Abstract 203: Cardiomyocyte T-tubule Membrane Turns Over, Releasing BIN1 Containing Microparticles into Blood

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Bing Xu ◽  
Ying Fu ◽  
TingTing Hong
Keyword(s):  
Flow Cytometry ◽  
Adult Mouse ◽  
Cardiac Tissue ◽  
Muscle Protein ◽  
Three Dimensional ◽  
Super Resolution ◽  
Annexin V ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Fluorescent Imaging

Bridging integrator 1 (BIN1) is a cardiac muscle protein that folds cardiomyocyte T-tubule membrane. BIN1 is intrinsic to cardiac health, and is reduced in acquired heart failure. Interestingly, we have found that BIN1 is also blood available, and that plasma BIN1 correlates with cardiac function, suggesting cardiac origin of plasma BIN1. We found that low plasma BIN1 correlates with failing muscle and predicts ventricular arrhythmia. However, the paradigm does not exist for an intracellular membrane associate cardiomyocyte protein to be homeostatically turned over into blood. In this study, using a mouse model with cardiac specific deletion of Bin1 gene, we identified with biochemical techniques that plasma BIN1 levels directly correlate with cardiac tissue BIN1 levels, indicating cardiac origin. Furthermore, investigations using both super-resolution fluorescent imaging and flow cytometry analysis revealed that adult ventricular cardiomyocytes constantly release BIN1 into blood via membrane microparticle production. Microparticles are small membrane vesicles shed from plasma membrane of a variety of cell types including platelets, leukocytes, and endothelial cells. Using super-resolution three-dimensional stochastic optical reconstruction microscopy (3D-STORM), we found similar to the blood cells, isolated adult mouse cardiomyocytes release Annexin V positive microparticles with diameters ranging between 0.1 to 1.0 μm. These microparticles also carry BIN1 protein. Flow cytometry was also used to detect and quantify microparticles <1.0 μm in size from medium bathing a pure population of adult mouse cardiomyocytes. BIN1 microparticle release is proportional to actin stability and amount of T-tubule membrane folds. Compared to wild type cardiomyocytes, microparticle release is significantly reduced from myocytes with heterozygous deletion of Bin1 gene. These data indicate that cardiomyocyte membrane undergoes dynamic turnover, releasing T-tubule folds into blood as microparticles. Furthermore, plasma BIN1 can be used as a direct measure of cardiomyocyte health and reserve.

Cocaine’s Direct Effect on Either Sickle or Normal Erythrocytes Does Not Increase Phosphatidylserine Exposure, Calcium Pumping, or Scramblase Activity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3692-3692
Author(s):  
Kitty DeJong ◽  
Robert Hagar
Keyword(s):  
Flow Cytometry ◽  
Pulmonary Hypertension ◽  
Calcium Influx ◽  
Plasma Concentrations ◽  
Calcium Ionophore ◽  
Annexin V ◽  
Cell Types ◽  
Red Cell ◽  
Sickle Cells ◽  
Red Cell Membranes

Hypothesis: Cocaine directly affects the exposure of phosphatidylserine, calcium pumping, and scramblase activity in sickle cells. Background: Cocaine is highly associated with pulmonary hypertension in both normal and sickle cell disease patients. Given the growing interest in pulmonary hypertension as a major morbidity factor, the causes and determinants of pulmonary hypertension need to be elucidated to allow rational study designs. Phosphatidylserine (PS) exposure is known to occur in sickle cells and appears to be relevant for vascular damage. Although the mechanism underlying PS exposure is poorly understood, signal transduction processes appear to play a role. We wondered if cocaine could activate these processes and cause PS exposure, or interfere with the proteins involved in the transbilayer movement of PS. Methods: Sickle and normal erythrocytes were exposed to cocaine HCl (from a 10% topical solution, Roxane Labs) at concentrations between 100 and 1000 ng/ml, which is the range of determined plasma concentrations after cocaine use. PS exposure after cocaine treatment was measured by labeling with fluorescently conjugated annexin V (AV) and analysis by flow cytometry. Alterations in scramblase activity were determined by assessing the percentage of PS-exposing cells following loading of the cells with 0.1 mM calcium using calcium ionophore. Calcium influx and Ca-ATPase-mediated calcium efflux were monitored using the fluorescent probe Fluo4, and flippase activity was assessed using NBD-PS followed by analysis with flow cytometry. Results: Sickle cells differ from normal cells with respect to most of the measured parameters, such as having more PS-exposing cells, lower flippase activity, and alterations in calcium kinetics. However, cocaine did not have any effect on PS exposure, calcium-induced scrambling, calcium influx, calcium pump activity, or flippase activity. The cell scatter patterns did not show any gross changes in red cell shape or density after cocaine treatment. Discussion: The lack of effect implies that cocaine is working through other mechanisms than effects on red cell membranes. Therefore, future studies should focus on other vascular cell types and membrane pathways. Alternatively, cocaine metabolites should be evaluated for their activities on red cells.

