- SPT Single Particle Tracking
- PALM Photoactivation Localization Microscopy
- STORM Stochastic Optical Reconstruction Microscopy
- TIRF Total Internal Reflection Microscopy
- HILO Highly Inclined Laminated Optical Sheet Microscopy
Meckel
Membrane Dynamics
Dr. Tobias Meckel
(Junior Research Group)
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Research
Membrane dynamics in response to cell adhesion
The plasma membrane of living cells is is a highly dynamic structure, which is characterized by a continous reorganization of the proteins and lipids it is composed of. It hosts and provides for a diverse range of physiological processes such as barrier and transport functions, energy production, organelle biogenesis and cellular adhesion. In other words, it controls the exchange of matter, energy, and information of a cell with its environment.
In order to perform this structural and functional set of tasks, the molecular complexity of biomembranes is understandably high and reflected by the fact that around one third of the genome encodes for membrane proteins. But there's more:
Above all molecular variety, it is the highly dynamic reorganization of the membrane that defines its complex nature. The formation of small or large, short-lived or long-lived and homo- or heteromeric clusters of membrane components is what really enables the plasma membrane to meet its diverse set of demands. In other words, the plasma membrane is a structure with spatio-temporal dynamic heterogeneity.
We aim for a quantitative description of this heterogeneity using single molecule microscopy on mammalian and plant cell systems. In particular, we are interested in how attachement to an extracellular matrix (or cell wall) alters the dynamic properties of the plasma membrane.
Integrin receptors
Cellular adhesion to a surface, extracellular matrix or another cell is governed by the dynamics of membrane-cytoskeleton interactions and mediated by specific receptors such as cadherins, integrins, selectins or adhesins. Each type of cellular adhesion requires a particular constellation of actin binding proteins, intermediate filament proteins and other cytoskeletal components. All these functions are tighly coupled in that they both depend on and modulate the dynamic properties of the plasma membrane.
We are especially interested in the molecular distribution and and dynamics of both adhesion related and also unrelated proteins, as they present themself in the context of three-dimensional (3D) culture conditions. By following both sets of proteins in our studies, we obtain information (i) on how number, size, distribution and lifetime of adhesomes change with matrix conditions, and (i) on how cellular attachement to a support influences membrane located signalling pathways, which are not directly linked to adhesion. Both are required to help in the understanding of the complex relationship between a cell its surrounding.
Methods
Cell cultivation in 2, 2½, and 3 dimensions
The cultivation of adherent cells on a planar support (e.g. a flask or coverslip) is in most cases owing to the fact of a simplified handling and microscopic observation. Cellular behavior and morphology, however, are highly dependent on the characteristics of their extracellular environment as “living cells possess an exquisite sensitivity to both the chemical and physical characteristics of the surfaces to which they adhere” (Geiger et al. Nat Rev Mol Cell Biol, 2009). To that end we currently optimize the cultivation of mammalian cells in 3D matrices to make them compatible with high-resolution and automated high-throughput microscopy.
Further, in colaboration with the DFG cluster of excellence 259 Center of Smart Interfaces (CSI) and the LOEWE research focus “Soft Control” we exploit the virtues of microfluidics and smart polymers in order to investigate cell adhesion to substrates with defined chemical and physical properties.
Single Molecule Microscopy
To capture and quantify the dynamic heterogeneity of membrane components, a technique with sufficient spatial and temporal resolution is required. Single molecule microscopy meets this demand. The ability to localize individual proteins in a living cell with a precision of 20 or 60 nm (in 2D or 3D, respectively) with a time resolution of 10-50 ms allows us to precisely follow the assembly and mobility and membrane components.
Several modes of single molecule microscopy with different illumination schemes can be used on our setup:
Quantitative Confocal Laser Scanning Microscopy
Using various calibration schemes we put the abilities of a confocal setup to true quantitative use. Calibrations of 3D spatial dimensions or local fluorescence intensities in combination with self-developed image analysis routines enable us to calculate surface/volume relations of cells and organelles or quantify local protein numbers with near single molecule accuracy.
Spatially realistic Monte Carlo simulations of single molecule diffusion
Mcell is a programm that uses spatially realistic 3D models and specialized Monte Carlo algorithms to simulate the movements and reactions of molecules within and between cells. In collaboration with its inventors, we currently develop a framwork which extends the application range of the program. Rather than feeding Mcell with measured diffusion coefficients to yield simulations, we aim to reverse the process and use it to quantify diffusion of confocal or single molecule datasets. The advantage of this process is to bypass the often complex mathematics of continuum models with an adaptive stochastic method, which is adaptive to any 3D dataset.
Atomic Force Microscopy
Our AFM is compatible with both microscopy as well as live cell recordings in liquid environments. We use it to characterize the physical properties (i.e. elasticity, rigidity) of the extracellular environment and the cell in contact with it.