Koichiro M. Hirosawa

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. Under higher physiological expression conditions , homodimers coalesce to form hetero- and homo-GPI-AP oligomer rafts through raft-based lipid interactions. When CD59 was ligated, it formed stable oligomer rafts containing up to four CD59 molecules, which triggered intracellular Ca(2+) responses that were dependent on GPI anchorage and cholesterol, suggesting a key part played by transient homodimer rafts. Transient homodimer rafts are most likely one of the basic units for the organization and function of raft domains containing GPI-APs.

Ultrafast Diffusion of a Fluorescent Cholesterol Analog in Compartmentalized Plasma Membranes

Cholesterol distribution and dynamics in the plasma membrane (PM) are poorly understood. The recent development of Bodipy488‐conjugated cholesterol molecule (Bdp‐Chol) allowed us to study cholesterol behavior in the PM, using single fluorescent‐molecule imaging. Surprisingly, in the intact PM, Bdp‐Chol diffused at the fastest rate ever found for any molecules in the PM, with a median diffusion coefficient (D) of 3.4 µm2/second, which was ∼10 times greater than that of non‐raft phospholipid molecules (0.33 µm2/second), despite Bdp‐Chols probable association with raft domains. Furthermore, Bdp‐Chol exhibited no sign of entrapment in time scales longer than 0.5 milliseconds. In the blebbed PM, where actin filaments were largely depleted, Bdp‐Chol and Cy3‐conjugated dioleoylphosphatidylethanolamine (Cy3‐DOPE) diffused at comparable Ds (medians = 5.8 and 6.2 µm2/second, respectively), indicating that the actin‐based membrane skeleton reduces the D of Bdp‐Chol only by a factor of ∼2 from that in the blebbed PM, whereas it reduces the D of Cy3‐DOPE by a factor of ∼20. These results are consistent with the previously proposed model, in which the PM is compartmentalized by the actin‐based membrane‐skeleton fence and its associated transmembrane picket proteins for the macroscopic diffusion of all of the membrane molecules, and suggest that the probability of Bdp‐Chol passing through the compartment boundaries, once it enters the boundary, is ∼10× greater than that of Cy3‐DOPE. Since the compartment sizes are greater than those of the putative raft domains, we conclude that raft domains coexist with membrane‐skeleton‐induced compartments and are contained within them.

Raft-based sphingomyelin interactions revealed by new fluorescent sphingomyelin analogs

Sphingomyelin (SM) has been proposed to form cholesterol-dependent raft domains and sphingolipid domains in the plasma membrane (PM). How SM contributes to the formation and function of these domains remains unknown, primarily because of the scarcity of suitable fluorescent SM analogs. We developed new fluorescent SM analogs by conjugating a hydrophilic fluorophore to the SM choline headgroup without eliminating its positive charge, via a hydrophilic nonaethylene glycol linker. The new analogs behaved similarly to the native SM in terms of their partitioning behaviors in artificial liquid order-disorder phase-separated membranes and detergent-resistant PM preparations. Single fluorescent molecule tracking in the live-cell PM revealed that they indirectly interact with each other in cholesterol- and sphingosine backbone–dependent manners, and that, for ∼10–50 ms, they undergo transient colocalization-codiffusion with a glycosylphosphatidylinositol (GPI)-anchored protein, CD59 (in monomers, transient-dimer rafts, and clusters), in CD59-oligomer size–, cholesterol-, and GPI anchoring–dependent manners. These results suggest that SM continually and rapidly exchanges between CD59-associated raft domains and the bulk PM.

Selective Labeling of Proteins on Living Cell Membranes Using Fluorescent Nanodiamond Probes

The impeccable photostability of fluorescent nanodiamonds (FNDs) is an ideal property for use in fluorescence imaging of proteins in living cells. However, such an application requires highly specific labeling of the target proteins with FNDs. Furthermore, the surface of unmodified FNDs tends to adsorb biomolecules nonspecifically, which hinders the reliable targeting of proteins with FNDs. Here, we combined hyperbranched polyglycerol modification of FNDs with the β-lactamase-tag system to develop a strategy for selective imaging of the protein of interest in cells. The combination of these techniques enabled site-specific labeling of Interleukin-18 receptor alpha chain, a membrane receptor, with FNDs, which eventually enabled tracking of the diffusion trajectory of FND-labeled proteins on the membrane surface.

Cortical actin nodes: Their dynamics and recruitment of podosomal proteins as revealed by super-resolution and single-molecule microscopy

Electron tomography of the plasma membrane (PM) identified several layers of cortical actin meshwork running parallel to the PM cytoplasmic surface throughout the PM. Here, cortical actin structures and dynamics were examined in living cells, using super-resolution microscopy, with (x,y)- and z-resolutions of ~140 and ~400 nm, respectively, and single-molecule imaging. The super-resolution microscopy identified sub-micron-sized actin clusters that appeared identical by both phalloidin post-fixation staining and Lifeact-mGFP expression followed by fixation, and therefore, these actin clusters were named “actin-pl-clusters”. In live cells, the actin-pl-clusters visualized by Lifeact-mGFP linked two or more actin filaments in the fine actin meshwork, acting as a node of the meshwork, and dynamically moved on/along the meshwork in a myosin II-dependent manner. Their formation depended on the Arp2/3 activities, suggesting that the movements could involve both the myosin motor activity and actin polymerization-depolymerization. The actin-pl-clusters differ from the actin nodes/asters found previously after latrunculin treatments, since myosin II and filamin A were not colocalized with the actin-pl-clusters, and the actin-pl-clusters were much smaller than the previously reported nodes/asters. The Lifeact linked to a fluorescently-labeled transmembrane peptide from syntaxin4 (Lifeact-TM) expressed in the PM exhibited temporary immobilization in the PM regions on which actin-pl-clusters and stress fibers were projected, showing that ≥66% of actin-pl-clusters and 89% of stress fibers were located in close proximity (within 3.5 nm) to the PM cytoplasmic surface. Podosome-associated cytoplasmic proteins, Tks4, Tks5, cortactin, and N-WASP, were transiently recruited to actin-pl-clusters, and thus, we propose that actin-pl-clusters also represent “actin podosome-like clusters”.