scholarly journals Expansion microscopy facilitates quantitative super-resolution studies of cytoskeletal structures in kinetoplastid parasites

Open Biology ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 210131 ◽  
Author(s):  
Peter Gorilak ◽  
Martina Pružincová ◽  
Hana Vachova ◽  
Marie Olšinová ◽  
Marketa Schmidt Cernohorska ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Cell Biology ◽  
Leishmania Major ◽  
Three Dimensional ◽  
Super Resolution ◽  
Cell Types ◽  
Three Dimensional Reconstruction ◽  
Resolution Method ◽  
Entire Volume ◽  
Dimensional Reconstruction

Expansion microscopy (ExM) has become a powerful super-resolution method in cell biology. It is a simple, yet robust approach, which does not require any instrumentation or reagents beyond those present in a standard microscopy facility. In this study, we used kinetoplastid parasites Trypanosoma brucei and Leishmania major , which possess a complex, yet well-defined microtubule-based cytoskeleton, to demonstrate that this method recapitulates faithfully morphology of structures as previously revealed by a combination of sophisticated electron microscopy (EM) approaches. Importantly, we also show that due to the rapidness of image acquisition and three-dimensional reconstruction of cellular volumes ExM is capable of complementing EM approaches by providing more quantitative data. This is demonstrated on examples of less well-appreciated microtubule structures, such as the neck microtubule of T. brucei or the pocket, cytosolic and multivesicular tubule-associated microtubules of L. major . We further demonstrate that ExM enables identifying cell types rare in a population, such as cells in mitosis and cytokinesis. Three-dimensional reconstruction of an entire volume of these cells provided details on the morphology of the mitotic spindle and the cleavage furrow. Finally, we show that established antibody markers of major cytoskeletal structures function well in ExM, which together with the ability to visualize proteins tagged with small epitope tags will facilitate studies of the kinetoplastid cytoskeleton.

Activated Platelets Release Two Types of Membrane Vesicles: Microvesicles by Surface Shedding and Exosomes Derived From Exocytosis of Multivesicular Bodies and -Granules

Blood ◽  
1999 ◽  
Vol 94 (11) ◽  
pp. 3791-3799 ◽  
Author(s):  
Harry F.G. Heijnen ◽  
Anja E. Schiel ◽  
Rob Fijnheer ◽  
Hans J. Geuze ◽  
Jan J. Sixma
Keyword(s):  
Platelet Activation ◽  
Annexin V ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Multivesicular Bodies ◽  
Factor X ◽  
Activated Platelets ◽  
Immuno Electron Microscopy ◽  
Different Types ◽  
Platelet Aggregates

Platelet activation leads to secretion of granule contents and to the formation of microvesicles by shedding of membranes from the cell surface. Recently, we have described small internal vesicles in multivesicular bodies (MVBs) and -granules, and suggested that these vesicles are secreted during platelet activation, analogous to the secretion of vesicles termed exosomes by other cell types. In the present study we report that two different types of membrane vesicles are released after stimulation of platelets with thrombin receptor agonist peptide SFLLRN (TRAP) or -thrombin: microvesicles of 100 nm to 1 μm, and exosomes measuring 40 to 100 nm in diameter, similar in size as the internal vesicles in MVBs and -granules. Microvesicles could be detected by flow cytometry but not the exosomes, probably because of the small size of the latter. Western blot analysis showed that isolated exosomes were selectively enriched in the tetraspan protein CD63. Whole-mount immuno-electron microscopy (IEM) confirmed this observation. Membrane proteins such as the integrin chains IIb-β3 and β1, GPIb, and P-selectin were predominantly present on the microvesicles. IEM of platelet aggregates showed CD63+ internal vesicles in fusion profiles of MVBs, and in the extracellular space between platelet extensions. Annexin-V binding was mainly restricted to the microvesicles and to a low extent to exosomes. Binding of factor X and prothrombin was observed to the microvesicles but not to exosomes. These observations and the selective presence of CD63 suggest that released platelet exosomes may have an extracellular function other than the procoagulant activity, attributed to platelet microvesicles.

