Fig 5 - uploaded by Elias M Puchner
Content may be subject to copyright.
Benchmarking with endocytic/endosomal landmark proteins reveals distinct phases of PI3P production and vesicle fusion. (A) Colocalization of vesicles with landmark proteins of the endocytic pathway. Representative superresolution images (red, left column of each pair) and GFP images (green, right column of each pair) for each class of protein are shown. (B) Colocalization with endocytic pathway proteins reveals distinct subclasses of vesicles. FYVE-mEos2 colocalizes with GFP-tagged clathrin (green) and GTPases Vps21p (orange) and Ypt7p (purple). For each dataset, the median, upper, and lower quartiles are displayed. (C) Model of PI3P production on vesicles in endocytic pathway followed by fusion. Phases of endocytic/ endosomal protein association, enzymatic production of PI3P, and fusion to early and late endosomes (1-3) are described in B.
Source publication
Significance
We combined molecule counting and superresolution microscopy to observe the size, shape, and biomolecular composition of vesicles in the endocytic pathway. From a large number of steady-state snapshots, we follow the maturation of vesicles to endosomes. First, phosphatidylinositol 3-phosphate (PI3P) is generated on newly formed vesicle...
Contexts in source publication
Context 1
... To correlate our observed PI3P-size trajectory with the timeline of vesicle maturation, we performed two-color colocalization experiments between our PI3P reporter (in the superresolution channel) and landmark endocytic pathway pro- teins labeled with GFP (in the conventional uorescence chan- nel) (Fig. 5A). We tagged three endocytic pathway proteins with GFP: (i) clathrin, present from the initial stages of membrane invagination and endocytic vesicle formation (18); (ii) the small GTPase Vps21 (yeast homolog of Rab5), an interaction partner of the PI3-kinase that associates with incoming vesicles and early endosomes; and (iii) the ...
Context 2
... each of the three sets of experiments, we counted PI3P binding sites and measured the size of individual vesicles as well as the amount of each GFP-tagged landmark protein. By plotting those vesicles with signicant GFP signal ( Fig. 5B and Fig. S4), we observed the same characteristic distribution of size and PI3P content. These three landmark proteins denote three distinct regimes in our observed distribution. Clathrin associates with very low PI3P-content vesicles, whose size and morphology in- dicate that these structures are early endocytic vesicles. In con- trast, Vps21 ...
Context 3
... but both clathrin-and Vps21-associated regimes display tight regulation of vesicle size. Finally, Ypt7 associates with vesicles of signicantly larger size and containing the highest numbers of PI3P binding sites. This trend is also clear in the medians of each class and hand-drawn ellipses, which include more that 80% of data points of each class (Fig. 5B, crosses and shaded ellipses, respectively). In general, more small individual vesicles are observed than large, Ypt7-associated endosomes. Given that many individual vesicles fuse to form a larger endo- some, this observation is consistent with the conservation of lipid membrane along the maturation ...
Context 4
... maturation trajectory of vesicles suggests an initial phase of PI3P production on vesicles whose size is tightly regu- lated, followed by a later phase in which fusion of these vesicles occurs. Previously it has been shown that Vps21 mediates vesicle tethering (37), which precedes fusion. We are able to resolve tethered vesicles (e.g., in Fig. 5A, middle row and column); how- ever, consistent with the notion that fusion is fast, such events are very rarely observed. The PI3P content is highest in large, fused structures, which we interpret to be early and late endosomes based on their morphology and Vps21/Ypt7 association. Owing to the increase in surface area in these larger ...
Context 5
... are very rarely observed. The PI3P content is highest in large, fused structures, which we interpret to be early and late endosomes based on their morphology and Vps21/Ypt7 association. Owing to the increase in surface area in these larger structures, our ndings indicate that most PI3P production occurs before vesicles start to fuse. As seen in Fig. 5A, most of the large Ypt7-associated endosomes exhibit complex, irregular morphology, consistent with EM studies in yeast that resolved the structure of endosomes and multivesicular bodies (41). Owing to this complex morphology and 2D projection of the images, the calculated surface area of those large structures may be slightly ...
