Live-cell Single-particle Tracking Research Articles

Revealing the sub-organelle structure and rheological properties of the Escherichia coli nucleoid by live-cell single-particle tracking and Brownian dynamics simulation

Revealing the sub-organelle structure and rheological properties of the Escherichia coli nucleoid by live-cell single-particle tracking and Brownian dynamics simulation

Optimized Red-Absorbing Dyes for Imaging and Sensing.

Rhodamine dyes are excellent scaffolds for developing a broad range of fluorescent probes. A key property of rhodamines is their equilibrium between a colorless lactone and fluorescent zwitterion. Tuning the lactone-zwitterion equilibrium constant (KL-Z) can optimize dye properties for specific biological applications. Here, we use known and novel organic chemistry to prepare a comprehensive collection of rhodamine dyes to elucidate the structure-activity relationships that govern KL-Z. We discovered that the auxochrome substituent strongly affects the lactone-zwitterion equilibrium, providing a roadmap for the rational design of improved rhodamine dyes. Electron-donating auxochromes, such as julolidine, work in tandem with fluorinated pendant phenyl rings to yield bright, red-shifted fluorophores for live-cell single-particle tracking (SPT) and multicolor imaging. The N-aryl auxochrome combined with fluorination yields red-shifted Förster resonance energy transfer (FRET) quencher dyes useful for creating a new semisynthetic indicator to sense cAMP using fluorescence lifetime imaging microscopy (FLIM). Together, this work expands the synthetic methods available for rhodamine synthesis, generates new reagents for advanced fluorescence imaging experiments, and describes structure-activity relationships that will guide the design of future probes.

Single-molecule tracking with nonparametric Bayesian analysis reveals that histone modification protein complexes form specifically at heterochromatin sites.

Eukaryotic cells can establish distinct heritable phenotypic states with epigenetic modifications. Histone H3K9 methylation (H3K9me) is a conserved epigenetic modification that is associated with transcriptional silencing and heterochromatin formation. The diverse functions of heterochromatin depend on the evolutionarily conserved HP1 family of proteins that recognize and bind to H3K9me nucleosomes. Schizosaccharomyces pombe (fission yeast) have two HP1 homologs, Swi6 and Chp2, which play indispensable roles in regulating heterochromatin structure. Swi6 simultaneously binds to H3K9me and oligomerizes, which enables the spreading of heterochromatin and transcriptional silencing. On the other hand, Chp2 binds to H3K9me and recruits the Nucleosome Remodeling and Deacetylase complex, SHREC, through interaction with the histone remodeler Mit1. The putative H3K9 demethylase Epe1 interacts with Swi6 while the histone deacetylase Clr3 is a part of SHREC, and Epe1 and Clr3 are shown to have opposing effects on nucleosome turnover and heterochromatin formation. Structural biology and in vitro biochemistry assays have revealed the interactions between these histone modification proteins involved in the heterochromatin formation, however, there has not yet been any in vivo measurement of the interaction between these proteins. Here, we use live-cell single-particle tracking (SPT) and super-resolution microscopy to investigate the in vivo dynamics and spatial organization of histone modification proteins. With nonparametric Bayesian analysis of SPT trajectories, we measure distinct biophysical mobility states and map these states to the potential in vivo biochemical role. We propose a new mechanism for histone modification proteins in which the heterochromatin site is the primary location for protein complex formation, and H3K9me is a required substrate for the interaction.

Revealing the accessibility and surface morphology of the Escherichia coli nucleoid by genetically encoded live-cell single-particle tracking.

Membraneless organelles play a key role in the subcellular organization and regulation of bacterial cells. In particular, the bacterial genome is highly compacted into a membraneless organelle: the nucleoid. The structure and accessibility of the nucleoid significantly influences gene expression, and this morphology varies with growth phase and under various environmental conditions. Therefore, revealing how the DNA meshwork changes as a function of the growth phase will deepen our understanding of how bacteria regulate transcription in response to stress. To realize this goal, we applied single-particle tracking in E. coli and super-resolved the dynamics of an extrinsic 25-nm protein assembly with a well-defined dodecahedron structure: GFP-nanocages (Hsia et al., Nature 2016). By super-resolving the GFP-nanocage positions from log phase to late stationary phase, we observed an increasingly obvious exclusion of the probe by nucleoid, which indicates that the chromosome is compacting and becoming less accessible for gene expression. Our preliminary experiments also found a novel observation: the nucleoid size and compaction are not necessarily correlated. We hypothesize that at the intermediate stage of compaction, cells with small nucleoids may have a relatively low global compaction level because DNA chains are dragged by the force generated by DNA-NAP complex formation, leading to compacted rigid cores surrounded by a loosened DNA meshwork. By measuring the single-molecule step sizes at the periphery of the nucleoid, we understand the rigidity and smoothness of nucleoid surface at various phases. Furthermore, by analyzing the angle distribution within single-particle trajectories, we quantify the orientation of interactions with the nucleoid. The gene expression levels differ with cell growth stage (Verma et al., PLoS Genetics 2019). Our research provides a novel way of understanding how subcellular structure and gene expression are related.

