| 1. |
- Dong, Yan, et al.
(författare)
-
Probing Exocytosis at Single Cells using Electrochemistry
- 2010
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Ingår i: Chemical Cytometry: Ultrasensive Analysis of Single Cells (ed C. Lu). - Weinheim : Wiley-VCH Verlag GmbH & Co.. - 9783527324958 ; , s. 159-174
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Bokkapitel (övrigt vetenskapligt/konstnärligt)
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| 2. |
- Hanrieder, Jörg, 1980, et al.
(författare)
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Imaging mass spectrometry in neuroscience.
- 2013
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Ingår i: ACS chemical neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 4:5, s. 666-79
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Tidskriftsartikel (refereegranskat)abstract
- Imaging mass spectrometry is an emerging technique of great potential for investigating the chemical architecture in biological matrices. Although the potential for studying neurobiological systems is evident, the relevance of the technique for application in neuroscience is still in its infancy. In the present Review, a principal overview of the different approaches, including matrix assisted laser desorption ionization and secondary ion mass spectrometry, is provided with particular focus on their strengths and limitations for studying different neurochemical species in situ and in vitro. The potential of the various approaches is discussed based on both fundamental and biomedical neuroscience research. This Review aims to serve as a general guide to familiarize the neuroscience community and other biomedical researchers with the technique, highlighting its great potential and suitability for comprehensive and specific chemical imaging.
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| 3. |
- Lovric, Jelena, 1980, et al.
(författare)
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Correlative High-Resolution Imaging of Iron Uptake in Lung Macrophages
- 2022
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 94:37, s. 12798-12806
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Tidskriftsartikel (refereegranskat)abstract
- Detection of iron at the subcellular level in order to gain insights into its transport, storage, and therapeutic prospects to prevent cytotoxic effects of excessive iron accumulation is still a challenge. Nanoscale magnetic sector secondary ion mass spectrometry (SIMS) is an excellent candidate for subcellular mapping of elements in cells since it provides high secondary ion collection efficiency and transmission, coupled with high-lateral-resolution capabilities enabled by nanoscale primary ion beams. In this study, we developed correlative methodologies that implement SIMS high-resolution imaging technologies to study accumulation and determine subcellular localization of iron in alveolar macrophages. We employed transmission electron microscopy (TEM) and backscattered electron (BSE) microscopy to obtain structural information and high-resolution analytical tools, NanoSIMS and helium ion microscopy-SIMS (HIM-SIMS) to trace the chemical signature of iron. Chemical information from NanoSIMS was correlated with TEM data, while high-spatial-resolution ion maps from HIM-SIMS analysis were correlated with BSE structural information of the cell. NanoSIMS revealed that iron is accumulating within mitochondria, and both NanoSIMS and HIM-SIMS showed accumulation of iron in electrolucent compartments such as vacuoles, lysosomes, and lipid droplets. This study provides insights into iron metabolism at the subcellular level and has future potential in finding therapeutics to reduce the cytotoxic effects of excessive iron loading.
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| 4. |
- Lovric, Jelena, 1980, et al.
(författare)
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Nano Secondary Ion Mass Spectrometry Imaging of Dopamine Distribution Across Nanometer Vesicles
- 2017
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Ingår i: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 11:4, s. 3446-3455
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Tidskriftsartikel (refereegranskat)abstract
- We report an approach to spatially resolve the content across nanometer neuroendocrine vesicles in nerve-like cells by correlating super high-resolution mass spectrometry imaging, NanoSIMS, with transmission electron microscopy (TEM). Furthermore, intracellular electrochemical cytometry at nanotip electrodes is used to count the number of molecules in individual vesicles to compare to imaged amounts in vesicles. Correlation between the NanoSIMS and TEM provides nanometer resolution of the inner structure of these organelles. Moreover, correlation with electrochemical methods provides a means to quantify and relate vesicle neurotransmitter content and release, which is used to explain the slow transfer of dopamine between vesicular compartments. These nanoanalytical tools reveal that dopamine loading/unloading between vesicular compartments, dense core and halo solution, is a kinetically limited process. The combination of NanoSIMS and TEM has been used to show the distribution profile of newly synthesized dopamine across individual vesicles. Our findings suggest that the vesicle inner morphology might regulate the neurotransmitter release event during open and closed exocytosis from dense core vesicles with hours of equilibrium needed to move significant amounts of catecholamine from the protein dense core despite its nanometer size.
