Investigations of Intercellular Mitochondrial Transfer in Neural Cells by Applied Single Molecule Genotyping

2021 ◽  
Author(s):  
◽  
Matthew Rowe
Keyword(s):  
Cell Line ◽  
Single Molecule ◽  
Cellular Response ◽  
Rolling Circle Amplification ◽  
Metabolic Phenotype ◽  
Neural Cells ◽  
Mitochondrial Transfer ◽  
Mitochondrial Ribosome

<p>Over the past decade and a half, evidence for transfer of whole mitochondria between mammalian cells has emerged in the literature. The notion that mitochondria are restricted to the cell of origin has been overturned by this curious phenomenon, yet the physiological relevance of these transfer events remains unclear.   This thesis investigates intercellular mitochondrial transfer in co-cultures of neural cells in vitro, to understand whether neural cells placed under stress demonstrate an enhanced rate of intercellular mitochondrial transfer. This would implicate the phenomenon as a cellular response to stress.   Reliable techniques for quantitative study of intercellular mitochondrial transfer are limited so far in this field. To address this, a novel quantitative approach was developed to detect intercellular mitochondrial transfer, based on single molecule genotyping by target-primed rolling circle amplification. This enabled imaging of individual mitochondrial DNA molecules in situ, to detect those molecules which had moved between cells. Through this strategy, intercellular mitochondrial transfer was detected in new in vitro co-culture models.   Primary murine pericytes derived from brain microvessels, were found to readily transfer mitochondria to a murine astrocyte cell line in vitro. Cisplatin, a DNA damaging agent; and chloramphenicol, a mitochondrial ribosome inhibitor, used to induce acute cellular injuries in the murine astrocyte cell line. These injuries were characterised and found to induce apoptosis, cause changes in growth characteristics, mitochondrial gene expression, and alter the metabolic phenotype of the cells. A derivative of the astrocyte cell line which completely lacks mitochondrial respiration, was found to model a chronic metabolic injury.  As pericytes are prevalent throughout the brain, the pericyte/astrocyte co-culture model was selected to evaluate how the rate of intercellular mitochondrial transfer was altered, when the astrocytes were injured prior to co-culture. Through in situ single molecule genotyping and high throughput confocal microscopy, quantitative data was produced on how the rate of intercellular mitochondrial transfer was altered by injury in these models. The rate of intercellular mitochondrial transfer remained unaltered by chloramphenicol, however both cisplatin and the chronic metabolic injury model demonstrated reduced numbers of pericyte mitochondrial DNAs transferred into the injured astrocytes.   These studies demonstrate successful application of a novel approach to study intercellular mitochondrial transfer and enable quantitative studies of this phenomenon.</p>

Investigations of Intercellular Mitochondrial Transfer in Neural Cells by Applied Single Molecule Genotyping

2021 ◽  
Author(s):  
◽  
Matthew Rowe
Keyword(s):  
Cell Line ◽  
Single Molecule ◽  
Cellular Response ◽  
Rolling Circle Amplification ◽  
Metabolic Phenotype ◽  
Neural Cells ◽  
Mitochondrial Transfer ◽  
Mitochondrial Ribosome

