scholarly journals The TPLATE complex mediates membrane bending during plant clathrin–mediated endocytosis

2021 ◽  
Vol 118 (51) ◽  
pp. e2113046118
Author(s):  
Alexander Johnson ◽  
Dana A. Dahhan ◽  
Nataliia Gnyliukh ◽  
Walter A. Kaufmann ◽  
Vanessa Zheden ◽  
...  
Keyword(s):  
Turgor Pressure ◽  
Model Systems ◽  
Physiological Processes ◽  
Major Route ◽  
Flat Membrane ◽  
Membrane Bending ◽  
Intracellular Forces ◽  
Unique Mechanism

Clathrin-mediated endocytosis is the major route of entry of cargos into cells and thus underpins many physiological processes. During endocytosis, an area of flat membrane is remodeled by proteins to create a spherical vesicle against intracellular forces. The protein machinery which mediates this membrane bending in plants is unknown. However, it is known that plant endocytosis is actin independent, thus indicating that plants utilize a unique mechanism to mediate membrane bending against high-turgor pressure compared to other model systems. Here, we investigate the TPLATE complex, a plant-specific endocytosis protein complex. It has been thought to function as a classical adaptor functioning underneath the clathrin coat. However, by using biochemical and advanced live microscopy approaches, we found that TPLATE is peripherally associated with clathrin-coated vesicles and localizes at the rim of endocytosis events. As this localization is more fitting to the protein machinery involved in membrane bending during endocytosis, we examined cells in which the TPLATE complex was disrupted and found that the clathrin structures present as flat patches. This suggests a requirement of the TPLATE complex for membrane bending during plant clathrin–mediated endocytosis. Next, we used in vitro biophysical assays to confirm that the TPLATE complex possesses protein domains with intrinsic membrane remodeling activity. These results redefine the role of the TPLATE complex and implicate it as a key component of the evolutionarily distinct plant endocytosis mechanism, which mediates endocytic membrane bending against the high-turgor pressure in plant cells.

scholarly journals The TPLATE complex mediates membrane bending during plant clathrin-mediated endocytosis

2021 ◽  
Author(s):  
Alexander Johnson ◽  
Dana A Dahhan ◽  
Nataliia Gnyliukh ◽  
Walter A Kaufmann ◽  
Vanessa Zheden ◽  
...  
Keyword(s):  
Turgor Pressure ◽  
Plant Cells ◽  
Distinct Mechanism ◽  
Distinct Solution ◽  
Membrane Bending ◽  
Underlying Mechanisms ◽  
Endocytic Vesicles ◽  
Spherical Vesicles

AbstractClathrin-mediated endocytosis in plants is an essential process but the underlying mechanisms are poorly understood, not least because of the extreme intracellular turgor pressure acting against the formation of endocytic vesicles. In contrast to other models, plant endocytosis is independent of actin, indicating a mechanistically distinct solution. Here, by using biochemical and advanced microscopy approaches, we show that the plant-specific TPLATE complex acts outside of endocytic vesicles as a mediator of membrane bending. Cells with disrupted TPLATE fail to generate spherical vesicles, and in vitro biophysical assays identified protein domains with membrane bending capability. These results redefine the role of the TPLATE complex as a key component of the evolutionarily distinct mechanism mediating membrane bending against high turgor pressure to drive endocytosis in plant cells.One Sentence SummaryWhile plant CME is actin independent, we identify that the evolutionarily ancient octameric TPLATE complex mediates membrane bending against high turgor pressure in plant clathrin-mediated endocytosis.

scholarly journals Closer to Nature: The Role of MSCs in Recreating the Microenvironment of the Hematopoietic Stem Cell Niche in vitro

2022 ◽  
pp. 1-10
Author(s):  
Patrick Wuchter ◽  
Anke Diehlmann ◽  
Harald Klüter
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stem Cell Niche ◽  
Model Systems ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Hematopoietic Stem Cell Niche ◽  
Cell Niche

