| 1. |
- Benninger, R. K. P., et al.
(författare)
-
Fluorescence imaging of two-photon linear dichroism : Cholesterol depletion disrupts molecular orientation in cell membranes
- 2005
-
Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 88:1, s. 609-622
-
Tidskriftsartikel (refereegranskat)abstract
- The plasma membrane of cells is an ordered environment, giving rise to anisotropic orientation and restricted motion of molecules and proteins residing in the membrane. At the same time as being an organized matrix of defined structure, the cell membrane is heterogeneous and dynamic. Here we present a method where we use fluorescence imaging of linear dichroism to measure the orientation of molecules relative to the cell membrane. By detecting linear dichroism as well as fluorescence anisotropy, the orientation parameters are separated from dynamic properties such as rotational diffusion and homo energy transfer ( energy migration). The sensitivity of the technique is enhanced by using two-photon excitation for higher photo-selection compared to single photon excitation. We show here that we can accurately image lipid organization in whole cell membranes and in delicate structures such as membrane nanotubes connecting two cells. The speed of our wide-field imaging system makes it possible to image changes in orientation and anisotropy occurring on a subsecond timescale. This is demonstrated by time-lapse studies showing that cholesterol depletion rapidly disrupts the orientation of a fluorophore located within the hydrophobic region of the cell membrane but not of a surface bound probe. This is consistent with cholesterol having an important role in stabilizing and ordering the lipid tails within the plasma membrane.
|
|
| 2. |
|
|
| 3. |
- Benninger, Richard K. P., et al.
(författare)
-
Live Cell Linear Dichroism Imaging Reveals Extensive Membrane Ruffling within the Docking Structure of Natural Killer Cell Immune Synapses
- 2009
-
Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 96:2, s. L13-L15
-
Tidskriftsartikel (refereegranskat)abstract
- We have applied fluorescence imaging of two-photon linear dichroism to measure the subresolution organization of the cell membrane during formation of the activating (cytolytic) natural killer (NK) cell immune synapse (IS). This approach revealed that the NK cell plasma membrane is convoluted into ruffles at the periphery, but not in the center of a mature cytolytic NK cell IS. Time-lapse imaging showed that the membrane ruffles formed at the initial point of contact between NK cells and target cells and then spread radialy across the intercellular contact as the size of the IS increased, becoming absent from the center of the mature synapse. Understanding the role of such extensive membrane ruff ling in the assembly of cytolytic synapses is an intriguing new goal.
|
|
| 4. |
- Kowalewski, Jacob, 1978-
(författare)
-
Modeling and Data Analysis in Cellular Biophysics
- 2009
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cellular biophysics deals with the physical aspects of cell biology. This thesis presents a number of studies where mathematical models and data analysis can increase our understanding of this field. During recent years development in experimental methods and mathematical modeling have driven the amount of data and complexity in our understanding of cellular biology to a new level. This development has made it possible to describe cellular systems quantitatively where only qualitative descriptions were previously possible. To deal with the complex data and models that arise in this kind of research a combination of tools from physics and cell biology has to be applied; this constitutes a field we call cellular biophysics. The aim of this doctoral thesis is to develop novel approaches in this field. I present eight studies where quantitative modeling and analysis are involved. The first two studies concern cells interacting with their surrounding environment in the kidney. These cells sense fluid flow and respond with calcium (Ca2+) signals. The interaction between fluid and cells in renal tubular epithelium can be described by biomechanical models. This thesis describes a mathematical model of flow sensing by cilia with focus on the flow frequency response and time delay between the mechanical stress and the Ca2+ signaling response. Intracellular Ca2+ is kept at a very low level compared to the extracellular environment, while several intracellular compartments have higher Ca2+ concentration than the cytoplasm. This makes Ca2+ an efficient messenger for intracellular signaling, the process whereby signals are transduced from an extracellular stimulus to an intracellular activity such as gene expression. An important type of Ca2+ signaling is oscillations in intracellular Ca2+ concentration which occur due to the concerted interplay between different transport mechanisms within a cell. A study in this thesis examines ways to explain these mechanisms in terms of a mathematical model. Another study in the thesis reports that erythropoietin can regulate the water permeability of astrocytes and that it alters the pattern of Ca2+ oscillations in astrocytes. In this thesis the analysis of this Ca2+ signaling is described. Simulations described in one of the studies show how different geometries can affect the fluorescence recovery and that geometrically constrained reactions can trap diffusing receptors in dendritic spines. When separate time scales are present in a fluorescence revovery after photobleaching (FRAP) experiment the reaction and diffusion components can be studied separately. Applying single particle tracking methods to the migration trajectories of natural killer cells shows that there is a correlation between the formation of conjugates and transient confinement zones (TCZs) in these trajectories in vitro. TCZs are also present in in vivo experiments where they show strong similarities with the in vitro situation. This approach is a novel concept in data analysis methods for tracking immune cells.
|
|
| 5. |
- Manneberg, Otto, et al.
