve as a novel tool to induce disease resistance by modulating the regulatory role of vWF on ACE2 expression.

Croquemort elicits activation of the immune deficiency pathway in ticks

The immune deficiency (IMD) pathway directs host defense in arthropods upon bacterial infection. In Pancrustacea, peptidoglycan recognition proteins sense microbial moieties and initiate nuclear factor-κB-driven immune responses. Proteins that elicit the IMD pathway in non-insect arthropods remain elusive. Here, we show that an Ixodes scapularis homolog of croquemort (Crq), a CD36-like protein, promotes activation of the tick IMD pathway. Crq exhibits plasma membrane localization and binds the lipid agonist 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol. Crq regulates the IMD and jun N-terminal kinase signaling cascades and limits the acquisition of the Lyme disease spirochete B. burgdorferi. Additionally, nymphs silenced for crq display impaired feeding and delayed molting to adulthood due to a deficiency in ecdysteroid synthesis. Collectively, we establish a distinct mechanism for arthropod immunity outside of insects and crustaceans.

A novel effector RipBT contributes to Ralstonia solanacearum virulence on potato.

Ralstonia solanacearum is one of the most destructive plant-pathogenic bacteria, infecting more than 200 plant species, including potato (Solanum tuberosum) and many other solanaceous crops. R. solanacearum has numerous pathogenicity factors, and type III effectors secreted through type III secretion system (T3SS) are key factors to counteract host immunity. Here, we show that RipBT is a novel T3SS-secreted effector by using a cyaA reporter system. Transient expression of RipBT in Nicotiania benthamiana induced strong cell death in a plasma membrane-localization dependent manner. Notably, mutation of RipBT in R. solanacearum showed attenuated virulence on potato, while RipBT transgenic potato plants exhibited enhanced susceptibility to R. solanacearum. Interestingly, transcriptomic analyses suggest that RipBT may interfere with plant reactive oxygen species (ROS) metabolism during the R. solanacearum infection of potato roots. In addition, the expression of RipBT remarkably suppressed the flg22-induced pathogen-associated molecular pattern-triggered immunity responses, such as the ROS burst. Taken together, RipBT acts as a T3SS effector, promoting R. solanacearum infection on potato and presumably disturbing ROS homeostasis.

Simultaneous co-overexpression of Saccharomyces cerevisiae septins Cdc3 and Cdc10 drives pervasive, phospholipid-, and tag-dependent plasma membrane localization.

Septin proteins contribute to many eukaryotic processes involving cellular membranes. In the budding yeast Saccharomyces cerevisiae, septin hetero-oligomers interact with the plasma membrane (PM) almost exclusively at the future site of cytokinesis. While multiple mechanisms of membrane recruitment have been identified, including direct interactions with specific phospholipids and curvature-sensitive interactions via amphipathic helices, these do not fully explain why yeast septins do not localize all over the inner leaflet of the PM. While engineering an inducible split-yellow fluorescent protein (YFP) system to measure the kinetics of yeast septin complex assembly, we found that ectopic co-overexpression of two tagged septins, Cdc3 and Cdc10, resulted in nearly uniform PM localization, as well as perturbation of endogenous septin function. Septin localization and function in gametogenesis were also perturbed. PM localization required the C-terminal YFP fragment fused to the C terminus of Cdc3, the septin-associated kinases Cla4 and Gin4, and phosphotidylinositol-4,5-bis-phosphate (PI[4,5]P2 ), but not the putative PI(4,5)P2 -binding residues in Cdc3. Endogenous Cdc10 was recruited to the PM, likely contributing to the functional interference. PM-localized septins did not exchange with the cytosolic pool, indicative of stable polymers. These findings provide new clues as to what normally restricts septin localization to specific membranes.

Selective Visualization of Tumor Cell Membranes and Tumors with a Viscosity-Sensitive Plasma Membrane Probe.

Cancer is a worldwide health problem. Revealing the changes in the microenvironment after cell carcinogenesis is helpful to understand cancer and develop sensitive methods for cancer diagnosis. We developed herein a viscosity-responsive plasma membrane probe (TPA-S) that was successfully used to probe the viscosity difference between normal and tumor cell plasma membranes for the first time. The probe shows AIE properties with good water solubility, significant near-infrared (NIR) fluorescence responses to viscosity with high sensitivity, and excellent cell membrane location performance. With these features, our experiments showed that TPA-S could selectively visualize cancer cell plasma membranes, revealing that the plasma membrane of tumor cells is more viscous than that of normal cells. In addition, TPA-S was successfully applied to specifically light up tumors. Altogether, this work explored the changes of cell membrane viscosity after canceration, provided a new method for selective visualization of tumor cells, and opened up a new approach for cancer diagnosis.