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| 5. |
- Piehowski, Paul D., et al.
(författare)
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Freeze-Etching and Vapor Matrix Deposition for ToF-SIMS Imaging of Single Cells
- 2008
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 24:15, s. 7906-7911
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Tidskriftsartikel (refereegranskat)abstract
- Freeze-etching, the practice of removing excess surface water from a sample through sublimation into the vacuum of the analysis environment, has been extensively used in conjunction with electron microscopy. Here, we apply this technique to time-of-flight secondary-ion mass spectrometry (ToF-SIMS) imaging of cryogenically preserved single cells. By removing the excess water which condenses onto the sample in vacuo, a uniform surface is produced that is ideal for imaging by static SIMS. We demonstrate that the conditions employed to remove deposited water do not adversely affect cell morphology and do not redistribute molecules in the topmost surface layers. In addition, we found water can be controllably redeposited onto the sample at temperatures below −100 °C in vacuum. The redeposited water increases the ionization of characteristic fragments of biologically interesting molecules 2-fold without loss of spatial resolution. The utilization of freeze-etch methodology will increase the reliability of cryogenic sample preparations for SIMS analysis by providing greater control of the surface environment. Using these procedures, we have obtained high quality spectra with both atomic bombardment as well as C60+ cluster ion bombardment.
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| 6. |
- Piehowski, Paul D., et al.
(författare)
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MS/MS Methodology To Improve Subcellular Mapping of Cholesterol Using TOF-SIMS
- 2008
-
Ingår i: Anal. Chem.. - : American Chemical Society (ACS). ; 80:22, s. 8662-8667
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Tidskriftsartikel (refereegranskat)abstract
- Time-of-flight secondary ion mass spectrometry (TOF-SIMS) can be utilized to map the distribution of various molecules on a surface with submicrometer resolution. Much of its biological application has been in the study of membrane lipids, such as phospholipids and cholesterol. Cholesterol is a particularly interesting molecule due to its involvement in numerous biological processes. For many studies, the effectiveness of chemical mapping is limited by low signal intensity from various biomolecules. Because of the high energy nature of the SIMS ionization process, many molecules are identified by detection of characteristic fragments. Commonly, fragments of a molecule are identified using standard samples, and those fragments are used to map the location of the molecule. In this work, MS/MS data obtained from a prototype C60+/quadrupole time-of-flight mass spectrometer was used in conjunction with indium LMIG imaging to map previously unrecognized cholesterol fragments in single cells. A model system of J774 macrophages doped with cholesterol was used to show that these fragments are derived from cholesterol in cell imaging experiments. Examination of relative quantification experiments reveals that m/z 147 is the most specific diagnostic fragment and offers a 3-fold signal enhancement. These findings greatly increase the prospects for cholesterol mapping experiments in biological samples, particularly with single cell experiments. In addition, these findings demonstrate the wealth of information that is hidden in the traditional TOF-SIMS spectrum.
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| 7. |
- Piehowski, Paul D, et al.
(författare)
-
Time-of-flight secondary ion mass spectrometry imaging of subcellular lipid heterogeneity: Poisson counting and spatial resolution.
- 2009
-
Ingår i: Analytical chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 81:14, s. 5593-602
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Tidskriftsartikel (refereegranskat)abstract
- Mass spectrometric imaging is a powerful tool to interrogate biological complexity. One such technique, time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging, has been successfully utilized for subcellular imaging of cell membrane components. In order for this technique to provide insight into biological processes, it is critical to characterize the figures of merit. Because a SIMS instrument counts individual events, the precision of the measurement is controlled by counting statistics. As the analysis area decreases, the number of molecules available for analysis diminishes. This becomes critical when imaging subcellular features; it limits the information obtainable, resulting in images with only a few counts of interest per pixel. Many features observed in low intensity images are artifacts of counting statistics, making validation of these features crucial to arriving at accurate conclusions. With TOF-SIMS imaging, the experimentally attainable spatial resolution is a function of the molecule of interest, sample matrix, concentration, primary ion, instrument transmission, and spot size of the primary ion beam. A model, based on Poisson statistics, has been developed to validate SIMS imaging data when signal is limited. This model can be used to estimate the effective spatial resolution and limits of detection prior to analysis, making it a powerful tool for tailoring future investigations. In addition, the model allows comparison of pixel-to-pixel intensity and can be used to validate the significance of observed image features. The implications and capabilities of the model are demonstrated by imaging the cell membrane of resting RBL-2H3 mast cells.
