PubMed Health⌕ Search

Biomedical subjects

Takahiro Fujiwara

Publications and source records attributed to Takahiro Fujiwara.

15 recordsLinked to original sources
PubMed2025

Live-Cell Monitoring and Omics Analysis of Liquid-Solid Transitions of Biomolecular Condensates.

Biomolecular condensates, or so-called membraneless organelles, transition from liquid into more solid-like states over time, contributing to the development of pathological conditions. The present study proposes a simple method using photoactive yellow protein (PYP) and its specific fluorescent covalent ligands to distinguish between the liquid and solid states of protein condensates in live cells. The method, compatible with fluorescence-activated cell sorting (FACS), correlates the stiffness of specific protein condensates with their accessibility to PYP ligands, enabling quantitative multicolor monitoring of condensate solidification. We applied this technique to 12 phase-separating proteins and their mutants, finding that TDP-43, particularly its A315T mutant linked to familial amyotrophic lateral sclerosis, most readily forms solid aggregates. Furthermore, this FACS-compatible strategy enabled the isolation of distinct cell populations based on condensate states, allowing for subsequent proteomic and transcriptomic analyses. Our findings demonstrate that condensate solidification is accompanied by the upregulated expression of extracellular matrix proteins, suggesting a previously unrecognized link between solid aggregate formation and extracellular matrix hardening.

Humans↗
PubMed2006

Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography.

Three-dimensional images of the undercoat structure on the cytoplasmic surface of the upper cell membrane of normal rat kidney fibroblast (NRK) cells and fetal rat skin keratinocytes were reconstructed by electron tomography, with 0.85-nm-thick consecutive sections made approximately 100 nm from the cytoplasmic surface using rapidly frozen, deeply etched, platinum-replicated plasma membranes. The membrane skeleton (MSK) primarily consists of actin filaments and associated proteins. The MSK covers the entire cytoplasmic surface and is closely linked to clathrin-coated pits and caveolae. The actin filaments that are closely apposed to the cytoplasmic surface of the plasma membrane (within 10.2 nm) are likely to form the boundaries of the membrane compartments responsible for the temporary confinement of membrane molecules, thus partitioning the plasma membrane with regard to their lateral diffusion. The distribution of the MSK mesh size as determined by electron tomography and that of the compartment size as determined from high speed single-particle tracking of phospholipid diffusion agree well in both cell types, supporting the MSK fence and MSK-anchored protein picket models.

Actin Cytoskeleton↗
PubMed2006

Impact of convective flow on the cellular uptake and transfection activity of lipoplex and adenovirus.

An in vitro cell culture model that mimics in vivo extracellular environment would be useful in developing in vivo gene delivery system. In the present study, a parallel flow model was applied to investigate the impact of convective flow on cellular uptake and transfection activity in endothelial cells. LipofectAMINE PLUS and adenovirus were used as model vectors, which bind cells via electrostatic- and ligand-receptor interactions, respectively. Whereas a convective flow increased the total amount of vector passing through the flow chamber by 3 orders of magnitude, uptake was increased by less than 10-fold, suggesting that the flow severely inhibited cellular uptake by reducing the retention time in the chamber and/or by diminishing the affinity between the cell and vector. Moreover, the uptake of both vectors was increased in a shear stress-dependent manner to a comparable extent, suggesting that the effect of flow on the cellular uptake was not significant. In contrast, transfection efficiency (TE), expressed as the transfection activity normalized by the cellular uptake of vectors was dramatically stimulated by shear stress, only when LipofectAMINE PLUS was used. Since the activities of the CMV promoter were unaffected by a shear stress, it is possible that altered intracellular trafficking may responsible for the improvement in lipoplex-mediated TE, presumably related to the cellular uptake pathway.

Adenoviridae↗
PubMed2005

Rapid hop diffusion of a G-protein-coupled receptor in the plasma membrane as revealed by single-molecule techniques.