<b><i>Background:</i></b> The stem cell niche in human bone marrow provides scaffolds, cellular frameworks and essential soluble cues to support the stemness of hematopoietic stem and progenitor cells (HSPCs). To decipher this complex structure and the corresponding cellular interactions, a number of in vitro model systems have been developed. The cellular microenvironment is of key importance, and mesenchymal stromal cells (MSCs) represent one of the major cellular determinants of the niche. Regulation of the self-renewal and differentiation of HSPCs requires not only direct cellular contact and adhesion molecules, but also various cytokines and chemokines. The C-X-C chemokine receptor type 4/stromal cell-derived factor 1 axis plays a pivotal role in stem cell mobilization and homing. As we have learned in recent years, to realistically simulate the physiological in vivo situation, advanced model systems should be based on niche cells arranged in a three-dimensional (3D) structure. By providing a dynamic rather than static setup, microbioreactor systems offer a number of advantages. In addition, the role of low oxygen tension in the niche microenvironment and its impact on hematopoietic stem cells need to be taken into account and are discussed in this review. <b><i>Summary:</i></b> This review focuses on the role of MSCs as a part of the bone marrow niche, the interplay between MSCs and HSPCs and the most important regulatory factors that need to be considered when engineering artificial hematopoietic stem cell niche systems. <b><i>Conclusion:</i></b> Advanced 3D model systems using MSCs as niche cells and applying microbioreactor-based technology are capable of simulating the natural properties of the bone marrow niche more closely than ever before.

scholarly journals MicroRNAs in the tumour microenvironment: big role for small players

2013 ◽  
Vol 20 (5) ◽  
pp. R257-R267 ◽  
Author(s):  
Patsy Soon ◽  
Hippokratis Kiaris
Keyword(s):  
Clinical Characteristics ◽  
Experimental Studies ◽  
Anticancer Therapy ◽  
Tumour Microenvironment ◽  
Regulatory Role ◽  
Tumour Stroma ◽  
Physiological Processes ◽  
Non Coding Rnas

MicroRNAs (miRNAs) represent a class of small non-coding RNAs with an important regulatory role in various physiological processes as well as in several pathologies including cancers. It is noteworthy that recent evidence suggests that the regulatory role of miRNAs during carcinogenesis is not limited to the cancer cells but they are also implicated in the activation of tumour stroma and its transition into a cancer-associated state. Results from experimental studies involving cells culturedin vitroand mice bearing experimental tumours, corroborated by profiling of clinical cancers for miRNA expression, underline this role and identify miRNAs as a potent regulator of the crosstalk between cancer and stroma cells. Considering the fundamental role of the tumour microenvironment in determining both the clinical characteristics of the disease and the efficacy of anticancer therapy, miRNAs emerge as an attractive target bearing important prognostic and therapeutic significance during carcinogenesis. In this article, we will review the available results that underline the role of miRNAs in tumour stroma biology and emphasise their potential value as tools for the management of the disease.

scholarly journals Characterization of the Urease Operon of Brucella abortus and Assessment of Its Role in Virulence of the Bacterium

2006 ◽  
Vol 75 (2) ◽  
pp. 774-780 ◽  
Author(s):  
Félix J. Sangari ◽  
Asunción Seoane ◽  
María Cruz Rodríguez ◽  
Jesús Agüero ◽  
Juan M. García Lobo
Keyword(s):  
Brucella Abortus ◽  
Urease Activity ◽  
Strong Acid ◽  
Oral Route ◽  
Open Reading Frames ◽  
Human Brucellosis ◽  
Major Route

ABSTRACT Most members of the genus Brucella show strong urease activity. However, the role of this enzyme in the pathogenesis of Brucella infections is poorly understood. We isolated several Tn5 insertion mutants deficient in urease activity from Brucella abortus strain 2308. The mutations of most of these mutants mapped to a 5.7-kbp DNA region essential for urease activity. Sequencing of this region, designated ure1, revealed the presence of seven open reading frames corresponding to the urease structural proteins (UreA, UreB, and UreC) and the accessory proteins (UreD, UreE, UreF, and UreG). In addition to the urease genes, another gene (cobT) was identified, and inactivation of this gene affected urease activity in Brucella. Subsequent analysis of the previously described sequences of the genomes of Brucella spp. revealed the presence of a second urease cluster, ure2, in all them. The ure2 locus was apparently inactive in B. abortus 2308. Urease-deficient mutants were used to evaluate the role of urease in Brucella pathogenesis. The urease-producing strains were found to be resistant in vitro to strong acid conditions in the presence of urea, while urease-negative mutants were susceptible to acid treatment. Similarly, the urease-negative mutants were killed more efficiently than the urease-producing strains during transit through the stomach. These results suggested that urease protects brucellae during their passage through the stomach when the bacteria are acquired by the oral route, which is the major route of infection in human brucellosis.

