Ultrafast single-molecule imaging reveals focal adhesion nano-architecture and molecular dynamics.

Using our newly developed ultrafast camera described in the companion paper, we reduced the data acquisition periods required for photoactivation/photoconversion localization microscopy (PALM, using mEos3.2) and direct stochastic reconstruction microscopy (dSTORM, using HMSiR) by a factor of ≈30 compared with standard methods, for much greater view-fields, with localization precisions of 29 and 19 nm, respectively, thus opening up previously inaccessible spatiotemporal scales to cell biology research. Simultaneous two-color PALM-dSTORM and PALM-ultrafast (10 kHz) single fluorescent-molecule imaging-tracking has been realized.

AMPA receptors in the synapse turnover by monomer diffusion.

The number and subunit compositions of AMPA receptors (AMPARs), hetero- or homotetramers composed of four subunits GluA1-4, in the synapse is carefully tuned to sustain basic synaptic activity. This enables stimulation-induced synaptic plasticity, which is central to learning and memory. The AMPAR tetramers have been widely believed to be stable from their formation in the endoplasmic reticulum until their proteolytic decomposition.

Native prion protein homodimers are destabilized by oligomeric amyloid β 1-42 species as shown by single-molecule imaging.

Prion proteins (PrPc) are receptors for amyloid β 1-42 (Aβ1-42) oligomers, but we do not know the impact of Aβ1-42 binding to PrPc on the interaction of membrane-bound PrPc with molecules that regulate downstream biological pathways. Stability of the PrPc dimeric complex and subsequent intermolecular interactions with membranous or cytoplasmic molecules are important for physiological functions of PrPc including neuroprotection. The principal aim of this study was to determine whether homodimer lifetime of PrPc is affected by the presence of Aβ1-42 oligomers.

Dynamic Meso-Scale Anchorage of GPI-Anchored Receptors in the Plasma Membrane: Prion Protein vs. Thy1.

The central mechanism for the transmission of the prion protein misfolding is the structural conversion of the normal cellular prion protein to the pathogenic misfolded prion protein, by the interaction with misfolded prion protein. This process might be enhanced due to the homo-dimerization/oligomerization of normal prion protein. However, the behaviors of normal prion protein in the plasma membrane have remained largely unknown.

Chemical labelling for visualizing native AMPA receptors in live neurons.

The location and number of neurotransmitter receptors are dynamically regulated at postsynaptic sites. However, currently available methods for visualizing receptor trafficking require the introduction of genetically engineered receptors into neurons, which can disrupt the normal functioning and processing of the original receptor. Here we report a powerful method for visualizing native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) which are essential for cognitive functions without any genetic manipulation.

Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane.

The mechanisms by which the diffusion rate in the plasma membrane (PM) is regulated remain unresolved, despite their importance in spatially regulating the reaction rates in the PM. Proposed models include entrapment in nanoscale noncontiguous domains found in PtK2 cells, slow diffusion due to crowding, and actin-induced compartmentalization. Here, by applying single-particle tracking at high time resolutions, mainly to the PtK2-cell PM, we found confined diffusion plus hop movements (termed "hop diffusion") for both a nonraft phospholipid and a transmembrane protein, transferrin receptor, and equal compartment sizes for these two molecules in all five of the cell lines used here (actual sizes were cell dependent), even after treatment with actin-modulating drugs.

Transient GPI-anchored protein homodimers are units for raft organization and function.

Advanced single-molecule fluorescent imaging was applied to study the dynamic organization of raft-associated glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the plasma membrane and their stimulation-induced changes. In resting cells, virtually all of the GPI-APs are mobile and continually form transient (~200 ms) homodimers (termed homodimer rafts) through ectodomain protein interactions, stabilized by the presence of the GPI-anchoring chain and cholesterol. Heterodimers do not form, suggesting a fundamental role for the specific ectodomain protein interaction.

Archipelago architecture of the focal adhesion: membrane molecules freely enter and exit from the focal adhesion zone.

The focal adhesion (FA) is an integrin-based structure built in/on the plasma membrane, mechanically linking the extracellular matrix with the termini of actin stress fibers, providing key scaffolds for the cells to migrate in tissues. The FA was considered as a micron-scale, massive assembly of various proteins, although its formation and decomposition occur quickly in several to several 10 s of minutes. The mechanism of rapid FA regulation has been a major mystery in cell biology.