Sugar transporter ZmSWEET1b is responsible for assimilate allocation and salt stress response in maize

Sugar efflux transporter SWEET family is involved in multiple biological processes, from nectar secretion, pollen fertility to seed filling. Although roles of SWEETs in abiotic stress adaption have been revealed mainly in reference organism Arabidopsis, cereal crops SWEETs responses to abiotic stimulation remain largely elusive. Here, we report the characterization of maize SWEET family member ZmSWEET1b, with emphasis on its response to salinity stress. ZmSWEET1b is a canonical sugar transporter, characteristic of seven transmembrane helices and plasma membrane localization. ZmSWEET1b and its rice ortholog OsSWEET1b in phylogenetic clade I underwent convergent selection during evolution. Two independent knockout lines were created by the CRISPR/Cas9 method to functionally characterized ZmSWEET1b. Sucrose and fructose contents are significantly decreased in ZmSWEET1b knockout lines. Mature leaves of ZmSWEET1b-edited lines exhibit chlorosis, reminiscent of senescence-like phenotype. Ears and seeds of ZmSWEET1b knockout lines are small. Upon salinity treatment, ZmSWEET1b-edited lines become more wilted. Transcriptional abundance of genes for Na+ efflux from roots to the rhizosphere, including ZmSOS1, ZmH+-ATPASE 2, and ZmH+-ATPASE 8, is decreased in salt-treated ZmSWEET1b knockout lines. These findings indicate that convergently selected sugar transporter ZmSWEET1b is important for maize plant development and responses to salt stress. The manipulation of ZmSWEET1b may represent a feasible way forward in the breeding of salinity tolerant ideotypes through the optimization of assimilate allocation.

Biological and clinical impact of membrane EGFR expression in a subgroup of OC patients from the phase IV ovarian cancer MITO-16A/MANGO-OV2A trial

BackgroundValidated prognostic biomarkers for anti-angiogenic therapy using the anti-VEGF antibody Bevacizumab in ovarian cancer (OC) patients are still an unmet clinical need. The EGFR can contribute to cancer-associated biological mechanisms in OC cells including angiogenesis, but its targeting gave disappointing results with less than 10% of OC patients treated with anti-EGFR compounds showing a positive response, likely due to a non adequate selection and stratification of EGFR-expressing OC patients.MethodsEGFR membrane expression was evaluated by immunohistochemistry in a cohort of 310 OC patients from the MITO-16A/MANGO-OV2A trial, designed to identify prognostic biomarkers of survival in patients treated with first line standard chemotherapy plus bevacizumab. Statistical analyses assessed the association between EGFR and clinical prognostic factors and survival outcomes. A single sample Gene Set Enrichment-like and Ingenuity Pathway Analyses were applied to the gene expression profile of 195 OC samples from the same cohort. In an OC in vitro model, biological experiments were performed to assess specific EGFR activation.ResultsBased on EGFR-membrane expression, three OC subgroups of patients were identified being the subgroup with strong and homogeneous EGFR membrane localization, indicative of possible EGFR out/in signalling activation, an independent negative prognostic factor for overall survival of patients treated with an anti-angiogenic agent. This OC subgroup resulted statistically enriched of tumors of histotypes different than high grade serous lacking angiogenic molecular characteristics. At molecular level, among the EGFR-related molecular traits identified to be activated only in this patients’ subgroup the crosstalk between EGFR with other RTKs also emerged. In vitro, we also showed a functional cross-talk between EGFR and AXL RTK; upon AXL silencing, the cells resulted more sensitive to EGFR targeting with erlotinib.ConclusionsStrong and homogeneous cell membrane localization of EGFR, associated with specific transcriptional traits, can be considered a prognostic biomarker in OC patients and could be useful for a better OC patients’ stratification and the identification of alternative therapeutic target/s in a personalized therapeutic approach.