Context 6
... vesicles within a cell and extrinsic noise owing to variation between cells. Certain endo- cytic proteins involved in lipid modication and protein sorting are expressed at very low levels [e.g., Fab1 (149 copies per cell) or Vps27 (172 copies per cell) (45)], creating potentially large stochastic variations between vesicles and endosomes (Fig. S5). In contrast, the higher expression level of our PI3P reporter results in a lower coefcient of variation (see also high correlation Fig. 3B) and thus less intrinsic noise than some other endocytic pro- teins. By synthetically introducing noise in different nodes, future experiments on modied endocytic pathways could reveal which parts ...
Context 7
... S4). Images were background-subtracted and corrected for uneven illumination, and the total GFP intensity per vesicle was calculated (SI Materials and Methods). Only vesicles with more than two FYVE-mEos2 molecules were analyzed (SI Materials and Methods). Vesicles with a signif- icant GFP signal (above the 90% threshold, Fig. S4) are plotted in Fig. 5B. Tables S1-S3) were grown at 30 °C overnight in synthetic SD medium, diluted 1:50 in SD, and grown for 3-4 h to log phase in the dark. To avoid bleaching and preactivation of mEos2, exposure of the cells to light was mini- mized for all following steps. The chambered borosilicate cov- erglasses (eight-well, Lab-Tek II, 155409; ...
Context 8
... a theoretical elliptical shape with a main axis ratio of 2:1. For vesicles with a very low number of phosphatidylinositol 3-phosphate (PI3P) binding sites (<10) the actual size of vesicles may be underestimated owing to an uneven distribution of mEos2 molecules. Also for very large and uneven multivesicular structures (e.g., YPT7 associated in Fig. 5A), the surface area is underestimated to some extent. However, this only compresses the endocytic trajectory for large structures and does not change its branching ...
Context 9
... used in this study results in undercounting owing to temporal overlap. In such dense samples either the data recording time needs to be en- larged and 405-nm activation lowered to avoid temporal overlap or the total number of uorescent bursts needs to be used as an ensemble value to calculate the number of molecules. For colocalization studies (Figs. 3 and 5), we combined conven- tional quantitative uorescence imaging with GFP and super- resolution with mEos2. Because xed yeast cells exhibit too high uorescent and blinking background in the GFP channel for single-molecule imaging with available photoswitchable uores- cent proteins (PAFPs), the combined conventional approach is a robust ...
Context 10
... vesicles above and below the focal plane are visible, whereas in the single-molecule mEos2 channel only a thin slice of about 500 nm thickness is imaged. Therefore, we used the mEos2 channel as a guide and analyzed only those vesicles showing at least three mEos2 mol- ecules. The colocalization of the FYVE domain with the three protein markers (Fig. S4 and Fig. 5) corresponds well to the current model of the endocytic pathway. Clathrin exhibits a clear anticorrelation with the FYVE domain: It only colocalizes with vesicles having small amounts of FYVE molecules (<50) and vesicles with a high number of FYVE molecules (>50) show no colocalization, except one outlier slightly above the GFP thresh- ...
Context 11
... (<50) and vesicles with a high number of FYVE molecules (>50) show no colocalization, except one outlier slightly above the GFP thresh- old (Fig. S4). Vps21 is associated with vesicles and early endo- somes having an intermediate number of FYVE molecules, and YPT7 is only associated with structures having the highest amount of FYVE domains. In Fig. 5B only vesicles with a clear colocalization signal above the 90% threshold (Fig. S4A) are displayed and cover the whole endocytic ...
Context 12
... (Upper). In contrast, the strain expressing FYVE-mEos2 alone shows low cross-talk into the GFP channel (<10%), which is barely above the background uorescence even for hundreds of mEos2 molecules (Lower). After mEos2 imaging the faint signal from the brightness vesicles disappears, showing that all mEos2 molecules were photoactivated and imaged. Fig. S5. Low expression levels of endocytic proteins can lead to large relative variations between vesicles. Using previously published expression levels for several endocytic proteins (7), we calculate the vesicle-to-vesicle coefcient of variation assuming random distribution of these proteins between ∼50 vesicles per cell. We note that the ...
Similar publications
The ΔF508 mutant form of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR) that is normally degraded by the ER-associated degradative pathway can be rescued to the cell surface through low-temperature (27°C) culture or small molecular corrector treatment. However, it is unstable on the cell surface, and rapidly internalized and t...