Mechanisms governing target search and binding dynamics of hypoxia-inducible factors.

Transcription factors (TFs) are classically attributed a modular construction, containing well-structured sequence-specific DNA-binding domains (DBDs) paired with disordered activation domains (ADs) responsible for protein-protein interactions targeting co-factors or the core transcription initiation machinery. However, this simple division of labor model struggles to explain why TFs with identical DNA-binding sequence specificity determined in vitro exhibit distinct binding profiles in vivo. The family of hypoxia-inducible factors (HIFs) offer a stark example: aberrantly expressed in several cancer types, HIF-1α and HIF-2α subunit isoforms recognize the same DNA motif in vitro - the hypoxia response element (HRE) - but only share a subset of their target genes in vivo, while eliciting contrasting effects on cancer development and progression under certain circumstances. To probe the mechanisms mediating isoform-specific gene regulation, we used live-cell single particle tracking (SPT) to investigate HIF nuclear dynamics and how they change upon genetic perturbation or drug treatment. We found that HIF-α subunits and their dimerization partner HIF-1β exhibit distinct diffusion and binding characteristics that are exquisitely sensitive to concentration and subunit stoichiometry. Using domain-swap variants, mutations, and a HIF-2α specific inhibitor, we found that although the DBD and dimerization domains are important, another main determinant of chromatin binding and diffusion behavior is the AD-containing intrinsically disordered region (IDR). Using Cut&Run and RNA-seq as orthogonal genomic approaches, we also confirmed IDR-dependent binding and activation of a specific subset of HIF target genes. These findings reveal a previously unappreciated role of IDRs in regulating the TF search and binding process that contribute to functional target site selectivity on chromatin.

Nucleosome breathing facilitates cooperative binding of pluripotency factors Sox2 and Oct4 to DNA

Critical lineage commitment events are staged by multiple transcription factors (TFs) binding to their cognate motifs, often positioned at nucleosome-enriched regions of chromatin. The underlying mechanism remains elusive due to difficulty in disentangling the heterogeneity in chromatin states. Using a novel coarse-grained model and molecular dynamics simulations, here we probe the association of Sox2 and Oct4 proteins that show clustered binding at the entry-exit region of a nucleosome. The model captures the conformational heterogeneity of nucleosome breathing dynamics that features repeated wrap-unwrap transitions of a DNA segment from one end of the nucleosome. During the dynamics, DNA forms bulges that diffuse stochastically and may regulate the target search dynamics of a protein by nonspecifically interacting with it. The overall search kinetics of the TF pair follows a “dissociation-compensated-association” mechanism, where Oct4 binding is facilitated by the association of Sox2. The cooperativity stems from a change in entropy caused by an alteration in the nucleosome dynamics upon TF binding. The binding pattern is consistent with a live-cell single-particle tracking experiment, suggesting the mechanism observed for clustered binding of a TF pair, which is a hallmark of cis-regulatory elements, has broader implications in understanding gene regulation in a complex chromatin environment.

Effects of Transcription-Dependent Physical Perturbations on the Chromosome Dynamics in Living Cells.

Recent studies with single-particle tracking in live cells have revealed that chromatin dynamics are directly affected by transcription. However, how transcription alters the chromatin movements followed by changes in the physical properties of chromatin has not been elucidated. Here, we measured diffusion characteristics of chromatin by targeting telomeric DNA repeats with CRISPR-labeling. We found that transcription inhibitors that directly block transcription factors globally increased the movements of chromatin, while the other inhibitor that blocks transcription by DNA intercalating showed an opposite effect. We hypothesized that the increased mobility of chromatin by transcription inhibition and the decreased chromatin movement by a DNA intercalating inhibitor is due to alterations in chromatin rigidity. We also tested how volume confinement of nuclear space affects chromatin movements. We observed decreased chromatin movements under osmotic pressure and with overexpressed chromatin architectural proteins that compact chromatin.