Optimal HSF1 activation in response to acute cold stress in BAT requires nuclear TXNIP

While TXNIP (thioredoxin interacting protein) in the plasma membrane and vesicular location is known to negatively regulate cellular glucose uptake by facilitating glucose transporter endocytosis, the function of TXNIP in the nucleus is far less understood. Herein, we sought to determine the function of nuclear TXNIP invivo, using a new HA-tagged TXNIP knock-in mouse model. We observed that TXNIP can be found in the nucleus of a variety of cells from different tissues including hepatocytes (liver), enterocytes (small intestine), exocrine cells (pancreas), and brown adipocytes (BAT). Further investigations into the role of nuclear TXNIP in BAT revealed that cold stress rapidly and transiently activated HSF1 (heat shock factor 1). HSF1 interaction with TXNIP during its activation is required for optimal HSF1 directed cold shock response in BAT.

Loss-of-function of gynoecium-expressed phospholipase pPLAIIγ triggers maternal haploid induction in Arabidopsis.

Production of in planta haploid embryos that inherit chromosomes from only one parent can greatly increase breeding efficiency via quickly generating homozygous plants, called doubled haploid. One of the main players of in planta haploid induction is a pollen-specific phospholipase A, which is able, when mutated, to induce invivo haploid induction in numerous monocots. However, no functional orthologous gene has been identified in dicots plants. Here, we show that loss-of-function of gynoecium-expressed phospholipase AII (pPLAIIγ) triggers maternal haploid plants in Arabidopsis, at an average rate of 1.07%. Reciprocal crosses demonstrate that haploid plants are triggered from the female side and not from the pollen, and the haploid plants carry the maternal genome. Promoter activity of pPLAIIγ shows enriched expression in the funiculus of flower development stages 13 and 18, and pPLAIIγ fused to yellow fluorescent protein reveals a plasma-membrane localization Interestingly, the polar localized PIN1 at the basal plasma membrane of the funiculus was all internalized in pplaIIγ mutants, suggesting that altered PIN1 localization in female organ could play a role in maternal haploid induction.

Dynamic localization of the Na+-HCO3- co-transporter NBCn1 to the plasma membrane, centrosomes, spindle and primary cilia.

Finely tuned regulation of transport protein localization is vital for epithelial function. The Na+-HCO3- co-transporter NBCn1 (also known as SLC4A7) is a key contributor to epithelial pH homeostasis, yet the regulation of its subcellular localization is not understood. Here, we show that a predicted N-terminal β-sheet and short C-terminal α-helical motif are essential for NBCn1 plasma membrane localization in epithelial cells. This localization was abolished by cell-cell contact disruption, and co-immunoprecipitation (co-IP) and proximity ligation (PLA) revealed NBCn1 interaction with E-cadherin and DLG1, linking it to adherens junctions and the Scribble complex. NBCn1 also interacted with RhoA and localized to lamellipodia and filopodia in migrating cells. Finally, analysis of native and GFP-tagged NBCn1 localization, subcellular fractionation, co-IP with Arl13B and CEP164, and PLA of NBCn1 and tubulin in mitotic spindles led to the surprising conclusion that NBCn1 additionally localizes to centrosomes and primary cilia in non-dividing, polarized epithelial cells, and to the spindle, centrosomes and midbodies during mitosis. We propose that NBCn1 traffics between lateral junctions, the leading edge and cell division machinery in Rab11 endosomes, adding new insight to the role of NBCn1 in cell cycle progression.

A Leucine-Rich Repeat Receptor-like Kinase TaBIR1 Contributes to Wheat Resistance against Puccinia striiformis f. sp. tritici