The podocyte slit diaphragm (SD), responsible for blood filtration in vertebrates, is a major target of injury in chronic kidney disease. The damage includes severe morphological changes with destabilization of SDs and their replacement by junctional complexes between abnormally broadened foot processes. In Drosophila melanogaster, SDs are present...
Uncontrolled cell division is a hallmark of cancer. Deregulation of Wnt components has been linked to aberrant cell division by multiple mechanisms, including Wnt-mediated stabilisation of proteins signalling, which was notably observed in mitosis. Analysis of Wnt components revealed an unexpected role of B-cell CLL/lymphoma 9 (BCL9) in maintaining...
B lymphocytes play a critical role in adaptive immunity. Upon antigen binding, B cell receptors (BCR) cluster on the plasma membrane and are internalized by endocytosis. In this process, B cells capture diverse antigens in various contexts and concentrations. However, it is unclear whether the mechanism of BCR endocytosis changes in response to the...
The essential mammalian gene TACC3 is frequently mutated and amplified in cancers and its fusion products exhibit oncogenic activity in glioblastomas. TACC3 functions in mitotic spindle assembly and chromosome segregation. In particular, phosphorylation on S558 by the mitotic kinase, Aurora-A, promotes spindle recruitment of TACC3 and triggers the...
Citations
... Since the exposures of the camera sequentially follow one another, and the individual blinking events start stochastically, one such event is more likely to be captured on multiple consecutive camera frames resulting in several, less accurate localizations [14]. This could lead to an overestimation of the number of emitters, which is typically handled by grouping localizations within a given space and time window [15][16][17][18][19][20][21][22]. The disadvantage of grouping such localizations is that it can link different fluorophores to each other, especially on high-density datasets [23]. ...
In standard SMLM methods, the photoswitching of single fluorescent molecules and the data acquisition processes are independent, which leads to the detection of single molecule blinking events on several consecutive frames. This mismatch results in several data points with reduced localization precision, and it also increases the possibilities of overlapping. Here we discuss how the synchronization of the fluorophores’ ON state to the camera exposure time increases the average intensity of the captured point spread functions and hence improves the localization precision. Simulations and theoretical results show that such synchronization leads to fewer localizations with 15% higher sum signal on average, while reducing the probability of overlaps by 10%.
... This targeting requires the active GTP-form of Rab5 because it is only apparent when the GTP-preferring variant (Rab5(Q79L)) is used (Fig. 3a) and when the EE-SNAREs are present ( Supplementary Fig. S4a). To explain this unexpected result, we considered previous reports showing that the PtdIns(3)P concentration is directly related to endosome maturation, with higher concentrations promoting the transition from early to late endosomes 37,38 . We therefore asked whether in the presence of active Rab5 targeting specificity may be regulated by the concentration of PtdIns(3)P. ...
... Acute depletion of PtdIns(3)P reverts endosomes from more mature to immature stages 22 . In yeast, PtdIns(3)P is first detectable in the membrane directly after endocytosis and then increases by orders of magnitude during progress from early (Vps21-positive) to late (Ypt7-positive) endosomes 37 . Indeed, we observe that at Fig. 3 | In the presence of GTP-Rab5, PtdIns(3)P fine-tunes targeting of EE-SNARE liposomes in a concentration-dependent manner. ...
In the secretory pathway the destination of trafficking vesicles is determined by specific proteins that, with the notable exception of SNAREs, are recruited from soluble pools. Previously we have shown that microinjected proteoliposomes containing early or late endosomal SNAREs, respectively, are targeted to the corresponding endogenous compartments, with targeting specificity being dependent on the recruitment of tethering factors by some of the SNAREs. Here, we show that targeting of SNARE-containing liposomes is refined upon inclusion of polyphosphoinositides and Rab5. Intriguingly, targeting specificity is dependent on the concentration of PtdIns(3)P, and on the recruitment of PtdIns(3)P binding proteins such as rabenosyn-5 and PIKfyve, with conversion of PtdIns(3)P into PtdIns(3,5)P2 re-routing the liposomes towards late endosomes despite the presence of GTP-Rab5 and early endosomal SNAREs. Our data reveal a complex interplay between permissive and inhibitory targeting signals that sharpen a basic targeting and fusion machinery for conveying selectivity in intracellular membrane traffic.