Cell Patterning: Interaction of Cardiac Myocytes and Fibroblasts in Three-Dimensional Culture

2008 ◽  
Vol 14 (2) ◽  
pp. 117-125 ◽  
Author(s):  
Troy A. Baudino ◽  
Alex McFadden ◽  
Charity Fix ◽  
Joshua Hastings ◽  
Robert Price ◽  
...  
Keyword(s):  
Cardiac Tissue ◽  
Three Dimensional ◽  
Normal Growth ◽  
Cell Types ◽  
Cell Interactions ◽  
Cellular Interactions ◽  
Cell Layers ◽  
Cell Cell

Patterning of cells is critical to the formation and function of the normal organ, and it appears to be dependent upon internal and external signals. Additionally, the formation of most tissues requires the interaction of several cell types. Indeed, both extracellular matrix (ECM) components and cellular components are necessary for three-dimensional (3-D) tissue formationin vitro. Using 3-D cultures we demonstrate that ECM arranged in an aligned fashion is necessary for the rod-shaped phenotype of the myocyte, and once this pattern is established, the myocytes were responsible for the alignment of any subsequent cell layers. This is analogous to thein vivopattern that is observed, where there appears to be minimal ECM signaling, rather formation of multicellular patterns is dependent upon cell–cell interactions. Our 3-D culture of myocytes and fibroblasts is significant in that it modelsin vivoorganization of cardiac tissue and can be used to investigate interactions between fibroblasts and myocytes. Furthermore, we used rotational cultures to examine cellular interactions. Using these systems, we demonstrate that specific connexins and cadherins are critical for cell–cell interactions. The data presented here document the feasibility of using these systems to investigate cellular interactions during normal growth and injury.

Activated Platelets Release Two Types of Membrane Vesicles: Microvesicles by Surface Shedding and Exosomes Derived From Exocytosis of Multivesicular Bodies and -Granules

Blood ◽  
1999 ◽  
Vol 94 (11) ◽  
pp. 3791-3799 ◽  
Author(s):  
Harry F.G. Heijnen ◽  
Anja E. Schiel ◽  
Rob Fijnheer ◽  
Hans J. Geuze ◽  
Jan J. Sixma
Keyword(s):  
Platelet Activation ◽  
Annexin V ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Multivesicular Bodies ◽  
Factor X ◽  
Activated Platelets ◽  
Immuno Electron Microscopy ◽  
Different Types ◽  
Platelet Aggregates

Abstract Platelet activation leads to secretion of granule contents and to the formation of microvesicles by shedding of membranes from the cell surface. Recently, we have described small internal vesicles in multivesicular bodies (MVBs) and -granules, and suggested that these vesicles are secreted during platelet activation, analogous to the secretion of vesicles termed exosomes by other cell types. In the present study we report that two different types of membrane vesicles are released after stimulation of platelets with thrombin receptor agonist peptide SFLLRN (TRAP) or -thrombin: microvesicles of 100 nm to 1 μm, and exosomes measuring 40 to 100 nm in diameter, similar in size as the internal vesicles in MVBs and -granules. Microvesicles could be detected by flow cytometry but not the exosomes, probably because of the small size of the latter. Western blot analysis showed that isolated exosomes were selectively enriched in the tetraspan protein CD63. Whole-mount immuno-electron microscopy (IEM) confirmed this observation. Membrane proteins such as the integrin chains IIb-β3 and β1, GPIb, and P-selectin were predominantly present on the microvesicles. IEM of platelet aggregates showed CD63+ internal vesicles in fusion profiles of MVBs, and in the extracellular space between platelet extensions. Annexin-V binding was mainly restricted to the microvesicles and to a low extent to exosomes. Binding of factor X and prothrombin was observed to the microvesicles but not to exosomes. These observations and the selective presence of CD63 suggest that released platelet exosomes may have an extracellular function other than the procoagulant activity, attributed to platelet microvesicles.

scholarly journals Apoptotic Effects on HL60 Human Leukaemia Cells Induced by Lavandin Essential Oil Treatment