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| 8. |
- Ren, Lin, 1987, et al.
(författare)
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The evidence for open and closed exocytosis as the primary release mechanism
- 2016
-
Ingår i: Quarterly Reviews of Biophysics. - 1469-8994 .- 0033-5835. ; 49
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Tidskriftsartikel (refereegranskat)abstract
- Exocytosis is the fundamental process by which cells communicate with each other. The events that lead up to the fusion of a vesicle loaded with chemical messenger with the cell membrane were the subject of a Nobel Prize in 2013. However, the processes occurring after the initial formation of a fusion pore are very much still in debate. The release of chemical messenger has traditionally been thought to occur through full distention of the vesicle membrane, hence assuming exocytosis to be all or none. In contrast to the all or none hypothesis, here we discuss the evidence that during exocytosis the vesicle-membrane pore opens to release only a portion of the transmitter content during exocytosis and then close again. This open and closed exocytosis is distinct from kiss- and-run exocytosis, in that it appears to be the main content released during regular exocytosis. The evidence for this partial release via open and closed exocytosis is presented considering primarily the quantitative evidence obtained with amperometry.
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| 9. |
- Cans, Ann-Sofie, 1971, et al.
(författare)
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A Secretory Artificial Cell for Exocytosis
- 2013
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 104:2 Suppl 1
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The complexity of exocytosis has left the molecular details of the process unclear. We present a minimal, artificial secretory cell designed for amperometric studies of release of signalling molecules through the fusion pore of single vesicles. In replacement of SNARE-proteins, the cell model has been equipped with an analog composed of complimentary DNA constructs, one on the vesicle and one on the target membrane. The DNA constructs hybridize in a zipper-like fashion bringing about docking of the vesicles and following the addition of Ca2+, fusion of the vesicles is completed. Exocytotic events recorded from the artificial cell closely approximate exocytosis in live cells. The results together with simulations of vesicular release demonstrate that the lipid-based fusion pore initially retains stability and limits diffusion of the secreted molecules.
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| 10. |
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| 11. |
- Ding, J., et al.
(författare)
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Inhibition of HMGCoA Reductase Reveals An Unexpected Role for Cholesterol During PGC Migration in the Mouse.
- 2008
-
Ingår i: BMC developmental biology. - 1471-213X. ; 8:1
-
Tidskriftsartikel (refereegranskat)abstract
- ABSTRACT: BACKGROUND: Primordial germ cells (PGCs) are the embryonic precursors of the sperm and eggs. Environmental or genetic defects that alter PGC development can impair fertility or cause formation of germ cell tumors. RESULTS: We demonstrate a novel role for cholesterol during germ cell migration in mice. Cholesterol was measured in living tissue dissected from mouse embryos and was found to accumulate within the developing gonads as germ cells migrate to colonize these structures. Cholesterol synthesis was blocked in culture by inhibiting the activity of HMG CoA reductase (HMGCR) resulting in germ cell survival and migration defects. These defects were rescued by co-addition of isoprenoids and cholesterol, but neither compound alone was sufficient. In contrast, loss of the last or penultimate enzyme in cholesterol biosynthesis did not alter PGC numbers or position in vivo. However embryos that lack these enzymes do not exhibit cholesterol defects at the stage at which PGCs are migrating. This demonstrates that during gestation, the cholesterol required for PGC migration can be supplied maternally. CONCLUSIONS: In the mouse, cholesterol is required for PGC survival and motility. It may act cell-autonomously by regulating clustering of growth factor receptors within PGCs or non cell-autonomously by controlling release of growth factors required for PGC guidance and survival.
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| 12. |
- Karlsson, Roger, 1975, et al.