... Stochasticity poses crucial challenges in maintaining causal ordering and temporal specificity, i.e., a tight probability distribution of events in time. However, despite the emphasis on stochasticity in constituent dynamics in the vesicular transport system [18][19][20][21] , endosomal trafficking processes display an extraordinary degree of robustness and predictability in delivering cargo to specific intracellular destinations, and receptors transported through the endosomal system show reproducible signalling outcomes. These properties suggest that there exist mechanisms to counter the stochasticity of the constituent processes and thus achieve tight control over the maturation, trafficking, and dynamics of the intracellular transport system. ...
Endosomal maturation is critical for robust and timely cargo transport to specific cellular compartments. The most prominent model of early endosomal maturation involves a phosphoinositide-driven gain or loss of specific proteins on individual endosomes, emphasising an autonomous and stochastic description. However, limitations in fast, volumetric imaging long hindered direct whole cell-level measurements of absolute numbers of maturation events. Here, we use lattice light-sheet imaging and bespoke automated analysis to track individual very early (APPL1-positive) and early (EEA1-positive) endosomes over the entire population, demonstrating that direct inter-endosomal contact drives maturation between these populations. Using fluorescence lifetime, we show that this endosomal interaction is underpinned by asymmetric binding of EEA1 to very early and early endosomes through its N- and C-termini, respectively. In combination with agent-based simulation which supports a ‘trigger-and-convert’ model, our findings indicate that APPL1- to EEA1-positive maturation is driven not by autonomous events but by heterotypic EEA1-mediated interactions, providing a mechanism for temporal and population-level control of maturation.
... This kind of data architecture is ideal for cluster analysis [6,7] and molecule counting [8,9], but pattern recognition [10] and segmentation [11] require new approaches. Quantitative evaluations were used for the ligand-specific dimer formation of TLR4 receptors [12], the characterization of individual vesicles [13], the cluster formation of TNRF1 receptors [14] or the mapping of the sarcomeric H-zone and I-band complexes of indirect flight muscles (IFMs) of Drosophila melanogaster [15]. There are algorithms specifically written for system simulations [16,17], molecule localizations [18], and the quantitative evaluation [19] of SMLM images. ...
Localization based superresolution technique provides the highest spatial resolution in optical microscopy. The final image is formed by the precise localization of individual fluorescent dyes, therefore the quantification of the collected data requires special protocols, algorithms and validation processes. The effects of labelling density and structured background on the final image quality were studied theoretically using the TestSTORM simulator. It was shown that system parameters affect the morphology of the final reconstructed image in different ways and the accuracy of the imaging can be determined. Although theoretical studies help in the optimization procedure, the quantification of experimental data raises additional issues, since the ground truth data is unknown. Localization precision, linker length, sample drift and labelling density are the major factors that make quantitative data analysis difficult. Two examples (geometrical evaluation of sarcomere structures and counting the γH2AX molecules in DNA damage induced repair foci) have been presented to demonstrate the efficiency of quantitative evaluation experimentally.
... However, some proteins exhibit a different oligomeric state when expressed in the Xenopus system compared with mammalian expression systems (Krashia et al., 2010). For mammalian cells, the resolution limit of light microscopy can be circumvented using super-resolution microscopy methods (Durisic et al., 2014;Hummer et al., 2016;Lee et al., 2012;Nan et al., 2013;Nicovich et al., 2017;Puchner et al., 2013;Sengupta et al., 2011). SMLM increases resolution by reconstructing the precise positions of isolated fluorophore emissions detected over thousands of frames (Betzig et al., 2006;Rust et al., 2006). ...
The oligomeric state of plasma membrane proteins is the result of the interactions between individual proteins and an important determinant of their function. Most approaches used to address this question rely on extracting these complexes from their native environment, which may disrupt weaker interactions. Therefore, microscopy techniques have been increasingly used in recent years to determine oligomeric states in situ . Classical light microscopy suffers from insufficient resolution, but super-resolution methods such as single molecule localization microscopy (SMLM) can circumvent this problem. When using SMLM to determine oligomeric states of proteins, subunits are labeled with fluorescent proteins that only emit light following activation or conversion at different wavelengths. Typically, individual molecules are counted based on a binomial distribution analysis of emission events detected within the same diffraction-limited volume. This strategy requires low background noise, a high recall rate for the fluorescent tag and intensive post-imaging data processing. To overcome these limitations, we developed a new method based on SMLM to determine the oligomeric state of plasma membrane proteins. Our dual-color colocalization (DCC) approach allows for accurate in situ counting even with low efficiencies of fluorescent protein detection. In addition, it is robust in the presence of background signals and does not require temporal clustering of localizations from individual proteins within the same diffraction limited volume, which greatly simplifies data acquisition and processing. We used DCC-SMLM to resolve the controversy surrounding the oligomeric state of two SLC26 multifunctional anion exchangers and to determine the oligomeric state of four members of the SLC17 family of organic anion transporters.
