Mitochondrial Structure, Dynamics, and Physiology: Light Microscopy to Disentangle the Network.

Mitochondria serve as energetic and signaling hubs of the cell: This function results from the complex interplay between their structure, function, dynamics, interactions, and molecular organization. The ability to observe and quantify these properties often represents the puzzle piece critical for deciphering the mechanisms behind mitochondrial function and dysfunction. Fluorescence microscopy addresses this critical need and has become increasingly powerful with the advent of superresolution methods and context-sensitive fluorescent probes. In this review, we delve into advanced light microscopy methods and analyses for studying mitochondrial ultrastructure, dynamics, and physiology, and highlight notable discoveries they enabled.

DNA-PAINT adaptors make for efficient multiplexing.

Multiplexed super-resolution imaging offers a route to spatial proteomics; however, time-efficient mapping of many protein species has been challenging. Two recent works in Cell highlight SUM-PAINT and FLASH-PAINT, methods that leverage adaptor DNA strand design to combine advances in multiplexing with increases in speed of label exchange. These advances permit unbiased omics-style analyses to advance biological insights from super-resolution images.

Surface passivation and functionalisation for mass photometry.

Interferometric scattering (iSCAT) microscopy enables the label-free observation of biomolecules. Consequently, single-particle imaging and tracking with the iSCAT-based method known as mass photometry (MP) is a growing area of study. However, establishing reliable cover glass passivation and functionalisation methods is crucial to reduce nonspecific binding and prepare surfaces for in vitro single-molecule binding experiments. Existing protocols for fluorescence microscopy can contain strongly scattering or mobile components, which make them impractical for MP-based microscopy. In this study, we characterise several different surface coatings using MP. We present approaches for cover glass passivation using 3-aminopropyltriethoxysilane (APTES) and polyethylene glycol (PEG, 2k) along with functionalisation via a maleimide-thiol linker. These coatings are compatible with water or salt buffers, and show low background scattering; thus, we are able to measure proteins as small as 60 kDa. In this technical note, we offer a surface preparation suitable for in vitro experiments with MP.

Polyphosphate affects cytoplasmic and chromosomal dynamics in nitrogen-starved Pseudomonas aeruginosa.

Polyphosphate (polyP) synthesis is a ubiquitous stress and starvation response in bacteria. In diverse species, mutants unable to make polyP have a wide variety of physiological defects, but the mechanisms by which this simple polyanion exerts its effects remain unclear. One possibility is that polyP's many functions stem from global effects on the biophysical properties of the cell. We characterize the effect of polyphosphate on cytoplasmic mobility under nitrogen-starvation conditions in the opportunistic pathogen Pseudomonas aeruginosa. Using fluorescence microscopy and particle tracking, we quantify the motion of chromosomal loci and cytoplasmic tracer particles. In the absence of polyP and upon starvation, we observe a 2- to 10-fold increase in mean cytoplasmic diffusivity. 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. Concomitant with this larger freedom of motion in polyP-deficient cells, we observe decompaction of the nucleoid and an increase in the steady-state concentration of ATP. The dramatic polyP-dependent effects we observe on cytoplasmic transport properties occur under nitrogen starvation, but not carbon starvation, suggesting that polyP may have distinct functions under different types of starvation.

Chromosome organization shapes replisome dynamics in Caulobacter crescentus.

DNA replication in bacteria takes place on highly compacted chromosomes, where segregation, transcription, and repair must occur simultaneously. Within this dynamic environment, colocalization of sister replisomes has been observed in many bacterial species, driving the hypothesis that a physical linker may tether them together. However, replisome splitting has also been reported in many of the same species, leaving the principles behind replisome organization a long-standing puzzle. Here, by tracking the replisome ß-clamp subunit in live Caulobacter crescentus, we find that rapid DNA segregation can give rise to a second focus which resembles a replisome, but does not replicate DNA. Sister replisomes can remain colocalized, or split apart to travel along DNA separately upon disruption of chromosome inter-arm alignment. Furthermore, chromosome arm-specific replication-transcription conflicts differentially modify replication speed on the two arms, facilitate the decoupling of the two replisomes. With these observations, we conclude that the dynamic chromosome organization flexibly shapes the organization of sister replisomes, and we outline principles which can help to reconcile previously conflicting models of replisome architecture.

Food perception promotes phosphorylation of MFFS131 and mitochondrial fragmentation in liver.