(författare)
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Mechanics of lipid bilayer junctions affecting the size of a connecting lipid nanotube
- 2011
-
Ingår i: Nanoscale Research Letters. - : Springer Science and Business Media LLC. - 1931-7573 .- 1556-276X. ; 6
-
Tidskriftsartikel (refereegranskat)abstract
- In this study we report a physical analysis of the membrane mechanics affecting the size of the highly curved region of a lipid nanotube (LNT) that is either connected between a lipid bilayer vesicle and the tip of a glass microinjection pipette (tube-only) or between a lipid bilayer vesicle and a vesicle that is attached to the tip of a glass microinjection pipette (two-vesicle). For the tube-only configuration (TOC), a micropipette is used to pull a LNT into the interior of a surface-immobilized vesicle, where the length of the tube L is determined by the distance of the micropipette to the vesicle wall. For the two-vesicle configuration (TVC), a small vesicle is inflated at the tip of the micropipette tip and the length of the tube L is in this case determined by the distance between the two interconnected vesicles. An electrochemical method monitoring diffusion of electroactive molecules through the nanotube has been used to determine the radius of the nanotube R as a function of nanotube length L for the two configurations. The data show that the LNT connected in the TVC constricts to a smaller radius in comparison to the tube-only mode and that tube radius shrinks at shorter tube lengths. To explain these electrochemical data, we developed a theoretical model taking into account the free energy of the membrane regions of the vesicles, the LNT and the high curvature junctions. In particular, this model allows us to estimate the surface tension coefficients from R(L) measurements.
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| 13. |
- Keighron, Jacqueline, 1982, et al.
(författare)
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Amperometric Detection of Single Vesicle Acetylcholine Release Events from an Artificial Cell
- 2015
-
Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 6:1, s. 181-188
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Tidskriftsartikel (refereegranskat)abstract
- Acetylcholine is a highly abundant nonelectroactive neurotransmitter in the mammalian central nervous system. Neurochemical release occurs on the millisecond time scale, requiring a fast, sensitive sensor such as an enzymatic amperometric electrode. Typically, the enzyme used for enzymatic electrochemical sensors is applied in excess to maximize signal. Here, in addition to sensitivity, we have also sought to maximize temporal resolution, by designing a sensor that is sensitive enough to work at near monolayer enzyme coverage. Reducing the enzyme layer thickness increases sensor temporal resolution by decreasing the distance and reducing the diffusion time for the enzyme product to travel to the sensor surface for detection. In this instance, the sensor consists of electrodeposited gold nanoparticle modified carbon fiber microelectrodes (CFMEs). Enzymes often are sensitive to curvature upon surface adsorption; thus, it was important to deposit discrete nanoparticles to maintain enzyme activity while depositing as much gold as possible to maximize enzyme coverage. To further enhance sensitivity, the enzymes acetylcholinesterase (AChE) and choline oxidase (ChO) were immobilized onto the gold nanoparticles at the previously determined optimal ratio (1:10 AChE/ChO) for most efficient sequential enzymatic activity. This optimization approach has enabled the rapid detection to temporally resolve single vesicle acetylcholine release from an artificial cell. The sensor described is a significant advancement in that it allows for the recording of acetylcholine release on the order of the time scale for neurochemical release in secretory cells.
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| 14. |
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| 15. |
- Kurczy, Michael, 1980, et al.
(författare)
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Composition Based Strategies for Controlling Radii in Lipid Nanotubes
- 2014
-
Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:1
-
Tidskriftsartikel (refereegranskat)abstract
- Nature routinely carries out small-scale chemistry within lipid bound cells and organelles. Liposome–lipid nanotube networks are being developed by many researchers in attempt to imitate these membrane enclosed environments, with the goal to perform small-scale chemical studies. These systems are well characterized in terms of the diameter of the giant unilamellar vesicles they are constructed from and the length of the nanotubes connecting them. Here we evaluate two methods based on intrinsic curvature for adjusting the diameter of the nanotube, an aspect of the network that has not previously been controllable. This was done by altering the lipid composition of the network membrane with two different approaches. In the first, the composition of the membrane was altered via lipid incubation of exogenous lipids; either with the addition of the low intrinsic curvature lipid soy phosphatidylcholine (soy-PC) or the high intrinsic curvature lipid soy phosphatidylethanolamine (soy-PE). In the second approach, exogenous lipids were added to the total lipid composition during liposome formation. Here we show that for both lipid augmentation methods, we observed a decrease in nanotube diameter following soy-PE additions but no significant change in size following the addition of soy-PC. Our results demonstrate that the effect of soy-PE on nanotube diameter is independent of the method of addition and suggests that high curvature soy-PE molecules facilitate tube membrane curvature.
