High-speed Single-particle Tracking Research Articles

Small stepping motion of processive dynein revealed by load-free high-speed single-particle tracking

Cytoplasmic dynein is a dimeric motor protein which processively moves along microtubule. Its motor domain (head) hydrolyzes ATP and induces conformational changes of linker, stalk, and microtubule binding domain (MTBD) to trigger stepping motion. Here we applied scattering imaging of gold nanoparticle (AuNP) to visualize load-free stepping motion of processive dynein. We observed artificially-dimerized chimeric dynein, which has the head, linker, and stalk from Dictyostelium discoideum cytoplasmic dynein and the MTBD from human axonemal dynein, whose structure has been well-studied by cryo-electron microscopy. One head of a dimer was labeled with 30 nm AuNP, and stepping motions were observed with 100 μs time resolution and sub-nanometer localization precision at physiologically-relevant 1 mM ATP. We found 8 nm forward and backward steps and 5 nm side steps, consistent with on- and off-axes pitches of binding cleft between αβ-tubulin dimers on the microtubule. Probability of the forward step was 1.8 times higher than that of the backward step, and similar to those of the side steps. One-head bound states were not clearly observed, and the steps were limited by a single rate constant. Our results indicate dynein mainly moves with biased small stepping motion in which only backward steps are slightly suppressed.

Copy Number and Function of Individual Disordered Proteins in the Nuclear Pore Complex Revealed by Combining Auxin-Inducible Degron Strategy and High-Speed Single -Molecule Microscopy

Intrinsically disordered nucleoporins (Nup) rich in phenylalanine-glycine (FG) repeats form the selectivity barrier in nuclear pore complexes to mediate nucleocytoplasmic transport in eukaryotic cells. However, the definite copy number of an individual FG Nup and its specific roles in mediating nucleocytoplasmic transport remain controversial. Here we demonstrated an approach to count the copy number of individual FG Nup and determine the consequences of the absence of each FG Nup on the nucleocytoplasmic transport kinetics in live cells. First, with CRISPR/Cas9 technique, we generated stable human cell lines expressing FG Nups fused with fluorescent proteins and degron tags, and then utilized auxin-inducible degron (AID) strategies for selective and rapid degradation of individual FG-Nups. Then we used high-speed single-molecule microscopy to directly quantify the copy number for each FG Nup in individual NPCs, as well as track nuclear export of firefly luciferase mRNPs at the presence or absence of a specific FG Nup.Our results revealed significant deviations on the copy number and facilitating roles for some of the FG Nups from previous measurements. For example, we found that in DLD-1 cells, all of the three nuclear basket FG Nups, Nup153, Nup50 and Tpr, have consistently shown an average of 8 copies in over 300 NPCs from 50 live cells, in contrast with previous studies, where 16-32 copies were estimated. Moreover, high-speed single-particle tracking of nuclear export of mRNAs in live cells in the absence of Nup153 at the NPCs, showed approximately a 4-fold reduction in their nuclear export efficiency, while the nuclear export times of successful mRNPs export events with and without the presence of Nup153 in the NPCs showed almost no differences.

Combined High-Speed Single Particle Tracking of Membrane Proteins and Superresolution of Membrane-associated Structures

Combined High-Speed Single Particle Tracking of Membrane Proteins and Superresolution of Membrane-associated Structures

Nanoscopic compartmentalization of membrane protein motion at the axon initial segment.

The axon initial segment (AIS) is enriched in specific adaptor, cytoskeletal, and transmembrane molecules. During AIS establishment, a membrane diffusion barrier is formed between the axonal and somatodendritic domains. Recently, an axonal periodic pattern of actin, spectrin, and ankyrin forming 190-nm-spaced, ring-like structures has been discovered. However, whether this structure is related to the diffusion barrier function is unclear. Here, we performed single-particle tracking time-course experiments on hippocampal neurons during AIS development. We analyzed the mobility of lipid-anchored molecules by high-speed single-particle tracking and correlated positions of membrane molecules with the nanoscopic organization of the AIS cytoskeleton. We observe a strong reduction in mobility early in AIS development. Membrane protein motion in the AIS plasma membrane is confined to a repetitive pattern of ∼190-nm-spaced segments along the AIS axis as early as day in vitro 4, and this pattern alternates with actin rings. Mathematical modeling shows that diffusion barriers between the segments significantly reduce lateral diffusion along the axon.

Organization of the axon initial segment: Actin like a fence.

