Visualization, Quantification, and Statistical Evaluation of Dynamics for DNA-Binding Proteins in Bacteria by Single-Molecule Tracking.

All DNA-binding proteins in vivo exist as a population of freely diffusing molecules and of DNA-bound molecules. The molecules bound to DNA can be split into specifically/tightly and nonspecifically bound proteins. Single-molecule tracking (SMT) is a method allowing to visualize protein dynamics in living cells, revealing their behavior in terms of mode of motion, diffusion coefficient/speed, change of dwell times, and unveiling preferred subcellular sites of dwelling. Bleaching-type SMT or fluorescent protein-tagged SMT involves rapid laser-induced bleaching of most fluorophore-labeled molecules. The remaining single fluorescent proteins are then continuously tracked. The trajectories of several fluorescent molecules per cell for a population of cells are analyzed and combined to permit a robust analysis of average behavior of single molecules in live cells, including analyses of protein dynamics in mutant cells or cells exposed to changes in environmental conditions.In this chapter, we describe the preparation of Bacillus subtilis cells, the recording of movies of those cells expressing a monomeric variant of a yellow fluorescent protein (mNeonGreen) fused to a protein of choice, and the subsequent curation of the movie data including the statistical analysis of the protein dynamics. We present a short overview of the analysis program SMTracker 2.0, highlighting its ability to analyze SMT data by non-expert scientists.

United under stress: High-speed transport network emerging at bacterial living edge.

Individuals tend to move freely when there is enough room but would act collectively for their survival under external stress. In the case of living cells, for instance, when a drop of low-density flagellated bacterial solution is transferred onto the agar surface, the initially disordered movement of individual bacteria would be replaced with coordinated cell swarming after a lag phase of a few hours. Here, we study how such cooperation is established while overcoming the disorder at the onset of the lag phase with single nanoparticle tracking. Upon the spreading of the droplet, the bacteria in the solution cluster and align near the almost immobilized contact line confining the drop, forming a narrow ring of cells. As individual cells move in and out of the ring continuously, certain flow patterns emerge in the inter-bacterial fluid. We reveal high-speed long-distance unidirectional flows with definite chirality along the outside of the ring, along the inside of the ring and across the ring. We speculate that these flows enable the fast and efficient transport, facilitating the communication and unification of the bacterial community.

Pre-ribosomal particles from nucleoli to cytoplasm.

The analysis of nucleocytoplasmic transport of proteins and messenger RNA has been the focus of advanced microscopic approaches. Recently, it has been possible to identify and visualize individual pre-ribosomal particles on their way through the nuclear pore complex using both electron and light microscopy. In this review, we focused on the transport of pre-ribosomal particles in the nucleus on their way to and through the pores.

Defect-Engineered Metal-Organic Frameworks as Nanocarriers for Pharmacotherapy: Insights into Intracellular Dynamics at The Single Particle Level.

Nanoscale Metal-Organic Frameworks (nanoMOFs) are widely implemented in a host of assays involving drug delivery, biosensing catalysis, and bioimaging. However, the cell pathways and cell fate remain poorly understood. Here, a new fluorescent nanoMOF integrating ATTO 655 into surface defects of colloidal UiO-66 is synthesized, allowing to track the spatiotemporal localization of Single nanoMOF in live cells. density functional theory reveals the stronger binding of ATTO 655 to the Zr6 cluster nodes compared with phosphate and Alendronate Sodium. Parallelized tracking of the spatiotemporal localization of thousands of nanoMOFs and analysis using machine learning platforms reveals whether nanoMOFs remain outside as well as their cellular internalization pathways. To quantitatively assess their colocalization with endo/lysosomal compartments, a colocalization proxy approach relying on the nanoMOF detection of particles in one channel to the signal in the corresponding endo/lysosomal compartments channel, considering signal versus local background intensity ratio and signal-to-noise ratio is developed. This strategy mitigates colocalization value inflation from high or low signal expression in endo/lysosomal compartments. The results accurately measure the nanoMOFs' colocalization from early to late endosomes and lysosomes and emphasize the importance of understanding their intracellular dynamics based on single-particle tracking for optimal and safe drug delivery.

DNA choreography: correlating mobility and organization of DNA across different resolutions from loops to chromosomes.