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| 16. |
- Kurczy, Michael, 1980, et al.
(författare)
-
Mass spectrometry imaging of mating Tetrahymena show that changes in cell morphology regulate lipid domain formation
- 2010
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 107:7, s. 2751-2756
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Tidskriftsartikel (refereegranskat)abstract
- Mass spectrometry imaging has been used here to suggest that changes in membrane structure drive lipid domain formation in mating single-cell organisms. Chemical studies of lipid bilayers in both living and model systems have revealed that chemical composition is coupled to localized membrane structure. However, it is not clear if the lipids that compose the membrane actively modify membrane structure or if structural changes cause heterogeneity in the surface chemistry of the lipid bilayer. We report that time-of-flight secondary ion mass spectrometry images of mating Tetrahymena thermophila acquired at various stages during mating demonstrate that lipid domain formation, identified as a decrease in the lamellar lipid phosphatidylcholine, follows rather than precedes structural changes in the membrane. Domains are formed in response to structural changes that occur during cell-to-cell conjugation. This observation has wide implications in all membrane processes.
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| 17. |
- Kurczy, Michael, 1980, et al.
(författare)
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Nanotome Cluster Bombardment to Recover Spatial Chemistry After Preparation of Biological Samples for SIMS Imaging
- 2010
-
Ingår i: Journal of the American Society for Mass Spectrometry. - : American Chemical Society (ACS). - 1044-0305 .- 1879-1123. ; 21:5, s. 833-836
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Tidskriftsartikel (refereegranskat)abstract
- A C-60(+) cluster ion projectile is employed for sputter cleaning biological surfaces to reveal spatio-chemical information obscured by contamination overlayers. This protocol is used as a supplemental sample preparation method for time of flight secondary ion mass spectrometry (ToF-SIMS) imaging of frozen and freeze-dried biological materials. Following the removal of nanometers of material from the surface using sputter cleaning, a frozen-patterned cholesterol film and a freeze-dried tissue sample were analyzed using ToF-SIMS imaging. In both experiments, the chemical information was maintained after the sputter dose, due to the minimal chemical damage caused by C-60(+) bombardment. The damage to the surface produced by freeze-drying the tissue sample was found to have a greater effect on the loss of cholesterol signal than the sputter-induced damage. In addition to maintaining the chemical information, sputtering is not found to alter the spatial distribution of molecules on the surface. This approach removes artifacts that might obscure the surface chemistry of the sample and are common to many biological sample preparation schemes for ToF-SIMS imaging.
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| 18. |
- Kurczy, Michael, 1980, et al.
(författare)
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Relative quantification of cellular sections with molecular depth profiling ToF-SIMS imaging
- 2008
-
Ingår i: Applied Surface Science. - : Elsevier BV. - 0169-4332. ; 255:4, s. 1158-1161
-
Tidskriftsartikel (refereegranskat)abstract
- We report the use of secondary ion mass spectrometry (SIMS) imaging to quantify the relative difference in the amount of lipid between two sections, the plasma membrane and the cytoplasm, of single cells from two different populations. Cells were each labeled with lipophillic dyes, frozen, fractured and analyzed in a ToF-SIMS mass spectrometer equipped with a 40 keV C60+ ion source. In addition to identifying cells from separate populations, the lipophilic dyes can be used as a marker for the outer leaflet of the cell membrane and therefore as a depth finder. Here, we show that it is possible to compare the amount of lipids with particular headgroups in the cell membrane of a treated cell to the membrane of a control cell. Following erosion of the cell membranes, the amount of the two specific lipid head groups in the cytoplasm of the treated cell can be compared to those lipids in a control cell. Here we take the first step in this experimental design and display the ability to analyze multiple sections of frozen cells following a single fracture.
