Final Report Summary - ISOLATE (Developing single cell technologies for systems biology)
Summary description of the project objectives
Development of single cell measurement techniques represents the key enabling technique to ultimately understand important complex biological behaviours. The education of highly qualified specialists with multidisciplinary skills is a pivotal prerequisite for addressing these biological problems and, even more importantly, for transferring this knowledge into marketable products.
ISOLATE represents an Initial Training Network involving eleven trainees (nine ESRs, two ERs). A range of methodologies provide unique training to fellows in single cell analyses and investigate complex biological phenomena. A high degree of multi-disciplinarity and the inclusion of industrial stakeholders make ISOLATE especially suitable for a training program ranging from forefront micro-technology, bioengineering, biophysics, analytical chemistry and systems biology.
ISOLATE develops novel technology for single cell cultivation, handling and analysis and utilize these technological capabilities to answer complex biological questions. The biological questions to be answered are connected to the complex processes of metabolism and signalling –areas with recognized importance for health and disease. ISOLATE develops microfluidics-based devices for single cell cultivation, highly-sophisticated optical methods for protein analysis on the single cell level, and tools for metabolite analysis and for visualizing signalling processes in single cells.
ISOLATE is focused on the following specific research objectives (ROs):
RO1: To develop an array-based microfluidic cultivation device for yeast cells
RO2: To develop an optical tweezer-based microfluidic cultivation device for yeast cells
RO3: To develop nano-biosensors to measure temporal behaviour of metabolites in single cells
RO4: To develop a mass-spectrometry based method for single yeast cell metabolomics
RO5: To develop an approach for imaging of kinase activity in single yeast cells
RO6: To develop imaging tools for single-molecule optical proteomics in single yeast cells
RO7: To understand metabolic oscillations in single yeast cells
RO8: To understand the system properties of glucose-induced signal transduction through the AMP-activated Snf1signalling kinase in yeast
Description of the work performed and the main results achieved since the beginning of the project
ISOLATE has made progress in developing key methods for single cell handling, imaging as well as measurements from single cell extracts.
We developed a microfluidic cell-culture chip that enables the trapping, cultivation and release of selected individual cells. Single cells can be trapped in a microfluidic channel using mild suction at defined cell immobilization orifices, where they can be cultivated under controlled environmental conditions. Cells of interest can then be individually and independently released for further downstream analysis by applying a negative dielectrophoretic force via the respective electrodes located at each immobilization site.
We have progressed in developing an advanced experimental system with holographic optical tweezer-based cell positioning and with improved imaging. Holographic optical tweezers allow multiple objects or cells to be trapped in 3D simultaneously which improve throughput and the diversity of analysis. Generating tweezer-based cell arrays is possible but several issues are still remaining to be addressed before the system can be used productively.
We have developed a single molecule fluorescence microscope capable of millisecond time scale imaging of proteins in single cells and also have designed and implemented advanced imaging analysis software catered for in vivo single-molecule single-cell microscopy. By means of a light profile meter the spinning disk confocal system was calibrated to obtain a uniform illumination over the sample area of interest, assuring the uniform coverage of the focal plane of interest. Developing an automatized setup that can ensemble the spinning disk with the optical tweezers, microfluidic pumps and inverted microscope is one of the current main tasks.
The improved slimfield method for optimised imaging and the first version of the low-light imaging software were reported in the first periodic report. Completion of these deliverables facilitated successful development of a second version of low-light imaging software. This new software has been designed, coded in MATLAB and fully implemented on the slimfield system using real in vivo data obtained from single S. cerevisiae cells using novel genomic fusions of Mig1-GFP.
Given that we have been able to observe a reduced ATP consumption rate in a yeast mutant lacking the Snf4 gene compared to a wild type we believe it will be feasible to image kinase activity in single cells using a fluorescence microscope. However, this entails detectors with very high sensitivity. One way to overcome this problem may be to average signals from many cells and/ or repeats. This is currently under investigation.
