Periodic Reporting for period 2 - CAPTUR3D (CAPTURING THE PHYSICS OF LIFE ON 3D-TRAFFICKING SUBCELLULAR NANOSYSTEMS)
Periodo di rendicontazione: 2022-09-01 al 2024-02-29
Our cells receive, process and emit signals which are fundamental to our life, using structures (also known as "organelles") that are less than one millionth of a metre in size and are continuously moving. Which physical principles govern the behavior of these subcellular nanosystems? How do they achieve controlled movements across the crowded intracellular world? Which is the structural and functional organization of their surface and their lumen? In the attempt to provide an answer, current optical microscopy methods fail to subtract the natural 3D movement of these nanosystems while preserving the spatial and temporal resolution required to probe their structure and function at the molecular level.
CAPTUR3D will tackle this bottleneck. An excitation light-beam will be focused in a periodic orbit around the nanosystem of interest and used to localize its position with unprecedented spatial (~10 nm) and temporal (~micro-milliseconds) resolution. Such privileged observation point will push biophysical investigations to a new level. For the first time, state-of-the-art imaging technologies and analytical tools (e.g. fluorescence correlation spectroscopy), will be used to perform molecular investigations on a moving, nanoscopic reference system.
This innovative strategy will be applied to the insulin secretory granule (ISG), a microscopic structure responsible for the regulation of glucose levels in our blood, the malfunction of which is a distinctive factor in the pathophysiology of Diabetes. Key open issues at the ISG level are selected, namely: (i) ISG-environment interactions and their role in directing ISG trafficking, (ii) ISG-membrane organization, (iii) ISG-lumen structural and functional organization, (iv) ISG alterations in type-2 diabetes (T2D). These issues will be tackled directly within human-derived Langherans islets.
First of all, CAPTUR3D is an opportunity to move faster to Diabetes precision medicine. This is one of the greatest challenges in Europe and worldwide: Diabetes prevention and treatment will be pursued successfully only if the molecular mechanisms leading to β-cell failure are identified, comprehended and targeted. Beyond the case study, CAPTUR3D can contribute to promote a paradigm shift in the way we address the vast amount of information still hidden behind a plethora of dynamic nanostructures in our cells, pushing ahead the frontier of current knowledge on living-matter physiopathology
CAPTUR3D will tackle this bottleneck. An excitation light-beam will be focused in a periodic orbit around the nanosystem of interest and used to localize its position with unprecedented spatial (~10 nm) and temporal (~micro-milliseconds) resolution. Such privileged observation point will push biophysical investigations to a new level. For the first time, state-of-the-art imaging technologies and analytical tools (e.g. fluorescence correlation spectroscopy), will be used to perform molecular investigations on a moving, nanoscopic reference system.
This innovative strategy will be applied to the insulin secretory granule (ISG), a microscopic structure responsible for the regulation of glucose levels in our blood, the malfunction of which is a distinctive factor in the pathophysiology of Diabetes. Key open issues at the ISG level are selected, namely: (i) ISG-environment interactions and their role in directing ISG trafficking, (ii) ISG-membrane organization, (iii) ISG-lumen structural and functional organization, (iv) ISG alterations in type-2 diabetes (T2D). These issues will be tackled directly within human-derived Langherans islets.