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 538 ◽  
Author(s):  
Valentina Laghezza Masci ◽  
Elisa Ovidi ◽  
Anna Rita Taddei ◽  
Giovanni Turchetti ◽  
Antonio Tiezzi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Essential Oil ◽  
Annexin V ◽  
Cell Types ◽  
Dependent Manner ◽  
Linalyl Acetate ◽  
Hl60 Cells ◽  
Morphological Alterations ◽  
Apoptotic Process ◽  
Human Leukaemia

Recent scientific investigations have reported a number of essential oils to interfere with intracellular signalling pathways and to induce apoptosis in different cancer cell types. In this paper, Lavandin Essential Oil (LEO), a natural sterile hybrid obtained by cross-breeding L. angustifolia × L. latifolia, was tested on human leukaemia cells (HL60). Based on the MTT results, the reduced cell viability of HL60 cells was further investigated to determine whether cell death was related to the apoptotic process. HL60 cells treated for 24 h with LEO were processed by flow cytometry, and the presence of Annexin V was measured. The activation of caspases-3 was evaluated by western blot and immunofluorescence techniques. Treated cells were also examined by scanning and transmission electron microscopy to establish the possible occurrence of morphological alterations during the apoptotic process. LEO main compounds, such as linalool, linalyl acetate, 1,8-cineole, and terpinen-4-ol, were also investigated by MTT and flow cytometry analysis. The set of obtained results showed that LEO treatments induced apoptosis in a dose-dependent, but not time-dependent, manner on HL60 cells, while among LEO main compounds, both terpinen-4-ol and linalyl acetate were able to induce apoptosis.

scholarly journals Predicting three-dimensional genome organization with chromatin states

2018 ◽  
Author(s):  
Yifeng Qi ◽  
Bin Zhang
Keyword(s):  
Genome Organization ◽  
De Novo ◽  
Three Dimensional ◽  
Super Resolution ◽  
Cell Types ◽  
Quantitative Agreement ◽  
Chromosome Conformation ◽  
Chromatin Loops ◽  
Chromatin Folding ◽  
Super Resolution Microscopy

ABSTRACTWe introduce a computational model to simulate chromatin structure and dynamics. Starting from one-dimensional genomics and epigenomics data that are available for hundreds of cell types, this model enables de novo prediction of chromatin structures at five-kilo-base resolution. Simulated chromatin structures recapitulate known features of genome organization, including the formation of chromatin loops, topologically associating domains (TADs) and compartments, and are in quantitative agreement with chromosome conformation capture experiments and super-resolution microscopy measurements. Detailed characterization of the predicted structural ensemble reveals the dynamical flexibility of chromatin loops and the presence of cross-talk among neighboring TADs. Analysis of the model’s energy function uncovers distinct mechanisms for chromatin folding at various length scales.

SEM of Hepatocytes and Biliary Passages in Goldfish Liver

1977 ◽  
Vol 35 ◽  
pp. 556-557
Author(s):  
Waykin Nopanitaya ◽  
Joe W. Grisham ◽  
Johnny L. Carson
Keyword(s):  
Carassius Auratus ◽  
Cell Layer ◽  
Three Dimensional ◽  
Cell Types ◽  
Biliary System ◽  
Fish Food ◽  
Commercial Fish ◽  
Goldfish Carassius Auratus ◽  
Commercial Fish Food

An interesting feature of the goldfish liver is the morphology of the hepatic plate, which is always formed by a two-cell layer of hepatocytes. Hepatic plates of the goldfish liver contain an infrequently seen second type of cell, in the centers of plates between two hepatocytes. A TEH study by Yamamoto (1) demonstrated ultrastructural differences between hepatocytes and centrally located cells in hepatic plates; the latter were classified as ductule cells of the biliary system. None of the previous studies clearly showed a three-dimensional organization of the two cell types described. In the present investigation we utilize SEM to elucidate the arrangement of hepatocytes and bile ductular cells in intralobular plates of goldfish liver.Livers from young goldfish (Carassius auratus), about 6-10 cm, fed commercial fish food were used for this study. Hepatic samples were fixed in 4% buffered paraformaldehyde, cut into pieces, fractured, osmicated, CPD, mounted Au-Pd coated, and viewed by SEM at 17-20 kV. Our observations were confined to the ultrastructure of biliary passages within intralobular plates, ductule cells, and hepatocytes.

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