Given the unexpected difficulties in selecting the aptamer binding fructose 1,6-bisphosphate and its characterization in terms of structure, Kd, etc. the selections of aptamers for the second and third nanosensor have been delayed and only just completed. The aptamer clones for the third nano-sensor are awaiting further characterization (structure, Kd, etc.). It is expected that this sensor will be available within the next 6-9 months
ISOLATE used available technology and new technological advances to address biological questions that can only be studied in single cells. For instance, by employing Mig1 nuclear-cytosolic shuttling we have observed sensing thresholds, distinct dynamic behaviours under different metabolic conditions and linked the dynamic behaviour to glucose uptake into yeast cells. Using single molecule tracking we found that Mig1 takes different oligomeric forms under different conditions, which may help find the transcription factor to locate its target sequences in the nucleus. We also found that, although Mig1 is nuclear and cytoplasmic, respectively, under high and low glucose conditions, a fraction of Mig1 is always present in the nucleus and dynamic traffic occurs at all times.
Furthermore, we wish to understand oscillations in central metabolism on the single cell level using the technology developed in this network. The ATP FRET sensor, the pH ratiometric sensor and NAD(P)H autofluorescence have been successfully applied to study metabolism at a single cell level. Metabolic oscillations turned out to be an intrinsic autonomous property of metabolism while the pH levels tend to remain well buffered within single yeast cells. In addition, the single cell resolution provided by the aforementioned technologies, allowed the characterization of rare individual cells in heterogeneous, yet isogenic, yeast cell populations, offering insights on the contribution of metabolism to the emergence of cell-to-cell variability under constant environmental conditions. For this purpose, Peredox, a metabolic biosensor previously used only for mammalian experimental systems, as well as TRACK, a metabolic biosensor specific for Trehalose-6-phosphate, have been also adopted and utilized for single yeast cell research. The auxin inducible degron, in combination with traditional knock-out strategies were used to investigate the role of signalling pathways and molecular mechanisms for the generation and maintenance of metabolic oscillations, dynamically and in single cells.
Development of single cell measurement techniques represents the key enabling technique to ultimately understand important complex biological behaviours. The education of highly qualified specialists with multidisciplinary skills is a pivotal prerequisite for addressing these biological problems and, even more importantly, for transferring this knowledge into marketable products.
ISOLATE represents an Initial Training Network involving eleven trainees (nine ESRs, two ERs). A range of methodologies provide unique training to fellows in single cell analyses and investigate complex biological phenomena. A high degree of multi-disciplinarity and the inclusion of industrial stakeholders make ISOLATE especially suitable for a training program ranging from forefront micro-technology, bioengineering, biophysics, analytical chemistry and systems biology.
ISOLATE develops novel technology for single cell cultivation, handling and analysis and utilize these technological capabilities to answer complex biological questions. The biological questions to be answered are connected to the complex processes of metabolism and signalling –areas with recognized importance for health and disease. ISOLATE develops microfluidics-based devices for single cell cultivation, highly-sophisticated optical methods for protein analysis on the single cell level, and tools for metabolite analysis and for visualizing signalling processes in single cells.
ISOLATE is focused on the following specific research objectives (ROs):
RO1: To develop an array-based microfluidic cultivation device for yeast cells
RO2: To develop an optical tweezer-based microfluidic cultivation device for yeast cells
RO3: To develop nano-biosensors to measure temporal behaviour of metabolites in single cells
RO4: To develop a mass-spectrometry based method for single yeast cell metabolomics
RO5: To develop an approach for imaging of kinase activity in single yeast cells
RO6: To develop imaging tools for single-molecule optical proteomics in single yeast cells
RO7: To understand metabolic oscillations in single yeast cells
RO8: To understand the system properties of glucose-induced signal transduction through the AMP-activated Snf1signalling kinase in yeast
Description of the work performed and the main results achieved since the beginning of the project
ISOLATE has made progress in developing key methods for single cell handling, imaging as well as measurements from single cell extracts.
