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Eukaryotic genomes store information on many levels, including their linear DNA sequence, the posttranslational modifications of its constituents (epigenetic modifications), and its three-dimensional folding. Understanding how this information is stored and read requires multidisciplinary collaborations from many branches of science beyond biology, including physics, chemistry, and computer science. Concurrent recent developments in all these areas have enabled researchers to image the genome with unprecedented spatial and temporal resolution. In this review, we focus on what single-molecule imaging and tracking of individual proteins in live cells have taught us about chromatin structure and dynamics. Starting with the basics of single-molecule tracking (SMT), we describe some advantages over in situ imaging techniques and its current limitations. Next, we focus on single-nucleosome studies and what they have added to our current understanding of the relationship between chromatin dynamics and transcription. In celebration of Robert Feulgen's ground-breaking discovery that allowed us to start seeing the genome, we discuss current models of chromatin structure and future challenges ahead. [ABSTRACT FROM AUTHOR]

The translation of messenger RNA (mRNA) into proteins represents the culmination of gene expression. Recent technological advances have revolutionized our ability to investigate this process with unprecedented precision, enabling the study of translation at the single-molecule level in real time within live cells. In this review, we provide an overview of single-mRNA translation reporters. We focus on the core technology, as well as the rapid development of complementary probes, tags, and accessories that enable the visualization and quantification of a wide array of translation dynamics. We then highlight notable studies that have utilized these reporters in model systems to address key biological questions. The high spatiotemporal resolution of these studies is shedding light on previously unseen phenomena, uncovering the full heterogeneity and complexity of translational regulation. [ABSTRACT FROM AUTHOR]

Autoblinking is a widespread phenomenon and exhibits high level of intensity in some bacteria. In Deinococcus radiodurans (D. radiodurans), strong autoblinking was found to be indistinguishable from PAmCherry and greatly prevented single-molecule tracking of proteins of interest. Here we employed the bright photoswitchable fluorescent protein mMaple3 to label PprI, one essential DNA repair factor, and characterized systematically the fluorescence intensity and bleaching kinetics of both autoblinking and PprI-mMaple3 molecules within cells grown under three different conditions. Under minimal media, we can largely separate autoblinking from mMaple3 molecules and perform reliably single-molecule tracking of PprI in D. radiodurans, by means of applying signal-to-noise ratio and constraining the minimal length for linking the trajectories. We observed three states of PprI molecules, which bear different subcellular localizations and distinct functionalities. Our strategy provides a useful means to study the dynamics and distributions of proteins of interest in bacterial cells with high level of autoblinking. [ABSTRACT FROM AUTHOR]

Cells must tightly regulate their gene expression programs and yet rapidly respond to acute biochemical and biophysical cues within their environment. This information is transmitted to the nucleus through various signaling cascades, culminating in the activation or repression of target genes. Transcription factors (TFs) are key mediators of these signals, binding to specific regulatory elements within chromatin. While live-cell imaging has conclusively proven that TF–chromatin interactions are highly dynamic, how such transient interactions can have long-term impacts on developmental trajectories and disease progression is still largely unclear. In this review, we summarize our current understanding of the dynamic nature of TF functions, starting with a historical overview of early live-cell experiments. We highlight key factors that govern TF dynamics and how TF dynamics, in turn, affect downstream transcriptional bursting. Finally, we conclude with open challenges and emerging technologies that will further our understanding of transcriptional regulation. [ABSTRACT FROM AUTHOR]

Nanog and Oct4 are core transcription factors that form part of a gene regulatory network to regulate hundreds of target genes for pluripotency maintenance in mouse embryonic stem cells (ESCs). To understand their function in the pluripotency maintenance, we visualised and quantified the dynamics of single molecules of Nanog and Oct4 in a mouse ESCs during pluripotency loss. Interestingly, Nanog interacted longer with its target loci upon reduced expression or at the onset of differentiation, suggesting a feedback mechanism to maintain the pluripotent state. The expression level and interaction time of Nanog and Oct4 correlate with their fluctuation and interaction frequency, respectively, which in turn depend on the ESC differentiation status. The DNA viscoelasticity near the Oct4 target locus remained flexible during differentiation, supporting its role either in chromatin opening or a preferred binding to uncondensed chromatin regions. Based on these results, we propose a new negative feedback mechanism for pluripotency maintenance via the DNA condensation state‐dependent interplay of Nanog and Oct4. Synopsis: The transcription factors Nanog and Oct4 are core pluripotency regulators in embryonic stem cells (ESCs). Here, single‐molecule live imaging in mouse ESCs shows that interaction of Nanog and Oct4 with their target loci is regulated by the mechanical properties of DNA and suggests a new negative feedback mechanism for pluripotency maintenance. Nanog interaction with its target loci is prolonged upon reduced Nanog expression and at the onset of differentiation.DNA viscoelasticity measurements show that DNA remains flexible near the Oct4 target locus as differentiation begins.Dissociation rates of both Nanog and Oct4 correlate with the mechanical properties of DNA near their binding sites.Expression levels of Nanog and Oct4 affect the local flexibility of DNA and their DNA interaction probability. [ABSTRACT FROM AUTHOR]

Single particle tracking (SPT) enables the investigation of biomolecular dynamics at a high temporal and spatial resolution in living cells, and the analysis of these SPT datasets can reveal biochemical interactions and mechanisms. Still, how to make the best use of these tracking data for a broad set of experimental conditions remains an analysis challenge in the field. Here, we develop a new SPT analysis framework: NOBIAS (NOnparametric Bayesian Inference for Anomalous Diffusion in Single-Molecule Tracking), which applies nonparametric Bayesian statistics and deep learning approaches to thoroughly analyze SPT datasets. In particular, NOBIAS handles complicated live-cell SPT data for which: the number of diffusive states is unknown, mixtures of different diffusive populations may exist within single trajectories, symmetry cannot be assumed between the x and y directions, and anomalous diffusion is possible. NOBIAS provides the number of diffusive states without manual supervision, it quantifies the dynamics and relative populations of each diffusive state, it provides the transition probabilities between states, and it assesses the anomalous diffusion behavior for each state. We validate the performance of NOBIAS with simulated datasets and apply it to the diffusion of single outer-membrane proteins in Bacteroides thetaiotaomicron. Furthermore, we compare NOBIAS with other SPT analysis methods and find that, in addition to these advantages, NOBIAS is robust and has high computational efficiency and is particularly advantageous due to its ability to treat experimental trajectories with asymmetry and anomalous diffusion.

