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.

Prod1 is a protein that regulates limb regeneration in salamanders by determining the direction of limb growth. Prod1 is attached to the membrane by a glycosylphosphatidylinositol (GPI) anchor, but the role of membrane anchoring in the limb regeneration process is poorly understood. In this study, we investigated the functional role of the anchoring of Prod1 to the membrane by using its synthetic mimics in combination with solid‐state NMR spectroscopy and fluorescent microscopy techniques. Anchoring did not affect the three‐dimensional structure of Prod1 but did induce aggregation by aligning the molecules and drastically reducing the molecular motion on the two‐dimensional membrane surface. Interestingly, aggregated Prod1 interacted with Prod1 molecules tethered on the surface of opposing membranes, inducing membrane adhesion. Our results strongly suggest that anchoring of the salamander‐specific protein Prod1 assists cell adhesion in the limb regeneration process.

Prod1 is aprotein that regulates limb regeneration in salamanders by determining the direction of limb growth. Prod1 is attached to the membrane by aglycosylphosphatidylinositol (GPI) anchor,but the role of membrane anchoring in the limb regeneration process is poorly understood. In this study,w ei nvestigated the functional role of the anchoring of Prod1 to the membrane by using its synthetic mimics in combination with solid-state NMR spectroscopya nd fluorescent microscopytechniques.Anchoring did not affect the threedimensional structure of Prod1 but did induce aggregation by aligning the molecules and drastically reducing the molecular motion on the two-dimensional membrane surface.I nterestingly,a ggregated Prod1 interacted with Prod1 molecules tethered on the surface of opposing membranes,i nducing membrane adhesion. Our results strongly suggest that anchoring of the salamander-specific protein Prod1 assists cell adhesion in the limb regeneration process.

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