What prevents the movement of membrane molecules between axonal and somatodendritic domains is unclear. In this issue, Albrecht et. al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201603108) demonstrate via high-speed single-particle tracking and superresolution microscopy that lipid-anchored molecules in the axon initial segment are confined to membrane domains separated by periodically spaced actin rings.

Nanoscopic substructures of raft-mimetic liquid-ordered membrane domains revealed by high-speed single-particle tracking.

Lipid rafts are membrane nanodomains that facilitate important cell functions. Despite recent advances in identifying the biological significance of rafts, nature and regulation mechanism of rafts are largely unknown due to the difficulty of resolving dynamic molecular interaction of rafts at the nanoscale. Here, we investigate organization and single-molecule dynamics of rafts by monitoring lateral diffusion of single molecules in raft-containing reconstituted membranes supported on mica substrates. Using high-speed interferometric scattering (iSCAT) optical microscopy and small gold nanoparticles as labels, motion of single lipids is recorded via single-particle tracking (SPT) with nanometer spatial precision and microsecond temporal resolution. Processes of single molecules partitioning into and escaping from the raft-mimetic liquid-ordered (Lo) domains are directly visualized in a continuous manner with unprecedented clarity. Importantly, we observe subdiffusion of saturated lipids in the Lo domain in microsecond timescale, indicating the nanoscopic heterogeneous molecular arrangement of the Lo domain. Further analysis of the diffusion trajectory shows the presence of nano-subdomains of the Lo phase, as small as 10 nm, which transiently trap the lipids. Our results provide the first experimental evidence of non-uniform molecular organization of the Lo phase, giving a new view of how rafts recruit and confine molecules in cell membranes.

High-Speed Single Particle Tracking on Model Lipid Membranes

Interferometric scattering detection microscopy (iSCAT) is a powerful method for single particle tracking (SPT) experiments. Recently, we reported on the use of iSCAT for visualizing the diffusion of gold nanoparticles (GNPs) as small as 5nm attached to lipids in model membranes with nanometer lateral precision and at up to 1 MHz frame rate. Exploiting this high spatial and temporal resolution, we revealed transient nanoscopic confinements of nanometer size on millisecond time scales on supported lipid bilayers (SLBs) [1]. To avoid artifacts caused by the glass-lipid interface, we have extended our studies to giant unilamellar vesicles (GUVs). Here, we demonstrate one of the unique capabilities of iSCAT, namely high axial resolution in tracking the displacement of a nanoparticle. We present three-dimensional trajectories of GNP-labeled lipids and unlabeled virus-like particles diffusing on a GUV membrane [2].[1] C.-L. Hsieh, S. Spindler, J. Ehrig, V. Sandoghdar, J. Phys. Chem. B 118 (2014).[2] S. Spindler, H. Stein, J. Ehrig, V. Sandoghdar, in preparation.

Nano-Substructures of raft-Mimetic Liquid-Ordered Membrane Domains Revealed by High-Speed Single-Particle Tracking

Biological membrane domains of distinct molecular compositions and organizations facilitate many important membrane functions. Lipid rafts are specialized membrane domains of liquid-ordered (Lo) phase that are found to be essential for various membrane processes. Despite of recent advances in identifying the biological significance of rafts, the dynamic nature and regulation mechanism of rafts are largely unknown due to the lack of technology to resolve the transient molecular interaction with rafts at the nanoscale. In this work, we investigate how rafts recruit and trap molecules by monitoring lateral diffusion of single lipids in raft-containing reconstituted membranes at ultrahigh spatiotemporal resolution. Using high-speed interferometric scattering (iSCAT) optical microscopy and small gold nanoparticles as labels, the motion of single lipids in the membrane is recorded via single-particle tracking (SPT) with 3 nm spatial precision and 20 μs temporal resolution. The processes of single molecules partitioning into and escaping from the raft-mimetic Lo domains are directly visualized in a continuous manner with unprecedented clarity. Surprisingly, we observe anomalous subdiffusion of saturated lipids in the Lo domain in microsecond timescale, indicating the nanoscopic heterogeneous molecular arrangement of the Lo domain. Further analysis of the diffusion trajectory shows the presence of nano-subdomains of the Lo phase, as small as 10 nm, that transiently trap the saturated lipids. Our data provide the first experimental evidence of non-uniform molecular organization of the Lo phase and show its critical role in determining molecular dynamics in the Lo phase. Our results also imply that the raft-associated molecules could be trapped between nano-subdomains of densely packed saturated lipids, surrounded by high density of cholesterol. The nanoscopic membrane environment created by the nano-subdomains of rafts is thus unique, which illustrates the essentials of lipid rafts of Lo phase in cell membrane.