This special feature of Open Biology, titled ‘Advances in Quantitative Bioimaging’, proposes an overview of the latest advancements in quantitative bioimaging techniques and their wide-ranging applications. The articles cover various topics, including modern imaging methods that enable visualization on a nanoscale, such as super-resolution microscopy and single-particle analysis. These techniques offer unparalleled insights into complex molecular structures and dynamic cellular processes in situ, such as mapping nuclear pore proteins or tracking single histone deposition events throughout the cell cycle. The articles presented in this edition showcase cutting-edge quantitative imaging techniques coupled with advanced computational analysis capable of precisely measuring biological structures and processes. Examples range from correlating calcium release events to underlying protein organization in heart cells to pioneering tools for categorizing changes in microglia morphology under various conditions. This editorial highlights how these advancements are revolutionizing our understanding of living systems, while acknowledging challenges that must be addressed to fully exploit the potential of these emerging technologies, such as improving molecular probes, algorithms and correlation protocols.

A model-based method is used to extract a short-lived state in the rotation kinetics of the F1-ATPase of a bacterial species, Paracoccus denitrificans (PdF1). Imaged as a single molecule, PdF1 takes large 120ø steps during it rotation. The apparent lack of further substeps in the trajectories not only renders the rotation of PdF1 unlike that of other F-ATPases, but also hinders the establishment of its mechano-chemical kinetic scheme. We addressed these challenges using the angular velocity extracted from the single-molecule trajectories and compare it with its theoretically calculated counterpart. The theory-experiment comparison indicate the presence of a 20μs lifetime state, 40o after ATP binding. We identify a kinetic cycle in which this state is a three-nucleotide occupancy state prior to ADP release from another site. A similar state was also reported in our earlier study of the Thermophilic bacillus F1-ATPase (lifetime ∼10μs), suggesting thereby a common mechanism for removing a nucleotide release bottleneck in the rotary mechanism.

Imaging the dynamics and interactions of biomolecules at the single-molecule level in live cells and reconstituted systems has generated unprecedented knowledge about the biomolecular processes underlying many cellular functions. To achieve the speed and sensitivity needed to detect and follow individual molecules, these experiments typically require custom-built microscopes or custom modifications of commercial systems. The costs of such single-molecule microscopes, their technical complexity and the lack of open-source documentation on how to build custom setups therefore limit the accessibility of single-molecule imaging techniques. To advance the adaptation of dynamic single-molecule imaging by a wider community, we present the “K2”: an open-source, simultaneous triple-color total internal reflection fluorescence (TIRF) microscope specifically designed for live-cell and single-molecule imaging. We explain our design considerations and provide step-by-step building instructions, parts list and full CAD models. The modular design of this TIRF microscope allows users to customize it to their scientific and financial needs, or to re-use parts of our design to improve the capabilities of their existing setups without necessarily having to build a full copy of the K2 microscope.

This study aimed to expand our understanding of myelin basic protein (MBP), a key component of central nervous system myelin, by developing a protocol to track and quantifying individual MBP particles during oligodendrocyte (OL) differentiation. MBP particle directionality, confinement, and diffusion were tracked by rapid TIRF and HILO imaging of Dendra2 tagged MBP in three stages of mouse oligodendroglia: OL precursors, early myelinating OLs, and mature myelinating OLs. The directionality and confinement of MBP particles increased at each stage consistent with progressive transport toward, and recruitment into, emerging myelin structures. Unexpectedly, diffusion data presented a more complex pattern with subpopulations of the most diffusive particles disappearing at the transition between the precursor and early myelinating stage, before reemerging in the membrane sheets of mature OLs. This diversity of particle behaviors, which would be undetectable by conventional ensemble-averaged methods, are consistent with a multifunctional view of MBP involving roles in myelin expansion and compaction. [ABSTRACT FROM AUTHOR]

This study aimed to expand our understanding of myelin basic protein (MBP), a key component of central nervous system myelin, by developing a protocol to track and quantifying individual MBP particles during oligodendrocyte (OL) differentiation. MBP particle directionality, confinement, and diffusion were tracked by rapid TIRF and HILO imaging of Dendra2 tagged MBP in three stages of mouse oligodendroglia: OL precursors, early myelinating OLs, and mature myelinating OLs. The directionality and confinement of MBP particles increased at each stage consistent with progressive transport toward, and recruitment into, emerging myelin structures. Unexpectedly, diffusion data presented a more complex pattern with subpopulations of the most diffusive particles disappearing at the transition between the precursor and early myelinating stage, before reemerging in the membrane sheets of mature OLs. This diversity of particle behaviors, which would be undetectable by conventional ensemble-averaged methods, are consistent with a multifunctional view of MBP involving roles in myelin expansion and compaction.