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| 19. |
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| 20. |
- Lanekoff, Ingela, 1975, et al.
(författare)
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An in situ fracture device to image lipids in single cells using ToF-SIMS
- 2011
-
Ingår i: SURFACE AND INTERFACE ANALYSIS. - : Wiley. - 0142-2421 .- 1096-9918. ; 43:1-2, s. 257-260
-
Tidskriftsartikel (refereegranskat)abstract
- Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) imaging yields molecule-specific images showing the spatial distribution of specific lipids with submicron resolution, making it a potentially powerful method for studying a variety of biological questions in single cells. In order to make possible the analysis of hydrated cells in vacuum, we have designed a device for in situ freeze-fracture of cell samples. PC12 cells are frozen between two silicon shards in a sandwich geometry, and the fracture is conducted under vacuum inside the analysis chamber of the instrument. After freeze-fracture, ToF-SIMS analysis was carried out using a Bi cluster ion source, providing high-resolution images of single cells. We also show that when combining freeze-fracture and cluster SIMS imaging, low abundant biologically important lipids, specifically PE, PC and non-SM PC, can be imaged in single cells. Finally, K+ ion localization was used as a diagnostic to identify fracture planes through the cell for these fractured samples.
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| 21. |
- Lanekoff, Ingela, 1975, et al.
(författare)
-
Time of Flight Mass Spectrometry Imaging of Samples Fractured In Situ with a Spring-Loaded Trap System.
- 2010
-
Ingår i: Analytical chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 82:15, s. 6652-6659
-
Tidskriftsartikel (refereegranskat)abstract
- An in situ freeze fracture device featuring a spring-loaded trap system has been designed and characterized for time of flight secondary ion mass spectrometry (TOF SIMS) analysis of single cells. The device employs the sandwich assembly, which is typically used in freeze fracture TOF SIMS experiments to prepare frozen, hydrated cells for high-resolution SIMS imaging. The addition of the spring-loaded trap system to the sandwich assembly offers two advances to this sample preparation method. First, mechanizing the fracture by adding a spring standardizes each fracture by removing the need to manually remove the top of the sandwich assembly with a cryogenically cooled knife. A second advance is brought about because the top of the sandwich is not discarded after the sandwich assembly has been fractured. This results in two imaging surfaces effectively doubling the sample size and providing the unique ability to image both sections of a cell bifurcated by the fracture. Here, we report TOF SIMS analysis of freeze fractured rat pheochromocytoma (PC12) cells using a Bi cluster ion source. This work exhibits the ability to obtain single cell chemical images with subcellular lateral resolution from cells preserved in an ice matrix. In addition to preserving the cells, the signal from lipid fragment ions rarely identified in single cells are better observed in the freeze-fractured samples for these experiments. Furthermore, using the accepted argument that K(+) signal indicates a cell that has been fractured though the cytoplasm, we have also identified different fracture planes of cells over the surface. Coupling a mechanized freeze fracture device to high-resolution cluster SIMS imaging will provide the sensitivity and resolution as well as the number of trials required to carry out biologically relevant SIMS experiments.
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| 22. |
- Mellander, Lisa J., et al.
(författare)
-
Two modes of exocytosis in an artificial cell
- 2014
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 4
-
Tidskriftsartikel (refereegranskat)abstract
- The details of exocytosis, the vital cell process of neuronal communication, are still under debate with two generally accepted scenarios. The first mode of release involves secretory vesicles distending into the cell membrane to release the complete vesicle contents. The second involves partial release of the vesicle content through an intermittent fusion pore, or an opened or partially distended fusion pore. Here we show that both full and partial release can be mimicked with a single large-scale cell model for exocytosis composed of material from blebbing cell plasma membrane. The apparent switching mechanism for determining the mode of release is demonstrated to be related to membrane tension that can be differentially induced during artificial exocytosis. These results suggest that the partial distension mode might correspond to an extended kiss-and-run mechanism of release from secretory cells, which has been proposed as a major pathway of exocytosis in neurons and neuroendocrine cells.
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| 23. |
- Najafinobar, Neda, 1985, et al.
