Content-aware frame interpolation (CAFI): deep learning-based temporal super-resolution for fast bioimaging.

The development of high-resolution microscopes has made it possible to investigate cellular processes in 3D and over time. However, observing fast cellular dynamics remains challenging because of photobleaching and phototoxicity. Here we report the implementation of two content-aware frame interpolation (CAFI) deep learning networks, Zooming SlowMo and Depth-Aware Video Frame Interpolation, that are highly suited for accurately predicting images in between image pairs, therefore improving the temporal resolution of image series post-acquisition. We show that CAFI is capable of understanding the motion context of biological structures and can perform better than standard interpolation methods. We benchmark CAFI's performance on 12 different datasets, obtained from four different microscopy modalities, and demonstrate its capabilities for single-particle tracking and nuclear segmentation. CAFI potentially allows for reduced light exposure and phototoxicity on the sample for improved long-term live-cell imaging. The models and the training and testing data are available via the ZeroCostDL4Mic platform.

Collective signalling drives rapid jumping between cell states.

Development can proceed in 'fits and starts', with rapid transitions between cell states involving concerted transcriptome-wide changes in gene expression. However, it is not clear how these transitions are regulated in complex cell populations, in which cells receive multiple inputs. We address this issue using Dictyostelium cells undergoing development in their physiological niche. A continuous single cell transcriptomics time series identifies a sharp 'jump' in global gene expression marking functionally different cell states. By simultaneously imaging the physiological dynamics of transcription and signalling, we show the jump coincides with the onset of collective oscillations of cAMP. Optogenetic control of cAMP pulses shows that different jump genes respond to distinct dynamic features of signalling. Late jump gene expression changes are almost completely dependent on cAMP, whereas transcript changes at the onset of the jump require additional input. The coupling of collective signalling with gene expression is a potentially powerful strategy to drive robust cell state transitions in heterogeneous signalling environments. Based on the context of the jump, we also conclude that sharp gene expression transitions may not be sufficient for commitment.

Age-related changes in the local milieu of inflamed tissues cause aberrant neutrophil trafficking and subsequent remote organ damage.

Aging is associated with dysregulated immune functions. Here, we investigated the impact of age on neutrophil diapedesis. Using confocal intravital microscopy, we found that in aged mice, neutrophils adhered to vascular endothelium in inflamed tissues but exhibited a high frequency of reverse transendothelial migration (rTEM). This retrograde breaching of the endothelium by neutrophils was governed by enhanced production of the chemokine CXCL1 from mast cells that localized at endothelial cell (EC) junctions. Increased EC expression of the atypical chemokine receptor 1 (ACKR1) supported this pro-inflammatory milieu in aged venules. Accumulation of CXCL1 caused desensitization of the chemokine receptor CXCR2 on neutrophils and loss of neutrophil directional motility within EC junctions. Fluorescent tracking revealed that in aged mice, neutrophils undergoing rTEM re-entered the circulation and disseminated to the lungs where they caused vascular leakage. Thus, neutrophils stemming from a local inflammatory site contribute to remote organ damage, with implication to the dysregulated systemic inflammation associated with aging.

Cytoplasmic cleavage of IMPA1 3' UTR is necessary for maintaining axon integrity.

The 3' untranslated regions (3' UTRs) of messenger RNAs (mRNAs) are non-coding sequences involved in many aspects of mRNA metabolism, including intracellular localization and translation. Incorrect processing and delivery of mRNA cause severe developmental defects and have been implicated in many neurological disorders. Here, we use deep sequencing to show that in sympathetic neuron axons, the 3' UTRs of many transcripts undergo cleavage, generating isoforms that express the coding sequence with a short 3' UTR and stable 3' UTR-derived fragments of unknown function. Cleavage of the long 3' UTR of Inositol Monophosphatase 1 (IMPA1) mediated by a protein complex containing the endonuclease argonaute 2 (Ago2) generates a translatable isoform that is necessary for maintaining the integrity of sympathetic neuron axons. Thus, our study provides a mechanism of mRNA metabolism that simultaneously regulates local protein synthesis and generates an additional class of 3' UTR-derived RNAs.

The fate of cells undergoing spontaneous DNA damage during development.