ABSTRACT The membrane-embedded injectisome, the structural component of the virulence-associated type III secretion system (T3SS), is used by Gram-negative bacterial pathogens to inject species-specific effector proteins into eukaryotic host cells. The cytosolic injectisome proteins are required for export of effectors and display both stationary, injectisome-bound populations and freely diffusing cytosolic populations. How the cytosolic injectisome proteins interact with each other in the cytosol and associate with membrane-embedded injectisomes remains unclear. Here, we utilized three-dimensional (3D) single-molecule tracking to resolve distinct cytosolic complexes of injectisome proteins in living Yersinia enterocolitica cells. Tracking of the enhanced yellow fluorescent protein (eYFP)-labeled ATPase YeSctN and its regulator, YeSctL, revealed that these proteins form a cytosolic complex with each other and then further with YeSctQ. YeSctNL and YeSctNLQ complexes can be observed both in wild-type cells and in ΔsctD mutants, which cannot assemble injectisomes. In ΔsctQ mutants, the relative abundance of the YeSctNL complex is considerably increased. These data indicate that distinct cytosolic complexes of injectisome proteins can form prior to injectisome binding, which has important implications for how injectisomes are functionally regulated. IMPORTANCE Injectisomes are membrane-embedded, multiprotein assemblies used by bacterial pathogens to inject virulent effector proteins into eukaryotic host cells. Protein secretion is regulated by cytosolic proteins that dynamically bind and unbind at injectisomes. However, how these regulatory proteins interact with each other remains unknown. By measuring the diffusion rates of single molecules in living cells, we show that cytosolic injectisome proteins form distinct oligomeric complexes with each other prior to binding to injectisomes. We additionally identify the molecular compositions of these complexes and quantify their relative abundances. Quantifying to what extent cytosolic proteins exist as part of larger complexes in living cells has important implications for deciphering the complexity of biomolecular mechanisms. The results and methods reported here are thus relevant for advancing our understanding of how injectisomes and related multiprotein assemblies, such as bacterial flagellar motors, are functionally regulated.

Living erythrocyte (red blood cell, RBC) membranes present rich ultrastructural and dynamic details, which require synchronous super‐resolution imaging and single‐molecule tracking to be revealed. Yet, it poses a serious challenge to achieve these dual functions in a single probe, due to the rigid and conflicting photophysical demands of the different techniques. Herein, we rationally developed a far‐red boron dipyrromethene membrane probe with blinking capability and persistent single‐molecule emission, and constructed a microfluidic platform for noninvasive trapping and long‐term imaging of RBCs. By combining them, multi‐dimensional super‐resolution reconstructions and single‐molecule tracking were achieved at the molecular scale on living human RBC membranes in a high‐throughput manner. Our integrated system defines a quantitative method for analyzing RBC membranes under physiological and pathological conditions, improving precision and revealing new perspectives for future disease diagnostics. [ABSTRACT FROM AUTHOR]

Mechanistic details of the signal recognition particle (SRP)-mediated insertion of membrane proteins have been described from decades of in vitro biochemical studies. However, the dynamics of the pathway inside the living cell remain obscure. By combining in vivo single-molecule tracking with numerical modeling and simulated microscopy, we have constructed a quantitative reaction-diffusion model of the SRP cycle. Our results suggest that the SRP-ribosome complex finds its target, the membrane-bound translocon, through a combination of three-dimensional (3D) and 2D diffusional search, together taking on average 750 ms. During this time, the nascent peptide is expected to be elongated only 12 or 13 amino acids, which explains why, in Escherichia coli, no translation arrest is needed to prevent incorrect folding of the polypeptide in the cytosol. We also found that a remarkably high proportion (75%) of SRP bindings to ribosomes occur in the cytosol, suggesting that the majority of target ribosomes bind SRP before reaching the membrane. In combination with the average SRP cycling time, 2.2 s, this result further shows that the SRP pathway is capable of targeting all substrate ribosomes to translocons. [ABSTRACT FROM AUTHOR]

Fluorescence correlation spectroscopy (FCS) has been widely used to investigate molecular diffusion behavior in various samples. The use of the maximum entropy method (MEM) for FCS data analysis provides a unique means to determine multiple distinct diffusion coefficients without a priori assumption of their number. Comparison of the MEM-based FCS method (MEM-FCS) with another method will reveal its utility and advantage as an analytical tool to investigate diffusion dynamics. Herein, we measured diffusion of fluorescent probes doped into nanostructured thin films using MEM-FCS, and validated the results with single molecule tracking (SMT) data. The efficacy of the MEM code employed was first demonstrated by analyzing simulated FCS data for systems incorporating one and two diffusion modes with broadly distributed diffusion coefficients. The MEM analysis accurately afforded the number of distinct diffusion modes and their mean diffusion coefficients. These results contrasted with those obtained by fitting the simulated data to conventional two-component and anomalous diffusion models, which yielded inaccurate estimates of the diffusion coefficients. Subsequently, the MEM analysis was applied to FCS data acquired from hydrophilic dye molecules incorporated into microphase-separated polystyrene-block-poly(ethylene oxide) (PS-b-PEO) thin films characterized under a water-saturated N2 atmosphere. The MEM analysis revealed distinct fast and slow diffusion components attributable to molecules diffusing on the film surface and inside the film, respectively. SMT studies of the same materials yielded trajectories for mobile molecules that appear to follow the curved PEO microdomains. Diffusion coefficients obtained from the SMT data were consistent with those obtained for the slow diffusion component detected by MEM-FCS. These results highlight the utility of MEM-FCS and SMT for gaining complementary information on molecular diffusion processes in heterogeneous material systems. [ABSTRACT FROM AUTHOR]

The glucocorticoid and mineralocorticoid receptors (GR and MR, respectively) have distinct, yet overlapping physiological and pathophysiological functions. There are indications that both receptors interact functionally and physically, but the precise role of this interdependence is poorly understood. Here, we analyzed the impact of GR coexpression on MR genome-wide transcriptional responses and chromatin binding upon activation by aldosterone and glucocorticoids, both physiological ligands of this receptor. Transcriptional responses of MR in the absence of GR result in fewer regulated genes. In contrast, coexpression of GR potentiates MR-mediated transcription, particularly in response to aldosterone, both in cell lines and in the more physiologically relevant model of mouse colon organoids. MR chromatin binding is altered by GR coexpression in a locus- and ligand-specific way. Single-molecule tracking of MR suggests that the presence of GR contributes to productive binding of MR/aldosterone complexes to chromatin. Together, our data indicate that coexpression of GR potentiates aldosterone-mediated MR transcriptional activity, even in the absence of glucocorticoids., Competing Interests: Competing interests statement:The authors declare no competing interest.