High-Speed Single-Particle Tracking Reveals Lipid Dynamics in Heterogeneous Raft-Containing Membranes

Phase coexistence and domain formation in lipid bilayers are intriguing phenomena and play essential roles in many cell functions. In particular, coexistence of liquid-ordered and liquid-disordered phases in membranes of ternary lipid composition has drawn great attention, as it underlies the concept of “lipid rafts” in cell membranes. Most biological significance of raft domains is originated from their capability of recruiting certain molecules into the domain through interaction between lipids and proteins. However, experimental evidence of how such interaction takes place at single-molecule level has been missing because it requires high spatiotemporal resolution for observing rapid and nanoscopic motion of individual molecules in raft-containing membranes.In this work, we investigated the dynamics of single lipid molecules in raft-containing bilayer membranes by high-speed optical imaging. An advanced microscope system was developed for tracking individual small gold nanoparticles (20 nm in diameter) attaching to the head groups of single lipid molecules (through biotin-avidin conjugation). The linear scattering signal from the nanoparticles was detected by optical interference where optimal spatial localization precision down to a few nanometers can be achieved at very high speed (50,000 frames per second). Furthermore, the photostable linear scattering signal offers unlimited observation time. Using this technique, we recorded precise and long diffusion trajectories of single lipid molecules in raft-containing supported lipid bilayers (DOPC/sphingomyelin/cholesterol, 40:40:20 mol ratios). Meanwhile, the micron-sized raft domains in the membrane were observed in fluorescence by adding a trace amount of dye-labeled DOPE that marks the non-raft regions. By correlating the detailed trajectories of single probe lipids recorded at high-speed to the corresponding fluorescence image that contours the raft domains, we reveal how a lipid molecule goes across the raft boundary and how its diffusion characteristics changes due to the raft domains with unprecedented clarity.

High-speed single-particle tracking of GM1 in model membranes reveals anomalous diffusion due to interleaflet coupling and molecular pinning.

The biological functions of the cell membrane are influenced by the mobility of its constituents, which are thought to be strongly affected by nanoscale structure and organization. Interactions with the actin cytoskeleton have been proposed as a potential mechanism with the control of mobility imparted through transmembrane “pickets” or GPI-anchored lipid nanodomains. This hypothesis is based on observations of molecular mobility using various methods, although many of these lack the spatiotemporal resolution required to fully capture all the details of the interaction dynamics. In addition, the validity of certain experimental approaches, particularly single-particle tracking, has been questioned due to a number of potential experimental artifacts. Here, we use interferometric scattering microscopy to track molecules labeled with 20–40 nm scattering gold beads with simultaneous <2 nm spatial and 20 μs temporal precision to investigate the existence and mechanistic origin of anomalous diffusion in bilayer membranes. We use supported lipid bilayers as a model system and demonstrate that the label does not influence time-dependent diffusion in the small particle limit (≤40 nm). By tracking the motion of the ganglioside lipid GM1 bound to the cholera toxin B subunit for different substrates and lipid tail properties, we show that molecular pinning and interleaflet coupling between lipid tail domains on a nanoscopic scale suffice to induce transient immobilization and thereby anomalous subdiffusion on the millisecond time scale.

Immobilization of GM1 Causes Pinning and Anomalous Diffusion of Cholera Toxin Bound GM1 Across a Bilayer Membrane

Small changes in the lateral organisation of components within the plasma membrane can cause dramatic effects on cellular function. As a result, the mobility of both transmembrane and single leaflet species has been the subject of intense study. A particularly appealing description of the cell membrane that has emerged in the past decade is that of the picket-fence model, based on high-speed single particle tracking of metallic nanoparticles on living cells. Nevertheless, there have been numerous reports that have questioned the validity of such approaches due to possible artefacts caused by the size of the label, the roughness of the membrane, or the precision of the imaging approach.Here, we used high-speed single particle tracking with interferometric scattering microscopy (iSCAT) with 1 nm spatial and 10 microsecond temporal accuracy of metallic nano particles as small as 20 nm to investigate the existence, role and mechanistic origin of time-dependent mobilities of membrane components in lipid bilayers. Using supported lipid bilayers as a model system, we demonstrate that surface-immobilization of GM1 on the lower leaflet induces anomalous subdiffusion of cholera toxin-bound GM1 in the upper leaflet. At the same time we show, by chemically modifying the lipid chain of GM1, that interleaflet coupling is a necessary element for the observed sub-diffusive behaviour. The observations are in excellent agreement with a simple barrier and valley model responsible for the onset of anomalous diffusion, in this case caused by pinned GM1 molecules on the lower leaflet. Our results demonstrate the validity of high-speed tracking using nanoparticle labels and present a general mechanism for communication across lipid bilayers in the absence of transmembrane components.