Diffusion of a G-protein coupled receptor, mu-opioid receptor (muOR), in the plasma membrane was tracked by single-fluorescent molecule video imaging and high-speed single-particle tracking. At variance with a previous publication, where gold-tagged muOR was found to be totally confined within a domain, which in turn underwent very slow diffusion itself, we found that muOR undergoes rapid hop diffusion over membrane compartments (210-nm and 730-nm nested double compartments in the case of normal rat kidney cell line), which are likely delimited by the actin-based membrane-skeleton "fence or corrals" and its associated transmembrane protein "pickets", at a rate comparable to that for transferrin receptor (every 45 and 760 ms on average, respectively), suggesting that the fence and picket models may also be applicable to G-protein coupled receptors. Further, we found that strong confinement of gold-labeled muOR could be induced by the prolonged on-ice preincubation of the gold probe with the cells, showing that this procedure should be avoided in future single-particle tracking experiments. Based on the dense, long trajectories of muOR obtained by high-speed single-particle tracking, the membrane compartments apposed and adjoined to each other could be defined that are delimited by rather straight boundaries, consistent with the involvement of actin filaments in membrane compartmentalization.

Actins↗
PubMed2005

Single-molecule tracking of membrane molecules: plasma membrane compartmentalization and dynamic assembly of raft-philic signaling molecules.

Tracking single molecules in the plasma membrane in live cells is becoming a useful technique for studying the spatial-temporal control of membrane molecular processes, such as signal transduction and the formation of large molecular complexes. In this review, three topics largely based on recent single-molecule observations are described, with a special emphasis on the results that are considered to be difficult to obtain using conventional methods monitoring the ensemble-averaged behavior of molecules. First, we describe the high-speed single-molecule tracking data, mostly obtained by our group that necessitated the paradigm shift of the plasma membrane structure, from the two-dimensional continuum fluid model to the compartmentalized fluid model. Second, we try to present a synthetic view of the cell membrane, which contains raft and other microdomains as well as being partitioned into small compartments. Furthermore, we present our working hypothesis, based on the literature, how large, stabilized rafts may be formed, after ligation or crosslinking, from small/unstable "reserve" rafts present in the steady-state cells. Finally, we explain our initial application of single-molecule fluorescence imaging for studies of the creation of T-cell receptor signaling complexes (immunological synapses or SMACS), by observing the recruitment of single Lck molecules as an initial approach. This revealed that the assembly of Lck at the T-cell receptor cluster site, observed by conventional fluorescence microscopy, actually represents dynamic concentrations of Lck molecules, entering and exiting the cluster domain rapidly, with the aid of the raft domains.

Animals↗
PubMed2005

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.

Actins↗
PubMed2004

Detection of non-Brownian diffusion in the cell membrane in single molecule tracking.

Molecules undergo non-Brownian diffusion in the plasma membrane, but the mechanism behind this anomalous diffusion is controversial. To characterize the anomalous diffusion in the complex system of the plasma membrane and to understand its underlying mechanism, single-molecule/particle methods that allow researchers to avoid ensemble averaging have turned out to be highly effective. However, the intrinsic problems of time-averaging (resolution) and the frequency of the observations have not been explored. These would not matter for the observations of simple Brownian particles, but they do strongly affect the observation of molecules undergoing anomalous diffusion. We examined these effects on the apparent motion of molecules undergoing simple, totally confined, or hop diffusion, using Monte Carlo simulations of particles undergoing short-term confined diffusion within a compartment and long-term hop diffusion between these compartments, explicitly including the effects of time-averaging during a single frame of the camera (exposure time) and the frequency of observations (frame rate). The intricate relationships of these time-related experimental parameters with the intrinsic diffusion parameters have been clarified, which indicated that by systematically varying the frame time and rate, the anomalous diffusion can be clearly detected and characterized. Based on these results, single-particle tracking of transferrin receptor in the plasma membrane of live PtK2 cells were carried out, varying the frame time between 0.025 and 33 ms (0.03-40 kHz), which revealed the hop diffusion of the receptor between 47-nm (average) compartments with an average residency time of 1.7 ms, with the aid of single fluorescent-molecule video imaging.

