Effect of tolytoxin on tunneling nanotube formation and function.
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ABSTRACT:
SUBMITTER: Dilsizoglu Senol A
PROVIDER: S-EPMC6450976 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Tunneling nanotubes (TNTs) are cellular extensions enabling cytosol-to-cytosol intercellular interaction between numerous cell types including macrophages. Previous studies of hematopoietic stem and progenitor cell (HSPC) transplantation for the lysosomal storage disorder cystinosis have shown that HSPC-derived macrophages form TNTs to deliver cystinosin-bearing lysosomes to cystinotic cells, leading to tissue preservation. Here, we explored if macrophage polarization to either proinflammatory M1-like M(LPS/IFN?) or anti-inflammatory M2-like M(IL-4/IL-10) affected TNT-like protrusion formation, intercellular transport and, ultimately, the efficacy of cystinosis prevention. We designed new automated image processing algorithms used to demonstrate that LPS/IFN? polarization decreased bone marrow-derived macrophages (BMDMs) formation of protrusions, some of which displayed characteristics of TNTs, including cytoskeletal structure, 3D morphology and size. In contrast, co-culture of macrophages with cystinotic fibroblasts yielded more frequent and larger protrusions, as well as increased lysosomal and mitochondrial intercellular trafficking to the diseased fibroblasts. Unexpectedly, we observed normal protrusion formation and therapeutic efficacy following disruption of anti-inflammatory IL-4/IL-10 polarization in vivo by transplantation of HSPCs isolated from the Rac2-/- mouse model. Altogether, we developed unbiased image quantification systems that probe mechanistic aspects of TNT formation and function in vitro, while HSPC transplantation into cystinotic mice provides a complex in vivo disease model. While the differences between polarization cell culture and mouse models exemplify the oversimplicity of in vitro cytokine treatment, they simultaneously demonstrate the utility of our co-culture model which recapitulates the in vivo phenomenon of diseased cystinotic cells stimulating thicker TNT formation and intercellular trafficking from macrophages. Ultimately, we can use both approaches to expand the utility of TNT-like protrusions as a delivery system for regenerative medicine.
| S-EPMC6787037 | biostudies-literature
Project description:In this study, we demonstrated that hypoxic conditions stimulated an increase in tunneling nanotube (TNT) formation in chemoresistant ovarian cancer cells (SKOV3, C200).We found that suppressing the mTOR pathway using either everolimus or metformin led to suppression of TNT formation in vitro, verifying TNTs as a potential target for cancer-directed therapy. Additionally, TNT formation was detected in co-cultures including between platinum-resistant SKOV3 cells, between SKOV3 cells and platinum-chemosensitive A2780 cells, and between SKOV3 cells cultured with benign ovarian epithelial (IOSE) cells; these findings indicate that TNTs are novel conduits for malignant cell interactions and tumor cell interactions with other cells in the microenvironment. When chemoresistant C200 and parent chemosensitive A2780 cells were co-cultured, chemoresistant cells displayed a higher likelihood of TNT formation to each other than to chemosensitive malignant or benign epithelial cells. Hypoxia-induced TNT formation represents a potential mechanism for intercellular communication in ovarian cancer and other forms of invasive refractory cancers.
| S-EPMC5190014 | biostudies-literature
Project description:Tunneling nanotubes (TNTs) are membrane conduits that mediate long-distance intercellular cross-talk in several organisms and play vital roles during development, pathogenic transmission, and cancer metastasis. However, the molecular mechanisms of TNT formation and function remain poorly understood. The protein MSec (also known as TNF?-induced protein 2 (TNFAIP2) and B94) is essential for TNT formation in multiple cell types. Here, using affinity protein purification, mass spectrometric identification, and confocal immunofluorescence microscopy assays, we found that MSec interacts with the endoplasmic reticulum (ER) chaperone ERp29. siRNA-mediated ERp29 depletion in mammalian cells significantly reduces TNT formation, whereas its overexpression induces TNT formation, but in a strictly MSec-dependent manner. ERp29 stabilized MSec protein levels, but not its mRNA levels, and the chaperone activity of ERp29 was required for maintaining MSec protein stability. Subcellular ER fractionation and subsequent limited proteolytic treatment suggested that MSec is associated with the outer surface of the ER. The ERp29-MSec interaction appeared to require the presence of other bridging protein(s), perhaps triggered by post-translational modification of ERp29. Our study implicates MSec as a target of ERp29 and reveals an indispensable role for the ER in TNT formation, suggesting new modalities for regulating TNT numbers in cells and tissues.