<p>Over the past decade and a half, evidence for transfer of whole mitochondria between mammalian cells has emerged in the literature. The notion that mitochondria are restricted to the cell of origin has been overturned by this curious phenomenon, yet the physiological relevance of these transfer events remains unclear.   This thesis investigates intercellular mitochondrial transfer in co-cultures of neural cells in vitro, to understand whether neural cells placed under stress demonstrate an enhanced rate of intercellular mitochondrial transfer. This would implicate the phenomenon as a cellular response to stress.   Reliable techniques for quantitative study of intercellular mitochondrial transfer are limited so far in this field. To address this, a novel quantitative approach was developed to detect intercellular mitochondrial transfer, based on single molecule genotyping by target-primed rolling circle amplification. This enabled imaging of individual mitochondrial DNA molecules in situ, to detect those molecules which had moved between cells. Through this strategy, intercellular mitochondrial transfer was detected in new in vitro co-culture models.   Primary murine pericytes derived from brain microvessels, were found to readily transfer mitochondria to a murine astrocyte cell line in vitro. Cisplatin, a DNA damaging agent; and chloramphenicol, a mitochondrial ribosome inhibitor, used to induce acute cellular injuries in the murine astrocyte cell line. These injuries were characterised and found to induce apoptosis, cause changes in growth characteristics, mitochondrial gene expression, and alter the metabolic phenotype of the cells. A derivative of the astrocyte cell line which completely lacks mitochondrial respiration, was found to model a chronic metabolic injury.  As pericytes are prevalent throughout the brain, the pericyte/astrocyte co-culture model was selected to evaluate how the rate of intercellular mitochondrial transfer was altered, when the astrocytes were injured prior to co-culture. Through in situ single molecule genotyping and high throughput confocal microscopy, quantitative data was produced on how the rate of intercellular mitochondrial transfer was altered by injury in these models. The rate of intercellular mitochondrial transfer remained unaltered by chloramphenicol, however both cisplatin and the chronic metabolic injury model demonstrated reduced numbers of pericyte mitochondrial DNAs transferred into the injured astrocytes.   These studies demonstrate successful application of a novel approach to study intercellular mitochondrial transfer and enable quantitative studies of this phenomenon.</p>

scholarly journals Enlisting the Ixodes scapularis Embryonic ISE6 Cell Line to Investigate the Neuronal Basis of Tick—Pathogen Interactions

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 70
Author(s):  
Lourdes Mateos-Hernández ◽  
Natália Pipová ◽  
Eléonore Allain ◽  
Céline Henry ◽  
Clotilde Rouxel ◽  
...  
Keyword(s):  
Cell Line ◽  
Peripheral Nerves ◽  
Ixodes Scapularis ◽  
Embryonic Cell ◽  
Cell Subpopulation ◽  
In Vivo Experiments

Neuropeptides are small signaling molecules expressed in the tick central nervous system, i.e., the synganglion. The neuronal-like Ixodes scapularis embryonic cell line, ISE6, is an effective tool frequently used for examining tick–pathogen interactions. We detected 37 neuropeptide transcripts in the I. scapularis ISE6 cell line using in silico methods, and six of these neuropeptide genes were used for experimental validation. Among these six neuropeptide genes, the tachykinin-related peptide (TRP) of ISE6 cells varied in transcript expression depending on the infection strain of the tick-borne pathogen, Anaplasma phagocytophilum. The immunocytochemistry of TRP revealed cytoplasmic expression in a prominent ISE6 cell subpopulation. The presence of TRP was also confirmed in A. phagocytophilum-infected ISE6 cells. The in situ hybridization and immunohistochemistry of TRP of I. scapularis synganglion revealed expression in distinct neuronal cells. In addition, TRP immunoreaction was detected in axons exiting the synganglion via peripheral nerves as well as in hemal nerve-associated lateral segmental organs. The characterization of a complete Ixodes neuropeptidome in ISE6 cells may serve as an effective in vitro tool to study how tick-borne pathogens interact with synganglion components that are vital to tick physiology. Therefore, our current study is a potential stepping stone for in vivo experiments to further examine the neuronal basis of tick–pathogen interactions.

scholarly journals Synthetic and genetic dimers as quantification ruler for single-molecule counting with PALM

2019 ◽  
Vol 30 (12) ◽  
pp. 1369-1376 ◽  
Author(s):  
Tim N. Baldering ◽  
Marina S. Dietz ◽  
Karl Gatterdam ◽  
Christos Karathanasis ◽  
Ralph Wieneke ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Fusion Proteins ◽  
Fluorescent Proteins ◽  
Superresolution Microscopy ◽  
Superresolution Imaging ◽  
Molecular Information ◽  
Kinetics Of