Plant cell surface-localized receptor-like kinases (RLKs) recognize invading pathogens and transduce the immune signals inside host cells, subsequently triggering immune responses to fight off pathogen invasion. Nonetheless, our understanding of the role of RLKs in wheat resistance to the biotrophic fungus Puccinia striiformis f. sp. tritici (Pst) remains limited. During the differentially expressed genes in Pst infected wheat leaves, a Leucine-repeat receptor-like kinase (LRR-RLK) gene TaBIR1 was significantly upregulated in the incompatible wheat-Pst interaction. qRT-PCR verified that TaBIR1 is induced at the early infection stage of Pst. The transient expression of TaBIR1-GFP protein in N. bentamiana cells and wheat mesophyll protoplasts revealed its plasma membrane location. The knockdown of TaBIR1 expression by VIGS (virus induced gene silencing) declined wheat resistance to stripe rust, resulting in reduced reactive oxygen species (ROS) production, callose deposition, and transcripts of pathogenesis-related genes TaPR1 and TaPR2, along with increased Pst infection area. Ectopic overexpression of TaBIR1 in N. benthamiana triggered constitutive immune responses with significant cell death, callose accumulation, and ROS production. Moreover, TaBIR1 triggered immunity is dependent on NbBAK1, the silencing of which significantly attenuated the defense response triggered by TaBIR1. TaBIR1 interacted with the NbBAK1 homologues in wheat, co-receptor TaSERK2 and TaSERK5, the transient expression of which could restore the impaired defense due to NbBAK1 silencing. Taken together, TaBIR1 is a cell surface RLK that contributes to wheat stripe rust resistance, probably as a positive regulator of plant immunity in a BAK1-dependent manner.

Hormonal regulation of non-cystic fibrosis transmembrane conductance regulator ion channels in the endocervix

To characterize ion channel expression and localization in the endocervix under different hormonal conditions using a nonhuman primate primary endocervical epithelial cell model. Experimental. University-based, translational science laboratory. We cultured and treated conditionally reprogrammed primary rhesus macaque endocervix cells with estradiol and progesterone and measured gene expression changes for several known ion channel and ion channel regulators of mucus secreting epithelia. Using both rhesus macaque endocervical samples and human samples, we localized channels in the endocervix using immunohistochemistry. The relative abundance of transcripts was evaluated using real-time polymerase chain reaction. Immunostaining results were evaluated qualitatively. Compared with controls, we found that estradiol increased gene expression for ANO6, NKCC1, CLCA1, and PDE4D. Progesterone down-regulated gene expression for ANO6, SCNN1A, SCNN1B, NKCC1, and PDE4D (P≤.05). Immunohistochemistry confirmed endocervical cell membrane localization of ANO1, ANO6, KCNN4, LRR8CA, and NKCC1. We found several ion channels and ion channel regulators that are hormonally sensitive in the endocervix. These channels, therefore, may play a role in the cyclic fertility changes in the endocervix and could be further investigated as targets for future fertility and contraceptive studies.

Potassium Activates mTORC2-dependent SGK1 Phosphorylation to Stimulate Epithelial Sodium Channel: Role in Rapid Renal Responses to Dietary Potassium.

Rapid renal responses to ingested potassium are essential to prevent hyperkalemia and also play a central role in blood pressure regulation. Although local extracellular K + concentration in kidney tissue is increasingly recognized as an important regulator of K + secretion, the underlying mechanisms that are relevant in vivo remain controversial. To assess the role of the signaling kinase mTOR complex-2 (mTORC2), the authors compared the effects of K + administered by gavage in wild-type mice and knockout mice with kidney tubule-specific inactivation of mTORC2. They found that mTORC2 is rapidly activated to trigger K + secretion and maintain electrolyte homeostasis. Downstream targets of mTORC2 implicated in epithelial sodium channel regulation (SGK1 and Nedd4-2) were concomitantly phosphorylated in wild-type, but not knockout, mice. These findings offer insight into electrolyte physiologic and regulatory mechanisms. Increasing evidence implicates the signaling kinase mTOR complex-2 (mTORC2) in rapid renal responses to changes in plasma potassium concentration [K + ]. However, the underlying cellular and molecular mechanisms that are relevant in vivo for these responses remain controversial. We used Cre-Lox-mediated knockout of rapamycin-insensitive companion of TOR (Rictor) to inactivate mTORC2 in kidney tubule cells of mice. In a series of time-course experiments in wild-type and knockout mice, we assessed urinary and blood parameters and renal expression and activity of signaling molecules and transport proteins after a K + load by gavage. A K + load rapidly stimulated epithelial sodium channel (ENaC) processing, plasma membrane localization, and activity in wild-type, but not in knockout, mice. Downstream targets of mTORC2 implicated in ENaC regulation (SGK1 and Nedd4-2) were concomitantly phosphorylated in wild-type, but not knockout, mice. We observed differences in urine electrolytes within 60 minutes, and plasma [K + ] was greater in knockout mice within 3 hours of gavage. Renal outer medullary potassium (ROMK) channels were not acutely stimulated in wild-type or knockout mice, nor were phosphorylation of other mTORC2 substrates (PKC and Akt). The mTORC2-SGK1-Nedd4-2-ENaC signaling axis is a key mediator of rapid tubule cell responses to increased plasma [K + ] in vivo . The effects of K + on this signaling module are specific, in that other downstream mTORC2 targets, such as PKC and Akt, are not acutely affected, and ROMK and Large-conductance K + (BK) channels are not activated. These findings provide new insight into the signaling network and ion transport systems that underlie renal responses to K +in vivo .