Single-molecule tracking (SMT) has emerged as the dominant technology to investigate the dynamics of chromatin-transcription factor (TF) interactions. How long a TF needs to bind to a regulatory site to elicit a transcriptional response is a fundamentally important question. However, highly divergent estimates of TF binding have been presented in the literature, stemming from differences in photobleaching correction and data analysis. TF movement is often interpreted as specific or non-specific association with chromatin, yet the dynamic nature of the chromatin polymer is often overlooked. In this perspective, we highlight how recent SMT studies have reshaped our understanding of TF dynamics, chromatin mobility, and genome organization in the mammalian nucleus, focusing on the technical details and biological implications of these approaches. In a remarkable convergence of fixed and live-cell imaging, we show how super-resolution and SMT studies of chromatin have dovetailed to provide a convincing nanoscale view of genome organization., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

One important factor that promotes the colonization of the upper digestive system of the human pathogen Helicobacter pylori is its helical cell shape. The bacteria cell shape is predominantly defined by its peptidoglycan cell wall. In rod-shaped species, PG synthesis is mediated by two dynamic molecular machines that facilitate growth along the perpendicular axis and the septum, called the elongasome and the divisome, respectively. Furthermore, many bacteria evolved additional mechanisms to locally change PG synthesis patterns to generate diverse cell shapes. Recent work characterizing cell shape mutants of Helicobacter pylori revealed a novel mechanism for the generation of a twisted helix from a rod, including PG-modifying enzymes as well as additional proteins such as the bactofilin homolog CcmA or the membrane proteins Csd5 and Csd7. In this study, we investigate the localization and dynamics of CcmA and Csd7 using live-cell imaging. We also address the question of how these change in the presence or absence of the putative interaction partners. [ABSTRACT FROM AUTHOR]

Multicomponent efflux complexes constitute a primary mechanism for Gram-negative bacteria to expel toxic molecules for survival. As these complexes traverse the periplasm and link inner and outer membranes, it remains unclear how they operate efficiently without compromising periplasmic plasticity. Combining single-molecule superresolution imaging and genetic engineering, we study in living Escherichia coli cells the tripartite efflux complex CusCBA of the resistance-nodulation-division family that is essential for bacterial resistance to drugs and toxic metals. We find that CusCBA complexes are dynamic structures and shift toward the assembled form in response to metal stress. Unexpectedly, the periplasmic adaptor protein CusB is a key metal-sensing element that drives the assembly of the efflux complex ahead of the transcription activation of the cus operon for defending against metals. This adaptor protein-mediated dynamic pump assembly allows the bacterial cell for efficient efflux upon cellular demand while still maintaining periplasmic plasticity; this could be broadly relevant to other multicomponent efflux systems.

One important factor that promotes the colonization of the upper digestive system of the human pathogen Helicobacter pylori is its helical cell shape. The bacteria cell shape is predominantly defined by its peptidoglycan cell wall. In rod-shaped species, PG synthesis is mediated by two dynamic molecular machines that facilitate growth along the perpendicular axis and the septum, called the elongasome and the divisome, respectively. Furthermore, many bacteria evolved additional mechanisms to locally change PG synthesis patterns to generate diverse cell shapes. Recent work characterizing cell shape mutants of Helicobacter pylori revealed a novel mechanism for the generation of a twisted helix from a rod, including PG-modifying enzymes as well as additional proteins such as the bactofilin homolog CcmA or the membrane proteins Csd5 and Csd7. In this study, we investigate the localization and dynamics of CcmA and Csd7 using live-cell imaging. We also address the question of how these change in the presence or absence of the putative interaction partners.

Bacteria are dependent on rapid alterations in gene expression. A prerequisite for rapid adaptations is efficient RNA turnover, with endonuclease RNase Y playing a crucial role in mRNA stability as well as in maturation. In Bacillus subtilis, RNase Y in turn interacts with the so-called "Y-complex" consisting of three proteins, which play important functions in sporulation, natural transformation and biofilm formation. It is thought that the Y-complex acts as an accessory factor in RNase Y regulation but might also have independent functions. Using single-molecule tracking, we show that all three Y-complex proteins exhibit three distinct mobilities, including movement through the cytosol and confined motion, predominantly at membrane-proximal sites but also within the cell center. A transcriptional arrest leads to a strong change in localization and dynamics of YmcA, YlbF and YaaT, supporting their involvement in global RNA degradation. However, Y-complex proteins show distinguishable protein dynamics, and the deletion of yaaT or ylbF shows a minor effect on the dynamics of YmcA. Cell fractionation reveals that YaaT displays a mixture of membrane association and presence in the cytosol, while YlbF and YmcA do not show direct membrane attachment. Taken together, our experiments reveal membrane-associated and membrane-independent activities of Y-complex proteins and a dynamic interplay between them with indirect membrane association of YmcA and YlbF via YaaT. [ABSTRACT FROM AUTHOR]

Owing to their unique abilities to manipulate, label, and image individual molecules in vitro and in cellulo , single-molecule techniques provide previously unattainable access to elementary biological processes. In imaging, single-molecule fluorescence resonance energy transfer (smFRET) and protein-induced fluorescence enhancement in vitro can report on conformational changes and molecular interactions, single-molecule pull-down (SiMPull) can capture and analyze the composition and function of native protein complexes, and single-molecule tracking (SMT) in live cells reveals cellular structures and dynamics. In labeling, the abilities to specifically label genomic loci, mRNA, and nascent polypeptides in cells have uncovered chromosome organization and dynamics, transcription and translation dynamics, and gene expression regulation. In manipulation, optical tweezers, integration of single-molecule fluorescence with force measurements, and single-molecule force probes in live cells have transformed our mechanistic understanding of diverse biological processes, ranging from protein folding, nucleic acids-protein interactions to cell surface receptor function. Ha et al., highlights recent advances in popular technologies for imaging, labeling, and manipulating DNA, RNA, proteins, and cellular structures, organization, and dynamics at the resolution of single molecules in vitro and in cellulo. [ABSTRACT FROM AUTHOR]

The GABAB receptor is a typical G protein–coupled receptor, and its functional impairment is related to a variety of diseases. While the premise of GABAB receptor activation is the formation of heterodimers, the receptor also forms a tetramer on the cell membrane. Thus, it is important to study the effect of the GABAB receptor aggregation state on its activation and signaling. In this study, we have applied single-molecule photobleaching step counting and single-molecule tracking methods to investigate the formation and change of GABAB dimers and tetramers. A single-molecule stoichiometry assay of the wild-type and mutant receptors revealed the key sites on the interface of ligand-binding domains of the receptor for its dimerization. Moreover, we found that the receptor showed different aggregation behaviors at different conditions. Our results offered new evidence for a better understanding of the molecular basis for GABAB receptor aggregation and activation.