How membrane proteins oligomerize determines their function. Superresolution microscopy can report on protein clustering and extract quantitative molecular information. Here, we evaluate the blinking kinetics of four photoactivatable fluorescent proteins for quantitative single-molecule microscopy. We identified mEos3.2 and mMaple3 to be suitable for molecular quantification through blinking histogram analysis. We designed synthetic and genetic dimers of mEos3.2 as well as fusion proteins of monomeric and dimeric membrane proteins as reference structures, and we demonstrate their versatile use for quantitative superresolution imaging in vitro and in situ. We further found that the blinking behavior of mEos3.2 and mMaple3 is modified by a reducing agent, offering the possibility to adjust blinking parameters according to experimental needs.

scholarly journals An undifferentiated variant derived from the human acute myelogenous leukemia cell line (KG-1)

Blood ◽  
1980 ◽  
Vol 56 (2) ◽  
pp. 265-273 ◽  
Author(s):  
HP Koeffler ◽  
R Billing ◽  
AJ Lusis ◽  
R Sparkes ◽  
DW Golde
Keyword(s):  
Cell Line ◽  
Acute Myelogenous Leukemia ◽  
Cellular Response ◽  
Leukemia Cell ◽  
Hla Antigens ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Leukemia Cell Line ◽  
Acute Myelogenous

Abstract A variant subline (KG-1a) of the human acute myelogenous leukemia (AML) cell line (KG-1) has been isolated. The cells retain the same constitutive markers as the parent line, including HLA antigens, isoenzymes, and karyotype. The cells from the subline are morphologically and histochemically undifferentiated blast cells, while the parent cells and several of its clones are at the myeloblast and promyelocyte stages of development. The variant cells do not respond to colony-stimulating factor (CSF), and they do not express the human la antigen, nor a recently characterized AML antigen. The parent KG-1 cells are stimulated to proliferate in the presence of CSF and the cells express the la and AML antigen. Variant AML cell lines, such as KG-1a, will be useful in vitro models for investigating cellular response to CSF and for studying antigen expression in leukemic cells.

Lenalidomide Effects on NK Function in Patients with Myelodysplastic Syndrome (MDS).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3437-3437 ◽  
Author(s):  
P.K. Epling-Burnette ◽  
Fanqi Bai ◽  
Jeffrey S. Painter ◽  
Julie Y. Djeu ◽  
Alan F. List
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cellular Response ◽  
Nk Cell ◽  
Leukemia Cell ◽  
T Test ◽  
Leukemia Cell Line ◽  
Immunomodulatory Effects

Abstract Lenalidomide, which is a 4-amino-glutarimide analogue of thalidomide, has significant erythropoietic activity in patients with lower-risk MDS (List et al, NEJM, 351:26,2004). Although its precise target of action in MDS is not known, lenalidomide modulates cellular response to varied stimuli including inhibition of angiogenic response and endotoxin induction of inflammatory cytokines and enhancement of antigen-induced immunologic response and erythropoietin receptor signaling. Clinical investigations in multiple myeloma indicate that the immunomodulatory effects of thalidomide and lenalidomide extends to the expansion of natural killer (NK) cells. We recently found that MDS patients have defective NK function, araising in part to reduced expression of activating NK receptors (NKRs) NKp30, CD244 (2B4), and NKG2D. To determine if lenalidomide may restore NK function in MDS, we investigated the effects of in vitro treatment with lenalidomide on NK function and phenotype. Lytic function was studied using peripheral blood mononuclear cells (PBMCs) as effector cells and the leukemia cell line, K562, as a target in standard 4-hr 51Cr-release assays at 12:1 and 25:1 effector:target (E:T) ratios. Among eight MDS patient’s specimens evaluated, five patients had significant increase in tumor lysis after treatment with 1 μM lenalidomide for 72 hours (p ≤ 0.01, T-test). In similar experiments using PBMCs from normal donors, we found that NK lysis of K562 was 42% ± 15 (25:1 E:T ratio) pre-treatment which increased to 71% ± 17 (25:1 E:T ratio) after treatment, which was statistically significant (p ≤ 0.01, T-test). We also examined the in vitro effects of lenalidomide on lytic activity by the NK cell lines, NK92 and NKL, which was significantly increased after drug treatment. To discern the mechanisms of lenalidomide action in NK cell lines and normal NK cells, we evaluated NKR display by flow cytometry, and NKR function by antibody redirected cytotoxicity using the FcγR+ murine mastocytoma (P815) target cell line. Using NK92 and NKL cells, treatment with lenalidomide 1 μM for 72 hrs increased lysis by anti-NKG2D and anti-CD244 activating antibodies. We also found that NKG2D surface expression was increased on normal NK cells after lenalidomide treatment in vitro. These results suggest that some MDS patients may have improved NK function through the immunomodulatory effects of lenalidomide. The relationship between in vitro NK responsiveness to lenalidomide and in vivo hematological response warrants investigation in patients with MDS.

