Periodic Reporting for period 4 - Piko (Revealing the adaptive internal organization and dynamics of bacteria and mitochondria)
Okres sprawozdawczy: 2024-04-01 do 2025-09-30
Living systems are composed of complex mixtures of biomolecules, structured into functional units. Their internal dynamics help to determine the kinetics of reactions that allow cells to respond to external stimuli and adapt to changing conditions. Bacteria respond to nutrient deprivation, or starvation, by forming granules and slowing internal transport. Mitochondria respond to starvation by shifting their metabolic program, a change that is concomitant with changes in their shape and interconnectivity. Yet, little is known about the physical nature of these changes. Insights from soft matter physics, particularly liquid–liquid phase separation (LLPS) and biophysical instabilities, offer a relevant conceptual framework. In both bacteria and mitochondria, cells organize biochemical reactions by forming dynamic, membrane-less compartments whose properties depend on crowding, metabolism, and cellular architecture.
The project Piko aimed to measure and model the internal organization and dynamics of bacteria and mitochondria. A major obstacle to this goal is the tiny size of these compartments, which lie at the edge of an ordinary microscope’s resolution. Thus, we also developed new tools that go beyond the diffraction limit of light, to track the dynamics of granules and other tracer particles over long times.
Our studies revealed that both bacterial and mitochondrial organization emerge from dynamic interactions between genome architecture, phase separation, membrane remodeling, and physical instabilities, positioning complex fluid behavior as a unifying principle of cellular organization and adaptability.
As a broader outcome, these fundamental studies have important implications for human health and disease. Bacteria populate our bodies, assisting basic functions such as digestion. Yet, pathogenic strains are a leading cause of illness. Mitochondria provide the energy currency of our bodies, ATP. When this capacity is disrupted, it can lead to disease.
The project Piko aimed to measure and model the internal organization and dynamics of bacteria and mitochondria. A major obstacle to this goal is the tiny size of these compartments, which lie at the edge of an ordinary microscope’s resolution. Thus, we also developed new tools that go beyond the diffraction limit of light, to track the dynamics of granules and other tracer particles over long times.
Our studies revealed that both bacterial and mitochondrial organization emerge from dynamic interactions between genome architecture, phase separation, membrane remodeling, and physical instabilities, positioning complex fluid behavior as a unifying principle of cellular organization and adaptability.
As a broader outcome, these fundamental studies have important implications for human health and disease. Bacteria populate our bodies, assisting basic functions such as digestion. Yet, pathogenic strains are a leading cause of illness. Mitochondria provide the energy currency of our bodies, ATP. When this capacity is disrupted, it can lead to disease.
We studied how the physical organization of bacterial cells changes under stress and during DNA replication. In one line of work, we found that during nitrogen starvation, bacteria used the polymer polyphosphate to reorganize their internal environment. When cells could not make polyphosphate, their interiors became dramatically more fluid: molecules and chromosomes moved much faster, the DNA became less compact, and energy levels increased. These effects occurred specifically under nitrogen starvation, revealing that polyphosphate helped tune the cell’s physical state in a context-dependent way, beyond its traditional role as an energy or phosphate store. In parallel, we investigated how bacteria organize the machinery that copies their DNA. Using live-cell imaging, we found that the two DNA-replicating machines could either stay together or move apart, depending on how the chromosome was arranged. When chromosome alignment was disrupted or conflicts with gene expression arose, the two replication machines truly separated. Together, our findings showed that bacterial chromosome dynamics actively shape cellular organization and function, resolving long-standing debates about how replication is structured inside cells.
We investigated how mitochondria organize their structure, genetic material, and gene expression, and found that physical considerations play a central role in mitochondrial function. First, we discovered that mitochondria divide in two distinct ways with different purposes. When division occurred near the ends of mitochondria, it separated damaged components into small fragments that were targeted for recycling, supporting quality control. When division occurred at the middle, it produced healthy new mitochondria, enabling growth and proliferation. These two modes relied on different molecular partners, revealing how cells independently regulate mitochondrial repair versus biogenesis—an insight relevant to many diseases linked to mitochondrial dysfunction.
We also uncovered how mitochondria organize RNA needed for gene expression. Using super-resolution imaging, we showed that mitochondrial RNA granules are tiny, fluid-like compartments with dense RNA cores surrounded by proteins. These granules exchanged components rapidly and could fuse, behaving like liquid droplets. Their even distribution depended on normal mitochondrial movement and division; when these dynamics were disrupted, granules clustered abnormally. This demonstrated that mitochondrial shape changes are essential for properly distributing the machinery that expresses mitochondrial genes.