The stochastic trajectories of molecules in living cells, as well as the dynamics in many other complex systems, often exhibit memory in their path over long periods of time. In addition, these systems can show dynamic heterogeneities due to which the motion changes along the trajectories. Such effects manifest themselves as spatiotemporal correlations. Despite the broad occurrence of heterogeneous complex systems in nature, their analysis is still quite poorly understood and tools to model them are largely missing. We contribute to tackling this problem by employing an integral representation of Mandelbrot’s fractional Brownian motion that is compliant with varying motion parameters while maintaining long memory. Two types of switching fractional Brownian motion are analysed, with transitions arising from a Markovian stochastic process and scale-free intermittent processes. We obtain simple formulas for classical statistics of the processes, namely the mean squared displacement and the power spectral density. Further, a method to identify switching fractional Brownian motion based on the distribution of displacements is described. A validation of the model is given for experimental measurements of the motion of quantum dots in the cytoplasm of live mammalian cells that were obtained by single-particle tracking.

Messenger RNA molecules have been localized to different positions in cells and have been followed by time-lapse microscopy. We have used MS2-mVenus–labeled mRNA and single-particle tracking to obtain information on the dynamics of single-mRNA molecules in real time. Using single-molecule tracking, we show that several mRNA molecules visualized via two MS2-binding sites and MS2-mVenus expressed in Bacillus subtilis cells show free diffusion through the entire cell and constrained motion predominantly close to the cell membrane and at the polar regions of the cells. Because constrained motion of mRNAs likely reflects molecules complexed with ribosomes, our data support the idea that translation occurs at sites surrounding the nucleoids. Squared displacement analyses show the existence of at least two distinct populations of molecules with different diffusion constants or possibly of three populations, for example, freely mobile mRNAs, mRNAs in transition complexes, or in complex with polysomes. Diffusion constants between differently sized mRNAs did not differ dramatically and were much lower than that of cytosolic proteins. These data agree with the large size of mRNA molecules and suggest that, within the viscous cytoplasm, size variations do not translate into mobility differences. However, at observed diffusion constants, mRNA molecules would be able to reach all positions within cells in a frame of seconds. We did not observe strong differences in the location of confined motion for mRNAs encoding mostly soluble or membrane proteins, indicating that there is no strong bias for localization of membrane protein-encoding transcripts for the cell membrane. [ABSTRACT FROM AUTHOR]

For decades, we have relied on population and time-averaged snapshots of dynamic molecular scale events to understand how genes are regulated during development and beyond. The advent of techniques to observe single-molecule kinetics in increasingly endogenous contexts, progressing from in vitro studies to living embryos, has revealed how much we have missed. Here, we provide an accessible overview of the rapidly expanding family of technologies for single-molecule tracking (SMT), with the goal of enabling the reader to critically analyse single-molecule studies, as well as to inspire the application of SMT to their own work. We start by overviewing the basics of and motivation for SMT experiments, and the trade-offs involved when optimizing parameters. We then cover key technologies, including fluorescent labelling, excitation and detection optics, localization and tracking algorithms, and data analysis. Finally, we provide a summary of selected recent applications of SMT to study the dynamics of gene regulation. [ABSTRACT FROM AUTHOR]

Messenger RNA molecules have been localized to different positions in cells and have been followed by time-lapse microscopy. We have used MS2-mVenus–labeled mRNA and single-particle tracking to obtain information on the dynamics of single-mRNA molecules in real time. Using single-molecule tracking, we show that several mRNA molecules visualized via two MS2-binding sites and MS2-mVenus expressed in Bacillus subtilis cells show free diffusion through the entire cell and constrained motion predominantly close to the cell membrane and at the polar regions of the cells. Because constrained motion of mRNAs likely reflects molecules complexed with ribosomes, our data support the idea that translation occurs at sites surrounding the nucleoids. Squared displacement analyses show the existence of at least two distinct populations of molecules with different diffusion constants or possibly of three populations, for example, freely mobile mRNAs, mRNAs in transition complexes, or in complex with polysomes. Diffusion constants between differently sized mRNAs did not differ dramatically and were much lower than that of cytosolic proteins. These data agree with the large size of mRNA molecules and suggest that, within the viscous cytoplasm, size variations do not translate into mobility differences. However, at observed diffusion constants, mRNA molecules would be able to reach all positions within cells in a frame of seconds. We did not observe strong differences in the location of confined motion for mRNAs encoding mostly soluble or membrane proteins, indicating that there is no strong bias for localization of membrane protein-encoding transcripts for the cell membrane.

Gonadal steroid 17β-estradiol (E2) exerts rapid, non-genomic effects on neurons and strictly regulates learning and memory through altering glutamatergic neurotransmission and synaptic plasticity. However, its non-genomic effects on AMPARs are not well understood. Here, we analyzed the rapid effect of E2 on AMPARs using single-molecule tracking and super-resolution imaging techniques. We found that E2 rapidly decreased the surface movement of AMPAR via membrane G protein-coupled estrogen receptor 1 (GPER1) in neurites in a dose-dependent manner. The cortical actin network played a pivotal role in the GPER1 mediated effects of E2 on the surface mobility of AMPAR. E2 also decreased the surface movement of AMPAR both in synaptic and extrasynaptic regions on neurites and increased the synaptic dwell time of AMPARs. Our results provide evidence for understanding E2 action on neuronal plasticity and glutamatergic neurotransmission at the molecular level.

