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Ken Ritchie

Publications and source records attributed to Ken Ritchie.

5 recordsLinked to original sources
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↗