Dynamic actin-mediated nano-scale clustering of CD44 regulates its meso-scale organization at the plasma membrane

Abstract

Transmembrane adhesion receptors at the cell surface, such as CD44, are often equipped with modules to interact with the extracellular matrix (ECM) and the intracellular cytoskeletal machinery. CD44 has been recently shown to compartmentalize the membrane into domains by acting as membrane pickets, facilitating the function of signaling receptors. While spatial organization and diffusion studies of membrane proteins are usually conducted separately, here we combine observations of organization and diffusion by using high spatio-temporal resolution imaging on living cells to reveal a hierarchical organization of CD44. CD44 is present in a meso-scale meshwork pattern where it exhibits enhanced confinement and is enriched in nanoclusters of CD44 along its boundaries. This nanoclustering is orchestrated by the underlying cortical actin dynamics. Interaction with actin is mediated by specific segments of the intracellular domain. This influences the organization of the protein at the nano-scale, generating a selective requirement for formin over Arp2/3-based actin-nucleation machinery. The extracellular domain and its interaction with elements of ECM do not influence the meso-scale organization, but may serve to reposition the meshwork with respect to the ECM. Taken together, our results capture the hierarchical nature of CD44 organization at the cell surface, with active cytoskeleton-templated nanoclusters localized to a meso-scale meshwork pattern.

FIGURE 1:

CD44 exhibits a nonrandom distribution at the plasma membrane at multiple spatiotemporal scales. (a) Schematic of a standard isoform of CD44 showing key domains of the protein, namely, the ECD, the Tm, and the ICD. (b) Schematic of SNAP-CD44-GFP and representative dynamic cartography of CD44 obtained at sublabeling conditions (∼30 nM). Each dot corresponds to the (x, y) coordinates (with subpixel accuracy) of individual receptors as they diffuse on the cell membrane. The (x, y) coordinates over 50 sequential frames are collapsed and overlaid into a single fluorescence frame. (c) Cartography of SNAP-CD44-GFP obtained at higher labeling conditions (∼50–100 nM); (x, y) coordinates from 1000 frames (1,354,066 localizations) collapsed in a single map with a zoomed-in ROI (c´). (d) Simulated cartography with similar number of localizations as in c, distributed in a random manner, and enlarged ROI. (e) Cartography construction of (x, y) coordinates in the marked ROI in c from 50 consecutive frames obtained at two different experimental time windows, between 30–35 s (magenta, e) and 90–95 s (green, e´) and merged image (right, e´´). Blue arrowheads highlight regions of confinement, and white dots represent persistent confinement regions or sites revisited by the receptor. (f) Cartography obtained from 400 frames (∼6.7 s) of DC-SPT data obtained by colabeling CD44 with JF549-cpSNAP ligand and JF646 SNAP ligand. Green and red dots correspond to localizations with the two different dyes indicating interparticle distances between 200 and 500 nm and black dots correspond to interparticle distances <200 nm. Zoomed-in ROIs depict the indicated reconstructed trajectories; f´ shows the subset corresponding to localization of particles with 0–200 nm interparticle distance (black in f) where the interparticle distance corresponds to <100 nm (blue circles) and 100–200 nm (gray circles). Note the <100 nm colocalization events always correspond to regions where localizations at the larger length scale of 100–200 and 200–500 nm interparticle distance are also found (indicated by purple arrowheads in f and f´). (g) Frequency distribution of step sizes of particles from trajectories wherein particles exhibit colocalization (red), temporary arrest (green), mobile (blue), and from the full trajectories (black) (27,856 trajectories). (h) Frequency distribution of duration of colocalization of particles identified in f. Note the lifetime of colocalization is in the range of <100 ms. (i) The 2D plot of all colocalized particles (<200 nm) obtained from the trajectories identified in f, where purple arrowheads indicate colocalization events that occur repeatedly at the same spot over the time period of observation. Color LUT bar indicates observation time from 0 to 6.7 s.

