scholarly journals A Na,K-ATPase–Fodrin–Actin Membrane Cytoskeleton Complex is Required for Endothelial Fenestra Biogenesis

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1387
Author(s):  
Meihua Ju ◽  
Sofia Ioannidou ◽  
Peter Munro ◽  
Olli Rämö ◽  
Helena Vihinen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Composition ◽  
Loss Of Function ◽  
Membrane Pores ◽  
Membrane Cytoskeleton ◽  
Vascular Endothelia ◽  
Biochemical Analyses ◽  
Pore Protein

Fenestrae are transcellular plasma membrane pores that mediate blood–tissue exchange in specialised vascular endothelia. The composition and biogenesis of the fenestra remain enigmatic. We isolated and characterised the protein composition of large patches of fenestrated plasma membrane, termed sieve plates. Loss-of-function experiments demonstrated that two components of the sieve plate, moesin and annexin II, were positive and negative regulators of fenestra formation, respectively. Biochemical analyses showed that moesin is involved in the formation of an actin–fodrin submembrane cytoskeleton that was essential for fenestra formation. The link between the fodrin cytoskeleton and the plasma membrane involved the fenestral pore protein PV-1 and Na,K-ATPase, which is a key regulator of signalling during fenestra formation both in vitro and in vivo. These findings provide a conceptual framework for fenestra biogenesis, linking the dynamic changes in plasma membrane remodelling to the formation of a submembrane cytoskeletal signalling complex.

scholarly journals Stepping stone: a cytohesin adaptor for membrane cytoskeleton restraint in the syncytial Drosophila embryo

2015 ◽  
Vol 26 (4) ◽  
pp. 711-725 ◽  
Author(s):  
Jiangshu Liu ◽  
Donghoon M. Lee ◽  
Cao Guo Yu ◽  
Stephane Angers ◽  
Tony J. C. Harris
Keyword(s):  
Plasma Membrane ◽  
Coiled Coil ◽  
Drosophila Embryo ◽  
Loss Of Function ◽  
Interacting Protein ◽  
Stepping Stone ◽  
Membrane Cytoskeleton ◽  
Coiled Coil Domain

Cytohesin Arf-GEFs are conserved plasma membrane regulators. The sole Drosophila cytohesin, Steppke, restrains Rho1-dependent membrane cytoskeleton activity at the base of plasma membrane furrows of the syncytial embryo. By mass spectrometry, we identified a single major Steppke-interacting protein from syncytial embryos, which we named Stepping stone (Sstn). By sequence, Sstn seems to be a divergent homologue of the mammalian cytohesin adaptor FRMD4A. Our experiments supported this relationship. Specifically, heterophilic coiled-coil interactions linked Sstn and Steppke in vivo and in vitro, whereas a separate C-terminal region was required for Sstn localization to furrows. Sstn mutant and RNAi embryos displayed abnormal, Rho1-dependent membrane cytoskeleton expansion from the base of pseudocleavage and cellularization furrows, closely mimicking Steppke loss-of-function embryos. Elevating Sstn furrow levels had no effect on the steppke phenotype, but elevating Steppke furrow levels reversed the sstn phenotype, suggesting that Steppke acts downstream of Sstn and that additional mechanisms can recruit Steppke to furrows. Finally, the coiled-coil domain of Steppke was required for Sstn binding and in addition homodimerization, and its removal disrupted Steppke furrow localization and activity in vivo. Overall we propose that Sstn acts as a cytohesin adaptor that promotes Steppke activity for localized membrane cytoskeleton restraint in the syncytial Drosophila embryo.

scholarly journals Phosphatidylethanolamine critically supports internalization of cell-penetrating protein C inhibitor

2007 ◽  
Vol 179 (4) ◽  
pp. 793-804 ◽  
Author(s):  
Petra Baumgärtner ◽  
Margarethe Geiger ◽  
Susanne Zieseniss ◽  
Julia Malleier ◽  
James A. Huntington ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein C ◽  
Docking Studies ◽  
Loss Of Function ◽  
Membrane Translocation ◽  
Nuclear Targeting ◽  
Cell Penetrating ◽  
Protein C Inhibitor

Although their contribution remains unclear, lipids may facilitate noncanonical routes of protein internalization into cells such as those used by cell-penetrating proteins. We show that protein C inhibitor (PCI), a serine protease inhibitor (serpin), rapidly transverses the plasma membrane, which persists at low temperatures and enables its nuclear targeting in vitro and in vivo. Cell membrane translocation of PCI necessarily requires phosphatidylethanolamine (PE). In parallel, PCI acts as a lipid transferase for PE. The internalized serpin promotes phagocytosis of bacteria, thus suggesting a function in host defense. Membrane insertion of PCI depends on the conical shape of PE and is associated with the formation of restricted aqueous compartments within the membrane. Gain- and loss-of-function mutations indicate that the transmembrane passage of PCI requires a branched cavity between its helices H and D, which, according to docking studies, precisely accommodates PE. Our findings show that its specific shape enables cell surface PE to drive plasma membrane translocation of cell-penetrating PCI.

