Periodic Reporting for period 2 - Piko (<br/>Revealing the adaptive internal organization and dynamics of bacteria and mitochondria)
Okres sprawozdawczy: 2021-04-01 do 2022-09-30
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.
The project Piko aims 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 are also developing new tools that go beyond the diffraction limit of light, to track the dynamics of granules and other tracer particles over long times.
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 aims 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 are also developing new tools that go beyond the diffraction limit of light, to track the dynamics of granules and other tracer particles over long times.
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.
Objective 1: Smart iSIM
We developed the feedback control for our iSIM, incorporating a neural network that can recognize bacterial and mitochondrial divisions. Our setup adapts acquisitions on-the-fly by switching between a slow imaging rate while detecting the onset of events, and a fast imaging rate during their progression. Thus, we capture mitochondrial and bacterial divisions at imaging rates that match their dynamic timescales, while extending overall imaging durations. Because EDA allows the microscope to respond specifically to complex biological events, it acquires data enriched in relevant content. This manuscript is posted as a preprint on bioRXiv, and has undergone a round of review at Nature Methods. We have revised and resubmitted the manuscript, and expect to receive a decision soon.
Objective 2: Low-photon tracking
We made progress toward constructing a low-photon microscope for tracking subcellular objects over long periods of time.
Objective 3a: Bacteria; polyP granule dynamics
We focused so far on the dynamics of polyP granules under starvation and exponential growth conditions. We characterized the effect of polyphosphate on cytoplasmic mobility under nitrogen-starvation conditions in the opportunistic pathogen Pseudomonas aeruginosa. Using fluorescence microscopy and particle tracking, we characterized the motion of chromosomal loci and free tracer particles in the cytoplasm. In the absence of polyP and upon starvation, we observed an increase in mobility both for chromosomal loci and for tracer particles. Tracer particles reveal that polyP also modulates the partitioning between a ’more mobile’ and a ’less mobile’ population: small particles in cells unable to make polyP are more likely to be ‘mobile’ and explore more of the cytoplasm, particularly during starvation. We speculate that this larger freedom of motion may be a consequence of nucleoid decompaction, which we also observe in starved cells deficient in polyP.
Objective 3b: Bacteria, starvation glassy dynamics
We synthesized and characterized new FDAAs compatible with STORM imaging. We demonstrated the incorporation of our probes, and their utility for visualizing PG at the nanoscale in gram-negative, gram-positive, and mycobacteria species. This improved FDAA toolkit will further endow researchers with a nanoscale perspective on the spatial distribution of PG biosynthesis.
Objective 4: Mitochondrial dynamics
We completed two studies on mitochondrial dynamics, looking at the organization of mitochondrially-encoded RNAs and mitochondrial division.
We developed the feedback control for our iSIM, incorporating a neural network that can recognize bacterial and mitochondrial divisions. Our setup adapts acquisitions on-the-fly by switching between a slow imaging rate while detecting the onset of events, and a fast imaging rate during their progression. Thus, we capture mitochondrial and bacterial divisions at imaging rates that match their dynamic timescales, while extending overall imaging durations. Because EDA allows the microscope to respond specifically to complex biological events, it acquires data enriched in relevant content. This manuscript is posted as a preprint on bioRXiv, and has undergone a round of review at Nature Methods. We have revised and resubmitted the manuscript, and expect to receive a decision soon.
Objective 2: Low-photon tracking
We made progress toward constructing a low-photon microscope for tracking subcellular objects over long periods of time.
Objective 3a: Bacteria; polyP granule dynamics
We focused so far on the dynamics of polyP granules under starvation and exponential growth conditions. We characterized the effect of polyphosphate on cytoplasmic mobility under nitrogen-starvation conditions in the opportunistic pathogen Pseudomonas aeruginosa. Using fluorescence microscopy and particle tracking, we characterized the motion of chromosomal loci and free tracer particles in the cytoplasm. In the absence of polyP and upon starvation, we observed an increase in mobility both for chromosomal loci and for tracer particles. Tracer particles reveal that polyP also modulates the partitioning between a ’more mobile’ and a ’less mobile’ population: small particles in cells unable to make polyP are more likely to be ‘mobile’ and explore more of the cytoplasm, particularly during starvation. We speculate that this larger freedom of motion may be a consequence of nucleoid decompaction, which we also observe in starved cells deficient in polyP.
Objective 3b: Bacteria, starvation glassy dynamics
We synthesized and characterized new FDAAs compatible with STORM imaging. We demonstrated the incorporation of our probes, and their utility for visualizing PG at the nanoscale in gram-negative, gram-positive, and mycobacteria species. This improved FDAA toolkit will further endow researchers with a nanoscale perspective on the spatial distribution of PG biosynthesis.
Objective 4: Mitochondrial dynamics
We completed two studies on mitochondrial dynamics, looking at the organization of mitochondrially-encoded RNAs and mitochondrial division.
Mitochondrial gene expression organization and division control. (Rey et al., Nature Cell Biol, 2020)
Mitochondrial RNA is found in mitochondrial RNA granules (MRGs), together with RNA-binding proteins and mitoribosome assembly factors. We investigated the molecular organization and biophysical properties of MRGs using super-resolution and electron microscopies. Our study demonstrates that liquid condensates are a motif that extends to mitochondria, while revealing important aspects specific to mitochondria. We found that mitochondrial RNA granules are liquid condensates comprising protein organized around an RNA core. Positioning of these membraneless organelles is dependent on mitochondrial dynamics and remodeling. We propose that condensation of RNA and proteins allows their positioning inside the mitochondrial organelle, with implications for inheritance and disease.
Mitochondrial division patterning and fate. (Kleele et al., Nature, 2021)
We captured hundreds of mitochondrial divisions and measured their physiological and molecular signatures. We discovered that mitochondria do not divide at random positions; instead, we find two geometrically and mechanistically distinct types of mitochondrial fission, one serving biogenesis of new mitochondria, and the other one serving quality control and subsequent mitophagy. Different organelle contacts and molecular adaptors are implicated in these two types of fission.
Mitochondrial RNA is found in mitochondrial RNA granules (MRGs), together with RNA-binding proteins and mitoribosome assembly factors. We investigated the molecular organization and biophysical properties of MRGs using super-resolution and electron microscopies. Our study demonstrates that liquid condensates are a motif that extends to mitochondria, while revealing important aspects specific to mitochondria. We found that mitochondrial RNA granules are liquid condensates comprising protein organized around an RNA core. Positioning of these membraneless organelles is dependent on mitochondrial dynamics and remodeling. We propose that condensation of RNA and proteins allows their positioning inside the mitochondrial organelle, with implications for inheritance and disease.
Mitochondrial division patterning and fate. (Kleele et al., Nature, 2021)
We captured hundreds of mitochondrial divisions and measured their physiological and molecular signatures. We discovered that mitochondria do not divide at random positions; instead, we find two geometrically and mechanistically distinct types of mitochondrial fission, one serving biogenesis of new mitochondria, and the other one serving quality control and subsequent mitophagy. Different organelle contacts and molecular adaptors are implicated in these two types of fission.