scholarly journals GABAA receptor currents in the dorsal motor nucleus of the vagus in females: influence of ovarian cycle and 5α-reductase inhibition

2019 ◽  
Vol 122 (5) ◽  
pp. 2130-2141
Author(s):  
Erica L. Littlejohn ◽  
Liliana Espinoza ◽  
Monica M. Lopez ◽  
Bret N. Smith ◽  
Carie R. Boychuk
Keyword(s):  
Sex Differences ◽  
Motor Neurons ◽  
Ovarian Cycle ◽  
Receptor Activity ◽  
Motor Nucleus ◽  
Gabaergic Neurotransmission ◽  
Physiological Processes ◽  
Dorsal Motor Nucleus

The dorsal motor nucleus of the vagus (DMV) contains the preganglionic motor neurons important in the regulation of glucose homeostasis and gastrointestinal function. Despite the role of sex in the regulation of these processes, few studies examine the role of sex and/or ovarian cycle in the regulation of synaptic neurotransmission to the DMV. Since GABAergic neurotransmission is critical to normal DMV function, the present study used in vitro whole cell patch-clamping to investigate whether sex differences exist in GABAergic neurotransmission to DMV neurons. It additionally investigated whether the ovarian cycle plays a role in those sex differences. The frequency of phasic GABAA receptor-mediated inhibitory postsynaptic currents in DMV neurons from females was lower compared with males, and this effect was TTX sensitive and abolished by ovariectomy (OVX). Amplitudes of GABAergic currents (both phasic and tonic) were not different. However, females demonstrated significantly more variability in the amplitude of both phasic and tonic GABAA receptor currents. This difference was eliminated by OVX in females, suggesting that these differences were related to reproductive hormone levels. This was confirmed for GABAergic tonic currents by comparing females in two ovarian stages, estrus versus diestrus. Female mice in diestrus had larger tonic current amplitudes compared with those in estrus, and this increase was abolished after administration of a 5α-reductase inhibitor but not modulation of estrogen. Taken together, these findings demonstrate that DMV neurons undergo GABAA receptor activity plasticity as a function of sex and/or sex steroids. NEW & NOTEWORTHY Results show that GABAergic signaling in dorsal vagal motor neurons (DMV) demonstrates sex differences and fluctuates across the ovarian cycle in females. These findings are the first to demonstrate that female GABAA receptor activity in this brain region is modulated by 5α-reductase-dependent hormones. Since DMV activity is critical to both glucose and gastrointestinal homeostasis, these results suggest that sex hormones, including those synthesized by 5α-reductase, contribute to visceral, autonomic function related to these physiological processes.

scholarly journals Antibody response to the 45 kDa Candida albicans antigen in an animal model and potential role of the antigen in adherence

2008 ◽  
Vol 57 (12) ◽  
pp. 1466-1472 ◽  
Author(s):  
Helena Bujdáková ◽  
Ema Paulovičová ◽  
Silvia Borecká-Melkusová ◽  
Juraj Gašperík ◽  
Soňa Kucharíková ◽  
...  
Keyword(s):  
Animal Model ◽  
Candida Albicans ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Vaccine Development ◽  
Laser Scanning Microscopy ◽  
Model Systems ◽  
Confocal Laser

The Candida antigen CR3-RP (complement receptor 3-related protein) is supposed to be a ‘mimicry’ protein because of its ability to bind antibody directed against the α subunit of the mammalian CR3 (CD11b/CD18). This study aimed to (i) investigate the specific humoral isotypic response to immunization with CR3-RP in vivo in a rabbit animal model, and (ii) determine the role of CR3-RP in the adherence of Candida albicans in vitro using the model systems of buccal epithelial cells (BECs) and biofilm formation. The synthetic C. albicans peptide DINGGGATLPQ corresponding to 11 amino-acids of the CR3-RP sequence DINGGGATLPQALXQITGVIT, determined by N-terminal sequencing, was used for immunization of rabbits to obtain polyclonal anti-CR3-PR serum and for subsequent characterization of the humoral isotypic response of rabbits. A significant increase of IgG, IgA and IgM anti-CR3-RP specific antibodies was observed after the third (P<0.01) and the fourth (P<0.001) immunization doses. The elevation of IgA levels suggested peptide immunomodulation of the IgA1 subclass, presumably in coincidence with Candida epithelial adherence. Blocking CR3-RP with polyclonal anti-CR3-RP serum reduced the ability of Candida to adhere to BECs, in comparison with the control, by up to 35 % (P<0.001), and reduced biofilm formation by 28 % (P<0.001), including changes in biofilm thickness and integrity detected by confocal laser scanning microscopy. These properties of CR3-RP suggest that it has potential for future vaccine development.