The dynamics of DNA in the cell nucleus plays a role in cellular processes and fates but the interplay of DNA mobility with the hierarchical levels of DNA organization is still underexplored. Here, we made use of DNA replication to directly label genomic DNA in an unbiased genome-wide manner. This was followed by live-cell time-lapse microscopy of the labeled DNA combining imaging at different resolutions levels simultaneously and allowing one to trace DNA motion across organization levels within the same cells. Quantification of the labeled DNA segments at different microscopic resolution levels revealed sizes comparable to the ones reported for DNA loops using 3D super-resolution microscopy, topologically associated domains (TAD) using 3D widefield microscopy, and also entire chromosomes. By employing advanced chromatin tracking and image registration, we discovered that DNA exhibited higher mobility at the individual loop level compared to the TAD level and even less at the chromosome level. Additionally, our findings indicate that chromatin movement, regardless of the resolution, slowed down during the S phase of the cell cycle compared to the G1/G2 phases. Furthermore, we found that a fraction of DNA loops and TADs exhibited directed movement with the majority depicting constrained movement. Our data also indicated spatial mobility differences with DNA loops and TADs at the nuclear periphery and the nuclear interior exhibiting lower velocity and radius of gyration than the intermediate locations. On the basis of these insights, we propose that there is a link between DNA mobility and its organizational structure including spatial distribution, which impacts cellular processes.

OneFlowTraX: a user-friendly software for super-resolution analysis of single-molecule dynamics and nanoscale organization.

Super-resolution microscopy (SRM) approaches revolutionize cell biology by providing insights into the nanoscale organization and dynamics of macromolecular assemblies and single molecules in living cells. A major hurdle limiting SRM democratization is post-acquisition data analysis which is often complex and time-consuming. Here, we present OneFlowTraX, a user-friendly and open-source software dedicated to the analysis of single-molecule localization microscopy (SMLM) approaches such as single-particle tracking photoactivated localization microscopy (sptPALM). Through an intuitive graphical user interface, OneFlowTraX provides an automated all-in-one solution for single-molecule localization, tracking, as well as mobility and clustering analyses. OneFlowTraX allows the extraction of diffusion and clustering parameters of millions of molecules in a few minutes. Finally, OneFlowTraX greatly simplifies data management following the FAIR (Findable, Accessible, Interoperable, Reusable) principles. We provide a detailed step-by-step manual and guidelines to assess the quality of single-molecule analyses. Applying different fluorophores including mEos3.2, PA-GFP, and PATagRFP, we exemplarily used OneFlowTraX to analyze the dynamics of plant plasma membrane-localized proteins including an aquaporin, the brassinosteroid receptor Brassinosteroid Insensitive 1 (BRI1) and the Receptor-Like Protein 44 (RLP44).

Mucus-Inspired Self-Healing Hydrogels: A Protective Barrier for Cells against Viral Infection.

Mucus is a dynamic biological hydrogel, composed primarily of the glycoprotein mucin, exhibits unique biophysical properties and forms a barrier protecting cells against a broad-spectrum of viruses. Here, this work develops a polyglycerol sulfate-based dendronized mucin-inspired copolymer (MICP-1) with ≈10% repeating units of activated disulfide as cross-linking sites. Cryo-electron microscopy (Cryo-EM) analysis of MICP-1 reveals an elongated single-chain fiber morphology. MICP-1 shows potential inhibitory activity against many viruses such as herpes simplex virus 1 (HSV-1) and SARS-CoV-2 (including variants such as Delta and Omicron). MICP-1 produces hydrogels with viscoelastic properties similar to healthy human sputum and with tuneable microstructures using linear and branched polyethylene glycol-thiol (PEG-thiol) as cross-linkers. Single particle tracking microrheology, electron paramagnetic resonance (EPR) and cryo-scanning electron microscopy (Cryo-SEM) are used to characterize the network structures. The synthesized hydrogels exhibit self-healing properties, along with viscoelastic properties that are tuneable through reduction. A transwell assay is used to investigate the hydrogel's protective properties against viral infection against HSV-1. Live-cell microscopy confirms that these hydrogels can protect underlying cells from infection by trapping the virus, due to both network morphology and anionic multivalent effects. Overall, this novel mucin-inspired copolymer generates mucus-mimetic hydrogels on a multi-gram scale. These hydrogels can be used as models for disulfide-rich airway mucus research, and as biomaterials.

Expanding the Palette of SWIR Emitting Nanoparticles Based on Au Nanoclusters for Single-Particle Tracking Microscopy.