Conserved ATP-dependent chromatin remodelers establish and maintain genome-wide chromatin architectures of regulatory DNA during cellular lifespan, but the temporal interactions between remodelers and chromatin targets have been obscure. We performed live-cell single-molecule tracking for RSC, SWI/SNF, CHD1, ISW1, ISW2, and INO80 remodeling complexes in budding yeast and detected hyperkinetic behaviors for chromatin-bound molecules that frequently transition to the free state for all complexes. Chromatin-bound remodelers display notably higher diffusion than nucleosomal histones, and strikingly fast dissociation kinetics with 4–7 s mean residence times. These enhanced dynamics require ATP binding or hydrolysis by the catalytic ATPase, uncovering an additional function to its established role in nucleosome remodeling. Kinetic simulations show that multiple remodelers can repeatedly occupy the same promoter region on a timescale of minutes, implicating an unending ‘tug-of-war’ that controls a temporally shifting window of accessibility for the transcription initiation machinery.

During transcription elongation, NusG aids RNA polymerase by inhibiting pausing, promoting anti-termination on rRNA operons, coupling transcription with translation on mRNA genes, and facilitating Rho-dependent termination. Despite extensive work, the in vivo functional allocation and spatial distribution of NusG remain unknown. Using single-molecule tracking and super-resolution imaging in live E. coli cells, we found NusG predominantly in a chromosome-associated population (binding to RNA polymerase in elongation complexes) and a slowly diffusing population complexed with the 30S ribosomal subunit; the latter provides a "30S-guided" path for NusG into transcription elongation. Only ∼10% of NusG is fast diffusing, with its mobility suggesting non-specific interactions with DNA for >50% of the time. Antibiotic treatments and deletion mutants revealed that chromosome-associated NusG participates mainly in rrn anti-termination within phase-separated transcriptional condensates and in transcription-translation coupling. This study illuminates the multiple roles of NusG and offers a guide on dissecting multi-functional machines via in vivo imaging. [Display omitted] • There is very little free NusG in the cell, with most NusG forming large complexes • Any free NusG interacts non-specifically with the chromosome >50% of the time • Most NusG regulates anti-termination on rrn genes, where NusG is found in condensates • NusG binds to free 30S subunits, helping initiate transcription-translation coupling El Sayyed et al. used single-molecule imaging in live E. coli cells to identify rrn anti-termination (with NusG in transcriptional condensates) and transcription-translation coupling as the main processes occupying NusG in vivo. The work also revealed previously unknown NusG interactions with the free 30S ribosomal subunit and the chromosome. [ABSTRACT FROM AUTHOR]

Gene regulation relies on transcription factors (TFs) exploring the nucleus searching their targets. So far, most studies have focused on how fast TFs diffuse, underestimating the role of nuclear architecture. We implemented a single-molecule tracking assay to determine TFs dynamics. We found that c-Myc is a global explorer of the nucleus. In contrast, the positive transcription elongation factor P-TEFb is a local explorer that oversamples its environment. Consequently, each c-Myc molecule is equally available for all nuclear sites while P-TEFb reaches its targets in a position-dependent manner. Our observations are consistent with a model in which the exploration geometry of TFs is restrained by their interactions with nuclear structures and not by exclusion. The geometry-controlled kinetics of TFs target-search illustrates the influence of nuclear architecture on gene regulation, and has strong implications on how proteins react in the nucleus and how their function can be regulated in space and time.

The plasma membrane consists of a diverse mixture of molecules that dynamically assemble into a highly non-random organization. The formation of nanoscale domains in the membrane is of particular interest as these domains underlie critical cellular functions. Single-molecule tracking is a powerful method to detect and quantify molecular motion at high temporal and spatial resolution and has therefore been instrumental in understanding mechanisms that underlie membrane organization. In single-molecule trajectories, regions of temporal confinement can be determined that might reveal interesting biophysical interactions important for domain formation. However, analytical methods for the detection of temporal confinement in single-molecule trajectories depend on a variety of parameters that heavily depend on experimental factors and the influence of these factors on the performance of confinement detection are not well understood. Here, we present elaborate confinement analyses on simulated random walks and trajectories that display transient confined behavior to optimize the parameters for different experimental conditions. Furthermore, we demonstrate a heatmap visualization tool that allows spatial mapping of confinement hotspots relative to subcellular markers. Using these optimized tools, we reliably detected subdiffusive behavior of different membrane components and observed differences in the confinement behavior of two types of glutamate receptors in neurons. This study will help in further understanding the dynamic behavior of the complex membrane and its role in cellular functioning.

Cadherin transmembrane proteins are responsible for intercellular adhesion in all biological tissues and modulate tissue morphogenesis, cell motility, force transduction, and macromolecular transport. The protein-mediated adhesions consist of adhesive trans interactions and lateral cis interactions. Although theory suggests cooperativity between cis and trans bonds, direct experimental evidence of such cooperativity has not been demonstrated. Here, the use of superresolution microscopy, in conjunction with intermolecular single-molecule Förster resonance energy transfer, demonstrated the mutual cooperativity of cis and trans interactions. Results further demonstrate the consequent assembly of large intermembrane junctions, using a biomimetic lipid bilayer cell adhesion model. Notably, the presence of cis interactions resulted in a nearly 30-fold increase in trans-binding lifetimes between epithelial-cadherin extracellular domains. In turn, the presence of trans interactions increased the lifetime of cis bonds. Importantly, comparison of trans-binding lifetimes of small and large cadherin clusters suggests that this cooperativity is primarily due to allostery. The direct quantitative demonstration of strong mutual cooperativity between cis and trans interactions at intermembrane adhesions provides insights into the long-standing controversy of how weak cis and trans interactions act in concert to create strong macroscopic cell adhesions. [ABSTRACT FROM AUTHOR]

The spread of antibiotic resistance is turning many of the currently used antibiotics less effective against common infections. To address this public health challenge, it is critical to enhance our understanding of the mechanisms of action of these compounds. Aminoglycoside drugs bind the bacterial ribosome, and decades of results from in vitro biochemical and structural approaches suggest that these drugs disrupt protein synthesis by inhibiting the ribosome's translocation on the messenger RNA, as well as by inducing miscoding errors. So far, however, we have sparse information about the dynamic effects of these compounds on protein synthesis inside the cell. In the present study, we measured the effect of the aminoglycosides apramycin, gentamicin, and paromomycin on ongoing protein synthesis directly in live Escherichia coli cells by tracking the binding of dye-labeled transfer RNAs to ribosomes. Our results suggest that the drugs slow down translation elongation two- to fourfold in general, and the number of elongation cycles per initiation event seems to decrease to the same extent. Hence, our results imply that none of the drugs used in this study cause severe inhibition of translocation. [ABSTRACT FROM AUTHOR]