Animals↗
PubMed2004

Fluorescence imaging for monitoring the colocalization of two single molecules in living cells.

The interaction, binding, and colocalization of two or more molecules in living cells are essential aspects of many biological molecular processes, and single-molecule technologies for investigating these processes in live cells, if successfully developed, would become very powerful tools. Here, we developed simultaneous, dual-color, single fluorescent molecule colocalization imaging, to quantitatively detect the colocalization of two species of individual molecules. We first established a method for spatially correcting the two full images synchronously obtained in two different colors, and then for overlaying them with an accuracy of 13 nm. By further assessing the precision of the position determination, and the signal/noise and signal/background ratios, we found that two single molecules in dual color can be colocalized to within 64-100 nm (68-90% detectability) in the membrane of cells for GFP and Alexa633. The detectability of true colocalization at the molecular level and the erroneous inclusion of incidental approaches of two molecules as colocalization have to be compromised at different levels in each experiment, depending on its purpose. This technique was successfully demonstrated in living cells in culture, monitoring colocalization of single molecules of E-cadherin fused with GFP diffusing in the plasma membrane with single molecules of Alexa633 conjugated to anti-E-cadherin Fab externally added to the culture medium. This work established a benchmark for monitoring the colocalization of two single molecules, which can be applied to wide ranges of studies for molecular interactions, both at the levels of single molecules and collections of molecules.

Fluorescent Dyes↗
PubMed2004

Single-molecule imaging analysis of Ras activation in living cells.

A single-molecule fluorescence resonance energy transfer (FRET) method has been developed to observe the activation of the small G protein Ras at the level of individual molecules. KB cells expressing H- or K-Ras fused with YFP (donor) were microinjected with the fluorescent GTP analogue BodipyTR-GTP (acceptor), and the epidermal growth factor-induced binding of BodipyTR-GTP to YFP-(H or K)-Ras was monitored by single-molecule FRET. On activation, Ras diffusion was greatly suppressed/immobilized, suggesting the formation of large, activated Ras-signaling complexes. These complexes may work as platforms for transducing the Ras signal to effector molecules, further suggesting that Ras signal transduction requires more than simple collisions with effector molecules. GAP334-GFP recruited to the membrane was also stationary, suggesting its binding to the signaling complex. The single-molecules FRET method developed here provides a powerful technique to study the signal-transduction mechanisms of various G proteins.

Boron Compounds↗
PubMed2004

Ultrafine membrane compartments for molecular diffusion as revealed by single molecule techniques.

Plasma membrane compartments, delimited by transmembrane proteins anchored to the membrane skeleton (anchored-protein picket model), would provide the membrane with fundamental mosaicism because they would affect the movement of practically all molecules incorporated in the cell membrane. Understanding such basic compartmentalized structures of the cell membrane is critical for further studies of a variety of membrane functions. Here, using both high temporal-resolution single particle tracking and single fluorescent molecule video imaging of an unsaturated phospholipid, DOPE, we found that plasma membrane compartments generally exist in various cell types, including CHO, HEPA-OVA, PtK2, FRSK, HEK293, HeLa, T24 (ECV304), and NRK cells. The compartment size varies from 30 to 230 nm, whereas the average hop rate of DOPE crossing the boundaries between two adjacent compartments ranges between 1 and 17 ms. The probability of passing a compartment barrier when DOPE is already at the boundary is also cell-type dependent, with an overall variation by a factor of approximately 7. These results strongly indicate the necessity for the paradigm shift of the concept on the plasma membrane: from the two-dimensional fluid continuum model to the compartmentalized membrane model in which its constituent molecules undergo hop diffusion over the compartments.

Animals↗
PubMed2003

Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization.