Single-molecule localization microscopy has proved promising to unravel the dynamics and molecular architecture of thin biological samples down to nanoscales. For applications in complex, thick biological tissues shifting single-particle emission wavelengths to the shortwave infrared (SWIR also called NIR II) region between 900 to 2100 nm, where biological tissues are more transparent is key. To date, mainly single-walled carbon nanotubes (SWCNTs) enable such applications, but they are inherently 1D objects. Here, 0D ultra-small luminescent gold nanoclusters (AuNCs, <3 nm) and ≈25 nm AuNC-loaded-polymeric particles that can be detected at the single-particle level in the SWIR are presented. Thanks to high brightness and excellent photostability, it is shown that the dynamics of the spherical polymeric particles can be followed at the single-particle level in solution at video rates for minutes. We compared single particle tracking of AuNC-loaded-polymeric particles with that of SWCNT diffusing in agarose gels demonstrating the specificity and complementarity of diffusion properties of these SWIR-emitting nano-objects when exploring a complex environment. This extends the library of photostable SWIR emitting nanomaterials to 0D nano-objects of variable size for single-molecule localization microscopy in the second biological window, opening unprecedented possibilities for mapping the structure and dynamics of complex biological systems.

Nanoparticle Anisotropy Increases Targeting Interactions on Live-Cell Membranes.

This paper describes how branch lengths of anisotropic nanoparticles can affect interactions between grafted ligands and cell-membrane receptors. Using live-cell, single-particle tracking, we found that DNA aptamer-gold nanostar nanoconstructs with longer branches showed improved binding efficacy to human epidermal growth factor receptor 2 (HER2) on cancer cell membranes. Inhibiting nanoconstruct-HER2 binding promoted nonspecific interactions, which increased the rotational speed of long-branched nanoconstructs but did not affect that of short-branched constructs. Bivariate analysis of the rotational and translational dynamics showed that longer branch lengths increased the ratio of targeting to nontargeting interactions. We also found that longer branches increased the nanoconstruct-cell interaction times before internalization and decreased intracellular trafficking velocities. Differences in binding efficacy revealed by single-particle dynamics can be attributed to the distinct protein corona distributions on short- and long-branched nanoconstructs, as validated by transmission electron microscopy. Minimal protein adsorption at the high positive curvature tips of long-branched nanoconstructs facilitated binding of DNA aptamer ligands to HER2. Our study reveals the significance of nanoparticle branch length in regulating local chemical environment and interactions with live cells at the single-particle level.

Identification of Early Stage Liver Fibrosis by Modifications in the Interstitial Space Diffusive Microenvironment Using Fluorescent Single-Walled Carbon Nanotubes.

During liver fibrosis, recurrent hepatic injuries lead to the accumulation of collagen and other extracellular matrix components in the interstitial space, ultimately disrupting liver functions. Early stages of liver fibrosis may be reversible, but opportunities for diagnosis at these stages are currently limited. Here, we show that the alterations of the interstitial space associated with fibrosis can be probed by tracking individual fluorescent single-walled carbon nanotubes (SWCNTs) diffusing in that space. In a mouse model of early liver fibrosis, we find that nanotubes generally explore elongated areas, whose lengths decrease as the disease progresses, even in regions where histopathological examination does not reveal fibrosis yet. Furthermore, this decrease in nanotube mobility is a purely geometrical effect as the instantaneous nanotube diffusivity stays unmodified. This work establishes the promise of SWCNTs both for diagnosing liver fibrosis at an early stage and for more in-depth studies of the biophysical effects of the disease.

Neural Tracing Protein-Functionalized Nanoparticles Capable of Fast Retrograde Axonal Transport in Live Neurons.

Neural tracing proteins like horseradish peroxidase-conjugated wheat germ agglutinin (WGA-HRP) can target the central nervous system (CNS) through anatomic retrograde transport without crossing the blood-brain barrier (BBB). Conjugating WGA-HRP to nanoparticles may enable the creation of BBB-bypassing nanomedicine. Microfluidics and two-photon confocal microscopy is applied to screen nanocarriers for transport efficacy and gain mechanistic insights into their interactions with neurons. Protein modification of gold nanoparticles alters their cellular uptake at the axonal terminal and activates fast retrograde transport. Trajectory analysis of individual endosomes carrying the nanoparticles reveals a run-and-pause pattern along the axon with endosomes carrying WGA-HRP-conjugated gold nanoparticles exhibiting longer run duration and faster instantaneous velocity than those carrying nonconjugated nanoparticles. The results offer a mechanistic explanation of the different axonal transport dynamics as well as a cell-based functional assay of neuron-targeted nanoparticles with the goal of developing BBB-bypassing nanomedicine for the treatment of nervous system disorders.

Full-Length Single Protein Molecules Tracking and Counting in Thin Silicon Channels.