Bacteria are dependent on rapid alterations in gene expression. A prerequisite for rapid adaptations is efficient RNA turnover, with endonuclease RNase Y playing a crucial role in mRNA stability as well as in maturation. In Bacillus subtilis, RNase Y in turn interacts with the so-called “Y-complex” consisting of three proteins, which play important functions in sporulation, natural transformation and biofilm formation. It is thought that the Y-complex acts as an accessory factor in RNase Y regulation but might also have independent functions. Using single-molecule tracking, we show that all three Y-complex proteins exhibit three distinct mobilities, including movement through the cytosol and confined motion, predominantly at membrane-proximal sites but also within the cell center. A transcriptional arrest leads to a strong change in localization and dynamics of YmcA, YlbF and YaaT, supporting their involvement in global RNA degradation. However, Y-complex proteins show distinguishable protein dynamics, and the deletion of yaaT or ylbF shows a minor effect on the dynamics of YmcA. Cell fractionation reveals that YaaT displays a mixture of membrane association and presence in the cytosol, while YlbF and YmcA do not show direct membrane attachment. Taken together, our experiments reveal membrane-associated and membrane-independent activities of Y-complex proteins and a dynamic interplay between them with indirect membrane association of YmcA and YlbF via YaaT.

Cell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shaped Escherichia coli, two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell: the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-like cell shape, aPBP function is less well understood. aPBPs were previously hypothesized to either work in concert with the Rod complex or to independently repair cell-wall defects. First, we demonstrate through modulation of enzyme levels that aPBPs do not contribute to rod-like cell shape but are required for mechanical stability, supporting their independent activity. By combining measurements of cell-wall stiffness, cell-wall insertion, and PBP1b motion at the single-molecule level, we then present evidence that PBP1b, the major aPBP, contributes to cell-wall integrity by repairing cell wall defects.

Abstract Background Proteins within the cytoplasmic membrane display distinct localization patterns and arrangements. While multiple models exist describing the dynamics of membrane proteins, to date, there have been few systematic studies, particularly in bacteria, to evaluate how protein size, number of transmembrane domains, and temperature affect their diffusion, and if conserved localization patterns exist. Results We have used fluorescence microscopy, single-molecule tracking (SMT), and computer-aided visualization methods to obtain a better understanding of the three-dimensional organization of bacterial membrane proteins, using the model bacterium Bacillus subtilis. First, we carried out a systematic study of the localization of over 200 B. subtilis membrane proteins, tagged with monomeric mVenus-YFP at their original gene locus. Their subcellular localization could be discriminated in polar, septal, patchy, and punctate patterns. Almost 20% of membrane proteins specifically localized to the cell poles, and a vast majority of all proteins localized in distinct structures, which we term microdomains. Dynamics were analyzed for selected membrane proteins, using SMT. Diffusion coefficients of the analyzed transmembrane proteins did not correlate with protein molecular weight, but correlated inversely with the number of transmembrane helices, i.e., transmembrane radius. We observed that temperature can strongly influence diffusion on the membrane, in that upon growth temperature upshift, diffusion coefficients of membrane proteins increased and still correlated inversely to the number of transmembrane domains, following the Saffman–Delbrück relation. Conclusions The vast majority of membrane proteins localized to distinct multimeric assemblies. Diffusion of membrane proteins can be suitably described by discriminating diffusion coefficients into two protein populations, one mobile and one immobile, the latter likely constituting microdomains. Our results show there is high heterogeneity and yet structural order in the cell membrane, and provide a roadmap for our understanding of membrane organization in prokaryotes.

Tumor cell‐surface markers are usually overexpressed or mutated protein receptors for which spatiotemporal regulation differs between and within cancers. Single‐molecule fluorescence imaging can profile individual markers in different cellular contexts with molecular precision. However, standard single‐molecule imaging methods based on overexpressed genetically encoded tags or cumbersome probes can significantly alter the native state of receptors. We introduce a live‐cell points accumulation for imaging in nanoscale topography (PAINT) method that exploits aptamers as minimally invasive affinity probes. Localization and tracking of individual receptors are based on stochastic and transient binding between aptamers and their targets. We demonstrated single‐molecule imaging of a model tumor marker (EGFR) on a panel of living cancer cells. Affinity to EGFR was finely tuned by rational engineering of aptamer sequences to define receptor motion and/or native receptor density. [ABSTRACT FROM AUTHOR]

The central dogma teaches us that DNA makes RNA, which in turn makes proteins, the main building blocks of the cell. But this over simplified linear transmission of information overlooks the vast majority of the genome produces RNAs that do not encode proteins and the myriad ways that RNA regulates cellular functions. Historically, one of the challenges in illuminating RNA biology has been the lack of tools for visualizing RNA in live cells. But clever approaches for exploiting RNA binding proteins, in vitro RNA evolution, and chemical biology have resulted in significant advances in RNA visualization tools in recent years. This review provides an overview of current tools for tagging RNA with fluorescent probes and tracking their dynamics, localization andfunction in live mammalian cells. This review article summarizes the current tools that have been developed to light up RNA in live mammalian cells. Such tools permit researchers to dissect the localization, biochemical processing, and dynamics of RNA in real time. [ABSTRACT FROM AUTHOR]

Once described as mere "bags of enzymes," bacterial cells are in fact highly organized, with many macromolecules exhibiting nonuniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid-liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in Escherichia coli. Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrient-rich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components. Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and demonstrates that this process can serve as a mechanism for intracellular organization in prokaryotes and eukaryotes alike. [ABSTRACT FROM AUTHOR]