scholarly journals Development and Characterisation of a Novel NF-κB Reporter Cell Line for Investigation of Neuroinflammation

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Marie-Theres Zeuner ◽  
Thomas Vallance ◽  
Sakthivel Vaiyapuri ◽  
Graeme S. Cottrell ◽  
Darius Widera
Keyword(s):  
Cell Line ◽  
Glioma Cell Line ◽  
Neural Cells ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Necrosis Factor Alpha ◽  
In Vitro Drug Screening ◽  
Anti Inflammatory ◽  
Cost Efficient

Aberrant activation of the transcription factor NF-κB, as well as uncontrolled inflammation, has been linked to autoimmune diseases, development and progression of cancer, and neurological disorders like Alzheimer’s disease. Reporter cell lines are a valuable state-of-the art tool for comparative analysis of in vitro drug screening. However, a reporter cell line for the investigation of NF-κB-driven neuroinflammation has not been available. Thus, we developed a stable neural NF-κB-reporter cell line to assess the potency of proinflammatory molecules and peptides, as well as anti-inflammatory pharmaceuticals. We used lentivirus to transduce the glioma cell line U251-MG with a tandem NF-κB reporter construct containing GFP and firefly luciferase allowing an assessment of NF-κB activity via fluorescence microscopy, flow cytometry, and luminometry. We observed a robust activation of NF-κB after exposure of the reporter cell line to tumour necrosis factor alpha (TNFα) and amyloid-β peptide [1-42] as well as to LPS derived from Salmonella minnesota and Escherichia coli. Finally, we demonstrate that the U251-NF-κB-GFP-Luc reporter cells can be used for assessing the anti-inflammatory potential of pharmaceutical compounds using Bay11-7082 and IMD0354. In summary, our newly generated cell line is a robust and cost-efficient tool to study pro- and anti-inflammatory potential of drugs and biologics in neural cells.

scholarly journals Preparation of long single-strand DNA concatemers for high-level fluorescence in situ hybridization

2021 ◽  
Author(s):  
Dongjian Cao ◽  
Sa Wu ◽  
Caili Xi ◽  
Dong Li ◽  
Kaiheng Zhu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Proteins ◽  
Signal To Noise Ratio ◽  
Rolling Circle Amplification ◽  
Simultaneous Detection ◽  
Single Strand ◽  
Detection Methods ◽  
Rolling Circle ◽  
High Level