Finally, we identified a physical mechanism that spaces mitochondrial DNA evenly along the organelle. We found that mitochondria frequently undergo a reversible shape instability called pearling, in which tubules briefly form evenly spaced beads. This process pulled DNA clusters apart and positioned them at regular intervals, ensuring reliable inheritance and uniform gene expression. Calcium triggered pearling, while internal membrane structure preserved the spacing afterward. Together, our findings showed that mitochondrial organization emerges from dynamic physical processes, rather than fixed molecular scaffolds, revealing new principles underlying cellular health.
We investigated how mitochondria organize their structure, genetic material, and gene expression, and found that physical considerations play a central role in mitochondrial function. First, we discovered that mitochondria divide in two distinct ways with different purposes. When division occurred near the ends of mitochondria, it separated damaged components into small fragments that were targeted for recycling, supporting quality control. When division occurred at the middle, it produced healthy new mitochondria, enabling growth and proliferation. These two modes relied on different molecular partners, revealing how cells independently regulate mitochondrial repair versus biogenesis—an insight relevant to many diseases linked to mitochondrial dysfunction.
We also uncovered how mitochondria organize RNA needed for gene expression. Using super-resolution imaging, we showed that mitochondrial RNA granules are tiny, fluid-like compartments with dense RNA cores surrounded by proteins. These granules exchanged components rapidly and could fuse, behaving like liquid droplets. Their even distribution depended on normal mitochondrial movement and division; when these dynamics were disrupted, granules clustered abnormally. This demonstrated that mitochondrial shape changes are essential for properly distributing the machinery that expresses mitochondrial genes.
Finally, we identified a physical mechanism that spaces mitochondrial DNA evenly along the organelle. We found that mitochondria frequently undergo a reversible shape instability called pearling, in which tubules briefly form evenly spaced beads. This process pulled DNA clusters apart and positioned them at regular intervals, ensuring reliable inheritance and uniform gene expression. Calcium triggered pearling, while internal membrane structure preserved the spacing afterward. Together, our findings showed that mitochondrial organization emerges from dynamic physical processes, rather than fixed molecular scaffolds, revealing new principles underlying cellular health.
In addition to the biological investigations and findings described above, we developed new ways to make microscopy smarter, gentler, and more informative by rethinking how and when cells are imaged.
First, we created adaptive, event-driven microscopy methods that focused light only on moments when something biologically important was about to happen. By using deep learning to detect early signs of rare events directly from low-impact images, we dynamically adjusted imaging settings in real time. This allowed us to capture fast processes such as organelle contacts and mitochondrial division while dramatically reducing light damage and extending experiment duration.
We also introduced a new super-resolution technique that measured not just where biological structures were, but also their size and orientation at the nanoscale. By using patterns of light minima and modeling fluorescence as a continuous distribution, our approach achieved high precision with far fewer photons than existing methods. This made it possible to quantitatively map the organization of tiny biological assemblies, such as mitochondrial DNA nucleoids, with improved accuracy and accessibility.
Finally, we worked toward a label-free form of super-resolution microscopy by combining DNA-PAINT with mass photometry. Instead of relying on fluorescent tags, this approach detected molecules based on their mass, enabling multiplexed, high-resolution imaging without labels. By developing new surface chemistries compatible with mass photometry, we laid the groundwork for a new imaging pipeline that merges nanoscale resolution with minimal perturbation, opening the door to more native views of biological structure.
First, we created adaptive, event-driven microscopy methods that focused light only on moments when something biologically important was about to happen. By using deep learning to detect early signs of rare events directly from low-impact images, we dynamically adjusted imaging settings in real time. This allowed us to capture fast processes such as organelle contacts and mitochondrial division while dramatically reducing light damage and extending experiment duration.
We also introduced a new super-resolution technique that measured not just where biological structures were, but also their size and orientation at the nanoscale. By using patterns of light minima and modeling fluorescence as a continuous distribution, our approach achieved high precision with far fewer photons than existing methods. This made it possible to quantitatively map the organization of tiny biological assemblies, such as mitochondrial DNA nucleoids, with improved accuracy and accessibility.
Finally, we worked toward a label-free form of super-resolution microscopy by combining DNA-PAINT with mass photometry. Instead of relying on fluorescent tags, this approach detected molecules based on their mass, enabling multiplexed, high-resolution imaging without labels. By developing new surface chemistries compatible with mass photometry, we laid the groundwork for a new imaging pipeline that merges nanoscale resolution with minimal perturbation, opening the door to more native views of biological structure.