Metal detoxification is essential for bacteria's survival in adverse environments and their pathogenesis in hosts. Understanding the underlying mechanisms is crucial for devising antibacterial treatments. In the Gram-negative bacterium Escherichia coli, membrane-bound sensor CusS and its response regulator CusR together regulate the transcription of the cus operon that plays important roles in cells' resistance to copper/silver, and they belong to the two-component systems (TCSs) that are ubiquitous across various organisms and regulate diverse cellular functions. In vitro protein reconstitution and associated biochemical/physical studies have provided significant insights into the functions and mechanisms of CusS-CusR and related TCSs. Such studies are challenging regarding multidomain membrane proteins like CusS and also lack the physiological environment, particularly the native spatial context of proteins inside a cell. Here, we use stroboscopic single-molecule imaging and tracking to probe the dynamic behaviors of both CusS and CusR in live cells, in combination with protein- or residue-specific genetic manipulations. We find that copper stress leads to a cellular protein concentration increase and a concurrent mobilization of CusS out of clustered states in the membrane. We show that the mobilized CusS has significant interactions with CusR for signal transduction and that CusS's affinity toward CusR switches on upon sensing copper at the interfacial metal-binding sites in CusS's periplasmic sensor domains, prior to ATP binding and autophosphorylation at CusS's cytoplasmic kinase domain(s). The observed CusS mobilization upon stimulation and its surprisingly early interaction with CusR likely ensure an efficient signal transduction by providing proper conformation and avoiding futile cross talks. [ABSTRACT FROM AUTHOR]

Transcription coactivators are proteins or protein complexes that mediate transcription factor (TF) function. However, they lack DNA-binding capacity, prompting the question of how they engage target loci. Three non-exclusive hypotheses have been posited: coactivators are recruited by complexing with TFs, by binding histones through epigenetic reader domains, or by partitioning into condensates through their extensive intrinsically disordered regions. Using p300 as a prototypical coactivator, we systematically mutated its annotated domains and show by single-molecule tracking in live U2OS cells that coactivator-chromatin binding depends entirely on combinatorial binding of multiple TF-interaction domains. Furthermore, we demonstrate that acetyltransferase activity opposes p300-chromatin association and that the N-terminal TF-interaction domains regulate that activity. Single TF-interaction domains are insufficient for chromatin binding and regulation of catalytic activity, implying a principle that we speculate could broadly apply to eukaryotic gene regulation: a TF must act in coordination with other TFs to recruit coactivator activity. [Display omitted] • The structured Core domain of p300 is dispensable for chromatin engagement • p300-chromatin binding requires multiple TF-interaction domains • p300 KAT activity opposes chromatin binding • p300 KAT activity may be regulated by its N-terminal TF-interaction domains Ferrie et al. demonstrate through live-cell single-molecule imaging that the coactivator p300, a central player in eukaryotic gene regulation, carries out its function of integrating multiple transcription factor (TF) inputs at the level of chromatin binding. Their results support a TF- rather than histone-mediated model of coactivator recruitment. [ABSTRACT FROM AUTHOR]

Microtubules in cells consist of functionally diverse subpopulations carrying distinct post-translational modifications (PTMs). Akin to the histone code, the tubulin code regulates a myriad of microtubule functions, ranging from intracellular transport to chromosome segregation. However, how individual PTMs only occur on subsets of microtubules to contribute to microtubule specialization is not well understood. In particular, microtubule detyrosination, the removal of the C-terminal tyrosine on α-tubulin subunits, marks the stable population of microtubules and modifies how microtubules interact with other microtubule-associated proteins to regulate a wide range of cellular processes. Previously, we found that in certain cell types, only ∼30% of microtubules are highly enriched with the detyrosination mark and that detyrosination spans most of the length of a microtubule, often adjacent to a completely tyrosinated microtubule. How the activity of a cytosolic detyrosinase, vasohibin (VASH), leads to only a small subpopulation of highly detyrosinated microtubules is unclear. Here, using quantitative super-resolution microscopy, we visualized nascent microtubule detyrosination events in cells consisting of 1–3 detyrosinated α-tubulin subunits after nocodazole washout. Microtubule detyrosination accumulates slowly and in a dispersed pattern across the microtubule length. By visualizing single molecules of VASH in live cells, we found that VASH engages with microtubules stochastically on a short timescale, suggesting limited removal of tyrosine per interaction, consistent with the super-resolution results. Combining these quantitative imaging results with simulations incorporating parameters from our experiments, we provide evidence for a stochastic model for cells to establish a subset of detyrosinated microtubules via a detyrosination-stabilization feedback mechanism. [Display omitted] • Detyrosination accumulates stochastically on microtubules in cells • Nascent, cellular detyrosination occurs on 1–3 subunits at a time • Detyrosination is enriched on microtubule subsets by inducing microtubule stability • The degree of enrichment of detyrosination depends on various cellular inputs Tang et al. show that the enrichment of detyrosination on a subset of microtubules inside of a cell can arise from feedback between microtubule stability and stochastic VASH activity. The level of enrichment is determined by cell-type-dependent factors, such as VASH concentration, enzymatic rate, and microtubule dynamics. [ABSTRACT FROM AUTHOR]

The human MET receptor tyrosine kinase contributes to vertebrate development and cell proliferation. As a proto‐oncogene, it is a target in cancer therapies. MET is also relevant for bacterial infection by Listeria monocytogenes and is activated by the bacterial protein internalin B. The processes of ligand binding, receptor activation, and the diffusion behavior of MET within the plasma membrane as well as its interconnections with various cell components are not fully understood. We investigated the receptor diffusion dynamics using single‐particle tracking and imaging fluorescence correlation spectroscopy and elucidated mobility states of resting and internalin B‐bound MET. We show that internalin B‐bound MET exhibits lower diffusion coefficients and diffuses in a more confined area in the membrane. We report that the fraction of immobile receptors is larger for internalin B‐bound receptors than for resting MET. Results of single‐particle tracking in cells treated with various cytotoxins depleting cholesterol from the membrane and disrupting the actin cytoskeleton and microtubules suggest that cholesterol and actin influence MET diffusion dynamics, while microtubules do not have any effect.