Mucins and TS in T. cruzi plasma membrane.

(A) Two-color super-resolution (STORM) image based on single molecule localizations of mucins (magenta) and TS (green) in a T. cruzi trypomastigote. (B) Two-color diffraction-limited fluorescence image of the same parasite. (C) Zoom-in of a membrane region showing the localizations of mucins and TS. Localizations classified as clustered using DBSCAN are represented with dots while non-clustered localizations are represented with crosses. The estimation of the effective diameter of the clusters (φC) is depicted. (D, E) Distributions of effective cluster diameters of mucins (D, φM) and TS (E, φT). The average effective diameters are indicated in brackets, while the medians are marked with dotted lines and represented as Mφ.

Characterization of the spatial distribution of mucins and TS nanodomains.

(A) Schematic description of the cluster position randomization: within each experimentally observed area, the clusters found with DBSCAN were relocated in random positions ensuring that they do not overlap to avoid cluster merging. (B, C) Histogram and Cumulative Distribution Function (CDF) of first neighbor distances between the mass centers of experimental and randomized clusters of mucins (B, dMM) and TS (C, dTT). The CDFs of 100 randomized distributions are displayed in grey, with their average highlighted in black. A zoom-in is shown in the insets to better appreciate the differences between the curves.

Separation distances and overlap between mucins and TS nanodomains.

(A) Shuffling of experimental data: given two circular areas of equal diameter of T. cruzi membrane, mucin clusters from one area are combined with TS clusters from the other (and vice-versa). First neighbor distances from the mass centers of mucins clusters to TS clusters (dMT) and vice versa (dTM) are determined. (B, C) Histogram and CDF of first neighbor cross-distances (B, dMT – C, dTM). For each case, the CDFs of 100 randomized distributions are displayed in grey, with their average highlighted in black. (D) Example of 2D Gaussian rendering of an experimental cluster dataset: mucins clusters in magenta, TS clusters in green, overlap areas in blue. (E). Histogram and CDF of overlap areas between mucins and TS clusters for experimental and shuffled data.

Analysis of the spatial distribution of non-clustered mucins and TS molecules.

(A,B) Histograms of the first neighbor distances of non-clustered proteins of the same kind (A, dMM and dTT) and between mucins and TS (B, dMT and dTM). Mean values are reported in brackets and the median is shown as a dashed line. (C) Schematic of the sampling of the membrane left available by the clusters using square areas. In each one of the 184 circular areas analyzed, the membrane area not occupied by clusters was sampled by taking non-overlapping, non-repetitive squares with a side of 146 nm, ensuring that they contained both mucins and TS localizations. (D) Histogram and CDF of experimental dMT (magenta) and dTM (green) compared to the distance distributions obtained from shuffling mucins and TS localizations from different square areas (in black). Kolmogorov–Smirnov test p-values are 2.5x10-15 for M-TS and 3.9x10-14 for TS-M.

Dimensional analysis of non-clustered mucins and TS molecules.

(A) Functional dependence of the complementary cumulative distribution function (CCDF) of the distance to the first neighbor (d) for various point distributions. The log[-log(CCDF)] as a function of the log(d) is a linear function whose slope n (dimensionality) takes extreme values of 1 for a strictly linear arrangement (1D) and 2 for a random 2D distribution. Intermediate organizations such as a fibrillar networks present values of 1 < n < 2. (B) log[-log(CCDF)] vs. the experimentally observed first neighbor distances of mucins (dMM) and TS (dTT). Dotted lines are linear fits retrieving a value of n = 1.36 in both cases. (C) Schematic of molecules distributed over a mesh of N rectilinear fibers of width (w). Average values of n obtained from simulations randomly distributing a density of molecules equal to the experimentally observed for mucins and TS over N (2, 4, or 6) rectilinear fibers of various thicknesses (10, 15 or 20 nm) inside an area of 146 nm x 146 nm. For each condition, 10,000 simulations were performed. Results for other combinations of N and thickness are presented in Supplementary Figures 2B and 2C. (D) Schematic representations illustrate two scenarios: mucins and TS arranged on separate fibrillar scaffolds versus both sharing a common scaffold. The plots show cumulative distribution functions (CDFs) of first-neighbor cross-distances derived from simulations. In these simulations, molecules were randomly distributed at densities matching experimental observations across a mesh of four rectilinear fibers (10 nm wide) within a 146 × 146 nm area. Magenta and green curves represent CDFs of distances from mucins to TS and TS to mucins, respectively, when both proteins share the same scaffold. The black curve corresponds to the scenario where mucins and TS occupy distinct fibrillar scaffolds.

Comparative analysis of the native structural organization of membrane protein domains.

Migration patterns between (A) TS (α-SAPA), (B) labeled mucins (α-FLAG) and (C) TolT-A (α-TolT-A) proteins were compared by Western blot (WB) under non-denaturing conditions (BN-PAGE, 3-12% gradient). T. brucei VSG glycoprotein (α-VSG) was included in each run as dimeric control (∼100 kDa). For each lane, proteins corresponding to 10 x 106 tissue-culture-derived T. cruzi trypomastigotes (WT, CL Brener strain) or 1 x 106 bloodstream T. brucei trypomastigotes (427 strain) were used. SDS-PAGE runs are included in each case for comparison under denaturing conditions (left panels in each case).