Direct-laser writing for subnanometer focusing and single-molecule imaging

Two-photon direct laser writing is an additive fabrication process that utilizes two-photon absorption of tightly focused femtosecond laser pulses to implement spatially controlled polymerization of a liquid-phase photoresist. Two-photon direct laser writing is capable of nanofabricating arbitrary three-dimensional structures with nanometer accuracy. Here, we explore direct laser writing for high-resolution optical microscopy by fabricating unique 3D optical fiducials for single-molecule tracking and 3D single-molecule localization microscopy. By having control over the position and three-dimensional architecture of the fiducials, we improve axial discrimination and demonstrate isotropic subnanometer 3D focusing (<0.8 nm) over tens of micrometers using a standard inverted microscope. We perform 3D single-molecule acquisitions over cellular volumes, unsupervised data acquisition and live-cell single-particle tracking with nanometer accuracy.

CRISPR/Cas12a-mediated labeling of MET receptor enables quantitative single-molecule imaging of endogenous protein organization and dynamics.

SummarySingle-molecule localization microscopy (SMLM) reports on protein organization in cells with near-molecular resolution and in combination with stoichiometric labeling enables protein counting. Fluorescent proteins allow stoichiometric labeling of cellular proteins; however, most methods either lead to overexpression or are complex and time demanding. We introduce CRISPR/Cas12a for simple and efficient tagging of endogenous proteins with a photoactivatable protein for quantitative SMLM and single-particle tracking. We constructed a HEK293T cell line with the receptor tyrosine kinase MET tagged with mEos4b and demonstrate full functionality. We determine the oligomeric state of MET with quantitative SMLM and find a reorganization from monomeric to dimeric MET upon ligand stimulation. In addition, we measured the mobility of single MET receptors in vivo in resting and ligand-treated cells. The combination of CRISPR/Cas12a-assisted endogenous protein labeling and super-resolution microscopy represents a powerful tool for cell biological research with molecular resolution.

Citrullination of HP1\u03b3 chromodomain affects association with chromatin

BackgroundStem cell differentiation involves major chromatin reorganisation, heterochromatin formation and genomic relocalisation of structural proteins, including heterochromatin protein 1 gamma (HP1γ). As the principal reader of the repressive histone marks H3K9me2/3, HP1 plays a key role in numerous processes including heterochromatin formation and maintenance.ResultsWe find that HP1γ is citrullinated in mouse embryonic stem cells (mESCs) and this diminishes when cells differentiate, indicating that it is a dynamically regulated post-translational modification during stem cell differentiation. Peptidylarginine deiminase 4, a known regulator of pluripotency, citrullinates HP1γ in vitro. This requires R38 and R39 within the HP1γ chromodomain, and the catalytic activity is enhanced by trimethylated H3K9 (H3K9me3) peptides. Mutation of R38 and R39, designed to mimic citrullination, affects HP1γ binding to H3K9me3-containing peptides. Using live-cell single-particle tracking, we demonstrate that R38 and R39 are important for HP1γ binding to chromatin in vivo. Furthermore, their mutation reduces the residence time of HP1γ on chromatin in differentiating mESCs.ConclusionCitrullination is a novel post-translational modification of the structural heterochromatin protein HP1γ in mESCs that is dynamically regulated during mESC differentiation. The citrullinated residues lie within the HP1γ chromodomain and are important for H3K9me3 binding in vitro and chromatin association in vivo.

Single-particle tracking localization microscopy reveals nonaxonemal dynamics of intraflagellar transport proteins at the base of mammalian primary cilia.

