Fig 2: The switchable impact of MPP1 on the molecular mobility and confinement of SM in raft nanodomains. svFCS measurements and representative diffusion laws for (a) fluorescently labeled sphingomyelin (SM-BODIPY) and (b) phosphatidylcholine (PC-BODIPY) in HEL cells.

Fig 3: MPP1 forms both small nano- and large micrometer size complexes with flotillins in the PM of erythroid cells (a) Representative SIM^2 images showing organization of endogenous flotillin 2 (Flot2) in highly polarized preassembled caps (polarized cells) or small flotillin nanoclusters uniformly distributed along the PM (non-polarized cells) of control HEL cells (left). The blank rectangle corresponds to the zoomed region (right) of the selected area wheres the estimated sizes of flotillin complexes were indicated (also see Figure S1d). (b) Representative SIM^2 images showing co-localization of Flot2 and MPP1 in control HEL cells. Two types of MPP1-flotillin complexes are found, large polarized-micrometer size (in flotillin 2-caps) and small uniformly distributed nanometer-size clusters (in non-polarized cells). Size estimation is shown and described in Figure S3. Scale bar, 1 µm. Images represent the central z-stack.

Fig 4: MPP1 affects the dynamics and confinement of Thy-1 in raft nanodomains. The diffusion law of eGFP-Thy-1 transfected control and MPP1 KnD cells.

Fig 5: Model of the MPP1-flotillin dependent organization of raft nanodomains. MPP1 acts as a raft-sequestering molecule and organizes raft nanodomains due to mutual interactions with flotillins. The high-affinity binding of MPP1 to the pre-existing flotillin nanoclusters enhances their oligomerization and limits their lateral mobility in the PM, although the exact stoichiometry is not known. Such temporal immobilization triggers changes in the surrounding environment resulting in locally elicited concentration and confinement of other raft-associated molecules such as sphingomyelin and Thy-1. This rearmament drives the physicochemical changes in the PM, resembling the organization of more ordered domains reflecting functional raft nanodomains (for more details, see the text). MPP1-flotillin complexes act therefore as basic structural units organizing functional raft nanodomains.