FIGURE 2:

Meso-scale meshwork of CD44 colocalizes with regions enriched in CD44 nanoclusters. (a) Total GFP fluorescence intensity and anisotropy map of the SNAP-CD44-GFP protein expressed in COS-7 cells that exhibit low levels of surface CD44. Note that the anisotropy image shows regions of low anisotropy (blue) and high anisotropy (red), corresponding respectively, to regions enriched in or depleted of CD44 molecules in nano-scale proximity (CD44 nanoclusters). (b) Schematic depicting the methodology by which FRET based anisotropy maps was correlated to localization maps obtained from high-density single molecule imaging and cartography analysis. (c, c´, c´´) Representative ROI image depicting the anisotropy map overlaid with localizations from raw cartography images integrated over 40 frames (left), random localizations obtained from simulations (center), and detected localization hotspots (red dots) of SNAP-CD44-GFP (right). (d) Histogram of the anisotropy values for the ROI shown in c. Red vertical lines indicate the thresholds chosen to classify regions of low anisotropy (Low A), medium anisotropy (Medium A), and high anisotropy (High A), where medium anisotropy is binned around the median value of anisotropy in a given ROI. (e) Fraction of detected localizations in the localization hotpots in low, medium, and high anisotropy regions compared with simulated localizations. Each symbol in the plot corresponds to a single ROI, and the data are obtained from at least six different cells from > 15 ROIs. Difference between distributions has been tested using Kolmogorov–Smirnov test.

FIGURE 3:

ECD and ICD independently affect CD44 nanoclustering. Schematics (a,c, and e) depict CD44-GFP constructs expressed in CHO cells used to generate the corresponding intensity and anisotropy images in b, d, and f. Anisotropy vs. intensity plots show a significant increase in anisotropy in the truncated protein lacking the ECD (a, b; p < 10^-43), ICD (c, d; p < 10^-58), or when the construct lacking the ECD (data from the same experiment as a and b) is compared with one lacking both ECD and the ICD (e, f; p < 10^-77). All raw distributions are statistically significant by Mann–Whitney test for each condition. (The data are from one representative experiment. [b] CD44-GFP = 20 fields, CD44TmICD-GFP = 27 fields. [d] CD44-GFP = 25 fields, CD44ECDTm-GFP = 13 fields. [f] CD44Tm-GFP = 15 fields.)

FIGURE 4:

Extent of CD44 nanoclustering correlates with the strength of tethering on the cell membrane. (a) Schematic show SNAP-tagged constructs expressed in MEFs, utilized for SPT. (b) Representative trajectories for the indicated constructs show distinct diffusion characteristics of the different constructs. (c–e) Quantification of the (c) mobile fraction by escape probability method, (d) confinement radius (r trap), (e) and diffusion coefficients of the full length and the truncated mutants. The data are derived from at least six cells for each construct. Number of trajectories: SNAP-CD44-GFP = 2977; SNAP-CD44TmICD-GFP = 2783; SNAP-CD44Tm-GFP = 4744.

FIGURE 5:

Meso-scale organization of CD44 is determined primarily by interactions of the ICD. (a) Representative cartography maps of the indicated CD44 constructs expressed in MEFs obtained from imaging at 10 fps and accumulating the spatial coordinates of individual molecules over 2 s (20 frames). (b) Quantification of the confinement areas for the different constructs during 2 s. Black lines correspond to the mean value. (c) Relative fractions of confinement areas for the different constructs, classified as a function of the confinement length, i.e., d < 170 nm, 170 < d < 230 nm, or d > = 230 nm. (d) Fraction of localization events that belong to the meshwork for the different constructs and compared with the fraction of similar type of localizations measured from randomized localizations. The data are from one representative experiment. The experiment has been conducted at least twice with similar results. Data were obtained from a number of cells expressing SNAP-CD44-GFP (8), SNAP-CD44TmICD-GFP (11), or SNAP-CD44Tm-GFP (9). Difference between distributions was tested for significance using Kruskal–Wallis and post hoc test with Tukey–Kramer. (b) SNAP-CD44-GFP and SNAP-CD44TmICD-GFP: p = 0.258 → ns; SNAP-CD44-GFP and SNAP-CD44Tm-GFP: p < e^-9; SNAP-CD44TmICD-GFP and SNAP-CD44Tm-GFP: p < e^-9. (d) SNAP-CD44-GFP and SNAP-CD44TmICD-GFP: p = 0.8564 → ns; SNAP-CD44-GFP and SNAP-CD44Tm-GFP: p < 0.005; SNAP-CD44TmICD-GFP and SNAP-CD44Tm-GFP: p = 0.0218. SNAP-CD44Tm-GFP (n) = 9 cells, SNAP-CD44TmICD-GFP (n) = 11 cells, SNAP-CD44-GFP (n) = 8 cells.