scholarly journals MicroRNA-34a induces endothelial progenitor cell senescence and impedes its angiogenesis via suppressing silent information regulator 1

2010 ◽  
Vol 299 (1) ◽  
pp. E110-E116 ◽  
Author(s):  
Ting Zhao ◽  
Jian Li ◽  
Alex F. Chen
Keyword(s):  
Progenitor Cell ◽  
Stem Cell Markers ◽  
Endothelial Progenitor ◽  
Cell Markers ◽  
Physiological Processes ◽  
Cycle Arrest ◽  
Forkhead Box ◽  
Silent Information Regulator 1

Endothelial progenitor cells (EPCs) play an important role in angiogenesis, which is essential for numerous physiological processes as well as tumor growth. Several microRNAs (miRNAs) have been reported to be involved in angiogenesis. MiR-34a, recently reported as a tumor suppressor, has been found to target silent information regulator 1 (Sirt1), leading to cell cycle arrest or apoptosis. However, the role of miR-34a in EPC-mediated angiogenesis was unknown. The present study tested the hypothesis that miR-34a inhibits EPC-mediated angiogenesis by inducing senescence via suppressing Sirt1. Bone marrow-derived EPCs from adult male Spraque-Dawley rats were used. Results of flow cytometry showed that EPCs after 7 days of culture expressed both stem cell markers CD34 and CD133 and endothelial cell markers VEGFR-2 (flk-1) and VE-cadherin. MiR-34a was expressed in normal EPCs, and overexpression of miR-34a via its mimic transfection significantly increased its expression and impaired in vitro EPC angiogenesis. MiR-34a overexpression led to a significantly increased EPC senescence, paralleled with an ∼40% Sirt1 reduction. Furthermore, knockdown of Sirt1 by its siRNA resulted in diminished EPC angiogenesis and increased senescence. Finally, overexpression of miR-34a increased the level of Sirt1 effector-acetylated forkhead box O transcription factors 1 (FoxO1), an effect mimicked in EPCs following Sirt1 knockdown. In conclusion, miR-34a impairs EPC-mediated angiogenesis by induction of senescence via inhibiting Sirt1.

scholarly journals HIV Tat-Mediated Induction of Monocyte Transmigration Across the Blood–Brain Barrier: Role of Chemokine Receptor CXCR3

2021 ◽  
Vol 9 ◽  
Author(s):  
Fang Niu ◽  
Ke Liao ◽  
Guoku Hu ◽  
Shamsudheen Moidunny ◽  
Sabita Roy ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
Chemokine Receptor ◽  
Nuclear Translocation ◽  
Model Systems ◽  
Brain Barrier ◽  
Human Monocytes ◽  
Blood Brain ◽  
Hiv Tat

HIV trans-activator of transcription (Tat), one of the cytotoxic proteins secreted from HIV-infected cells, is also known to facilitate chemokine-mediated transmigration of monocytes into the brain leading, in turn, to neuroinflammation and thereby contributing to the development of HIV-associated neurocognitive disorders (HAND). The mechanism(s) underlying HIV Tat-mediated enhancement of monocyte transmigration, however, remain largely unknown. CXC chemokine receptor 3 (CXCR3) that is expressed by the peripheral monocytes is known to play a role in the monocyte influx and accumulation. In the present study, we demonstrate for the first time that exposure of human monocytes to HIV Tat protein resulted in upregulated expression of CXCR3 leading, in turn, to increased monocyte transmigration across the blood–brain barrier (BBB) both in the in vitro and in vivo model systems. This process involved activation of toll-like receptor 4 (TLR4), with downstream phosphorylation and activation of TANK-binding kinase 1 (TBK1), and subsequent phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3), ultimately leading to enhanced expression of CXCR3 in human monocytes. These findings imply a novel molecular mechanism underlying HIV Tat-mediated increase of monocyte transmigration across the BBB, while also implicating a novel role of CXCR3-dependent monocyte transmigration in HIV Tat-mediated neuroinflammation.