We developed a microfluidic cell-culture chip that enables the trapping, cultivation and release of selected individual cells. Single cells can be trapped in a microfluidic channel using mild suction at defined cell immobilization orifices, where they can be cultivated under controlled environmental conditions. Cells of interest can then be individually and independently released for further downstream analysis by applying a negative dielectrophoretic force via the respective electrodes located at each immobilization site.
We have progressed in developing an advanced experimental system with holographic optical tweezer-based cell positioning and with improved imaging. Holographic optical tweezers allow multiple objects or cells to be trapped in 3D simultaneously which improve throughput and the diversity of analysis. Generating tweezer-based cell arrays is possible but several issues are still remaining to be addressed before the system can be used productively.
We have developed a single molecule fluorescence microscope capable of millisecond time scale imaging of proteins in single cells and also have designed and implemented advanced imaging analysis software catered for in vivo single-molecule single-cell microscopy. By means of a light profile meter the spinning disk confocal system was calibrated to obtain a uniform illumination over the sample area of interest, assuring the uniform coverage of the focal plane of interest. Developing an automatized setup that can ensemble the spinning disk with the optical tweezers, microfluidic pumps and inverted microscope is one of the current main tasks.
The improved slimfield method for optimised imaging and the first version of the low-light imaging software were reported in the first periodic report. Completion of these deliverables facilitated successful development of a second version of low-light imaging software. This new software has been designed, coded in MATLAB and fully implemented on the slimfield system using real in vivo data obtained from single S. cerevisiae cells using novel genomic fusions of Mig1-GFP.
Given that we have been able to observe a reduced ATP consumption rate in a yeast mutant lacking the Snf4 gene compared to a wild type we believe it will be feasible to image kinase activity in single cells using a fluorescence microscope. However, this entails detectors with very high sensitivity. One way to overcome this problem may be to average signals from many cells and/ or repeats. This is currently under investigation.
Given the unexpected difficulties in selecting the aptamer binding fructose 1,6-bisphosphate and its characterization in terms of structure, Kd, etc. the selections of aptamers for the second and third nanosensor have been delayed and only just completed. The aptamer clones for the third nano-sensor are awaiting further characterization (structure, Kd, etc.). It is expected that this sensor will be available within the next 6-9 months
ISOLATE used available technology and new technological advances to address biological questions that can only be studied in single cells. For instance, by employing Mig1 nuclear-cytosolic shuttling we have observed sensing thresholds, distinct dynamic behaviours under different metabolic conditions and linked the dynamic behaviour to glucose uptake into yeast cells. Using single molecule tracking we found that Mig1 takes different oligomeric forms under different conditions, which may help find the transcription factor to locate its target sequences in the nucleus. We also found that, although Mig1 is nuclear and cytoplasmic, respectively, under high and low glucose conditions, a fraction of Mig1 is always present in the nucleus and dynamic traffic occurs at all times.
Furthermore, we wish to understand oscillations in central metabolism on the single cell level using the technology developed in this network. The ATP FRET sensor, the pH ratiometric sensor and NAD(P)H autofluorescence have been successfully applied to study metabolism at a single cell level. Metabolic oscillations turned out to be an intrinsic autonomous property of metabolism while the pH levels tend to remain well buffered within single yeast cells. In addition, the single cell resolution provided by the aforementioned technologies, allowed the characterization of rare individual cells in heterogeneous, yet isogenic, yeast cell populations, offering insights on the contribution of metabolism to the emergence of cell-to-cell variability under constant environmental conditions. For this purpose, Peredox, a metabolic biosensor previously used only for mammalian experimental systems, as well as TRACK, a metabolic biosensor specific for Trehalose-6-phosphate, have been also adopted and utilized for single yeast cell research. The auxin inducible degron, in combination with traditional knock-out strategies were used to investigate the role of signalling pathways and molecular mechanisms for the generation and maintenance of metabolic oscillations, dynamically and in single cells.