Liver mitochondria play a central role in metabolic adaptations to changing nutritional states, yet their dynamic regulation upon anticipated changes in nutrient availability has remained unaddressed. Here, we found that sensory food perception rapidly induced mitochondrial fragmentation in the liver through protein kinase B/AKT (AKT)-dependent phosphorylation of serine 131 of the mitochondrial fission factor (MFFS131). This response was mediated by activation of hypothalamic pro-opiomelanocortin (POMC)-expressing neurons. A nonphosphorylatable MFFS131G knock-in mutation abrogated AKT-induced mitochondrial fragmentation in vitro. In vivo, MFFS131G knock-in mice displayed altered liver mitochondrial dynamics and impaired insulin-stimulated suppression of hepatic glucose production. Thus, rapid activation of a hypothalamus-liver axis can adapt mitochondrial function to anticipated changes of nutritional state in control of hepatic glucose metabolism.

Label-Free Imaging of DNA Interactions with 2D Materials.

Two-dimensional (2D) materials offer potential as substrates for biosensing devices, as their properties can be engineered to tune interactions between the surface and biomolecules. Yet, not many methods can measure these interactions in a liquid environment without introducing labeling agents such as fluorophores. In this work, we harness interferometric scattering (iSCAT) microscopy, a label-free imaging technique, to investigate the interactions of single molecules of long dsDNA with 2D materials. The millisecond temporal resolution of iSCAT allows us to capture the transient interactions and to observe the dynamics of unlabeled DNA binding to a hexagonal boron nitride (hBN) surface in solution for extended periods (including a fraction of 10%, of trajectories lasting longer than 110 ms). Using a focused ion beam technique to engineer defects, we find that DNA binding affinity is enhanced at defects; when exposed to long lanes, DNA binds preferentially at the lane edges. Overall, we demonstrate that iSCAT imaging is a useful tool to study how biomolecules interact with 2D materials, a key component in engineering future biosensors.

Live-cell imaging powered by computation.

The proliferation of microscopy methods for live-cell imaging offers many new possibilities for users but can also be challenging to navigate. The prevailing challenge in live-cell fluorescence microscopy is capturing intra-cellular dynamics while preserving cell viability. Computational methods can help to address this challenge and are now shifting the boundaries of what is possible to capture in living systems. In this Review, we discuss these computational methods focusing on artificial intelligence-based approaches that can be layered on top of commonly used existing microscopies as well as hybrid methods that integrate computation and microscope hardware. We specifically discuss how computational approaches can improve the signal-to-noise ratio, spatial resolution, temporal resolution and multi-colour capacity of live-cell imaging.

Nanoscale organization of the endogenous ASC speck.

The NLRP3 inflammasome is a central component of the innate immune system. Its activation leads to formation of the ASC speck, a supramolecular assembly of the inflammasome adaptor protein ASC. Different models, based on ASC overexpression, have been proposed for the structure of the ASC speck. Using dual-color 3D super-resolution imaging (dSTORM and DNA-PAINT), we visualized the ASC speck structure following NLRP3 inflammasome activation using endogenous ASC expression. A complete structure was only obtainable by labeling with both anti-ASC antibodies and nanobodies. The complex varies in diameter between ∼800 and 1000 nm, and is composed of a dense core with emerging filaments. Dual-color confocal fluorescence microscopy indicated that the ASC speck does not colocalize with the microtubule-organizing center at late time points after Nigericin stimulation. From super-resolution images of whole cells, the ASC specks were sorted into a pseudo-time sequence indicating that they become denser but not larger during formation.

Event-driven acquisition for content-enriched microscopy.

A common goal of fluorescence microscopy is to collect data on specific biological events. Yet, the event-specific content that can be collected from a sample is limited, especially for rare or stochastic processes. This is due in part to photobleaching and phototoxicity, which constrain imaging speed and duration. We developed an event-driven acquisition framework, in which neural-network-based recognition of specific biological events triggers real-time control in an instant structured illumination microscope. 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 event-driven acquisition allows the microscope to respond specifically to complex biological events, it acquires data enriched in relevant content.

Fluorescent d-Amino Acids for Super-resolution Microscopy of the Bacterial Cell Wall.

Fluorescent d-amino acids (FDAAs) have previously been developed to enable in situ highlighting of locations of bacterial cell wall growth. Most bacterial cells lie at the edge of the diffraction limit of visible light; thus, resolving the precise details of peptidoglycan (PG) biosynthesis requires super-resolution microscopy after probe incorporation. Single molecule localization microscopy (SMLM) has stringent requirements on the fluorophore photophysical properties and therefore has remained challenging in this context. Here, we report the synthesis and characterization of new FDAAs compatible with one-step labeling and SMLM imaging. We demonstrate 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 endow researchers with a nanoscale perspective on the spatial distribution of PG biosynthesis for a broad range of bacterial species.

Flipper Probes for the Community.

This article describes four fluorescent membrane tension probes that have been designed, synthesized, evaluated, commercialized and applied to current biology challenges in the context of the NCCR Chemical Biology. Their names are Flipper-TR®, ER Flipper-TR®, Lyso Flipper-TR®, and Mito Flipper-TR®. They are available from Spirochrome.