scholarly journals Fracture healing is delayed in the absence of gasdermin signaling

2021 ◽  
Author(s):  
Gabriel Mbalaviele ◽  
Kai Sun ◽  
Chun Wang ◽  
Jianqiu Xiao ◽  
Michael D Brodt ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fracture Healing ◽  
Inflammatory Responses ◽  
Biomechanical Properties ◽  
Amino Terminal ◽  
Membrane Pores ◽  
Bone Injury ◽  
Bone Callus

Amino-terminal fragments from proteolytically cleaved gasdermins (GSDMs) form plasma membrane pores that enable the secretion of interleukin-1β (IL-1β) and IL-18. Excessive GSDM-mediated pore formation can compromise the integrity of the plasma membrane thereby causing the lytic inflammatory cell death, pyroptosis. We found that GSDMD and GSDME were the only GSDMs that were readily expressed in bone microenvironment. Therefore, we tested the hypothesis that GSDMD and GSDME are implicated in fracture healing owing to their role in the obligatory inflammatory response following injury. We found that bone callus volume and biomechanical properties of injured bones were significantly reduced in mice lacking either GSDM compared with wild-type (WT) mice, indicating that fracture healing was compromised in mutant mice. However, compound loss of GSDMD and GSDME did not exacerbate the outcomes, suggesting shared actions of both GSDMs in fracture healing. Mechanistically, bone injury induced IL-1β and IL-18 secretion in vivo, a response that was mimicked in vitro by bone debris and ATP, which function as inflammatory danger signals. Importantly, the secretion of these cytokines was attenuated in conditions of GSDMD deficiency. Finally, deletion of IL-1 receptor reproduced the phenotype of Gsdmd or Gsdme deficient mice, implying that inflammatory responses induced by the GSDM-IL-1 axis promote bone healing after fracture.

scholarly journals RNF141 interacts with KRAS to promote colorectal cancer progression

Oncogene ◽  
2021 ◽  
Author(s):  
Jiuna Zhang ◽  
Xiaoyu Jiang ◽  
Jie Yin ◽  
Shiying Dou ◽  
Xiaoli Xie ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Plasma Membrane ◽  
Tumor Growth ◽  
Cancer Progression ◽  
Critical Role ◽  
Ring Finger ◽  
Immunohistochemical Analysis ◽  
Bimolecular Fluorescence Complementation

AbstractRING finger proteins (RNFs) play a critical role in cancer initiation and progression. RNF141 is a member of RNFs family; however, its clinical significance, roles, and mechanism in colorectal cancer (CRC) remain poorly understood. Here, we examined the expression of RNF141 in 64 pairs of CRC and adjacent normal tissues by real-time PCR, Western blot, and immunohistochemical analysis. We found that there was more expression of RNF141 in CRC tissue compared with its adjacent normal tissue and high RNF141 expression associated with T stage. In vivo and in vitro functional experiments were conducted and revealed the oncogenic role of RNF141 in CRC. RNF141 knockdown suppressed proliferation, arrested the cell cycle in the G1 phase, inhibited migration, invasion and HUVEC tube formation but promoted apoptosis, whereas RNF141 overexpression exerted the opposite effects in CRC cells. The subcutaneous xenograft models showed that RNF141 knockdown reduced tumor growth, but its overexpression promoted tumor growth. Mechanistically, liquid chromatography-tandem mass spectrometry indicated RNF141 interacted with KRAS, which was confirmed by Co-immunoprecipitation, Immunofluorescence assay. Further analysis with bimolecular fluorescence complementation (BiFC) and Glutathione-S-transferase (GST) pull-down assays showed that RNF141 could directly bind to KRAS. Importantly, the upregulation of RNF141 increased GTP-bound KRAS, but its knockdown resulted in a reduction accordingly. Next, we demonstrated that RNF141 induced KRAS activation via increasing its enrichment on the plasma membrane not altering total KRAS expression, which was facilitated by the interaction with LYPLA1. Moreover, KRAS silencing partially abolished the effect of RNF141 on cell proliferation and apoptosis. In addition, our findings presented that RNF141 functioned as an oncogene by upregulating KRAS activity in a manner of promoting KRAS enrichment on the plasma membrane in CRC.

scholarly journals TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Chengwu Xiao ◽  
Wei Zhang ◽  
Meimian Hua ◽  
Huan Chen ◽  
Bin Yang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Prognostic Indicator ◽  
The Cancer Genome Atlas ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Protein Levels ◽  
Kaplan Meier ◽  
Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

scholarly journals β-elemene alleviates airway stenosis via the ILK/Akt pathway modulated by MIR143HG sponging miR-1275