Abstract Fluorescence in situ hybridization (FISH) is a powerful tool to visualize transcripts in fixed cells and tissues. Despite the recent advances in FISH detection methods, it remains challenging to achieve high-level FISH imaging with a simple workflow. Here, we introduce a method to prepare long single-strand DNA concatemers (lssDNAc) through a controllable rolling-circle amplification (CRCA). Prepared lssDNAcs were used to develop novel AmpFISH workflows. In addition, we present its applications in different scenarios. AmpFISH shows the following advantages: 1) enhanced FISH signal-to-noise ratio (SNR) up to 160-fold compared with single-molecule FISH; 2) simultaneous detection of FISH signals and fluorescent proteins or immunofluorescence (IF) in tissues; 3) simple workflows; and 4) cost-efficiency. In brief, AmpFISH provides convenient and versatile tools for sensitive RNA/DNA detection and to gain useful information on cellular molecules using simple workflows.

scholarly journals Propagation of Arthropod-Borne Rickettsia spp. in Two Mosquito Cell Lines

2007 ◽  
Vol 73 (20) ◽  
pp. 6637-6643 ◽  
Author(s):  
Joyce M. Sakamoto ◽  
Abdu F. Azad
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Spotted Fever ◽  
Culture Conditions ◽  
Spotted Fever Group ◽  
Rickettsia Felis ◽  
Transitional Group ◽  
Mosquito Cell Lines

ABSTRACT Rickettsiae are obligate intracellular alphaproteobacteria that include pathogenic species in the spotted fever, typhus, and transitional groups. The development of a standardized cell line in which diverse rickettsiae can be grown and compared would be highly advantageous to investigate the differences among and between pathogenic and nonpathogenic species of rickettsiae. Although several rickettsial species have been grown in tick cells, tick cells are more difficult to maintain and they grow more slowly than insect cells. Rickettsia-permissive arthropod cell lines that can be passaged rapidly are highly desirable for studies on arthropod-Rickettsia interactions. We used two cell lines (Aedes albopictus cell line Aa23 and Anopheles gambiae cell line Sua5B) that have not been used previously for the purpose of rickettsial propagation. We optimized the culture conditions to propagate one transitional-group rickettsial species (Rickettsia felis) and two spotted-fever-group rickettsial species (R. montanensis and R. peacockii) in each cell line. Both cell lines allowed the stable propagation of rickettsiae by weekly passaging regimens. Stable infections were confirmed by PCR, restriction digestion of rompA, sequencing, and the direct observation of bacteria by fluorescence in situ hybridization. These cell lines not only supported rickettsial growth but were also permissive toward the most fastidious species of the three, R. peacockii. The permissive nature of these cell lines suggests that they may potentially be used to isolate novel rickettsiae or other intracellular bacteria. Our results have important implications for the in vitro maintenance of uncultured rickettsiae, as well as providing insights into Rickettsia-arthropod interactions.

Bone Marrow Mesenchymal Stromal Cells Transfer Their Mitochondria to Acute Myeloid Leukaemia Blasts to Support Their Proliferation and Survival

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 772-772 ◽  
Author(s):  
Christopher Marlein ◽  
Lyubov Zaitseva ◽  
Rachel E Piddock ◽  
Stephen Robinson ◽  
Dylan R Edwards ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Stromal Cells ◽  
Mitochondrial Protein ◽  
Bone Marrow Microenvironment ◽  
Mitochondrial Mass ◽  
Mitochondrial Transfer ◽  
Acute Myeloid