Primary cilia play a vital role in cellular sensing and signaling. An essential component of ciliogenesis is intraflagellar transport (IFT), which is involved in IFT protein recruitment, axonemal engagement of IFT protein complexes, and so on. The mechanistic understanding of these processes at the ciliary base was largely missing, because it is challenging to observe the motion of IFT proteins in this crowded region using conventional microscopy. Here, we report short-trajectory tracking of IFT proteins at the base of mammalian primary cilia by optimizing single-particle tracking photoactivated localization microscopy for IFT88-mEOS4b in live human retinal pigment epithelial cells. Intriguingly, we found that mobile IFT proteins “switched gears” multiple times from the distal appendages (DAPs) to the ciliary compartment (CC), moving slowly in the DAPs, relatively fast in the proximal transition zone (TZ), slowly again in the distal TZ, and then much faster in the CC. They could travel through the space between the DAPs and the axoneme without following DAP structures. We further revealed that BBS2 and IFT88 were highly populated at the distal TZ, a potential assembly site. Together, our live-cell single-particle tracking revealed region-dependent slowdown of IFT proteins at the ciliary base, shedding light on staged control of ciliary homeostasis.

Live-cell single-particle tracking photoactivated localization microscopy of Cascade-mediated DNA surveillance.

Type I CRISPR-Cas systems utilize small CRISPR RNA (crRNA) molecules to scan DNA strands for target regions. Different crRNAs are bound by several CRISPR-associated (Cas) protein subunits that form the stable ribonucleoprotein complex Cascade. The Cascade-mediated DNA surveillance process requires a sufficient degree of base-complementarity between crRNA and target sequences and relies on the recognition of small DNA motifs, termed protospacer adjacent motifs. Recently, super-resolution microscopy and single-particle tracking methods have been developed to follow individual protein complexes in live cells. Here, we described how this technology can be adapted to visualize the DNA scanning process of Cascade assemblies in Escherichia coli cells. The activity of recombinant Type I-Fv Cascade complexes of Shewanella putrefaciens CN-32 serves as a model system that facilitates comparative studies for many of the diverse CRISPR-Cas systems.

Toward the "unravelling" of HIV: Host cell factors involved in HIV-1 core uncoating.

Early HIV-1 replication requires fusion of viral and cellular membranes that release a fullerene-shaped viral core structure, formed by a lattice of primarily hexameric capsid (CA) rings, into the cellular cytoplasm. The core contains positive-polarity single-strand viral genomic RNA and enzymes that synthesize viral double-strand DNA that is integrated into a host chromosome. Only HIV-1 that release intact cores into the cytoplasm are infectious [1], and disassembly of the core occurs by a regulated process called uncoating. For the purpose of this article, we define uncoating as any dissociation of CA from the viral core. However, the precise mechanism, timing, and location of uncoating is contentious. Recent live-cell single-particle tracking of infectious HIV-1 cores has provided unprecedented insight into uncoating kinetics [2]. One such study found that core integrity, measured by “leakiness” of a core-trapped fluorescent marker, was lost in the cytoplasm approximately 30 min after fusion (after first-strand transfer of reverse transcription) and that this was required for productive infection [3]. However, any such loss of core integrity must not allow innate cellular sensors, such as cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS), to identify and restrict retroviral DNA in the cytoplasm [4]. Another recent study found that a small amount of CA dissociates gradually from the core post-entry before the remaining core structure docks at the nuclear pore complex (NPC), leading to accelerated CA loss, nuclear entry, integration of viral DNA into host DNA, and productive infection [5]. As these two studies measure different events and are not mutually exclusive, it is our view, consistent with that expressed in the review by Francis and Melikyan [2], that HIV-1 uncoating may begin by the loss of core integrity and gradual CA dissociation in the cytoplasm, followed by CA-dependent nuclear docking and accelerated CA loss at the NPC. An alternative model of uncoating is that an intact core arrives at the NPC before uncoating [1]. Here, we discuss the host cell factors subverted by HIV-1 to regulate ordered uncoating in cells (Fig 1), with the caveat that the spatiotemporal specifics of where and when host factors interact with the core and affect CA uncoating requires further investigation. Open in a separate window Fig 1 Host cell factors regulate HIV-1 uncoating. The HIV-1 virion, which contains the CypA, ERK2, and eEF1A host proteins that regulate uncoating, binds and fuses with the host cell membrane, and the core is released into the cytoplasm. PDZD8 and CypA binds CA to stabilize cores to promote infection; however, infection is inhibited if CypA associates with MxB, in which MxB oligomers bind CA to hyperstabilize cores. Cellular eEF1A interacts with HIV-1 RT and facilitates uncoating. Dia1 and Dia2 bind CA–NC complexes to facilitate uncoating likely by localized microtubules stabilization. Dynein interacts with the core via the BICD2 adapter protein or direct interaction with IN, and kinesin-1 interacts with the core via the FEZ1 (phosphorylated by MARK2) adapter protein. This is important for transport of the replication complexes through the cytoplasm to MTOCs at the nuclear periphery as well as uncoating. Pin1 binds CA phosphorylated by MEK1/2 activated ERK2 during virion maturation to facilitate uncoating. MELK also phosphorylates CA to promote uncoating. HIV-1 RT binds eEF1A to stabilize the RTC. The NPC proteins Nup358 and Nup153 help import the PIC, and Nup358 is relocalized to the cytoplasm by KIF5B, while Nup153 prolongs CA association with PICs in the nucleus. TNPO3 localizes CPSF6 to the nucleus to prevent CPSF6-dependent hyperstable cores, and TNPO3 may also help complete CA dissociation from replication complexes in the nucleus. This figure does not attempt to illustrate the spatiotemporal specifics of where and when host factors interact with the core and regulate CA uncoating. Green text: promotes uncoating for optimal kinetics. Orange text: delays uncoating for optimal kinetics. Red text: causes hyperstable cores and inhibits infection. Only viral and host proteins relevant in uncoating and discussed in this article are included in the figure. CA, capsid; CPSF6, cleavage and polyadenylation specificity factor 6; CypA, cyclophilin A; BICD2, bicaudal D2; Dia1/2, diaphanous-related formins 1 and 2; eEF1A, eukaryotic translation elongation factor 1A; ERK2, extracellular signal-regulated kinase 2; FEZ1, fasciculation and elongation factor zeta 1; IN, integrase; KIF5B, kinesin-1 heavy chain; NC, nucleocapsid; NPC, nuclear pore complex; Nup, nucleoporin; MARK2, microtubule affinity-regulating kinase 2; MEK1/2, mitogen-activated protein 1 and 2; MELK, maternal embryonic leucine zipper kinase; MTOC, microtubule-organizing centers; MxB, myxovirus resistance protein B; PDZD8, PDZ domain-containing 8 protein; PIC, pre-integration complex; RT, reverse transcriptase; RTC, reverse transcription complexes; TNPO3, transportin 3.