Physics meets biology: The joining of two forces to further our understanding of cellular function.

Some biological questions are tough to solve through standard molecular and cell biological methods and naturally lend themselves to investigation by physical approaches. Below, a group of formally trained physicists discuss, among other things, how they apply physics to address biological questions and how physical approaches complement conventional biological approaches.

Distinct fission signatures predict mitochondrial degradation or biogenesis.

Mitochondrial fission is a highly regulated process that, when disrupted, can alter metabolism, proliferation and apoptosis1-3. Dysregulation has been linked to neurodegeneration3,4, cardiovascular disease3 and cancer5. Key components of the fission machinery include the endoplasmic reticulum6 and actin7, which initiate constriction before dynamin-related protein 1 (DRP1)8 binds to the outer mitochondrial membrane via adaptor proteins9-11, to drive scission12. In the mitochondrial life cycle, fission enables both biogenesis of new mitochondria and clearance of dysfunctional mitochondria through mitophagy1,13. Current models of fission regulation cannot explain how those dual fates are decided. However, uncovering fate determinants is challenging, as fission is unpredictable, and mitochondrial morphology is heterogeneous, with ultrastructural features that are below the diffraction limit. Here, we used live-cell structured illumination microscopy to capture mitochondrial dynamics. By analysing hundreds of fissions in African green monkey Cos-7 cells and mouse cardiomyocytes, we discovered two functionally and mechanistically distinct types of fission. Division at the periphery enables damaged material to be shed into smaller mitochondria destined for mitophagy, whereas division at the midzone leads to the proliferation of mitochondria. Both types are mediated by DRP1, but endoplasmic reticulum- and actin-mediated pre-constriction and the adaptor MFF govern only midzone fission. Peripheral fission is preceded by lysosomal contact and is regulated by the mitochondrial outer membrane protein FIS1. These distinct molecular mechanisms explain how cells independently regulate fission, leading to distinct mitochondrial fates.

Mitochondrial membrane tension governs fission.

During mitochondrial fission, key molecular and cellular factors assemble on the outer mitochondrial membrane, where they coordinate to generate constriction. Constriction sites can eventually divide or reverse upon disassembly of the machinery. However, a role for membrane tension in mitochondrial fission, although speculated, has remained undefined. We capture the dynamics of constricting mitochondria in mammalian cells using live-cell structured illumination microscopy (SIM). By analyzing the diameters of tubules that emerge from mitochondria and implementing a fluorescence lifetime-based mitochondrial membrane tension sensor, we discover that mitochondria are indeed under tension. Under perturbations that reduce mitochondrial tension, constrictions initiate at the same rate, but are less likely to divide. We propose a model based on our estimates of mitochondrial membrane tension and bending energy in living cells which accounts for the observed probability distribution for mitochondrial constrictions to divide.

Single-molecule localization microscopy.

Single-molecule localization microscopy (SMLM) describes a family of powerful imaging techniques that dramatically improve spatial resolution over standard, diffraction-limited microscopy techniques and can image biological structures at the molecular scale. In SMLM, individual fluorescent molecules are computationally localized from diffraction-limited image sequences and the localizations are used to generate a super-resolution image or a time course of super-resolution images, or to define molecular trajectories. In this Primer, we introduce the basic principles of SMLM techniques before describing the main experimental considerations when performing SMLM, including fluorescent labelling, sample preparation, hardware requirements and image acquisition in fixed and live cells. We then explain how low-resolution image sequences are computationally processed to reconstruct super-resolution images and/or extract quantitative information, and highlight a selection of biological discoveries enabled by SMLM and closely related methods. We discuss some of the main limitations and potential artefacts of SMLM, as well as ways to alleviate them. Finally, we present an outlook on advanced techniques and promising new developments in the fast-evolving field of SMLM. We hope that this Primer will be a useful reference for both newcomers and practitioners of SMLM.

3D Structure From 2D Microscopy Images Using Deep Learning.

Understanding the structure of a protein complex is crucial in determining its function. However, retrieving accurate 3D structures from microscopy images is highly challenging, particularly as many imaging modalities are two-dimensional. Recent advances in Artificial Intelligence have been applied to this problem, primarily using voxel based approaches to analyse sets of electron microscopy images. Here we present a deep learning solution for reconstructing the protein complexes from a number of 2D single molecule localization microscopy images, with the solution being completely unconstrained. Our convolutional neural network coupled with a differentiable renderer predicts pose and derives a single structure. After training, the network is discarded, with the output of this method being a structural model which fits the data-set. We demonstrate the performance of our system on two protein complexes: CEP152 (which comprises part of the proximal toroid of the centriole) and centrioles.