scholarly journals EOMES is responsible for WNT memory and can substitute for WNT in mesendoderm specification

2021 ◽  
Author(s):  
Anna Yoney ◽  
Lu Bai ◽  
Ali H. Brivanlou ◽  
Eric D Siggia
Keyword(s):  
Target Genes ◽  
Embryonic Stem ◽  
Model Systems ◽  
Mechanistic Basis ◽  
Vitro Model ◽  
Transcriptional Output ◽  
Accessible Chromatin ◽  
Pluripotency Maintenance

Embryogenesis is guided by a limited set of signaling pathways that are reused at different times and places throughout development. How a context dependent signaling response is generated has been a central question of developmental biology, which can now be addressed with in vitro model systems. Our previous work in human embryonic stem cells (hESCs) established that pre-exposure of cells to WNT/β-catenin signaling is sufficient to switch the output of ACTIVIN/SMAD2 signaling from pluripotency maintenance to mesendoderm (ME) differentiation. A body of previous literature has established the role of both pathways in ME differentiation. However, our work demonstrated that the two signals do not need to be present simultaneously and that hESCs have a means to record WNT signals. Here we demonstrate that hESCs have accessible chromatin at SMAD2 binding sites near pluripotency and ME-associated target genes and that WNT priming does not alter SMAD2 binding. Rather our results indicate that stable transcriptional output at ME genes results from WNT-dependent production of an additional SMAD2 co-factor, EOMES. We show that expression of EOMES can replace WNT signaling in ME differentiation, providing a mechanistic basis for WNT-priming and memory in early development.

PDIA3 Reduce Glioma-associated Macrophage/microglia Pro-tumor Activation trough IL-6-STAT3-PDIA3 Pathway.

2020 ◽  
Author(s):  
Marta Chiavari ◽  
Gabriella Maria Pia Ciotti ◽  
Francesco Canonico ◽  
Fabio Altieri ◽  
Pierluigi Navarra ◽  
...  
Keyword(s):  
Overall Survival ◽  
Endoplasmic Reticulum ◽  
The Cancer Genome Atlas ◽  
Main Function ◽  
Physiological Processes ◽  
Relevant Role ◽  
Cancer Genome Atlas ◽  
Thiol Oxidoreductase

Abstract Background: Glioblastoma (GB - grade IV glioma) is the most aggressive and common cancer of central nervous system with an overall survival of 14-16 months. The GB tumor microenvironment includes cells of the innate immune system identified as glioma-associated microglia/macrophages (GAMs). It is known that between GAMs and GB cells there is a double interaction, but the role of GAMs is still poorly characterized. The endoplasmic reticulum (ER) protein ERp57, also known as PDIA3, is a thiol oxidoreductase with main function related on glycoprotein folding in endoplasmic reticulum. However, PDIA3 shows different functions. In fact, the various subcellular localizations and binding partners of PDIA3 affect numerous physiological processes and diseases: different regulation and modulation of PDIA3 has been reported in multiple pathologies including neurodegenerative diseases and cancer. Methods: In the present work, we evaluated in both GB cells and microglia-macrophage cells the expression of PDIA3 using specimens collected after surgical from 18 GB patients. In addition, we studied in vitro microglia-glioma interaction to determine the role of PDIA3 in viability and the activation of both GB and microglia cells. The study was carried using PDIA3-silenced T98G cells and/or using a pharmacological inhibitor of PDIA3 activity (Punicalagin-PUN).Results: We initially investigated the role of the PDIA3 in GB survival by inquiring The Cancer Genome Atlas dataset. The results indicated that 352 out of 690 patients reported over-expression of PDIA3, which significantly correlated with a ~55% reduction of overall survival. Subsequently, for the first time, we investigated the PDIA3 expression in the tumor and the nearby parenchyma of 18 GB patients and our data showed a significant upregulation (15% vs 10%) of ERp57/PDIA3 in GAMs of tumor specimens respect the microglia present in parenchyma. In addition, we show that conditioned medium (CMs) obtained from both wild type T98G and PDIA3 silenced T98G induced an activation of microglia cells, but the PDIA3 silenced-T98G CMs significant limited the microglia pro-tumor activation probably through a IL-6-STAT3-PDIA3 dependent mechanism. Conclusion: Our data support the relevant role of PDIA3 expression in GB pathology and link the different activation of microglia to a mechanism a IL-6-STAT3-PDIA3 dependent.

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