Embryonic development involves extensive and often rapid cell proliferation. An unavoidable side effect of cell proliferation is DNA damage. The consequences of spontaneous DNA damage during development are not clear. Here, we define an approach to determine the effects of DNA damage on cell fate choice. Using single cell transcriptomics, we identified a subpopulation of Dictyostelium cells experiencing spontaneous DNA damage. Damaged cells displayed high expression of rad51, with the gene induced by multiple types of genotoxic stress. Using live imaging, we tracked high Rad51 cells from differentiation onset until cell fate assignment. High Rad51 cells were shed from multicellular structures, excluding damaged cells from the spore population. Cell shedding resulted from impaired cell motility and defective cell-cell adhesion, with damaged cells additionally defective in activation of spore gene expression. These data indicate DNA damage is not insulated from other aspects of cell physiology during development and multiple features of damaged cells prevent propagation of genetic error. Our approach is generally applicable for monitoring rare subpopulations during development, and permits analysis of developmental perturbations occurring within a physiological dynamic range.

Transition state dynamics during a stochastic fate choice.

The generation of multiple fates from a uniform cell population via self-organisation is a recurring feature in development and regeneration. However, for most self-organising systems, we have little understanding of the processes that allow cells to become different. One of the clearest examples of developmental self-organisation is shown by Dictyostelium, with cells segregating into two major fates, stalk and spore, within multicellular aggregates. To characterise the gene expression decisions that underlie this cell fate bifurcation, we carried out single cell transcriptomics on Dictyostelium aggregates. Our data show the transition of progenitors into prespore and prestalk cells occurs via distinct developmental intermediates. Few cells were captured switching between states, with minimal overlap in fate marker expression between cell types, suggesting states are discrete and transitions rapid. Surprisingly, fate-specific transcript dynamics were a small proportion of overall gene expression changes, with transcript divergence coinciding precisely with large-scale remodelling of the transcriptome shared by prestalk and prespore cells. These observations suggest the stepwise separation of cell identity is temporally coupled to global expression transitions common to both fates.

Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis.

Neutrophils require directional cues to navigate through the complex structure of venular walls and into inflamed tissues. Here we applied confocal intravital microscopy to analyze neutrophil emigration in cytokine-stimulated mouse cremaster muscles. We identified differential and non-redundant roles for the chemokines CXCL1 and CXCL2, governed by their distinct cellular sources. CXCL1 was produced mainly by TNF-stimulated endothelial cells (ECs) and pericytes and supported luminal and sub-EC neutrophil crawling. Conversely, neutrophils were the main producers of CXCL2, and this chemokine was critical for correct breaching of endothelial junctions. This pro-migratory activity of CXCL2 depended on the atypical chemokine receptor 1 (ACKR1), which is enriched within endothelial junctions. Transmigrating neutrophils promoted a self-guided migration response through EC junctions, creating a junctional chemokine "depot" in the form of ACKR1-presented CXCL2 that enabled efficient unidirectional luminal-to-abluminal migration. Thus, CXCL1 and CXCL2 act in a sequential manner to guide neutrophils through venular walls as governed by their distinct cellular sources.

Hydrocarbons Are Essential for Optimal Cell Size, Division, and Growth of Cyanobacteria.

Cyanobacteria are intricately organized, incorporating an array of internal thylakoid membranes, the site of photosynthesis, into cells no larger than other bacteria. They also synthesize C15-C19 alkanes and alkenes, which results in substantial production of hydrocarbons in the environment. All sequenced cyanobacteria encode hydrocarbon biosynthesis pathways, suggesting an important, undefined physiological role for these compounds. Here, we demonstrate that hydrocarbon-deficient mutants of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 exhibit significant phenotypic differences from wild type, including enlarged cell size, reduced growth, and increased division defects. Photosynthetic rates were similar between strains, although a minor reduction in energy transfer between the soluble light harvesting phycobilisome complex and membrane-bound photosystems was observed. Hydrocarbons were shown to accumulate in thylakoid and cytoplasmic membranes. Modeling of membranes suggests these compounds aggregate in the center of the lipid bilayer, potentially promoting membrane flexibility and facilitating curvature. In vivo measurements confirmed that Synechococcus sp. PCC 7002 mutants lacking hydrocarbons exhibit reduced thylakoid membrane curvature compared to wild type. We propose that hydrocarbons may have a role in inducing the flexibility in membranes required for optimal cell division, size, and growth, and efficient association of soluble and membrane bound proteins. The recent identification of C15-C17 alkanes and alkenes in microalgal species suggests hydrocarbons may serve a similar function in a broad range of photosynthetic organisms.

Cyanobacteria use micro-optics to sense light direction.

