Periodic Reporting for period 3 - CAPTUR3D (CAPTURING THE PHYSICS OF LIFE ON 3D-TRAFFICKING SUBCELLULAR NANOSYSTEMS)
Reporting period: 2024-03-01 to 2025-08-31
Our cells rely on a symphony of signals to sustain life, using continuously moving, nanoscale structures known as organelles. But what physical principles govern these nanosystems? How do they navigate the crowded intracellular world? What is the molecular organization of their surface and lumen? Answering these questions is a fundamental challenge, as current optical microscopy methods cannot compensate for the constant 3D motion of these organelles while preserving the high spatial and temporal resolution required to probe their molecular function.
CAPTUR3D will overcome this bottleneck. By rapidly orbiting an excitation light beam around a target nanosystem, our technology will track its position with unprecedented spatial (~10 nm) and temporal (~microseconds) resolution. This creates a "locked-on" observation point that will elevate biophysical investigations to a new level. For the first time, state-of-the-art analytical tools, such as fluorescence correlation spectroscopy, can be applied to perform molecular investigations on a moving, nanoscopic frame of reference.
We will apply this innovative strategy to the insulin secretory granule (ISG), the organelle responsible for regulating blood glucose levels and whose malfunction is a hallmark of Diabetes. We will address key unresolved questions, including: (i) the role of ISG-environment interactions in guiding granule trafficking, (ii) the molecular organization of the ISG membrane, (iii) the structural and functional composition of the ISG lumen, and (iv) the specific alterations ISGs undergo in type 2 diabetes (T2D). These questions will be tackled directly within human-derived islets of Langerhans.
Ultimately, CAPTUR3D will accelerate the path towards precision medicine for Diabetes, one of the greatest health challenges worldwide. The prevention and treatment of Diabetes can only succeed if the molecular mechanisms leading to β-cell failure are precisely identified and understood. Beyond this critical case study, CAPTUR3D will pioneer a paradigm shift in how we investigate the vast universe of dynamic nanostructures in our cells, advancing the frontiers of our knowledge in cell biology and human disease.
CAPTUR3D will overcome this bottleneck. By rapidly orbiting an excitation light beam around a target nanosystem, our technology will track its position with unprecedented spatial (~10 nm) and temporal (~microseconds) resolution. This creates a "locked-on" observation point that will elevate biophysical investigations to a new level. For the first time, state-of-the-art analytical tools, such as fluorescence correlation spectroscopy, can be applied to perform molecular investigations on a moving, nanoscopic frame of reference.
We will apply this innovative strategy to the insulin secretory granule (ISG), the organelle responsible for regulating blood glucose levels and whose malfunction is a hallmark of Diabetes. We will address key unresolved questions, including: (i) the role of ISG-environment interactions in guiding granule trafficking, (ii) the molecular organization of the ISG membrane, (iii) the structural and functional composition of the ISG lumen, and (iv) the specific alterations ISGs undergo in type 2 diabetes (T2D). These questions will be tackled directly within human-derived islets of Langerhans.
Ultimately, CAPTUR3D will accelerate the path towards precision medicine for Diabetes, one of the greatest health challenges worldwide. The prevention and treatment of Diabetes can only succeed if the molecular mechanisms leading to β-cell failure are precisely identified and understood. Beyond this critical case study, CAPTUR3D will pioneer a paradigm shift in how we investigate the vast universe of dynamic nanostructures in our cells, advancing the frontiers of our knowledge in cell biology and human disease.
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 of 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 (ExM) 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 $ \beta $-cells, uncovering a hitherto neglected reshaping of the intracellular landscape and providing a benchmark to interpret previous data and guide future studies.
In the last few months, the Exploitation phase was not only activated but significantly enhanced, leading to high-impact results. Specifically, the agreement to access a biobank of pancreatic tissues from both healthy and diabetic donors (Prof. Marchetti’s Group) has been concretized. This has enabled us to perform ExM experiments on human samples (3 donors per group), which have revealed clear structural alterations in key organelles, such as granules and mitochondria, in diabetic tissue. These results are the subject of a manuscript in preparation and were presented at the EASD 2025 conference.
Concurrently, the technological platform patented by the PI to test drug function has been successfully exploited. We measured the activity and biodistribution of a promising drug, baricitinib, confirming its beta-protective power. These findings have culminated in a major publication in the journal Science Advances. The potential of analyzing the intrinsic fluorescence of drugs, which is at the core of the patent, has become a strategic pillar of the project. It now constitutes the cornerstone of WP4 and the fundamental asset of the ERC Proof of Concept (PoC) project that stemmed from CAPTUR3D and was funded this year.
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 of 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 (ExM) 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 $ \beta $-cells, uncovering a hitherto neglected reshaping of the intracellular landscape and providing a benchmark to interpret previous data and guide future studies.
In the last few months, the Exploitation phase was not only activated but significantly enhanced, leading to high-impact results. Specifically, the agreement to access a biobank of pancreatic tissues from both healthy and diabetic donors (Prof. Marchetti’s Group) has been concretized. This has enabled us to perform ExM experiments on human samples (3 donors per group), which have revealed clear structural alterations in key organelles, such as granules and mitochondria, in diabetic tissue. These results are the subject of a manuscript in preparation and were presented at the EASD 2025 conference.