| S-EPMC6509506 | biostudies-literature
Project description:Tunneling NanoTubes (TNTs) are membrane conduits that mediate long distance intercellular crosstalk in several organisms and play vital roles during development, pathogenic transmission and cancer metastasis. However, the molecular mechanisms of TNT formation and function remain poorly understood. The protein MSec is essential for TNT formation in multiple cell types. We identified a novel interaction of MSec with the endoplasmic reticulum chaperone ERp29. ERp29 depletion in mammalian cells led to a significant reduction in TNT formation, while its over-expression induced TNT formation, but strictly in an MSec-dependent manner. ERp29 stabilized MSec protein levels but not its mRNA levels, and the chaperone activity of ERp29 was required for maintaining MSec protein stability. Our study implicates MSec as a new target of ERp29 and reveals an indispensable role for the endoplasmic reticulum in the biogenesis of TNTs, thus suggesting new modalities for regulating TNT numbers.
2019-03-15 | PXD012441 | Pride
Project description:Tunneling nanotubes (TNTs) connect distant cells and mediate cargo transfer for intercellular communication in physiological and pathological contexts. How cells generate these actin-mediated protrusions to span lengths beyond those attainable by canonical filopodia remains unknown. Through a combination of micropatterning, microscopy and optical tweezer-based approaches, we demonstrate that tunneling nanotubes formed through the outward extension of actin achieve distances greater than the mean length of filopodia, and that branched Arp2/3-dependent pathways attenuate the extent to which actin polymerizes in nanotubes, thus limiting their occurrence. Proteomic analysis using Epidermal growth factor receptor kinase substrate 8 (Eps8) as a positive effector of tunneling nanotubes showed that, upon Arp2/3 inhibition, proteins enhancing filament turnover and depolymerization were reduced and Eps8 instead exhibited heightened interactions with the inverted Bin/Amphiphysin/Rvs (I-BAR) domain protein IRSp53 that provides a direct connection with linear actin polymerases. Our data reveals how common protrusion players (Eps8 and IRSp53) form tunneling nanotubes, and that when competing pathways overutilizing such proteins and monomeric actin in Arp2/3 networks are inhibited, processes promoting linear actin growth dominate to favour tunneling nanotube formation.
| S-SCDT-10_15252-EMBJ_2023113761 | biostudies-other
Project description:BackgroundBy allowing intercellular communication between cells, tunneling nanotubes (TNTs) could play critical role in cancer progression. If TNT formation is known to require cytoskeleton remodeling, key mechanism controlling their formation remains poorly understood.MethodsThe cells of human bronchial (HBEC-3, A549) or mesothelial (H2452, H28) lines are transfected with different siRNAs (inactive, anti-RASSF1A, anti-GEFH1 and / or anti-Rab11). At 48 h post-transfection, i) the number and length of the nanotubes per cell are quantified, ii) the organelles, previously labeled with specific tracers, exchanged via these structures are monitored in real time between cells cultured in 2D or 3D and in normoxia, hypoxia or in serum deprivation condition.ResultsWe report that RASSF1A, a key-regulator of cytoskeleton encoded by a tumor-suppressor gene on 3p chromosome, is involved in TNTs formation in bronchial and pleural cells since controlling proper activity of RhoB guanine nucleotide exchange factor, GEF-H1. Indeed, the GEF-H1 inactivation induced by RASSF1A silencing, leads to Rab11 accumulation and subsequent exosome releasing, which in turn contribute to TNTs formation. Finally, we provide evidence involving TNT formation in bronchial carcinogenesis, by reporting that hypoxia or nutriment privation, two almost universal conditions in human cancers, fail to prevent TNTs induced by the oncogenic RASSF1A loss of expression.ConclusionsThis finding suggests for the first time that loss of RASSF1A expression could be a potential biomarker for TNTs formation, such TNTs facilitating intercellular communication favoring multistep progression of bronchial epithelial cells toward overt malignancy.