The Actin Cytoskeleton Controls the Activation of Invariant Natural Killer T Cells by Fine-Tuning CD1d Nanoscale Aggregation on Antigen Presenting Cells

Invariant Natural killer T (iNKT) cells are a subset of lipid-specific T cells, restricted by the MHC-I class like molecule CD1d. A recent study showed a correlation between the co-localization of lipid-loaded CD1d molecules with lipid rafts on the membrane of antigen presenting cells, and their capacity of eliciting secretion of Th1-cytokines by stimulated iNKT cells [1]. Thus, this study suggested that not only the structure of CD1d bound to an exogenous lipid could influence Cd1d-mediated immunity, but also its partitioning on the membrane. Here, we address the spatiotemporal behaviour of α-Galactosylceramide loaded CD1d complexes on the cell membrane of human myeloid cells using multiple colour high-speed single-particle tracking (100 Hz) combined with an iNKT T Cell Receptor-Qdot conjugate as imaging probe. Furthermore, we complement these studies using STED nanoscopy to obtain nanoscale images of CD1d spatial organization. Our results indicate a direct role of the actin cytoskeleton in actively segregating CD1d nanoclusters on the cell membrane resulting in an inhibition of the activation of iNKT cells [2]. As a whole, our work proposes a new paradigm of biophysical interaction between CD1d presenting cells and NKT cells which deviates significantly from classical MHCI/II complexes and CD8/CD4-T Cells interactions.[1] J. S. Im et al., Immunity, 30, 888, 2009.[2] J. A. Torreno-Pina et al., in preparation.

Single Molecule Applications of Quantum Dots

Fluorescent nanocrystals composed of semiconductor materials were first introduced for biological applications in the late 1990s. The focus of this review is to give a brief survey of biological applications of quantum dots (QDs) at the single QD sensitivity level. These are described as follows: 1) QD blinking and bleaching statistics, 2) the use of QDs in high speed single particle tracking with a special focus on how to design the biofunctional coatings of QDs which enable specific targeting to single proteins or lipids of interest, 3) a hybrid lipid-DNA analogue binding QDs which allows for tracking single lipids in lipid bilayers, 4) two-photon fluorescence correlation spectroscopy of QDs and 5) optical trapping and excitation of single QDs. In all of these applications, the focus is on the single particle sensitivity level of QDs. The high applicability of QDs in live cell imaging experiments held together with the prospects in localization microscopy and single molecule manipulation experiments gave QDs a promising future in single molecule research.

Single Molecule Applications of Quantum Dots

Fluorescent nanocrystals composed of semiconductor materials were first introduced for biological applications in the late 1990s. The focus of this review is to give a brief survey of biological applications of quantum dots (QDs) at the single QD sensitivity level. These are described as follows: 1) QD blinking and bleaching statistics, 2) the use of QDs in high speed single particle tracking with a special focus on how to design the biofunctional coatings of QDs which enable specific targeting to single proteins or lipids of interest, 3) a hybrid lipid-DNA analogue binding QDs which allows for tracking single lipids in lipid bilayers, 4) two-photon fluorescence correlation spectroscopy of QDs and 5) optical trapping and excitation of single QDs. In all of these applications, the focus is on the single particle sensitivity level of QDs. The high applicability of QDs in live cell imaging experiments held together with the prospects in localization microscopy and single molecule manipulation experiments gave QDs a promising future in single molecule research.