... For example, subsets of the localisation data sets can be produced using filters or by cropping using regions of interest on rendered images. Plugins for more in-depth analyses are also provided for techniques such as single-particle tracking, clustering and cluster visualisation [11][12][13] , pair-correlation photoactivated localisation microscopy (PC-PALM) [14][15][16][17] , time-correlated photoactivated localisation microscopy (tcPALM) 18 , cross-talk activation analysis 19 and Fourier image resolution 20 . Fitting and analyses capabilities are supported by a suite of calibration and modelling plugins which allow analysis of the noise and gain of electron multiplying charge-coupled device (EM-CCD) and scientific complementary metal-oxide-semiconductor (sCMOS) cameras for use in maximum likelihood fitting models, as well as construction of point spread function (PSF) models from fluorescent bead calibration images. ...
Single-molecule localisation microscopy (SMLM) uses software to extract super-resolved positions from microscope images of fluorescent molecules. These localisations can then be used to render super-resolution images or analysed to extract information about molecular behaviour. The GDSC SMLM software provides a set of tools for analysing SMLM data in a single cross-platform environment. The software identifies fluorescent molecules in raw microscope images and localises their positions using stages of spot detection, spot fitting and spot rejection. The resulting localisation data set can then be visualised, cropped and filtered. A suite of downstream analysis tools enable the user to perform single-particle tracking, cluster analysis and drift correction. In addition, GDSC SMLM also provides utility tools that enable modelling of EM-CCD and sCMOS cameras as well as point spread functions (PSFs) for data simulation. The software is written in Java and runs as a collection of plugins for the ImageJ software.
... In this regard, SMLM has been intensively studied to technically approach biomolecular resolution [7][8][9] and best image qualities [10][11][12] . Even though, SMLM meets great challenges in quantitative capability [13][14][15][16][17][18][19] , which hinders its widest application in biomedical research. ...
Single molecule localization microscopy (SMLM) is irreplaceable among super-resolution microscopies in revealing biological ultra-structures, given its unmatched high resolution. However, its sub-optimal quantitative capability, which is critical for characterizing true biomolecular organization of ultra-structures in cells, has hindered its widest application in biomedical research. Here, in SMLM imaging of cellular structures such as lipid rafts and microtubules with saturation labelling, we identified ultra-bright localizations, each of which is contributed by simultaneous emission of multiple molecules within a diffraction-limit region and has been regarded before as a regular localization from single molecule. Consistently, ultra-bright localizations are also observed in simulated SMLM imaging of endoplasmic reticulum or microtubules from public resource. Furthermore, after calibrating each ultrabright localization into multiple single-molecule localizations using the photon-number-based models, the density of total localizations shows linear correlation with the true molecule density, presenting SMLM with new reconstruction method as a quantitative analysis approach. Therefore, identification and dissection of ultra-bright localizations in SMLM enable the close and quantitative estimate of the true biomolecular organization.
... Indeed, we observed this power-law dependence with an exponent suggesting that the expression was limited by the diffusion of particles of a characteristic size (s) of ∼90 nm (Fig. 3b). This mesoscale length scale corresponds to many biological entities, for example, trafficking vesicles and mRNA ribonucleoprotein particles 25,30,31 (both are ∼100 nm). We note that if the process-limiting k exp was nuclear or if multiple processes were to be limited, then the limiting size could be a little different (50-150 nm), but would remain in the mesoscale range. ...