Phototropin phosphorylation of ROOT PHOTOTROPISM 2 and its role in mediating phototropism, leaf positioning, and chloroplast accumulation movement in Arabidopsis.

Directional movements impact the ability of plants to respond and adjust their growth accordingly to the prevailing light environment. The plasma-membrane associated protein, ROOT PHOTOTROPISM 2 (RPT2) is a key signalling component involved in chloroplast accumulation movement, leaf positioning, and phototropism, all of which are regulated redundantly by the ultraviolet/blue light-activated AGC kinases phototropin 1 and 2 (phot1 and phot2). We recently demonstrated that members of the NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3)/RPT2-like (NRL) family in Arabidopsis thaliana, including RPT2, are directly phosphorylated by phot1. However, whether RPT2 is a substrate for phot2, and the biological significance of phot phosphorylation of RPT2 remains to be determined. Here, we show that RPT2 is phosphorylated by both phot1 and phot2 at a conserved serine residue (S591) within the C-terminal region of the protein. Blue light triggered the association of 14-3-3 proteins with RPT2 consistent with S591 acting as a 14-3-3 binding site. Mutation of S591 had no effect on the plasma membrane localization of RPT2 but reduced its functionality for leafpositioning and phototropism. Moreover, our findings indicate that S591 phosphorylation within the C-terminus of RPT2 is required for chloroplast accumulation movement to low level blue light. Taken together, these findings further highlight the importance of the C-terminal region of NRL proteins and how its phosphorylation contributes to phot receptor signalling in plants.

Overview of the structure and function of the dopamine transporter and its protein interactions.

The dopamine transporter (DAT) plays an integral role in dopamine neurotransmission through the clearance of dopamine from the extracellular space. Dysregulation of DAT is central to the pathophysiology of numerous neuropsychiatric disorders and as such is an attractive therapeutic target. DAT belongs to the solute carrier family 6 (SLC6) class of Na+/Cl- dependent transporters that move various cargo into neurons against their concentration gradient. This review focuses on DAT (SCL6A3 protein) while extending the narrative to the closely related transporters for serotonin and norepinephrine where needed for comparison or functional relevance. Cloning and site-directed mutagenesis experiments provided early structural knowledge of DAT but our contemporary understanding was achieved through a combination of crystallization of the related bacterial transporter LeuT, homology modeling, and subsequently the crystallization of drosophila DAT. These seminal findings enabled a better understanding of the conformational states involved in the transport of substrate, subsequently aiding state-specific drug design. Post-translational modifications to DAT such as phosphorylation, palmitoylation, ubiquitination also influence the plasma membrane localization and kinetics. Substrates and drugs can interact with multiple sites within DAT including the primary S1 and S2 sites involved in dopamine binding and novel allosteric sites. Major research has centered around the question what determines the substrate and inhibitor selectivity of DAT in comparison to serotonin and norepinephrine transporters. DAT has been implicated in many neurological disorders and may play a role in the pathology of HIV and Parkinson's disease via direct physical interaction with HIV-1 Tat and α-synuclein proteins respectively.