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Guoying Zhang ◽  
Cheng Xue ◽  
Yiming Zeng
Keyword(s):  
Cell Cycle ◽  
Cell Viability ◽  
Cell Model ◽  
Protective Efficacy ◽  
Rabbit Model ◽  
Akt Pathway ◽  
Loss Of Function ◽  
Airway Stenosis

Abstract Background We have previously found that β-elemene could inhibit the viability of airway granulation fibroblasts and prevent airway hyperplastic stenosis. This study aimed to elucidate the underlying mechanism and protective efficacy of β-elemene in vitro and in vivo. Methods Microarray and bioinformatic analysis were used to identify altered pathways related to cell viability in a β-elemene-treated primary cell model and to construct a β-elemene-altered ceRNA network modulating the target pathway. Loss of function and gain of function approaches were performed to examine the role of the ceRNA axis in β-elemene's regulation of the target pathway and cell viability. Additionally, in a β-elemene-treated rabbit model of airway stenosis, endoscopic and histological examinations were used to evaluate its therapeutic efficacy and further verify its mechanism of action. Results The hyperactive ILK/Akt pathway and dysregulated LncRNA-MIR143HG, which acted as a miR-1275 ceRNA to modulate ILK expression, were suppressed in β-elemene-treated airway granulation fibroblasts; β-elemene suppressed the ILK/Akt pathway via the MIR143HG/miR-1275/ILK axis. Additionally, the cell cycle and apoptotic phenotypes of granulation fibroblasts were altered, consistent with ILK/Akt pathway activity. In vivo application of β-elemene attenuated airway granulation hyperplasia and alleviated scar stricture, and histological detections suggested that β-elemene's effects on the MIR143HG/miR-1275/ILK axis and ILK/Akt pathway were in line with in vitro findings. Conclusions MIR143HG and ILK may act as ceRNA to sponge miR-1275. The MIR143HG/miR-1275/ILK axis mediates β-elemene-induced cell cycle arrest and apoptosis of airway granulation fibroblasts by modulating the ILK/Akt pathway, thereby inhibiting airway granulation proliferation and ultimately alleviating airway stenosis.

scholarly journals Heterodimer Formation of the Homodimeric ABC Transporter OpuA

2021 ◽  
Vol 22 (11) ◽  
pp. 5912
Author(s):  
Patricia Alvarez-Sieiro ◽  
Hendrik R. Sikkema ◽  
Bert Poolman
Keyword(s):  
Abc Transporter ◽  
Conformational Dynamics ◽  
Host Organism ◽  
Affinity Tag ◽  
Practical Applications ◽  
Fundamental Research ◽  
Protein Multimerization ◽  
Biochemical Analyses

Many proteins have a multimeric structure and are composed of two or more identical subunits. While this can be advantageous for the host organism, it can be a challenge when targeting specific residues in biochemical analyses. In vitro splitting and re-dimerization to circumvent this problem is a tedious process that requires stable proteins. We present an in vivo approach to transform homodimeric proteins into apparent heterodimers, which then can be purified using two-step affinity-tag purification. This opens the door to both practical applications such as smFRET to probe the conformational dynamics of homooligomeric proteins and fundamental research into the mechanism of protein multimerization, which is largely unexplored for membrane proteins. We show that expression conditions are key for the formation of heterodimers and that the order of the differential purification and reconstitution of the protein into nanodiscs is important for a functional ABC-transporter complex.

scholarly journals Human Cd25+Cd4+ T Regulatory Cells Suppress Naive and Memory T Cell Proliferation and Can Be Expanded in Vitro without Loss of Function

2001 ◽  
Vol 193 (11) ◽  
pp. 1295-1302 ◽  
Author(s):  
Megan K. Levings ◽  
Romina Sangregorio ◽  
Maria-Grazia Roncarolo
Keyword(s):  
T Cell ◽  
Peripheral Blood ◽  
T Regulatory Cells ◽  
Transforming Growth Factor ◽  
Cytotoxic T Lymphocyte ◽  
Cell Receptor ◽  
Major Advance ◽  
Loss Of Function