First of all, CAPTUR3D is an opportunity to move faster to Diabetes precision medicine. This is one of the greatest challenges in Europe and worldwide: Diabetes prevention and treatment will be pursued successfully only if the molecular mechanisms leading to β-cell failure are identified, comprehended and targeted. Beyond the case study, CAPTUR3D can contribute to promote a paradigm shift in the way we address the vast amount of information still hidden behind a plethora of dynamic nanostructures in our cells, pushing ahead the frontier of current knowledge on living-matter physiopathology
CAPTUR3D activities are ideally divided into three major phases: Preparatory, Production, and Exploitation. After 30 months of activity, as can be viewed in the Gantt diagram, the Preparatory phase has been completed, while the other two phases have been activated and are being executed as planned. In more detail, the Preparatory phase was executed by optimizing sample-maintenance, sample-labelling, and sample-imaging procedures. Concerning these latter, unexpected new results were obtained on the metabolic response of α and β cells to glucose by studying tissue autofluorescence lifetime, which are in turn stimulating the development of new machine-learning-based strategies to discriminate α and β cells based on microscopy data. Approximately 12 months from the start, the Italian Ministry of Health blocked the use of tissue explants from human patients for research purposes. As a consequence, Project objectives were re-focused on cell models and an updated DoA approved in April 2022. The successful completion of the Preparatory Phase laid the ground to the ‘Production Phase’ which comprises three work packages aimed at addressing, at high spatiotemporal resolution, the molecular details of the insulin-granule surroundings, membrane, and lumen. The body of work performed so far produced results on each of these target environments. A few exemplary outcomes are worth of mention. First of all, we provided demonstration of successful feedback-based 3D orbital tracking of single insulin granules, being able to extract information on the dynamics of selected intra-granule molecules during granule trafficking. Second, we successfully tested a genetically-encoded pH biosensor targeted to the insulin granule lumen: the average pH pf the granule and its variation in response to glucose were measured and we now expect to measure pH fluctuations at the unprecedented speed of micro-milliseconds. Third, optical super-resolution in the form of Expansion Microscopy was introduced in the Project activities to compensate for the lack of STimulated Emission Depletion (STED): ExM was successfully applied to study the effect of pro-inflammatory cytokines on β-cells, uncovering an hitherto neglected reshaping of the intracellular landscape and providing a benchmark to interpret previous data and guide future studies aimed at evaluating β-cell protection against cytokines. In the last few months of this reporting period, the Exploitation phase was activated and actually potentiated to compensate for the lack of a T2D tissue samples (as detailed above). In particular, on one side we agreed with our collaborators (Prof. Marchetti’s Group) on activating the access to a tissue bank which comprises pancreatic tissues from both control and diabetic donors; on the other side we are exploiting ongoing collaborations and a new technological platform recently patented by the PI to test quantitatively the release and biological function of drugs in β cells.
The following progresses beyond the state of the art promoted by CAPTUR3D are worth of mention:
-First of all, in the preparatory phase of CAPTUR3D we obtained the first demonstration that α and β cell functional responses can be distinguished and measured in a living human islet, with no need to perturb its integrity/architecture (e.g. by cytofluorimetry-based approaches) or perturb its viability (e.g. by fixation-based procedures such as immunohistochemistry or electron microscopy). This is a major technological advancement with respect to existing strategies and definitively paves the way to investigations of the human islet with cell-type specificity and under different conditions of biomedical/clinical interest. Until the end of the project we envision to further refine α- and β-cell recognition by combining the established procedure Machine Learning tools
-We provided a first demonstration that the combination of fast fluorescence fluctuation spectroscopy and feedback-based 3D orbital tracking is a fast and robust approach to extract information on the dynamics of molecules enclosed within sub-cellular nanostructures (e.g. the insulin secretory granule) which are also moving in the complex 3D cellular environment. This is the basic measurement of the CAPTUR3D methodological framework and, as such, the most fundamental.
- We provided the first direct observation of β-cell damages induced by the inflammatory state typical of diabetic pathology (both type 1 and 2) by a combination of live-cell infrared microscopy and fixed-cell super resolution optical microscopy. It was well accepted that, during the onset of diabetic pathology, pancreatic cells suffer the inflammatory insult from cytokines but the details of what happens to the structural organization of cells had remained obscure so far. These results open new perspectives for the identification of pharmacological targets.
-First of all, in the preparatory phase of CAPTUR3D we obtained the first demonstration that α and β cell functional responses can be distinguished and measured in a living human islet, with no need to perturb its integrity/architecture (e.g. by cytofluorimetry-based approaches) or perturb its viability (e.g. by fixation-based procedures such as immunohistochemistry or electron microscopy). This is a major technological advancement with respect to existing strategies and definitively paves the way to investigations of the human islet with cell-type specificity and under different conditions of biomedical/clinical interest. Until the end of the project we envision to further refine α- and β-cell recognition by combining the established procedure Machine Learning tools
-We provided a first demonstration that the combination of fast fluorescence fluctuation spectroscopy and feedback-based 3D orbital tracking is a fast and robust approach to extract information on the dynamics of molecules enclosed within sub-cellular nanostructures (e.g. the insulin secretory granule) which are also moving in the complex 3D cellular environment. This is the basic measurement of the CAPTUR3D methodological framework and, as such, the most fundamental.
- We provided the first direct observation of β-cell damages induced by the inflammatory state typical of diabetic pathology (both type 1 and 2) by a combination of live-cell infrared microscopy and fixed-cell super resolution optical microscopy. It was well accepted that, during the onset of diabetic pathology, pancreatic cells suffer the inflammatory insult from cytokines but the details of what happens to the structural organization of cells had remained obscure so far. These results open new perspectives for the identification of pharmacological targets.