Emerging single-molecule protein sensing techniques are ushering in a transformative era in biomedical research. Nevertheless, challenges persist in realizing ultra-fast full-length protein sensing, including loss of molecular integrity due to protein fragmentation, biases introduced by antibodies affinity, identification of proteoforms, and low throughputs. Here, a single-molecule method for parallel protein separation and tracking is introduced, yielding multi-dimensional molecular properties used for their identification. Proteins are tagged by chemo-selective dual amino-acid specific labels and are electrophoretically separated by their mass/charge in custom-designed thin silicon channel with subwavelength height. This approach allows analysis of thousands of individual proteins within a few minutes by tracking their motion during the migration. The power of the method is demonstrated by quantifying a cytokine panel for host-response discrimination between viral and bacterial infections. Moreover, it is shown that two clinically-relevant splice isoforms of Vascular endothelial growth factor (VEGF) can be accurately quantified from human serum samples. Being non-destructive and compatible with full-length intact proteins, this method opens up ways for antibody-free single-protein molecule quantification.

Multicolor Long-Term Single-Particle Tracking Using 10 nm Upconverting Nanoparticles.

Single-particle tracking (SPT) is a powerful technique to unveil molecular behaviors crucial to the understanding of many biological processes, but it is limited by factors such as probe photostability and spectral orthogonality. To overcome these limitations, we develop upconverting nanoparticles (UCNPs), which are photostable over several hours at the single-particle level, enabling long-term multicolor SPT. We investigate the brightness of core-shell UCNPs as a function of inert shell thickness to minimize particle size while maintaining sufficient signal for SPT. We explore different rare-earth dopants to optimize for the brightest probes and find that UCNPs doped with 2% Tm3+/30% Yb3+, 10% Er3+/90% Yb3+, and 15% Tm3+/85% Yb3+ represent the optimal probes for blue, green, and near-infrared emission, respectively. The multiplexed 10 nm probes enable three-color single-particle tracking on live HeLa cells for tens of minutes using a single, near-infrared excitation source. These photostable and multiplexed probes open new avenues for numerous biological applications.

Targeted Single Particle Tracking with Upconverting Nanoparticles.

Single particle tracking (SPT) is a powerful technique for real-time microscopic visualization of the movement of individual biomolecules within or on the surface of living cells. However, SPT often suffers from the suboptimal performance of the photon-emitting labels used to tag the biomolecules of interest. For example, fluorescent dyes have poor photostability, while quantum dots suffer from blinking that hampers track acquisition and interpretation. Upconverting nanoparticles (UCNPs) have recently emerged as a promising anti-Stokes luminescent label for SPT. In this work, we demonstrated targeted SPT using UCNPs. For this, we synthesized 30 nm diameter doped UCNPs and coated them with amphiphilic polymers decorated with polyethylene glycol chains to make them water-dispersible and minimize their nonspecific interactions with cells. Coated UCNPs highly homogeneous in brightness (as confirmed by a single particle investigation) were functionalized by immunoglobulin E (IgE) using a biotin-streptavidin strategy. Using these IgE-UCNP SPT labels, we tracked high-affinity IgE receptors (FcεRI) on the membrane of living RBL-2H3 mast cells at 37 °C in the presence and absence of antigen and obtained good agreement with the literature. Moreover, we used the FcεRI-IgE receptor-antibody system to directly compare the performance of UCNP-based SPT labels to organic dyes (AlexaFluor647) and quantum dots (QD655). Due to their photostability as well as their backgroundless and continuous luminescence, SPT trajectories obtained with UCNP labels are no longer limited by the photophysics of the label but only by the dynamics of the system and, in particular, the movement of the label out of the field of view and/or focal plane.

Variable-Angle Epifluorescence Microscopy for Single-Particle Tracking in the Plant ER.

Single-particle tracking (SPT) of biomolecules in the plant endoplasmic reticulum has the potential to inform on the formation of protein-protein complexes, metabolons, and the transport of molecules through both the ER membrane and lumen. Plant cells are particularly challenging for observing and tracking single molecules due to their unique structure, size, and considerable autofluorescence. However, by using variable-angle or highly inclined epifluorescence microscopy (VAEM) and transient expression in tobacco, it is possible to observe single-particle dynamics in the ER. Selecting the appropriate fluorophore, and ensuring the correct fluorophore density in the ER, is essential for successful SPT. By using tuneable fluorophores, which can be photoconverted and photoactivated, it is possible to vary the density of visible fluorophores in the ER dynamically. Here we describe methods to prepare plant samples for VAEM and two methods for determining and analyzing single-particle tracks from VAEM time series.

Deep Learning-Assisted Automated Multidimensional Single Particle Tracking in Living Cells.