Active suppression by T regulatory (Tr) cells plays an important role in the downregulation of T cell responses to foreign and self-antigens. Mouse CD4+ Tr cells that express CD25 possess remarkable suppressive activity in vitro and in autoimmune disease models in vivo. Thus far, the existence of a similar subset of CD25+CD4+ Tr cells in humans has not been reported. Here we show that human CD25+CD4+ Tr cells isolated from peripheral blood failed to proliferate and displayed reduced expression of CD40 ligand (CD40L), in response to T cell receptor–mediated polyclonal activation, but strongly upregulated cytotoxic T lymphocyte–associated antigen (CTLA)-4. Human CD25+CD4+ Tr cells also did not proliferate in response to allogeneic antigen-presenting cells, but they produced interleukin (IL)-10, transforming growth factor (TGF)-β, low levels of interferon (IFN)-γ, and no IL-4 or IL-2. Importantly, CD25+CD4+ Tr cells strongly inhibited the proliferative responses of both naive and memory CD4+ T cells to alloantigens, but neither IL-10, TGF-β, nor CTLA-4 seemed to be directly required for their suppressive effects. CD25+CD4+ Tr cells could be expanded in vitro in the presence of IL-2 and allogeneic feeder cells and maintained their suppressive capacities. These findings that CD25+CD4+ Tr cells with immunosuppressive effects can be isolated from peripheral blood and expanded in vitro without loss of function represent a major advance towards the therapeutic use of these cells in T cell–mediated diseases.

scholarly journals Long noncoding RNA LINC00941 promotes pancreatic cancer progression by competitively binding miR-335-5p to regulate ROCK1-mediated LIMK1/Cofilin-1 signaling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jie Wang ◽  
Zhiwei He ◽  
Jian Xu ◽  
Peng Chen ◽  
Jianxin Jiang
Keyword(s):  
Pancreatic Cancer ◽  
Cell Growth ◽  
Cell Lines ◽  
Cancer Progression ◽  
Noncoding Rna ◽  
Epithelial Mesenchymal Transition ◽  
Loss Of Function ◽  
Mesenchymal Transition

AbstractAn accumulation of evidence indicates that long noncoding RNAs are involved in the tumorigenesis and progression of pancreatic cancer (PC). In this study, we investigated the functions and molecular mechanism of action of LINC00941 in PC. Quantitative PCR was used to examine the expression of LINC00941 and miR-335-5p in PC tissues and cell lines, and to investigate the correlation between LINC00941 expression and clinicopathological features. Plasmid vectors or lentiviruses were used to manipulate the expression of LINC00941, miR-335-5p, and ROCK1 in PC cell lines. Gain or loss-of-function assays and mechanistic assays were employed to verify the roles of LINC00941, miR-335-5p, and ROCK1 in PC cell growth and metastasis, both in vivo and in vitro. LINC00941 and ROCK1 were found to be highly expressed in PC, while miR-335-5p exhibited low expression. High LINC00941 expression was strongly associated with larger tumor size, lymph node metastasis, and poor prognosis. Functional experiments revealed that LINC00941 silencing significantly suppressed PC cell growth, metastasis and epithelial–mesenchymal transition. LINC00941 functioned as a molecular sponge for miR-335-5p, and a competitive endogenous RNA (ceRNA) for ROCK1, promoting ROCK1 upregulation, and LIMK1/Cofilin-1 pathway activation. Our observations lead us to conclude that LINC00941 functions as an oncogene in PC progression, behaving as a ceRNA for miR-335-5p binding. LINC00941 may therefore have potential utility as a diagnostic and treatment target in this disease.

scholarly journals Norepinephrine Protects against Methamphetamine Toxicity through β2-Adrenergic Receptors Promoting LC3 Compartmentalization

2021 ◽  
Vol 22 (13) ◽  
pp. 7232
Author(s):  
Gloria Lazzeri ◽  
Carla L. Busceti ◽  
Francesca Biagioni ◽  
Cinzia Fabrizi ◽  
Gabriele Morucci ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Light Chain ◽  
Adrenergic Receptors ◽  
Cell Damage ◽  
Protective Effects ◽  
Autophagy Flux ◽  
Brain Areas ◽  
Indirect Consequence

Norepinephrine (NE) neurons and extracellular NE exert some protective effects against a variety of insults, including methamphetamine (Meth)-induced cell damage. The intimate mechanism of protection remains difficult to be analyzed in vivo. In fact, this may occur directly on target neurons or as the indirect consequence of NE-induced alterations in the activity of trans-synaptic loops. Therefore, to elude neuronal networks, which may contribute to these effects in vivo, the present study investigates whether NE still protects when directly applied to Meth-treated PC12 cells. Meth was selected based on its detrimental effects along various specific brain areas. The study shows that NE directly protects in vitro against Meth-induced cell damage. The present study indicates that such an effect fully depends on the activation of plasma membrane β2-adrenergic receptors (ARs). Evidence indicates that β2-ARs activation restores autophagy, which is impaired by Meth administration. This occurs via restoration of the autophagy flux and, as assessed by ultrastructural morphometry, by preventing the dissipation of microtubule-associated protein 1 light chain 3 (LC3) from autophagy vacuoles to the cytosol, which is produced instead during Meth toxicity. These findings may have an impact in a variety of degenerative conditions characterized by NE deficiency along with autophagy impairment.

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