Abstract Background Survival of acute myeloid leukaemia (AML) blasts is established to be heavily dependent on the bone marrow microenvironment, where bone marrow mesenchymal stromal cells (BM-MSCs) are an important cell type. Contrary to the Warburg hypothesis, AML blasts rely on oxidative phosphorylation for survival and have increased mitochondrial levels compared to normal CD34+ progenitors. Current research is being directed at the biology behind how the bone marrow microenvironment supports the proliferation of the disease. With the knowledge that AML blasts have an increased mitochondrial mass and that BM-MSCs have the ability to be mitochondrial donors, we examined the BM-MSC AML blast interaction to determine if the increased mitochondrial mass was a result of inter-cellular mitochondrial transfer. Methods Primary AML blasts were obtained from patient bone marrow. Primary AML and normal BM-MSCs were isolated from patients bone marrow, with informed consent and under approval from the UK National Research Ethics Service (LRCEref07/H0310/146), using adherence. BM-MSCs were characterised using flow cytometry for expression of CD90+, CD73+, CD105+ and CD45-. Mitochondrial transfer was assessed in vitro using qPCR and MitoTracker staining based methods. A P0 OCI-AML3 cell line was created using a 40-day incubation with ethidium bromide, pyruvate and uridine. In vivo experiments using an NSG primary AML xenograft model were also carried out (in accordance with University of East Anglia ethics review board). For mechanistic determination, BM-MSCs with a mCherry mitochondrial labelled protein were created using a lentivirus. Levels of mitochondrial transfer were assessed by mCherry mitochondrial protein acquisition in the AML during co-culture with the BM-MSCs. Results We report that BM-MSCs support AML blast survival via the inter-cellular transfer of mitochondria from 'benign' to malignant cells. To examine this transfer we used primary AML blasts and BM-MSCs derived from patient bone marrow, along with AML cell lines. We found in vitro that primary AML blasts increase their mitochondrial mass, respiratory capacity and ATP production after co-culture with primary BM-MSCs. A P0 OCI-AML3 cell line, with mutated mitochondrial DNA (mtDNA), was generated using ethidium bromide treatment allowing mitochondrial transfer to be specifically analysed. mtDNA was restored in this cell line after co-culture with primary BM-MSCs. Further to this mouse mtDNA was detected in the P0 OCI-AML3 cells after co-culture with the mouse BM-MSC cell line (M2-10B4). Moreover, mitochondrial transfer was directly observed between primary BM-MSCs and primary AML blasts, visualised by the acquisition of a mCherry labelled mitochondrial protein. This transfer of mitochondria was one directional. Moreover, a reduction of mitochondrial transfer was observed in AML blasts upon the addition of cytochalasin to the co-culture, highlighting that mitochondrial transfer is at least in part facilitated through tunnelling nanotubes (TNTs). Finally, mitochondrial transfer was confirmed in vivo whereby murine mitochondria were transferred to human AML in a mouse xenografts model. Conclusion Here we show that the bone marrow microenvironment supports the AML blasts by donating mitochondria, which in turn enhances the oxidative phosphorylation and growth capacity of the blasts. Targeting the microenvironment is predicted to provide novel therapeutic approaches for the treatment of cancer. Disclosures Rushworth: Infinity Pharmaceuticals: Research Funding.

scholarly journals P07.01 Functional role of N-Myc in SHH type TP53 mutated MB’s metabolism

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii36-iii36
Author(s):  
K Yokogami
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Functional Role ◽  
Ex Vivo ◽  
Therapeutic Option ◽  
In Vitro Models ◽  
Metabolic Phenotype ◽  
Matched Pair

Abstract BACKGROUND Medulloblastoma is classified in 4 subgroups. Prognosis and therapeutic option was different from each subgroups. Thus, we need subgroup-specific in vitro models for investigating new therapeutic targets. Little established medulloblastoma cell-lines, which have been subgrouped is available. Especially, commercially available SHH type TP53 mutated cell-line is only DAOY. We established new cell lines 505CSC / 507FBS from the patient with SHH type with TP53 mutated MB. This matched pair cell line showed high expression of N-MYC in serum free conditioned medium. To know the functional role of N-MYC in MB, we used 507CSC and DAOY. MATERIAL AND METHODS Using chemical inhibitor of N-Myc in 507CSC and DAOY, proliferation assay, mRNA expression and measurements of ex-vivo metabolic phenotype were performed. RESULTS N-MYC inhibition leads to cell death in both cell lines. N-MYC regulated glucose, glutamine and methionine metabolism. Especially the targets were PKM2, GLS2, MAT2A, DNMT1 and 3A. CONCLUSION N-MYC is a target of therapy in a patient with SHH type TP53 mutated medulloblastoma.

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