CaMKII Control of Actin Cytoskeletal Dynamics

The calcium calmodulin dependent kinase (CaMKII) binds and organizes actin Binding is abolished by calcium calmodulin, triggering cytoskeletal remodeling with important implications for synaptic transmission in dendritic spines; CaMKII and actin-rich post-synaptic micro-compartments that expand within one minute upon synaptic stimulation to initiate long-term changes associated with learning. CaMKII organizes static F-actin bundles in-vitro, but fluorescence microscopy single particle tracking (SPT) of GFP-tagged CaMKII holoenzymes (GFP-CaMKII) in spines has not detected stimulus-dependent jumps in mobility consistent with un-bundling. However, changes in binding affinities, in response to osmotic and mechanical forces, might provoke diverse cytoskeletal CaMKII-actin architectures in vivo. To look for these, we initially used SPT in live cells and found both rat neuronal CaMKII isoforms bound RFP-actin labeled stress fibers weakly, comparable to G-actin, with strong binding to the fibers obtained only when multiple holoenzyme subunits are engaged (Khan et al. 2016. Biophys. J.111,395-408). We have now measured the binding of GFP-CaMKII molecules to rhodamine-phalloidin labeled, single F-actin filaments and characterized the architectural transitions of the formed filament networks to mechano-osmotic stress. CaMKII induced formation of mechanically-resilient networks with aster-like nodes. Cryo-electron tomography showed CaMKII localized in small clusters (n < 5) at the nodes. Although nodes disappeared rapidly upon addition of calcium calmodulin, the network structure took several minutes to dissipate. Both C. elegans and human CaMKIIs formed networks at stoichiometric concentrations, revealing an evolutionary-conserved interaction. At the micromolar concentrations investigated, CaMKII-actin bundles were sparsely distributed in the networks. Macromolecular crowding by addition of polyethylene glycol or force generated by addition of myosin motors plus ATP bundled the filaments; but CaMKII retarded, rather than assisted, this transition. CaMKII also protected against filament fragmentation by motor forces. We propose that stimulation-triggered disruption of spine CaMKII-actin interactions has two distinct effects, formation of a compliant F-actin network primarily due to loss of nodal contacts and polymerization of the released G-actin. We are simulating these processes in a 3-dimensional “virtual” spine to determine CaMKII, G and F-actin concentrations and affinities that best mimic the observed spine dynamics upon stimulation.