Multi-Color Single Particle Tracking of QD-IgE-FcεRI: Directly Correlating Oligomer Size with Receptor Mobility and Signaling

IgE binds to its high affinity receptor, FcεRI, expressed on mast cells and basophils. Crosslinking of IgE-FcεRI complexes leads to intracellular signaling and eventual immune response. Although much is known about this receptor family, the precise mechanism of signal initiation remains elusive. Previous single particle tracking (SPT) experiments have revealed that small, mobile receptors are signaling competent. However, high-speed SPT had typically been limited to two-color tracking, such that aggregates larger than dimers could not be distinguished. In order to better understand the relationship between receptor mobility, aggregate size and signaling, we have developed a novel high-speed hyperspectral line-scanning microscope (HSM) to perform multi-color SPT (mcSPT) of up to 8 colors of quantum dot (QD)- labeled IgE-FcεRI simultaneously on the surface of mast cells. The HSM is unique in its capability to acquire a 4D image (x,y,λ,t) of ∼30 microns⊥2 with 128 spectral channels at ∼30 fps. This combined with the advantageous characteristics of QDs (high quantum yield, single source excitation, narrow emission spectra) provides an exceptional platform for mcSPT. Using the HSM, we can determine receptor aggregate size from the spectral signature, and then correlate aggregate size with mobility. Comparison of aggregate mobility in the presence of the tyrosine kinase inhibitor, PP2, reveals the influence of adaptor protein recruitment on mobility. The HSM also allows for simultaneous measurement of calcium flux (Fluo-4) while performing mcSPT, providing a direct readout of signaling in response to receptor aggregate formation and mobility. These experiments not only address important questions in IgE-FcεRI signal initiation, but also provide new biophysical insight into the long-standing debate over the relationship between receptor oligomer size and mobility in the plasma membrane.

High-Speed Interferometric Scattering Microscopy of Receptor Mobility Reveals Anomalous Diffusion in Model Membranes

The diffusive behavior of membrane proteins has been reported to change with the timescale of the measurement. High-speed single-particle tracking studies have revealed that it is Brownian on time scales less than 100 μs, confined from 1 to 10 ms, and Brownian at longer times (>10 ms).1 This behavior has been interpreted as hop diffusion between submicron compartments in the plasma membrane. Due to the fundamental difficulty in achieving simultaneously high spatial and temporal resolution, these results have relied on a single experimental approach. Furthermore, attempts to use a complementary fluorescence technique to study anomalous diffusion have not reproduced the hop diffusion model.2Here we use interferometric scattering microscopy (iSCAT)3 to track the motion of the GM1 ganglioside receptor binding the B subunit of cholera toxin using 40-nm gold nanoparticle labels, in both supported lipid bilayers (SLBs) and droplet hydrogel bilayers (DHBs).4 In SLBs, in single trajectories containing >200,000 frames and providing simultaneous 10 μs temporal and sub-nm spatial precision, we observe a shift from Brownian diffusion at early time scales (30 - 100 μs) to confined diffusion (100 μs - 10 ms) back to Brownian diffusion (>10 ms). In contrast, GM1 diffusion in DHBs is Brownian at all timescales. These data suggest that the presence of a structural support on one side of the bilayer is sufficient to produce anomalous diffusion, even for diffusers that do not penetrate the membrane.1. Kusumi, A. et al. Annu. Rev. Biophys. Biomol. Struct.34, 351-378 (2005).2. Weiser, S. et al. Biophys. J. 92, 3719-3728 (2007).3. Ortega-Arroyo, J. and Kukura, P. Phys. Chem. Chem. Phys. (2012)DOI: 10.1039/c2cp41013c4. Thompson, J. R et al.Nano Lett.7, 3875-3878 (2007).

High-Speed Single Quantum Dot Imaging of in Live Cells Reveal Hop Diffusion

Ultra high-speed single particle tracking (image frame rates 40-50 kHz) experiments with 40 nm gold particles has indicated that lipids and proteins in the plasma membrane undergo hop-diffusion between nanometer sized compartments (Fujiwara et al. (2002) J Cell Biol. 157:1071-81). These findings have yet to be independently confirmed. In this work, we show that high-speed single particle tracking with quantum dots (QDs) and using a standard wide-field fluorescence microscope and an EMCCD is possible at image acquisition rates of up to ∼2000 Hz. The spatial precision in these experiments is ∼40 nm (as determined from the standard deviation of repeated position measurements of an immobile QD on a cell). Using this system, we show that membrane proteins and lipids, which have been exogenously labeled with functionalized QDs, show examples of three types of motion in the plasma membrane of live mouse embryo fibroblasts, 1) approximately free diffusion, 2) confined (immobile) diffusion, and 3) hop diffusion between compartments with a size of ∼100 nm diameter, and a lifetime of ∼100-200 milliseconds. In these experiments, we have used QDs that had a hydrodynamic radius (RH) of about 12-15 nm as determined by fluorescence correlation spectroscopy (FCS). While these QDs are very large compared to fluorescent dyes, they are 25-40% smaller in radius and are at least two fold smaller in volume than the 40 nm diameter gold particles used in ultra high speed SPT.