FIGURE 6:

CD44 nanoclustering is regulated by the underlying actomyosin machinery. Total intensity and anisotropy images of cells expressing CD44-GFP (a–c) expressed in CHO cells, either untreated or treated with actin polymerization stabilizer, Jas (a, Jas; 14 µM, 15 min; Con [n] = 10 fields, Treatment [n] = 22 fields), Myosin inhibition cocktail (b, MLY 20 µM; 60 min; Con [n] = 20 fields, Treatment [n] = 26 fields), Ezrin inhibitor (c, 25 µM; 60 min, Con [n] = 16 fields, Treatment [n] = 11 fields). Graphs show anisotropy values plotted against intensity collected from regions from the cells as detailed in experimental methods. In all conditions treatment with the indicated inhibitors show a significant difference in the recorded values of anisotropy (p < 10^-5), Difference between distributions has been tested for significance by Mann–Whitney tests. The data are from one representative experiment. Each experiment was conducted at least twice with similar results. (d) Schematic of CD44 and different deletion mutants for ezrin, ankyrin, and last 15 amino acids of the tail with the names of the constructs indicated next to its diagram. (e) Plot shows intensity vs. anisotropy distributions of the CD44 mutants in MCF-7 cells that exhibit low surface levels of CD44. (Distribution of anisotropy values were tested for significance using Mann–Whitney test and p < 10^-120 was obtained for CD44-GFP and CD44ECDTm-GFP; CD44-GFP [n] = 19 fields, CD44-ECDTm-GFP [n] = 15 fields, CD44-∆15GFP [n] = 16 fields, CD44-∆ERM-GFP [n] = 13 fields, CD44-∆EA-GFP [n] = 16 fields, CD44-∆Ank-GFP [n] = 17 fields.)

FIGURE 7:

Formin-mediated actin polymerization affect nano- as well as meso-scale distribution and turnover of CD44. (a, b) Total intensity and anisotropy images of cells expressing CD44-GFP expressed in CHO cells treated with formin inhibitor (SMIFH2 for 30 min; Con [n] = 19 fields, Treatment [n] = 13 fields, p < 10^-5). (c) Plot describing fraction of localizations detected on the meshwork in control cells compared with formin inhibited condition (p < e^-8). (d) Plot depicting time evolution of meso-scale domains on vehicle (DMSO) vs. formin inhibitor treatment. The x-axis depicts time as 2 s sliding window (depicted as frame number) and the y-axis depicts confinement area. (e) Plot depicting confinement area of the mesoscale domains in formin-perturbed cells compared with untreated ones do not exhibit detectable differences. (DMSO [n] = 12 cells, SMIFH2 [n] = 9 cells).

FIGURE 8:

Proposed model for plasma membrane organization of CD44. In the cell membrane an ROI is outlined to show the distribution of monomers as well as clusters of CD44 receptors. Nanoclustered receptors are shown coupled to actin cytoskeletal elements by adaptors such as ezrin/ankyrin (see zoomed-in nanocluster) interspersed with unattached CD44 molecules. The clusters of receptors are depicted as being driven by the action of formin polymerized actin filaments and myosin driven actin motility (molecules not depicted in the schematic). The meso-scale domains are CD44 localization hotspots identified in our experiment that are characterized by their close association with nanoclusters of the protein. The emerging meso-scale meshwork of the cell membrane receptor (depicted by the orange dotted line) may reflect the cytoskeletal meshwork juxtaposed to the plasma membrane.