Mapping Ceacam 1-4L's Nanoscale Spatial Distribution, Structure, Dynamics, and Association with Lipid Ordered Domains by Super-Resolution Microscopy

Super-resolution microscopy has provided novel quantitative insights into the nanoscale spatial distribution of membrane proteins, including the observation that some systems form micro- and nano-sized clusters. However, details of the protein's self-association state (monomer / dimer / oligomer) within the different cluster types are often missing. Since it has been shown that a protein's oligomeric form might be a crucial determinant of its function, it is important to have a technique capable of measuring both spatial distribution and association state. To that end, we have applied a correlated dSTORM/homo-FRET approach to characterize CEACAM 1-4L, the progenitor isoform of the carcinoembryonic antigen-related cellular adhesion molecule (CEACAMs) family. CEACAMs are cell surface glycoproteins involved in homo- and hetero-philic intercellular interactions that control cellular growth, differentiation, tumourigenesis, inflammation and infection. While CEACAM1-4L is known to exist as a monomer or oligomer with the former being hypothesized to be involved in activatory intercellular interactions, the basis for its spatial distribution and association state as a function of distribution remain poorly understood. Our correlated approach involved live cell homo-FRET studies of eYFP-CEACAM1-4L in HeLa cells followed by fixed cell dSTORM imaging performed using nanobody labeling with Alexa Fluor 647 (AF647) conjugated to an anti-eYFP single domain antibody. Cluster analysis of the dSTORM datasets using Voronoi tessellation revealed that the CEACAM1-4L nanoclusters were preferentially comprised of monomeric CEACAM1-4L while microclusters contained a heterogeneous distribution of CEACAM1-4L monomers and oligomers. To further characterize these clusters, super-resolution imaging using Alexa-647 labelled cholera toxin was performed to examine the co-localization of lipid ordered domains, which are known to drive membrane protein lateral distribution and clustering, with CEACAM1-4L. Finally, live-cell single particle tracking (SPT) of CEACAM1-4L was performed to investigate the putative confined, obstructed, free diffusion, and directed motion dynamics of CEACAM1-4L.

Viral Membrane Fusion and Nucleocapsid Delivery into the Cytoplasm are Distinct Events in Some Flaviviruses

Flaviviruses deliver their genome into the cell by fusing the viral lipid membrane to an endosomal membrane. The sequence and kinetics of the steps required for nucleocapsid delivery into the cytoplasm remain unclear. Here we dissect the cell entry pathway of virions and virus-like particles from two flaviviruses using single-particle tracking in live cells, a biochemical membrane fusion assay and virus infectivity assays. We show that the virus particles fuse with a small endosomal compartment in which the nucleocapsid remains trapped for several minutes. Endosomal maturation inhibitors inhibit infectivity but not membrane fusion. We propose a flavivirus cell entry mechanism in which the virus particles fuse preferentially with small endosomal carrier vesicles and depend on back-fusion of the vesicles with the late endosomal membrane to deliver the nucleocapsid into the cytoplasm. Virus entry modulates intracellular calcium release and phosphatidylinositol-3-phosphate kinase signaling. Moreover, the broadly cross-reactive therapeutic antibody scFv11 binds to virus-like particles and inhibits fusion.

Three-dimensional single-particle tracking in live cells: news from the third dimension

Single-particle tracking (SPT) is of growing importance in the biophysical community. It is used to investigate processes such as drug and gene delivery, viral uptake, intracellular trafficking or membrane-bound protein mobility. Traditionally, SPT is performed in two dimensions (2D) because of its technical simplicity. However, life occurs in three dimensions (3D) and many methods have been recently developed to track particles in 3D. Now, is the third dimension worth the effort? Here we investigate the differences between the 2D and 3D analyses of intracellular transport with the 3D development of a time-resolved mean square displacement (MSD) analysis introduced previously. The 3D trajectories, and the 2D projections, of fluorescent nanoparticles were obtained with an orbital tracking microscope in two different cell types: in Dictyostelium discoideum ameba and in adherent, more flattened HuH-7 human cells. As expected from the different 3D organization of both cells’ cytoskeletons, a third of the active transport was lost upon projection in the ameba whereas the identification of the active phases was barely affected in the HuH-7 cells. In both cell types, we found intracellular diffusion to be anisotropic and the diffusion coefficient values derived from the 2D analysis were therefore biased.