Concurrently, the technological platform patented by the PI to test drug function has been successfully exploited. We measured the activity and biodistribution of a promising drug, baricitinib, confirming its beta-protective power. These findings have culminated in a major publication in the journal Science Advances. The potential of analyzing the intrinsic fluorescence of drugs, which is at the core of the patent, has become a strategic pillar of the project. It now constitutes the cornerstone of WP4 and the fundamental asset of the ERC Proof of Concept (PoC) project that stemmed from CAPTUR3D and was funded this year.
The CAPTUR3D project has promoted several significant advances beyond the current state of the art:
We achieved the first non-invasive functional discrimination of α and β cells within intact, living human islets. This was accomplished by analyzing their intrinsic autofluorescence, removing the need for perturbative methods like cell sorting or fixation-based histology. This technological breakthrough paves the way for studying human islet pathophysiology with single-cell specificity in a native context. We envision further refining this method by integrating advanced Machine Learning tools.
We provided the first demonstration of combining feedback-based 3D orbital tracking with fluorescence fluctuation spectroscopy to study molecular dynamics inside motile organelles. This core methodological framework of CAPTUR3D allows us, for the first time, to measure molecular behavior within single insulin granules as they actively traffic through the complex 3D cellular environment, a feat previously unattainable.
We reported the first direct, multimodal microscopic evidence of ultrastructural damage to β-cells induced by pro-inflammatory cytokines, which mimic the inflammatory environment of diabetes. While it was known that cytokines harm β-cells, the specific details of the induced structural disorganization remained obscure. Our results reveal these details at high resolution, opening new perspectives for identifying novel pharmacological targets to protect the β-cell.
(NEW) For the first time, we characterized the intracellular dynamics of glucagon secretory granules (GSGs) in α-cells and performed a direct comparison with insulin granules. Our work uncovered a fundamental, previously unknown difference in their trafficking mechanisms: the actin cytoskeleton plays a distinct regulatory role in glucagon granule mobilization in α-cells compared to its role in insulin granule transport in β-cells. This finding challenges the assumption of a fully conserved secretory machinery between the two main islet cell types.
(NEW) We performed the first-ever calibrated, absolute pH measurement within individual insulin granules using a targeted genetic biosensor. By applying phasor-FLIM microscopy, we determined the average luminal pH to be ~5.8. Crucially, this high-precision technique allowed us to unveil a previously unknown spatial heterogeneity, demonstrating that granules located proximally to the plasma membrane are significantly more acidic than those in the cell interior. This adds a new layer of understanding to granule maturation and priming for secretion.
(NEW) Building on our methodological advancements, we are completing the first super-resolution analysis of human pancreatic tissue from diabetic donors using Expansion Microscopy (ExM). This work, currently being prepared for publication, will provide an unprecedented nanoscale view of how key organelles—specifically insulin granules and mitochondria—are structurally altered in the diabetic state compared to healthy tissue. This will offer novel insights into the cellular basis of the pathology.
We achieved the first non-invasive functional discrimination of α and β cells within intact, living human islets. This was accomplished by analyzing their intrinsic autofluorescence, removing the need for perturbative methods like cell sorting or fixation-based histology. This technological breakthrough paves the way for studying human islet pathophysiology with single-cell specificity in a native context. We envision further refining this method by integrating advanced Machine Learning tools.
We provided the first demonstration of combining feedback-based 3D orbital tracking with fluorescence fluctuation spectroscopy to study molecular dynamics inside motile organelles. This core methodological framework of CAPTUR3D allows us, for the first time, to measure molecular behavior within single insulin granules as they actively traffic through the complex 3D cellular environment, a feat previously unattainable.
We reported the first direct, multimodal microscopic evidence of ultrastructural damage to β-cells induced by pro-inflammatory cytokines, which mimic the inflammatory environment of diabetes. While it was known that cytokines harm β-cells, the specific details of the induced structural disorganization remained obscure. Our results reveal these details at high resolution, opening new perspectives for identifying novel pharmacological targets to protect the β-cell.
(NEW) For the first time, we characterized the intracellular dynamics of glucagon secretory granules (GSGs) in α-cells and performed a direct comparison with insulin granules. Our work uncovered a fundamental, previously unknown difference in their trafficking mechanisms: the actin cytoskeleton plays a distinct regulatory role in glucagon granule mobilization in α-cells compared to its role in insulin granule transport in β-cells. This finding challenges the assumption of a fully conserved secretory machinery between the two main islet cell types.
(NEW) We performed the first-ever calibrated, absolute pH measurement within individual insulin granules using a targeted genetic biosensor. By applying phasor-FLIM microscopy, we determined the average luminal pH to be ~5.8. Crucially, this high-precision technique allowed us to unveil a previously unknown spatial heterogeneity, demonstrating that granules located proximally to the plasma membrane are significantly more acidic than those in the cell interior. This adds a new layer of understanding to granule maturation and priming for secretion.
(NEW) Building on our methodological advancements, we are completing the first super-resolution analysis of human pancreatic tissue from diabetic donors using Expansion Microscopy (ExM). This work, currently being prepared for publication, will provide an unprecedented nanoscale view of how key organelles—specifically insulin granules and mitochondria—are structurally altered in the diabetic state compared to healthy tissue. This will offer novel insights into the cellular basis of the pathology.