Bacterial phototaxis was first recognized over a century ago, but the method by which such small cells can sense the direction of illumination has remained puzzling. The unicellular cyanobacterium Synechocystis sp. PCC 6803 moves with Type IV pili and measures light intensity and color with a range of photoreceptors. Here, we show that individual Synechocystis cells do not respond to a spatiotemporal gradient in light intensity, but rather they directly and accurately sense the position of a light source. We show that directional light sensing is possible because Synechocystis cells act as spherical microlenses, allowing the cell to see a light source and move towards it. A high-resolution image of the light source is focused on the edge of the cell opposite to the source, triggering movement away from the focused spot. Spherical cyanobacteria are probably the world's smallest and oldest example of a camera eye.

Cyanobacteria are blue-green bacteria that are abundant in the environment. Cyanobacteria in the oceans are among the world's most important oxygen producers and carbon dioxide consumers. Synechocystis is a spherical single-celled cyanobacterium that measures about three thousandths of a millimetre across. Because Synechocystis needs sunlight to produce energy, it is important for it to find places where the light is neither too weak nor too strong. Unlike some bacteria, Synechocystis can't swim, but it can crawl across surfaces. It uses this ability to move to places where the light conditions are better. It was already known that Synechocystis cells move towards a light source that is shone at them from one side, which implies that the cyanobacteria can "see" where the light is. But how can such a tiny cell accurately detect where light is coming from? Schuergers et al. tracked how Synechocystis moved in response to different light conditions, and found that the secret of "vision" in these cyanobacteria is that the cells act as tiny spherical lenses. When a light is shone at the cell, an image of the light source is focused at the opposite edge of the cell. Light-detecting molecules called photoreceptors respond to the focused image of the light source, and this provides the information needed to steer the cell towards the light. Although the details are different, and although a Synechocystis cell is in terms of volume about 500 billion times smaller than a human eyeball, vision in Synechocystis actually works by principles similar to vision in humans. Schuergers et al.'s findings open plenty of further questions, as other types of bacteria may also act as tiny lenses. More also remains to be learnt about how the cyanobacteria process visual information.

Single-molecule studies of the dynamics and interactions of bacterial OXPHOS complexes.

Although significant insight has been gained into biochemical, genetic and structural features of oxidative phosphorylation (OXPHOS) at the single-enzyme level, relatively little was known of how the component complexes function together in time and space until recently. Several pioneering single-molecule studies have emerged over the last decade in particular, which have illuminated our knowledge of OXPHOS, most especially on model bacterial systems. Here, we discuss these recent findings of bacterial OXPHOS, many of which generate time-resolved information of the OXPHOS machinery with the native physiological context intact. These new investigations are transforming our knowledge not only of the molecular arrangement of OXPHOS components in live bacteria, but also of the way components dynamically interact with each other in a functional state. These new discoveries have important implications towards putative supercomplex formation in bacterial OXPHOS in particular. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Conrad Mullineaux.

Independent mobility of proteins and lipids in the plasma membrane of Escherichia coli.

Fluidity is essential for many biological membrane functions. The basis for understanding membrane structure remains the classic Singer-Nicolson model, in which proteins are embedded within a fluid lipid bilayer and able to diffuse laterally within a sea of lipid. Here we report lipid and protein diffusion in the plasma membrane of live cells of the bacterium Escherichia coli, using Fluorescence Recovery after Photobleaching (FRAP) and Total Internal Reflection Fluorescence (TIRF) microscopy to measure lateral diffusion coefficients. Lipid and protein mobility within the membrane were probed by visualizing an artificial fluorescent lipid and a simple model membrane protein consisting of a single membrane-spanning alpha-helix with a Green Fluorescent Protein (GFP) tag on the cytoplasmic side. The effective viscosity of the lipid bilayer is strongly temperature-dependent, as indicated by changes in the lipid diffusion coefficient. Surprisingly, the mobility of the model protein was unaffected by changes in the effective viscosity of the bulk lipid, and TIRF microscopy indicates that it clusters in segregated, mobile domains. We suggest that this segregation profoundly influences the physical behaviour of the protein in the membrane, with strong implications for bacterial membrane function and bacterial physiology.

Single-molecule in vivo imaging of bacterial respiratory complexes indicates delocalized oxidative phosphorylation.