Characterization of a PIP Binding Site in the N-Terminal Domain of V-ATPase a4 and Its Role in Plasma Membrane Association

Vacuolar ATPases (V-ATPases) are multi-subunit ATP-dependent proton pumps necessary for cellular functions, including pH regulation and membrane fusion. The evidence suggests that the V-ATPase a-subunit's interaction with the membrane signaling lipid phosphatidylinositol (PIPs) regulates the recruitment of V-ATPase complexes to specific membranes. We generated a homology model of the N-terminal domain of the human a4 isoform (a4NT) using Phyre2.0 and propose a lipid binding domain within the distal lobe of the a4NT. We identified a basic motif, K234IKK237, critical for interaction with phosphoinositides (PIP), and found similar basic residue motifs in all four mammalian and both yeast a-isoforms. We tested PIP binding of wildtype and mutant a4NT in vitro. In protein lipid overlay assays, the double mutation K234A/K237A and the autosomal recessive distal renal tubular-causing mutation K237del reduced both PIP binding and association with liposomes enriched with PI(4,5)P2, a PIP enriched within plasma membranes. Circular dichroism spectra of the mutant protein were comparable to wildtype, indicating that mutations affected lipid binding, not protein structure. When expressed in HEK293, wildtype a4NT localized to the plasma membrane in fluorescence microscopy and co-purified with the microsomal membrane fraction in cellular fractionation experiments. a4NT mutants showed reduced membrane association and decreased plasma membrane localization. Depletion of PI(4,5)P2 by ionomycin caused reduced membrane association of the WT a4NT protein. Our data suggest that information contained within the soluble a4NT is sufficient for membrane association and that PI(4,5)P2 binding capacity is involved in a4 V-ATPase plasma membrane retention.

Infantile human labial glands: Distribution of aquaporins and claudins in the context of paracellular and transcellular pathways

Human labial glands consist of saliva-secreting cells which are formed by serous and predominantly mucous glandular cells. The following excretory duct system converts the isotonic saliva into a hypotonic fluid. Liquids are transported across the membrane of epithelial cells by paracellular or transcellular mode of action. We studied aquaporins (AQP) and tight junction proteins in the endpieces and duct system of human labial glands of 3–5-month-old infants for the first time. AQP1, AQP3, and AQP5 represent the transcellular transport; tight junction proteins like claudin-1, − 3, − 4, and − 7 regulate the permeability of the paracellular pathway.Specimens of 28 infants were included in this study and analyzed histologically. AQP1 was present in myoepithelial cells and in endothelial cells of small blood vessels. AQP3 showed basolateral plasmamembrane localization in glandular endpieces. AQP5 was localized at the apical cytomembrane in serous and mucous glandular cells and at the lateral membrane in serous cells. Ducts remained unstained with the antibody to AQP1, AQP3, and AQP5. Claudin-1, − 3, − 4, and − 7 were expressed mainly in the lateral plasmamembrane of serous glandular cells. In the ducts, claudin-1, − 4, and − 7 were detected at the basal cell layer, claudin-7 also at the lateral cytomembrane. Our findings provide new insights into the localization of epithelial barrier components necessary for regulating saliva-modification in infantile labial glands.

Antimicrobial Peptide Cec4 Eradicates Multidrug-Resistant Acinetobacter baumannii in vitro and in vivo.

Acinetobacter baumannii has become a major difficulty in the treatment of bacteria-associated infection. The previously reported antimicrobial peptide Cec4 exhibited good and stable activity against A. baumannii in vitro, but the mechanisms and effects in vivo are elusive. The effects of Cec4 on bacterial membrane permeability, membrane potential and bacterial reactive oxygen species were measured. The cell membrane localization of antimicrobial peptides was studied by fluorescence labelling. The ability of bacteria to develop resistance to antimicrobial peptides was studied by continuous induction, and transcriptome difference was analysed. The in vivo toxicity of Cec4 against nematodes and mice was studied, and the in vivo therapeutic potential of Cec4 against A. baumannii was assessed. Cec4 effectively cleared multidrug-resistant A. baumannii by altering bacterial cell membrane permeability, changing bacterial cell membrane polarity, and increasing bacterial intracellular reactive oxygen species. Cec4 affected the expression of the secretion system, outer membrane, and efflux pump genes of A. baumannii. In addition, the bacteria did not acquire stable drug-resistant ability. Cec4 at 1.024 mg/mL did not affect the proliferation of HeLa and HepG2 cells, and Cec4 at 45 mg/kg had little effect on the mortality of Caenorhabditis elegans, even the liver and kidney tissues of mouse. Most importantly, Cec4 could effectively improve the survival rates and reduce the bacterial load of various tissues in the mouse model of infection. In conclusion, Cec4 can damage the cell membrane of bacteria, and the bacteria is not easy to produce resistance to Cec4. Besides, Cec4 has good potential for the treatment of multidrug-resistant A. baumannii infections.