Glycosphingolipids and Non-Raft Phospholipids Exhibit Very Similar Dynamics in Single-Molecule Observations

The diffusion of typical raft-associated molecules, glycosphingolipids, GM1, GM3, GD1a, was observed using high-speed single-particle tracking (HS-SPT) at a temporal resolution of ∼6 μs. Each glycosphingolipid molecule was tagged with a 40-nm-gold particle conjugated by the respective Fab antibody or a Cy3-conjugated Fab antibody. At video rate, both probes exhibited the same diffusion behavior, undergoing apparent simple Brownian diffusion at the same effective diffusion coefficient (about 0.2 μm2/s). In HS-SPT observations, each glycosphingolipid molecule underwent actin-dependent hop diffusion over the membrane compartment of an average of 110 nm in diameter at an average frequency of once every 25 ms. Surprisingly, this behavior is the same as that of a typical non-raft phospholipid DOPE, qualitatively and quantitatively. These results are at variance with the previous FCS/FRAP studies. In addition, previous investigations concluded that GM1, labeled either with cholera toxin (CTX) in its headgroup or with Bodipy-FL on the alkyl chain, diffused much more slowly than non-raft phospholipid probes. Here, we found that CTX bound to GM1 in the plasma membrane diffused as fast as DOPE during the initial 0.5 s after its binding to the membrane, but slowed by a factor of 4 within 10 min after its binding. These results suggest that the slowed diffusion of GM1 found in previous observations would probably be due to the influences of CTX-induced GM1 crosslinking or of perturbation by Bodipy-FL on the alkyl chain. These results indicate that in the plasma membrane of non-stimulated cells, glycosphingolipids movements are not slowed by the possible presence of raft, further suggesting that the rafts in the steady-state cells are much smaller than the compartment size (110 nm) and/or short-lived (<<25 ms).

Both MHC Class II and its GPI-Anchored Form Undergo Hop Diffusion as Observed by Single-Molecule Tracking

Previously, investigations using single-fluorescent-molecule tracking at frame rates of up to 65Hz, showed that the transmembrane MHC class II protein and its GPI-anchored modified form expressed in CHO cells undergo simple Brownian diffusion, without any influence of actin depolymerization with cytochalasin D. These results are at apparent variance with the view that GPI-anchored proteins stay with cholesterol-enriched raft domains, as well as with the observation that both lipids and transmembrane proteins undergo short-term confined diffusion within a compartment and long-term hop diffusion between compartments. Here, this apparent discrepancy has been resolved by reexamining the same paradigm, by using both high-speed single-particle tracking (50kHz) and single fluorescent-molecule tracking (30Hz). Both molecules exhibited rapid hop diffusion between 40-nm compartments, with an average dwell time of 1–3ms in each compartment. Cytochalasin D hardly affected the hop diffusion, consistent with previous observations, whereas latrunculin A increased the compartment sizes with concomitant decreases of the hop rates, which led to an ∼50% increase in the median macroscopic diffusion coefficient. These results indicate that the actin-based membrane skeleton influences the diffusion of both transmembrane and GPI-anchored proteins.

Paradigm Shift of the Plasma Membrane Concept from the Two-Dimensional Continuum Fluid to the Partitioned Fluid: High-Speed Single-Molecule Tracking of Membrane Molecules

Recent advancements in single-molecule tracking methods with nanometer-level precision now allow researchers to observe the movement, recruitment, and activation of single molecules in the plasma membrane in living cells. In particular, on the basis of the observations by high-speed single-particle tracking at a frame rate of 40,000 frames s(1), the partitioning of the fluid plasma membrane into submicron compartments throughout the cell membrane and the hop diffusion of virtually all the molecules have been proposed. This could explain why the diffusion coefficients in the plasma membrane are considerably smaller than those in artificial membranes, and why the diffusion coefficient is reduced upon molecular complex formation (oligomerization-induced trapping). In this review, we first describe the high-speed single-molecule tracking methods, and then we critically review a new model of a partitioned fluid plasma membrane and the involvement of the actin-based membrane-skeleton "fences" and anchored-transmembrane protein "pickets" in the formation of compartment boundaries.