| S-EPMC6180646 | biostudies-literature
Project description:The tuberculosis (TB) bacillus, Mycobacterium tuberculosis (Mtb), and HIV-1 act synergistically; however, the mechanisms by which Mtb exacerbates HIV-1 pathogenesis are not well known. Using in vitro and ex vivo cell culture systems, we show that human M(IL-10) anti-inflammatory macrophages, present in TB-associated microenvironment, produce high levels of HIV-1. In vivo, M(IL-10) macrophages are expanded in lungs of co-infected non-human primates, which correlates with disease severity. Furthermore, HIV-1/Mtb co-infected patients display an accumulation of M(IL-10) macrophage markers (soluble CD163 and MerTK). These M(IL-10) macrophages form direct cell-to-cell bridges, which we identified as tunneling nanotubes (TNTs) involved in viral transfer. TNT formation requires the IL-10/STAT3 signaling pathway, and targeted inhibition of TNTs substantially reduces the enhancement of HIV-1 cell-to-cell transfer and overproduction in M(IL-10) macrophages. Our study reveals that TNTs facilitate viral transfer and amplification, thereby promoting TNT formation as a mechanism to be explored in TB/AIDS potential therapeutics.
| S-EPMC6733268 | biostudies-literature
Project description:A tunneling nanotube (TNT) is a newly discovered structure involved in cell-cell communication and is found in various types of cells. Here we identify S100A4 as an extracellular molecule and describe its role in attracting the growth direction of TNTs. Together with its putative receptor, receptor for advanced glycation end product, we demonstrate their involvement in TNT direction guidance. Our results further suggest a mechanism for direction guidance of TNTs. In TNT-initiating cells, p53 activates caspase-3, which leads to S100A4 cleavage and its subsequent decrease in cellular concentration. The decrease in cellular S100A4 induces the formation of a gradient of S100A4, from a low concentration in initiating cells toward a high concentration in target cells. This concentration gradient of S100A4 induces direction guidance for TNTs.
| S-EPMC3542613 | biostudies-literature
Project description:Acute myeloid leukemia (AML) is a bone marrow derived blood cancer where intercellular communication in the leukemic bone marrow participates in disease development, progression and chemoresistance. Tunneling nanotubes (TNTs) are intercellular communication structures involved in transport of cellular contents and pathogens, also demonstrated to play a role in both cell death modulation and chemoresistance. Here we investigated the presence of TNTs by live fluorescent microscopy and identified TNT formation between primary AML cells and in AML cell lines. We found that NF-κB activity was involved in TNT regulation and formation. Cytarabine downregulated TNTs and inhibited NF-κB alone and in combination with daunorubicin, providing additional support for involvement of the NF-κB pathway in TNT formation. Interestingly, daunorubicin was found to localize to lysosomes in TNTs connecting AML cells indicating a novel function of TNTs as drug transporting devices. We conclude that TNT communication could reflect important biological features of AML that may be explored in future therapy development.
| S-EPMC5352373 | biostudies-literature
Project description:The human T-cell leukemia virus type 1 (HTLV-1) is highly dependent on cell-to-cell interaction for transmission and productive infection. Cell-to-cell interactions through the virological synapse, biofilm-like structures and cellular conduits have been reported, but the relative contribution of each mechanism on HTLV-1 transmission still remains vastly unknown. The HTLV-1 protein p8 has been found to increase viral transmission and cellular conduits. Here we show that HTLV-1 expressing cells are interconnected by tunneling nanotubes (TNTs) defined as thin structures containing F-actin and lack of tubulin connecting two cells. TNTs connected HTLV-1 expressing cells and uninfected T-cells and monocytes and the viral proteins Tax and Gag localized to these TNTs. The HTLV-1 expressing protein p8 was found to induce TNT formation. Treatment of MT-2 cells with the nucleoside analog cytarabine (cytosine arabinoside, AraC) reduced number of TNTs and furthermore reduced TNT formation induced by the p8 protein. Intercellular transmission of HTLV-1 through TNTs provides a means of escape from recognition by the immune system. Cytarabine could represent a novel anti-HTLV-1 drug interfering with viral transmission.
| S-EPMC6057998 | biostudies-literature