The formation and maintenance of polarized distributions of membrane proteins in the cell membrane are key to the function of polarized cells. In polarized neurons, various membrane proteins are localized to the somatodendritic domain or the axon. Neurons control polarized delivery of membrane proteins to each domain, and in addition, they must also block diffusional mixing of proteins between these domains. However, the presence of a diffusion barrier in the cell membrane of the axonal initial segment (IS), which separates these two domains, has been controversial: it is difficult to conceive barrier mechanisms by which an even diffusion of phospholipids could be blocked. Here, by observing the dynamics of individual phospholipid molecules in the plasma membrane of developing hippocampal neurons in culture, we found that their diffusion was blocked in the IS membrane. We also found that the diffusion barrier is formed in neurons 7-10 days after birth through the accumulation of various transmembrane proteins that are anchored to the dense actin-based membrane skeleton meshes being formed under the IS membrane. We conclude that various membrane proteins anchored to the dense membrane skeleton function as rows of pickets, which even stop the overall diffusion of phospholipids, and may represent a universal mechanism for formation of diffusion barriers in the cell membrane.

Actin Cytoskeleton↗
PubMed2002

Phospholipids undergo hop diffusion in compartmentalized cell membrane.

The diffusion rate of lipids in the cell membrane is reduced by a factor of 5-100 from that in artificial bilayers. This slowing mechanism has puzzled cell biologists for the last 25 yr. Here we address this issue by studying the movement of unsaturated phospholipids in rat kidney fibroblasts at the single molecule level at the temporal resolution of 25 micros. The cell membrane was found to be compartmentalized: phospholipids are confined within 230-nm-diameter (phi) compartments for 11 ms on average before hopping to adjacent compartments. These 230-nm compartments exist within greater 750-nm-phi compartments where these phospholipids are confined for 0.33 s on average. The diffusion rate within 230-nm compartments is 5.4 microm2/s, which is nearly as fast as that in large unilamellar vesicles, indicating that the diffusion in the cell membrane is reduced not because diffusion per se is slow, but because the cell membrane is compartmentalized with regard to lateral diffusion of phospholipids. Such compartmentalization depends on the actin-based membrane skeleton, but not on the extracellular matrix, extracellular domains of membrane proteins, or cholesterol-enriched rafts. We propose that various transmembrane proteins anchored to the actin-based membrane skeleton meshwork act as rows of pickets that temporarily confine phospholipids.

Actins↗
PubMed2002

Relationship of lipid rafts to transient confinement zones detected by single particle tracking.

We examined the physical and chemical characteristics of transient confinement zones (TCZs) that are detected in single particle trajectories of molecules moving within the membrane of C3H 10T1/2 murine fibroblasts and their relationship to "rafts." We studied the lateral movement of different membrane molecules thought to partition to varying degrees into or out of the putative lipid domains known as rafts. We found that lipid analogs spend significantly less time in TCZs compared with Thy-1, a glycosylphosphatidylinositol-anchored protein, and GM1, a glycosphingolipid. For Thy-1, we found that zone abundance was markedly reduced by cholesterol extraction, suggesting that a major source of the observed temporary confinement is related to the presence of raft domains. More detailed analysis of particle trajectories reveals that zones can be revisited even tens of seconds after the original escape and that diffusion within the zones is reduced by a factor of approximately 2, consistent with the zone being a cholesterol-rich liquid-ordered phase. Surprisingly, transient confinement was not strongly temperature dependent. Overall, our data demonstrate that there are raft-related domains present in certain regions of the plasma membrane of C3H cells, which can persist for tens of seconds.

Animals↗
PubMedDate not supplied

The fence and picket structure of the plasma membrane of live cells as revealed by single molecule techniques (Review).

Models of the organization of the plasma membrane of live cells as discovered through diffusion measurements of integral membrane molecules (transmembrane and GPI-anchored proteins, and lipid) at the single molecule level are discussed. Diffusion of transmembrane protein and, indeed, even lipid is anomalous in that the molecules tend to diffuse freely in limited size compartments, with infrequent intercompartment transitions. This average residency time in a compartment is dependent on the diffusing species and on its state of oligomerization, becoming completely confined to a single compartment upon sufficient oligomerization. This will be of great importance in determining cellular mechanisms for controlling the random diffusive motion of membrane molecules and in understanding signalling processes.

Animals↗