The translational and rotational dynamics of anisotropic optical nanoprobes revealed in single particle tracking (SPT) experiments offer molecular-level information about cellular activities. Here, we report an automated high-speed multidimensional SPT system integrated with a deep learning algorithm for tracking the 3D orientation of anisotropic gold nanoparticle probes in living cells with high localization precision (<10 nm) and temporal resolution (0.9 ms), overcoming the limitations of rotational tracking under low signal-to-noise ratio (S/N) conditions. This method can resolve the azimuth (0°-360°) and polar angles (0°-90°) with errors of less than 2° on the experimental and simulated data under S/N of ∼4. Even when the S/N approaches the limit of 1, this method still maintains better robustness and noise resistance than the conventional pattern matching methods. The usefulness of this multidimensional SPT system has been demonstrated with a study of the motions of cargos transported along the microtubules within living cells.

FluoroTensor: Identification and tracking of colocalised molecules and their stoichiometries in multi-colour single molecule imaging via deep learning.

The identification of photobleaching steps in single molecule fluorescence imaging is a well-established procedure for analysing the stoichiometries of molecular complexes. Nonetheless, the method is challenging with protein fluorophores because of the high levels of noise, rapid bleaching and highly variable signal intensities, all of which complicate methods based on statistical analyses of intensities to identify bleaching steps. It has recently been shown that deep learning by convolutional neural networks can yield an accurate analysis with a relatively short computational time. We describe here an improved use of such an approach that detects bleaching events even in the first time point of observation, and we have included this within an integrated software package incorporating fluorescence spot detection, colocalisation, tracking, FRET and photobleaching step analyses of single molecules or complexes. This package, known as FluoroTensor, is written in Python with a self-explanatory user interface.

Redistribution of the glycocalyx exposes phagocytic determinants on apoptotic cells.

Phagocytes remove dead and dying cells by engaging "eat-me" ligands such as phosphatidylserine (PtdSer) on the surface of apoptotic targets. However, PtdSer is obscured by the bulky exofacial glycocalyx, which also exposes ligands that activate "don't-eat-me" receptors such as Siglecs. Clearly, unshielding the juxtamembrane "eat-me" ligands is required for the successful engulfment of apoptotic cells, but the mechanisms underlying this process have not been described. Using human and murine cells, we find that apoptosis-induced retraction and weakening of the cytoskeleton that anchors transmembrane proteins cause an inhomogeneous redistribution of the glycocalyx: actin-depleted blebs emerge, lacking the glycocalyx, while the rest of the apoptotic cell body retains sufficient actin to tether the glycocalyx in place. Thus, apoptotic blebs can be engaged by phagocytes and are targeted for engulfment. Therefore, in cells with an elaborate glycocalyx, such as mucinous cancer cells, this "don't-come-close-to-me" barrier must be removed to enable clearance by phagocytosis.

Real-Time Dissection of the Exosome Pathway for Influenza Virus Infection.

Exosomes play an important role in the spread of viral infections and immune escape. However, the exact ability and mechanisms by which exosomes produced during viral infections (vExos) infect host cells are still not fully understood. In this study, we developed a dual-color exosome labeling strategy that simultaneously labels the external and internal structures of exosomes with quantum dots to enable in situ monitoring of the transport process of vExos in live cells using the single-particle tracking technique. Our finding revealed that vExos contains the complete influenza A virus (IAV) genome and viral ribonucleoprotein complexes (vRNPs) proteins but lacks viral envelope proteins. Notably, these vExos have the ability to infect cells and produce progeny viruses. We also found that vExos are transported in three stages, slow-fast-slow, and move to the perinuclear region via microfilaments and microtubules. About 30% of internalized vExos shed the external membrane and release the internal vRNPs into the cytoplasm by fusion with endolysosomes. This study suggested that vExos plays a supporting role in IAV infection by assisting with IAV propagation in a virus-independent manner. It emphasizes the need to consider the infectious potential of vExos and draws attention to the potential risk of exosomes produced by viral infections.

Mapping Extracellular Space Features of Viral Encephalitis to Evaluate the Proficiency of Anti-Viral Drugs.

The extracellular space (ECS) is an important barrier against viral attack on brain cells, and dynamic changes in ECS microstructure characteristics are closely related to the progression of viral encephalitis in the brain and the efficacy of antiviral drugs. However, mapping the precise morphological and rheological features of the ECS in viral encephalitis is still challenging so far. Here, a robust approach is developed using single-particle diffusional fingerprinting of quantum dots combined with machine learning to map ECS features in the brain and predict the efficacy of antiviral encephalitis drugs. These results demonstrated that this approach can characterize the microrheology and geometry of the brain ECS at different stages of viral infection and identify subtle changes induced by different drug treatments. This approach provides a potential platform for drug proficiency assessment and is expected to offer a reliable basis for the clinical translation of drugs.