Focal adhesion membrane is dotted with protein islands and partitioned for molecular hop diffusion

Abstract

Using the ultrafast camera system and new theories for hop diffusion described in the companion paper, we for the first time demonstrated that membrane molecules undergo hop diffusion among the compartments in the bulk basal plasma membrane (PM), with virtually the same compartment sizes (108 nm) as those in the bulk apical PM and the same dwell lifetimes within a compartment (10 and 24 ms for the phospholipid and transferrin receptor [TfR], respectively), suggesting that the basic structures and molecular dynamics are very similar in the bulk regions of the apical and basal PMs. Ultrafast PALM and single-molecule imaging revealed that the focal adhesion (FA) is mostly a fluid membrane, partitioned into ~74-nm compartments where TfR and β3 integrin undergo hop diffusion, and that the FA membrane is sparsely dotted with 51-nm diameter paxillin islands, where many other FA proteins probably assemble (compartmentalized archipelago model). β3 integrin intermittently associates with the paxillin islands, dynamically linking them to the extracellular matrix.

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“…The entire bulk PM is compartmentalized (∼100 nm and in the range of 30-230 nm, depending on the cell line) by the actinbased membrane-skeleton "fences" and rows of TM-protein "pickets" anchored to and aligned along these fences (Figure 2Aa) (Fujiwara et al, 2002(Fujiwara et al, , 2016Murase et al, 2004;Trimble and Grinstein, 2015;Ostrowski et al, 2016;Freeman et al, 2018). In the compartmentalized PM, both TM proteins and lipids undergo short-term confined diffusion within a compartment plus occasional hop movements to an adjacent compartment, which is termed hop diffusion (Figure 2Aa) (Fujiwara et al, 2002(Fujiwara et al, , 2016(Fujiwara et al, , 2021aMurase et al, 2004;Kusumi et al, 2005Kusumi et al, , 2012aJaqaman and Grinstein, 2012;Xia et al, 2019). As a consequence, each molecule in the PM exhibits two diffusion coefficients: the microscopic diffusion coefficient (D micro : 3-10 µm 2 /s), describing the unhindered diffusion within a compartment, and the macroscopic diffusion coefficient (D MACRO : 0.1-1 µm 2 /s), determined by the compartment size and the hop frequency across intercompartmental boundaries composed of the picket fence.…”

Thiolation for Enhancing Photostability of Fluorophores at the Single‐Molecule Level

“…The relationship between the motion states of membrane proteins and their environment is critical [36] and can have a significant impact on biological processes. [37,38] In this study, we performed single-molecule tracking of CD56 in U2OS cells using a tsq2 conjugate with a CD56 antibody (Figure 5a-c, Supporting Information sections [11][12]. Characterizations of tsq2-CD56-antibody are shown in Figure S20-21.…”

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“…All single-molecule observations were performed at 37°C, using a microscope housed in a homemade environmental chamber. Fluorescently labelled molecules in cells were excited by total internal reflection (TIR) illumination (for those on the basal PM) or oblique-angle illumination (for those on the apical PM) using objective-lens-type TIRF microscopes, which were constructed on Each individual fluorescent spot in the image was identified and tracked by using an in-house computer program, as described previously 14,56,57,74 . The superimposition of images in different colours obtained by two separate cameras was conducted as described previously 75 .…”

“…Briefly, cells were initially fixed with 0.3% (v/v) glutaraldehyde in cytoskeleton buffer (150 mM NaCl, 5 mM EGTA, 5 mM glucose and 5 mM MgCl2, buffered with 10 mM MES at pH 6.1) containing 0.25% (v/v) Triton X-100 for 1 min, and then post-fixed with 2% (v/v) Sapphire 488-300 CW; 0.164 µW/µm 2 ). The PALM data acquisition was performed at 60 Hz for 14,560 frames with an image size of 512 x 512 pixels, using the high-speed camera system (Photron: mini AX200-II) described previously 56,57 . The final magnification was 250x (~80 nm/pixel; square pixels).…”