Cells that grow in confined spaces eventually build up mechanical compressive stress. This growth-induced pressure decreases cell growth. Growth-induced pressure is important in a multitude of contexts, including cancer1–3, microbial infections4 and biofouling5; yet, our understanding of its origin and molecular consequences remains limited. Here we combine microfluidic confinement of the yeast Saccharomyces cerevisiae6 with rheological measurements using genetically encoded multimeric nanoparticles7 to reveal that growth-induced pressure is accompanied with an increase in a key cellular physical property: macromolecular crowding. We develop a fully calibrated model that predicts how increased macromolecular crowding hinders protein expression and thus diminishes cell growth. This model is sufficient to explain the coupling of growth rate to pressure without the need for specific molecular sensors or signalling cascades. As molecular crowding is similar across all domains of life, this could be a deeply conserved mechanism of biomechanical feedback that allows environmental sensing originating from the fundamental physical properties of cells. Rheological measurements combined with a fully calibrated model show that growth-induced pressure increases macromolecular crowding, inhibiting protein expression and cell growth.
... 10 Typically, upon prolonged violet light illumination, green-to-red photoconversion remains incomplete, meaning that from a pool of PCFPs, only a limited fraction can ever be imaged in the red channel of the microscope. 4,9,11,12 In addition to unsuccessful protein folding or chromophore maturation, incomplete PCE's can be attributed to possible sub-populations within the PCFP pool unable to photoconvert, rapid photobleaching in the red state before detection can be made, or premature photobleaching in the green state, before photoconversion occurs. 4,9,11,12 In general, green-to-red photoconversion, the mechanism of which remains incompletely understood, 3 is in . ...
... 4,9,11,12 In addition to unsuccessful protein folding or chromophore maturation, incomplete PCE's can be attributed to possible sub-populations within the PCFP pool unable to photoconvert, rapid photobleaching in the red state before detection can be made, or premature photobleaching in the green state, before photoconversion occurs. 4,9,11,12 In general, green-to-red photoconversion, the mechanism of which remains incompletely understood, 3 is in . CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ...
... 8 Second, it complicates counting by quantitative PALM (qPALM). 12 In this technique the stoichiometry of protein complexes, typically non-resolvable spatially, can in principle still be determined by counting fluorescence bursts from each complex. [12][13][14][15][16] However, a limited PCE results in severe undercounting errors. ...
Green-to-red photoconvertible fluorescent proteins (PCFPs) are widely employed as markers in photoactivated localization microscopy (PALM). However, their highly complex photophysical behavior complicates their usage. The fact that only a limited fraction of a PCFP ensemble can form the photoconverted state upon near-UV light illumination, termed photoconversion efficiency (PCE), lowers the achievable spatial resolution in PALM and creates undercounting errors in quantitative counting applications. Here, we show that the PCE of mEos4b is not a fixed property of this PCFP, but strongly depends on illumination conditions. Attempts to reduce long-lived blinking in red mEos4b by application of 488 nm light leads to a reduction of the PCE. Furthermore, the PCE of mEos4b strongly depends on the applied 405-nm power density. A refined photophysical model of mEos4b accounts for the observed effects, involving nonlinear green-state photobleaching upon violet light illumination favored by photon absorption by a putative radical dark state.
TOC GRAPHICS
... Currently, single-molecule localization microscopy (SMLM) is the method of choice to study the organization of membrane receptors due to its ability to localize emitters (i.e., labeled receptors) with nanometer precision and its potential for quantitative assessments of protein distribution and number [17][18][19][20] . SMLM-based studies confirmed the earlier observations using TIRF microscopy that receptors and associated signaling molecules can cluster in nonstimulated and stimulated immune cells [21][22][23][24][25] . ...
Cells communicate with their environment via surface receptors, but nanoscopic receptor organization with respect to complex cell surface morphology remains unclear. This is mainly due to a lack of accessible, robust and high-resolution methods. Here, we present an approach for mapping the topography of receptors at the cell surface with nanometer precision. The method involves coating glass coverslips with glycine, which preserves the fine membrane morphology while allowing immobilized cells to be positioned close to the optical surface. We developed an advanced and simplified algorithm for the analysis of single-molecule localization data acquired in a biplane detection scheme. These advancements enable direct and quantitative mapping of protein distribution on ruffled plasma membranes with near isotropic 3D nanometer resolution. As demonstrated successfully for CD4 and CD45 receptors, the described workflow is a straightforward quantitative technique to study molecules and their interactions at the complex surface nanomorphology of differentiated metazoan cells. A super-resolution localisation-based method is shown to map receptor topography in immune cells, which allows quantitative study of molecules and their interactions at the complex surface nanomorphology of cells.