Chemiosmotic energy coupling through oxidative phosphorylation (OXPHOS) is crucial to life, requiring coordinated enzymes whose membrane organization and dynamics are poorly understood. We quantitatively explore localization, stoichiometry, and dynamics of key OXPHOS complexes, functionally fluorescent protein-tagged, in Escherichia coli using low-angle fluorescence and superresolution microscopy, applying single-molecule analysis and novel nanoscale co-localization measurements. Mobile 100-200nm membrane domains containing tens to hundreds of complexes are indicated. Central to our results is that domains of different functional OXPHOS complexes do not co-localize, but ubiquinone diffusion in the membrane is rapid and long-range, consistent with a mobile carrier shuttling electrons between islands of different complexes. Our results categorically demonstrate that electron transport and proton circuitry in this model bacterium are spatially delocalized over the cell membrane, in stark contrast to mitochondrial bioenergetic supercomplexes. Different organisms use radically different strategies for OXPHOS membrane organization, likely depending on the stability of their environment.

Dynamics and stoichiometry of a regulated enhancer-binding protein in live Escherichia coli cells.

Bacterial enhancer-dependent transcription systems support major adaptive responses and offer a singular paradigm in gene control analogous to complex eukaryotic systems. Here we report new mechanistic insights into the control of one-membrane stress-responsive bacterial enhancer-dependent system. Using millisecond single-molecule fluorescence microscopy of live cells we determine the localizations, two-dimensional diffusion dynamics and stoichiometries of complexes of the bacterial enhancer-binding ATPase PspF during its action at promoters as regulated by inner membrane interacting negative controller PspA. We establish that a stable repressive PspF-PspA complex is located in the nucleoid, transiently communicating with the inner membrane via PspA. The PspF as a hexamer stably binds only one of the two psp promoters at a time, suggesting that psp promoters will fire asynchronously and cooperative interactions of PspF with the basal transcription complex influence dynamics of the PspF hexamer-DNA complex and regulation of the psp promoters.

Experimental approaches for addressing fundamental biological questions in living, functioning cells with single molecule precision.

In recent years, single molecule experimentation has allowed researchers to observe biological processes at the sensitivity level of single molecules in actual functioning, living cells, thereby allowing us to observe the molecular basis of the key mechanistic processes in question in a very direct way, rather than inferring these from ensemble average data gained from traditional molecular and biochemical techniques. In this short review, we demonstrate the impact that the application of single molecule bioscience experimentation has had on our understanding of various cellular systems and processes, and the potential that this approach has for the future to really address very challenging and fundamental questions in the life sciences.

Measuring the stoichiometry of functional PspA complexes in living bacterial cells by single molecule photobleaching.

We report a general method based on wide-field fluorescence imaging of single molecule photobleaching and the Chung-Kennedy algorithm to measure the stoichiometry of functional protein complexes in living bacterial cells.

Clustering and dynamics of cytochrome bd-I complexes in the Escherichia coli plasma membrane in vivo.

The cytochrome bd-I complex of Escherichia coli is a respiratory terminal oxidase and an integral component of the cytoplasmic membrane. As with other respiratory components, the organization and dynamics of this complex in living membranes is unknown. We set out to visualize the distribution and dynamics of this complex in vivo. By exchanging cydB for cydB-gfpgcn4 on the E. coli chromosome, we produced a strain (YTL01) that expresses functional GFP-tagged cytochrome bd-I terminal oxidase complexes under wild-type genetic control. We imaged live YTL01 cells using video-rate epifluorescence and total internal reflection fluorescence (TIRF) microscopy in combination with fluorescence recovery after photobleaching (FRAP) and saw mobile spots of GFP fluorescence in plasma membranes. Numbers of GFP molecules per spot were quantified by step-wise photobleaching giving a broad distribution with a mean of approximately 76, indicating that cytochrome bd-I is concentrated in mobile patches in the E. coli plasma membrane. We hypothesize that respiration occurs in mobile membrane patches which we call 'respirazones'.

Are Escherichia coli OXPHOS complexes concentrated in specialized zones within the plasma membrane?

Most organisms are able to synthesize ATP by OXPHOS (oxidative phosphorylation). Mitochondria in eukaryotes perform OXPHOS in the inner mitochondrial membrane, whereas the plasma membrane is used by prokaryotes. However, whereas OXPHOS is a well-understood process at the biochemical level, relatively little is known about its operation at the level of the whole-organelle/cell. We observed that a fluorescently labelled terminal oxidase, the cytochrome bd complex, is heterogeneously distributed in the Escherichia coli plasma membrane. This observation forms the basis of a working hypothesis that patches of the E. coli plasma membrane ('respirazones') are dedicated to respiratory function by the high concentration of OXPHOS components in these zones relative to the adjacent membrane. The formulation and physiological significance of this hypothesis are discussed in this paper.