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Ken Ritchie

Publications and source records attributed to Ken Ritchie.

10 recordsLinked to original sources
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

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

Role of the membrane skeleton in creation of microdomains.

The membrane skeleton is a specialized part of the cytoskeleton that is in close proximity to the cell membrane with a protein composition and structure that differs from that of the bulk cytoskeleton. The membrane skeleton and various transmembrane proteins bound to it form a mosaic of compartments in the membrane that is responsible for the temporary confinement of membrane proteins and lipids and controls the rate of their repetitive hop movements between these membrane skeleton-based compartments, known as "hop diffusion", found by observation of single-molecule diffusion. Such hop diffusion has been found to be universal with compartment sizes that range from 30 to 700 nm, depending on the cell type. The part of the membrane skeleton that is directly involved in temporal confinement of membrane molecules has been successfully imaged by raster scanning a single membrane molecule using an optical trap (single molecule scanning optical force imaging). Such compartmentalization enables dynamic spatial regulation of signal transduction in the plasma membrane, by arresting signaling complexes of activated receptor molecules and enlarged, stabilized rafts within a compartment. Furthermore, high concentrations of the membrane skeleton and its associated immobile transmembrane proteins are involved in formation of the cell membrane polarity such as is found across the initial segment between the axon and the cell body in neurons. Argument is advanced that the creation of various membrane domains in the cell membrane must be influenced by the membrane skeleton.

Animals↗
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

Single-particle tracking image microscopy.

The techniques of single particle tracking (SPT) and optical force microscopy (OFM) as described above allow direct imaging of the motion of molecules in the membrane of live cells, and provide a means of controlling the movement by an almost noninvasive method. Combination of these techniques with other single-molecule methods, such as single-fluorophore imaging, allows direct comparison of motion at video rate (because faster than video rate imaging of fluorophore is still not generally feasible) to determine any effect due to the attached colloidal gold particle. Also, simultaneous use of the two techniques allows for monitoring two molecules, one at high time resolution. As such, the system can then be used in conjunction with green fluorescent protein (GFP) transfection to watch simultaneously the motion of an internal component of, say, a signaling pathway while seeing the motion of the transmembrane signaling receptor.

Microscopy↗
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↗
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↗