(författare)
-
Cholesterol Alters the Dynamics of Release in Protein Independent Cell Models for Exocytosis
- 2016
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6:Article number: 33702
-
Tidskriftsartikel (refereegranskat)abstract
- Neurons communicate via an essential process called exocytosis. Cholesterol, an abundant lipid in both secretory vesicles and cell plasma membrane can affect this process. In this study, amperometric recordings of vesicular dopamine release from two different artificial cell models created from a giant unilamellar liposome and a bleb cell plasma membrane, show that with higher membrane cholesterol the kinetics for vesicular release are decelerated in a concentration dependent manner. This reduction in exocytotic speed was consistent for two observed modes of exocytosis, full and partial release. Partial release events, which only occurred in the bleb cell model due to the higher tension in the system, exhibited amperometric spikes with three distinct shapes. In addition to the classic transient, some spikes displayed a current ramp or plateau following the maximum peak current. These post spike features represent neurotransmitter release from a dilated pore before constriction and show that enhancing membrane rigidity via cholesterol adds resistance to a dilated pore to re-close. This implies that the cholesterol dependent biophysical properties of the membrane directly affect the exocytosis kinetics and that membrane tension along with membrane rigidity can influence the fusion pore dynamics and stabilization which is central to regulation of neurochemical release.
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| 24. |
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| 25. |
- Nguyen, Tho D. K., et al.
(författare)
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Visualization of Partial Exocytotic Content Release and Chemical Transport into Nanovesicles in Cells
- 2022
-
Ingår i: Acs Nano. - : American Chemical Society (ACS). - 1936-0851 .- 1936-086X. ; 16:3, s. 4831-4842
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Tidskriftsartikel (refereegranskat)abstract
- For decades, "all-or-none"and "kiss-and-run"were thought to be the only major exocytotic release modes in cell-to-cell communication, while the significance of partial release has not yet been widely recognized and accepted owing to the lack of direct evidence for exocytotic partial release. Correlative imaging with transmission electron microscopy and NanoSIMS imaging and a dual stable isotope labeling approach was used to study the cargo status of vesicles before and after exocytosis; demonstrating a measurable loss of transmitter in individual vesicles following stimulation due to partial release. Model secretory cells were incubated with 13C-labeled l-3,4-dihydroxyphenylalanine, resulting in the loading of 13C-labeled dopamine into their vesicles. A second label, di-N-desethylamiodarone, having the stable isotope 127I, was introduced during stimulation. A significant drop in the level of 13C-labeled dopamine and a reduction in vesicle size, with an increasing level of 127I-, was observed in vesicles of stimulated cells. Colocalization of 13C and 127I- in several vesicles was observed after stimulation. Thus, chemical visualization shows transient opening of vesicles to the exterior of the cell without full release the dopamine cargo. We present a direct calculation for the fraction of neurotransmitter release from combined imaging data. The average vesicular release is 60% of the total catecholamine. An important observation is that extracellular molecules can be introduced to cells during the partial exocytotic release process. This nonendocytic transport process appears to be a general route of entry that might be exploited pharmacologically. © 2022 The Authors. Published by American Chemical Society.
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| 26. |
- Ostrowski, Sara G, et al.
(författare)
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Secondary Ion MS Imaging To Relatively Quantify Cholesterol in the Membranes of Individual Cells from Differentially Treated Populations
- 2007
-
Ingår i: Analytical chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 79:10, s. 3554-3560
-
Tidskriftsartikel (refereegranskat)abstract
- Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a well-established bioanalytical method for directly imaging the chemical distribution across single cells. Here we report a protocol for the use of SIMS imaging to comparatively quantify the relative difference in cholesterol level between the plasma membranes of two cells. It should be possible to apply this procedure to the study of other selected lipids. This development enables direct comparison of the chemical effects of different drug treatments and incubation conditions in the plasma membrane at the single-cell level. Relative, quantitative TOF-SIMS imaging has been used here to compare macrophage cells treated to contain elevated levels of cholesterol with respect to control cells. In situ fluorescence microscopy with two different membrane dyes was used to discriminate morphologically similar but differentially treated cells prior to SIMS analysis. SIMS images of fluorescently identified cells reveal that the two populations of cells have distinct outer leaflet membrane compositions with the membranes of the cholesterol-treated macrophages containing more than twice the amount of cholesterol of control macrophages. Relative quantification with SIMS to compare the chemical composition of single cells can provide valuable information about normal biological functions, causative agents of diseases, and possible therapies for diseases.
