Periodic Reporting for period 5 - CellStructure (Structural cell biology in situ using superresolution microscopy)
Periodo di rendicontazione: 2023-10-01 al 2024-05-31
Structural biology is the field that investigates the structure and dynamics of bioproteins to understand their function in the cell. Electron microscopy (EM) is the most popular technique in the field, but it is limited, as it cannot be used in living cells and struggles with identifying specific proteins in the dense cellular environment. Optical superresolution microscopy technologies optimally complement classical structural techniques because the fluorescent label that is attached to proteins of interest provides high contrast and specificity, and because they can be used in living cells to directly measure dynamics. The superresolution approach with currently the highest resolution is single-molecule localization microscopy (SMLM), in which proteins are labeled with fluorophores that can be switched between a dark and a bright state. By switching on only very few fluorophores at a time, we can determine their precise positions, and from the positions recorded over long times we can render a superresolution image.
The aim of the ERC-CoG grant CellStructure was to develop new superresolution technologies based on SMLM for structural biology to study protein assemblies with nanometer resolution in fixed and live cells and to use these technologies to study the fundamental process of endocytosis, by which cells take up molecules from the outside. Our technologies are shared openly with the community and will enable many labs to perform biological research that previously was just not possible because of the limited resolution. This will lead to a better understanding of the fundamental processes of life with its implications for understanding and treating diseases.
Specifically, the objectives of the project were a) to develop a method to count the precise copy number of proteins in an assembly, one of the most important parameters to understand their structure and function; b) to develop a microscope approach with nanometer 3D resolution for structural biology; c) to extend superresolution microscopy to many colors to investigate the relative arrangement of proteins and to integrate other modalities such as EM in a correlative way; d) to develop new data analysis tools to extract biological information from the measurements and e) to investigate the arrangements of proteins during endocytosis to understand their function.
The aim of the ERC-CoG grant CellStructure was to develop new superresolution technologies based on SMLM for structural biology to study protein assemblies with nanometer resolution in fixed and live cells and to use these technologies to study the fundamental process of endocytosis, by which cells take up molecules from the outside. Our technologies are shared openly with the community and will enable many labs to perform biological research that previously was just not possible because of the limited resolution. This will lead to a better understanding of the fundamental processes of life with its implications for understanding and treating diseases.
Specifically, the objectives of the project were a) to develop a method to count the precise copy number of proteins in an assembly, one of the most important parameters to understand their structure and function; b) to develop a microscope approach with nanometer 3D resolution for structural biology; c) to extend superresolution microscopy to many colors to investigate the relative arrangement of proteins and to integrate other modalities such as EM in a correlative way; d) to develop new data analysis tools to extract biological information from the measurements and e) to investigate the arrangements of proteins during endocytosis to understand their function.
To count proteins in cells, we developed a reference structure based on the nuclear pore complex, a channel to the nucleus. This not only allowed us to count precise copy numbers of other nuclear pore proteins and of proteins in the kinetochore, a protein machinery that separates the genetic material during cell division, the reference structure also became the gold standard for quality control in superresolution microscopy, used by many groups worldwide.
To increase the throughput of the notoriously slow SMLM we completely automated our microscopes so that they can acquire data around the clock without user intervention. To increase the resolution of SMLM, we developed a novel approach to 3D that uses a nearfield effect, by which a fluorophore can couple its emission directly into the substrate. In addition, we implemented a very complex microscope that uses interferometry to precisely localize fluorophores in 3D in entire cells. Finally, we integrated a new SMLM variant called MINFLUX in the lab. We established its use for fixed and live cell imaging and for tracking single proteins in cells. This allowed us to resolve for the first time the steps that a motor protein called kinesin takes while it transports molecules through a cell. Finally, we developed a new approach to MINFLUX that has the prospect to substantially simplify these still complex and expensive instruments.
We increased the number of fluorophores that can be imaged simultaneously in SMLM to four colors by new hardware and software technologies, that now allow us to precisely dissect the structure of complex protein machines. By developing technologies that directly correlate SMLM and EM, we hope to combine the best of both worlds and combine angstrom resolution and cellular context of EM with the molecular specificity of SMLM. To this end, we build an SMLM microscope that works under cryogenic temperatures and can image cells on an EM grid directly before the EM images are acquired.
We developed new algorithms and software to determine the positions of single fluorophores with a highest precision, even under challenging circumstances such as very high densities or multiple channels using new deep-learning approaches. Another important software development was an algorithm to extract specific structural parameters from the fluorophore positions, which can then be used to quantitatively answer biological questions. All our software developments are integrated into an open-source software platform.
All these tools now allowed us to see the process of endocytosis in a new light and to uncover unknown mechanisms. In the model organism yeast, we mapped out the positions of many endocytic proteins and discovered how the force is generated that pulls in the membrane. In human cells we investigated the mechanism how the protein coat around the growing vesicle is formed and could resolve a long-standing question the field.
To increase the throughput of the notoriously slow SMLM we completely automated our microscopes so that they can acquire data around the clock without user intervention. To increase the resolution of SMLM, we developed a novel approach to 3D that uses a nearfield effect, by which a fluorophore can couple its emission directly into the substrate. In addition, we implemented a very complex microscope that uses interferometry to precisely localize fluorophores in 3D in entire cells. Finally, we integrated a new SMLM variant called MINFLUX in the lab. We established its use for fixed and live cell imaging and for tracking single proteins in cells. This allowed us to resolve for the first time the steps that a motor protein called kinesin takes while it transports molecules through a cell. Finally, we developed a new approach to MINFLUX that has the prospect to substantially simplify these still complex and expensive instruments.
We increased the number of fluorophores that can be imaged simultaneously in SMLM to four colors by new hardware and software technologies, that now allow us to precisely dissect the structure of complex protein machines. By developing technologies that directly correlate SMLM and EM, we hope to combine the best of both worlds and combine angstrom resolution and cellular context of EM with the molecular specificity of SMLM. To this end, we build an SMLM microscope that works under cryogenic temperatures and can image cells on an EM grid directly before the EM images are acquired.
We developed new algorithms and software to determine the positions of single fluorophores with a highest precision, even under challenging circumstances such as very high densities or multiple channels using new deep-learning approaches. Another important software development was an algorithm to extract specific structural parameters from the fluorophore positions, which can then be used to quantitatively answer biological questions. All our software developments are integrated into an open-source software platform.
All these tools now allowed us to see the process of endocytosis in a new light and to uncover unknown mechanisms. In the model organism yeast, we mapped out the positions of many endocytic proteins and discovered how the force is generated that pulls in the membrane. In human cells we investigated the mechanism how the protein coat around the growing vesicle is formed and could resolve a long-standing question the field.
All new technologies greatly surpassed the state of the art and allowed us to create textbook knowledge about the important process of endocytosis.