(författare)
-
A three-dimensional ultrasonic cage for characterization of individual cells
- 2008
-
Ingår i: Applied Physics Letters. - : AIP Publishing. - 0003-6951 .- 1077-3118. ; 93, s. 063901-
-
Tidskriftsartikel (refereegranskat)abstract
- We demonstrate enrichment, controlled aggregation, and manipulation of microparticles and cells by an ultrasonic cage integrated in a microfluidic chip compatible with high-resolution optical microscopy. The cage is designed as a dual-frequency resonant filleted square box integrated in the fluid channel. Individual particles may be trapped three dimensionally, and the dimensionality of one-dimensional to three-dimensional aggregates can be controlled. We investigate the dependence of the shape and position of a microparticle aggregate on the actuation voltages and aggregate size, and demonstrate optical monitoring of individually trapped live cells with submicrometer resolution.
|
|
| 6. |
- Manneberg, Otto, et al.
(författare)
-
Flow-free transport of cells in microchannels by frequency-modulated ultrasound
- 2009
-
Ingår i: Lab on a Chip. - 1473-0197 .- 1473-0189. ; 9, s. 833-837
-
Tidskriftsartikel (refereegranskat)abstract
- We demonstrate flow-free transport of cells and particles by the use of frequency-modulated ultrasonic actuation of a microfluidic chip. Two different modulation schemes are combined: A rapid (1 kHz) linear frequency sweep around similar to 6.9 MHz is used for two-dimensional spatial stabilization of the force field over a 5 mm long inlet channel of constant cross section, and a slow (0.2-0.7 Hz) linear frequency sweep around similar to 2.6 MHz is used for flow-free ultrasonic transport and positioning of cells or particles. The method is used for controlling the motion and position of cells monitored with high-resolution optical microscopy, but can also be used more generally for improving the robustness and performance of ultrasonic manipulation micro-devices.
|
|
| 7. |
|
|
| 8. |
- McCann, Fiona E., et al.
(författare)
-
The activating NKG2D ligand MHC class I-related chain a transfers from target cells to NK cells in a manner that allows functional consequences
- 2007
-
Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 178:6, s. 3418-3426
-
Tidskriftsartikel (refereegranskat)abstract
- Recently, it has become apparent that surface proteins commonly transfer between immune cells in contact. Inhibitory receptors and ligands exchange between cells during NK cell surveillance and we report here that NK cells also acquire activating ligands from target cells. Specifically, the stress-inducible activating ligand for NKG2D, MHC class I-related chain A (MICA), transferred to NK cells upon conjugation with MICA-expressing target cells. Acquisition of MICA from target cells was dependent on cell contact and occurred after accumulation of MICA at the immunological synapse. Moreover, transfer of MICA was facilitated by specific molecular recognition via NKG2D and augmented by Src kinase signaling. Importantly, MICA associated with its new host NK cell membrane in an orientation that allowed engagement with NKG2D in trans and indeed could down-regulate NKG2D in subsequent homotypic interactions with other NK cells. MICA captured from target cells could subsequently transfer between NK cells and, more importantly, NK cell degranulation was triggered in such NK cell-NK cell interactions. Thus, NK cells can influence other NK cells with proteins acquired from target cells and our data specifically suggest that NK cells could lyse other NK cells upon recognition of activating ligands acquired from target cells. This mechanism could constitute an important function for immunoregulation of NK cell activity.
|
|
| 9. |
|
|
| 10. |
- Önfelt, Björn, et al.
(författare)
-
Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria
- 2006
-
Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 177:12, s. 8476-8483
-
Tidskriftsartikel (refereegranskat)abstract
- We report that two classes of membrane nanotubes between human monocyte-derived macrophages can be distinguished by their cytoskeletal structure and their functional properties. Thin membrane nanotubes contained only F-actin, whereas thicker nanotubes, i.e., those > similar to 0.7 mu m in diameter, contained both F-actin and microtubules. Bacteria could be trapped and surf along thin, but not thick, membrane nanotubes toward connected macrophage cell bodies. Once at the cell body, bacteria could then be phagocytosed. The movement of bacteria is aided by a constitutive flow of the nanotube surface because streptavidin-coated beads were similarly able to traffic along nanotubes between surface-biotinylated macrophages. Mitochondria. and intracellular vesicles, including late endosomes and lysosomes, could be detected within thick, but not thin, membrane nanotubes. Analysis from kymographs demonstrated that vesicles moved in a stepwise, bidirectional manner at similar to 1 mu m/s, consistent with their traffic being mediated by the microtubules found only in thick nanotubes. Vesicular traffic in thick nanotubes and surfing of beads along thin nanotubes were both stopped upon the addition of azide, demonstrating that both processes require ATP. However, microtubule destabilizing agents colchicine or nocodazole abrogated vesicular transport but not the flow of the nanotube surface, confirming that distinct cytoskeletal structures of nanotubes give rise to different functional properties. Thus, membrane nanotubes between macrophages are more complex than unvarying ubiquitous membrane tethers and facilitate several means for distal interactions between immune cells.
|
|
