We develop and emply biophysical techniques to study cellular processes with high precision and detail.
Living cells have the remarkable ability to sense environmental signals such as physical force or small molecules. This information is processed by intracellular signaling networks, which allow a cell to respond to the stimulus. Our research, at the intersection of physics and biology, aims to understand how these cellular signaling processes are regulated on different hierarchical length-scales by employing and further developing single molecule and super-resolution microscopy techniques.
On a mesoscopic length scale signaling proteins can self-assemble to complexes and intracellular organelles. We employ recently developed quantitative super-resolution microscopy to resolve these structures inside cells below the optical diffraction limit and to measure their biomolecular composition. This characterization allows to detect changes as organelles mature and to follow spatial re-arrangements of signaling proteins in response to stimuli. A primary focus of our lab is to investigate if and how such spatial re-arrangements can modulate the signaling strength of pathways.
Single Molecule Localization Microscopy (SMLM):
Location is the root of key questions for understanding how proteins dynamically regulate the cell's behavior. Where are they located inside the cell and what happens when this localization changes? Important examples include the polarization of proteins in a cell to establish a direction, the dynamic coating and un-coating of organelles with proteins and the formation of oligomeric signaling complexes. The discovery and application of fluorescent proteins (such as the Green Fluorescent Protein GFP) in combination with molecular genetics allows the distribution of a protein of interest to be observed within a cell using fluorescence microscopy. While conventional fluorescence microscopy gives valuable insights into biological and biophysical processes, a major drawback is the limited spatial resolution due to diffraction of light. Since cells are highly crowded with proteins, whose size is in the nanometer range, the point spread functions from individual fluorescent proteins overlap and blur the image.
Localization-based super-resolution microscopy techniques such as photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM) circumvent this problem by employing photo-switchable or photo-activatable fluorescent proteins fused to a protein of interest and by fitting their point-spread function to precisely determine their locations. In each frame of a super-resolution microscopy movie, only a very sparse subset of single proteins is converted to the detectable fluorescent state by low dosages of 405 nm light. This sub-set of proteins is subsequently excited and imaged until the fluorophores are bleached (see image to the right). In the next cycle, a new sparse sub-set of proteins is converted and imaged. In this way, individual proteins are stochastically activated and their point spread functions become well separated in time and space. The final super-resolution picture is a rendered image containing all protein localizations with an accuracy of typically 20nm.
Counting proteins in complexes below the optical diffraction limit:
Super-resolution microscopy techniques offer information about the local number of molecules within a spatial region, especially when photoactivatable fluorescent proteins (PAFPs) are used as the probe. However, they often have associated over- and undercounting errors that must be corrected. For example, the blinking of PAFPs - such as mEos2 - has been shown to cause overcounting artifacts that vary with experimental conditions and complicate quantitative image reconstruction. In addition, problems with protein misfolding or incomplete photoconversion will result in undetected fluorescent proteins and undercounting artifacts. In our experiments we employ our recently developed intracellular single-molecule superresolution microscopy calibration to correct for blinking by measuring the photo-physical parameters in cells under imaging conditions. By counting the number of molecules in protein complexes of defined stoichiometry, we then determine the fraction of undetected PAFPs; this allows us to calibrate and count absolute numbers of molecules.
Tracking Organelle Maturation:
Cells contain a variety of membrane-based organelles that execute a wide range of functions such as: membrane trafficking, receptor recycling, phagy- or autophagy, and cytoskeleton organization. The organelle’s morphology and biomolecular composition defines their identity and is central to the processes of building, modifying, trafficking, and recycling. Therefore, there is a biological importance to classify organelles based on both their size/structure and the number of regulatory biomolecules they contain.
Quantitative SR microscopy allows us to gain novel information about the relationship between the size/structure and biomolecular composition below the optical diffraction limit. Further, it allows us to track the structural changes and maturation of organelles. Recently, we developed and applied a quantitative super-resolution microscopy approach that tracked endosome maturation in yeast cells by measuring their size and biomolecular composition. We found that during endosome maturation, both organelle size/morphology and composition are dynamically regulated. Endocytic vesicles split off of the plasma membrane with a diameter of about 80 nm, fuse to form early endosomes, and then mature to late endosomes (figure right side).
It is well know that the production of phosphatidylinositol 3-phosphate (PI3P) from plasma membrane phospholipids is required for endocytosis and for membrane transport to early and late endosomes. We were interested in identifying if regulatory PI3P is produced on individual incoming vesicles or on matured endosomes. To address this question, we combined conventional co-localization microscopy using GFP with single molecule counting using mEos2 and SR microscopy.
The PI3P-binding domain of EEA1 fused to mEos2 served as a reporter of the number of accessible PI3P binding sites as well as the size of the vesicles. In addition, GFP fused to endocytic landmark proteins allowed for simultaneous determination of the stage of maturation (Figure left side). From a large number of steady-state snapshots, the maturation of vesicles to endosomes could be observed.
This new approach opened up the possibility of accurately measuring molecular stoichiometries across a few orders of magnitude while resolving organelles below the optical diffraction limit.
Cluster formation in cellular signaling:
Signaling molecules receive, process and transmit the information by means of biochemical reactions and conformational changes. While these processes play a major role in regulating the signaling output, additional layers of control exist. The complex spatial organization of biomolecules is one such layer of control; signaling proteins can change their activity by associating into di-, tri-, and higher order oligomeric complexes or dynamically coating organelles to define their state of maturation. By fusing a fluorescent protein to our protein of interest we can use quantitative SR microscopy to observe the biomolecular composition of such structures below the optical diffraction limit and track individual signaling proteins in living cells to quantify the dynamics of proteins during signaling in real time.
Additionally, we apply synthetic biology (including opto-genetics) and genetic engineering techniques to extend our understanding beyond the naturally occurring spatial organization of signaling proteins. By externally manipulating the localization and oligomeric state of signaling proteins with light (or small molecule ligands), we can systematically investigate how information transmission in cell signaling is mediated by protein complex formation and clustering. This approach, in combination with SR microscopy, expands our tools to externally control the activity of cells and explores potential biomedical application.