Insights into Cell Membrane Microdomain Organization from Live Cell Single Particle Tracking of the IgE High Affinity Receptor FcϵRI of Mast Cells

Current models propose that the plasma membrane of animal cells is composed of heterogeneous and dynamic microdomains known variously as cytoskeletal corrals, lipid rafts and protein islands. Much of the experimental evidence for these membrane compartments is indirect. Recently, live cell single particle tracking studies using quantum dot-labeled IgE bound to its high affinity receptor FcϵRI, provided direct evidence for the confinement of receptors within micrometer-scale cytoskeletal corrals. In this study, we show that an innovative time-series analysis of single particle tracking data for the high affinity IgE receptor, FcϵRI, on mast cells provides substantial quantitative information about the submicrometer organization of the membrane. The analysis focuses on the probability distribution function of the lengths of the jumps in the positions of the quantum dots labeling individual IgE FcϵRI complexes between frames in movies of their motion. Our results demonstrate the presence, within the micrometer-scale cytoskeletal corrals, of smaller subdomains that provide an additional level of receptor confinement. There is no characteristic size for these subdomains; their size varies smoothly from a few tens of nanometers to a over a hundred nanometers. In QD-IGE labeled unstimulated cells, jumps of less than 70 nm predominate over longer jumps. Addition of multivalent antigen to crosslink the QD-IgE-FcϵRI complexes causes a rapid slowing of receptor motion followed by a long tail of mostly jumps less than 70 nm. The reduced receptor mobility likely reflects both the membrane heterogeneity revealed by the confined motion of the monomeric receptor complexes and the antigen-induced cross linking of these complexes into dimers and higher oligomers. In both cases, the probability distribution of the jump lengths is well fit, from 10 nm to over 100 nm, by a novel power law. The fit for short jumps suggests that the motion of the quantum dots can be modeled as diffusion in a fractal space of dimension less than two.

A new Paradigm in Single-Particle Tracking in Live Cells: Onset of Ergodicity Breaking

Experimental single-particle tracking (SPT) is extensively used to study the dynamics of membrane proteins and lipids in living cells. Most studies show that molecular motion in the plasma membrane, as well as in the cytoplasm and the nucleus, undergoes anomalous subdiffusion. SPT data is usually analyzed in terms of the temporal mean square displacement because averaging along individual trajectories is more readily available than ensemble averages. However, in some intriguing physical phenomena ergodicity is broken and thus temporal averages do not converge to the ensemble measurements. Here we measured more than 1,000 Kv2.1 potassium channel trajectories in live human embryonic kidney (HEK) cells, analyzed their ergodic and non-ergodic properties and uncovered the physical mechanism underlying their anomalous diffusion pattern. We have labeled Kv2.1 channels with quantum dots (QDs) in HEK cells, imaged the cell basal membrane with total internal fluorescence microscopy and analyzed the individual channel trajectories. We found that the distributions of the two types of averages are clearly different. The temporal average yielded a much broader MSD than the ensemble-average. Nevertheless, the diffusion pattern of these channels follows anomalous subdiffusion in the lag time, i.e. the MSD is sublinear, both for the temporal and the ensemble averages. Our data reveal that two processes simultaneously coexist and only one of them is ergodic. The ergodicity breaking is found to be maintained by a set of anchoring points associated to the actin cytoskeleton. Control experiments indicate these effects are not induced by the quantum dots. These results are accurately modeled by a continuous time random walk on a fractal structure. When the actin cytoskeleton is disrupted, ergodicity is recovered. These experimental observations have direct biological implications in the dynamics of membrane proteins.

Detecting the conformation of individual proteins in live cells

We combined single molecule fluorescence resonance energy transfer (smFRET) with single particle tracking in live cells to detect the in vivo conformation of individual proteins. We site-specifically labeled recombinant SNARE proteins with a FRET donor and acceptor before microinjecting them into cultured cells. We observed that individual proteins rapidly incorporated into folded complexes at the cell membrane, demonstrating the potential of this method to reveal dynamic interactions within cells.