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| 27. |
- Ren, Lin, et al.
(författare)
-
The evidence for open and closed exocytosis as the primary release mechanism
- 2016
-
Ingår i: Quarterly Reviews of Biophysics. - : Cambridge University Press (CUP). - 0033-5835 .- 1469-8994. ; 49
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Tidskriftsartikel (refereegranskat)abstract
- Exocytosis is the fundamental process by which cells communicate with each other. The events that lead up to the fusion of a vesicle loaded with chemical messenger with the cell membrane were the subject of a Nobel Prize in 2013. However, the processes occurring after the initial formation of a fusion pore are very much still in debate. The release of chemical messenger has traditionally been thought to occur through full distention of the vesicle membrane, hence assuming exocytosis to be all or none. In contrast to the all or none hypothesis, here we discuss the evidence that during exocytosis the vesicle-membrane pore opens to release only a portion of the transmitter content during exocytosis and then close again. This open and closed exocytosis is distinct from kiss- and-run exocytosis, in that it appears to be the main content released during regular exocytosis. The evidence for this partial release via open and closed exocytosis is presented considering primarily the quantitative evidence obtained with amperometry.
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| 28. |
- Simonsson, Lisa, 1982, et al.
(författare)
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A functioning artificial secretory cell
- 2012
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 2, s. no. 824-
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Tidskriftsartikel (refereegranskat)abstract
- We present an amperometric study of content release from individual vesicles in an artificial secretory cell designed with the minimal components required to carry out exocytosis. Here, the membranes of the cell and vesicles are substituted for protein-free giant and large unilamellar vesicles respectively. In replacement of the SNARE-complex, the cell model was equipped with an analog composed of complimentary DNA constructs. The DNA constructs hybridize in a zipper-like fashion to bring about docking of the artificial secretory vesicles and following the addition of Ca2+ artificial exocytosis was completed. Exocytotic events recorded from the artificial cell closely approximate exocytosis in live cells. The results together with simulations of vesicular release demonstrate that the molecular flux in this model is attenuated and we suggest that this is the result of restricted diffusion through a semi-stable fusion pore or a partitioning of the signalling molecule out of the fused vesicle membrane.
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| 29. |
- Thomen, Aurélien, et al.
(författare)
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Subcellular Mass Spectrometry Imaging and Absolute Quantitative Analysis across Organelles
- 2020
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Ingår i: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 14:4, s. 4316-4325
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Tidskriftsartikel (refereegranskat)abstract
- Mass spectrometry imaging is a field that promises to become a mainstream bioanalysis technology by allowing the combination of single-cell imaging and subcellular quantitative analysis. The frontier of single-cell imaging has advanced to the point where it is now possible to compare the chemical contents of individual organelles in terms of raw or normalized ion signal. However, to realize the full potential of this technology, it is necessary to move beyond this concept of relative quantification. Here we present a nanoSIMS imaging method that directly measures the absolute concentration of an organelle-associated, isotopically labeled, pro-drug directly from a mass spectrometry image. This is validated with a recently developed nanoelectrochemistry method for single organelles. We establish a limit of detection based on the number of isotopic labels used and the volume of the organelle of interest, also offering this calculation as a web application. This approach allows subcellular quantification of drugs and metabolites, an overarching and previously unmet goal in cell science and pharmaceutical development.
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| 30. |
- Trouillon, Raphaël, 1982, et al.
(författare)
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Chemical Analysis of Single Cells
- 2013
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 85:2, s. 522-542
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Forskningsöversikt (refereegranskat)abstract
- In this Review, we provide an overview of methods developed for chemical analysis of single cells over the last two years. Many biological systems contain an ensemble of cells with heterogeneous chemistry; therefore, it is important to analyze them on an individual basis in order to elucidate the role each cell plays in the function of these systems. In clinical diagnostics, the development of extremely sensitive measurements, down to single cells, may provide the best ability for diagnoses. Single cell analysis has, in fact, been present for quite some time. Investigators in life sciences consider the cell as the unit of life and so the pursuit to quantify, image, and modulate the cell has been ongoing for decades.
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