Distribution Of Membrane Proteins Research Articles

Nanoscale manipulation of membrane curvature for probing endocytosis in live cells.

Clathrin-mediated endocytosis (CME) involves nanoscale bending and inward budding of the plasma membrane, by which cells regulate both the distribution of membrane proteins and the entry of extracellular species. Extensive studies have shown that CME proteins actively modulate the plasma membrane curvature. However, the reciprocal regulation of how the plasma membrane curvature affects the activities of endocytic proteins is much less explored, despite studies suggesting that membrane curvature itself can trigger biochemical reactions. This gap in our understanding is largely due to technical challenges in precisely controlling the membrane curvature in live cells. In this work, we use patterned nanostructures to generate well-defined membrane curvatures ranging from +50 nm to -500 nm radius of curvature. We find that the positively curved membranes are CME hotspots, and that key CME proteins, clathrin and dynamin, show a strong preference towards positive membrane curvatures with a radius <200 nm. Of ten CME-related proteins we examined, all show preferences for positively curved membrane. In contrast, other membrane-associated proteins and non-CME endocytic protein caveolin1 show no such curvature preference. Therefore, nanostructured substrates constitute a novel tool for investigating curvature-dependent processes in live cells.

Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy.

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, "flash-and-freeze," that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution.

Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, "flash-and-freeze," that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution.

Molecular Mechanisms of V-SNARE Function in Secretory Granule Exocytosis

Insulin is released by regulated exocytosis and docking of insulin granules at the plasma membrane, followed by the assembly of the secretory machinery is required for insulin release. Newly formed granules require a complex maturation process to become release-ready and the formation of these fusion competent mature secretory granules involves a series of discrete and unique events comprised of granule sorting, maturation, docking, priming, and finally Ca2+-dependent fusion. Soluble NSF attachment receptor (SNARE) proteins are the conserved core machinery required for all intracellular membrane fusion events. While SNAREs specifically control fusion, vesicular SNAREs (v-SNAREs) perform additional roles that are still elusive. In secretory cell types, v-SNAREs are pivotal for targeting vesicles to different locations, mediating both sorting and membrane protein distribution and reductions in v-SNAREs result in defective regulated exocytosis. Using total internal reflection fluorescence (TIRF) microscopy, we studied the role of VAMP8 in secretory granule exocytosis in pancreatic beta cells. We found that VAMP8 associates with early, late and recycling endosome compartment, rather than insulin containing granules. However, VAMP8 traffics and fuses at the plasma membrane specifically with the recycling endosomal compartment. VAMP8 expression compromised secretory granule fusion, whereas VAMP2 expression facilitated granule fusion, delineating a VAMP8-dependent fusion step between recycling endosomes and the plasma membrane. Truncation of the VAMP8 C-terminal region resulted in attenuated exocytosis and a change in secretion kinetics. Moreover, secreting insulin measurements are negatively correlated with increased VAMP8 copy number in human islets. We conclude that VAMP8 vesicles play an important role in granule genesis and sorting of essential membrane proteins regulating insulin secretion. Additionally, the TMD of VAMP8 may be necessary for initiating fusion upon stimulation and appears to have some direct interaction on insulin secretion. This work should fill critical gaps in our understanding of regulated secretion and the treatment of type-2 diabetes.

Single Molecule Fluroescence Approaches to Plasma Membrane Biophysics

The plasma membrane is the fundamental component of a cell. Not only is the plasma membrane the outer barrier of the cell, it also regulates many cellular functions including endocytosis, exocytosis, receptor signalling and cell migration. As essential as the plasma membrane is, methods to capture the structure of and the proteins within the membrane on biologically-relevant length scales remain lacking. A particularly vexing issue is whether membrane rafts - regions of locally-ordered lipids and associated proteins observed in model membrane systems - are present in intact cells. Much of the previous evidence for these rafts relies on invasive biochemical techniques and the little imaging evidence available shows that these regions are small (< 100 nanometres) and short-lived (< 100 milliseconds).While still limited by the diffraction limit of visible light, single-molecule fluorescence imaging allows such small and transient features to be detected. We will present three new approaches to investigating these structures in intact membranes. The first is a spectral fluorescence correlation spectroscopy approach demonstrating the presence of regions of local membrane order and disorder within live cell membranes. These regions have lifetimes of 5 to 25 ms and through simulations we can show that both probe diffusion and domain diffusion give rise to the observed fluctuations in emission intensity. The second technique is a self-calibrating image analysis method that is capable of capturing the coordination of local differences in membrane order with membrane protein distribution within intact cells. This approach demonstrates the presence of protein sorting within intact cells as well as the effect of expression level on effective protein sorting. The final technique is a 3D super-resolution method to map the nanometer-scale topography of the plasma membrane. We will show initial results demonstrating our ability to map these topographical features and their correlation with protein sorting.

Probing Cholesterol-Dependence of Integrin-Urokinase Receptor Complex Formation using Confocal Dual-Color Fluorescence Intensity Analysis

The modern view of a plasma membrane is that of a complex supramolecular assembly of notable lipid/protein heterogeneity, in which the local lipid environment may have a profound impact on membrane protein distribution and function. Yet, the underlying mechanisms of lipid-mediated regulation of membrane protein functionality remain elusive, in part due to the limitations of existing methodologies and the often small size and transient nature of lipid/protein heterogeneities in cellular membranes. Exemplary, we still have rather limited knowledge about the significance of lipid composition in hetero-protein complex formation in such membrane systems. To address this fascinating, but poorly understood topic, here we introduce a single molecule-sensitive confocal experimental strategy, comprised of dual-color fluorescence peak analysis and photon counting histogram (PCH) method, which not only allows the detection of hetero-protein complexes in well-defined lipid environments, but also provides accurate insight into the composition of such complexes in the membrane. By applying the described methodology, here we demonstrate the formation of complexes of αvβ3 integrins and GPI-anchored urokinase plasminogen activator receptors (uPAR) in cholesterol-containing model lipid mixtures. PCH analysis of fluorescence intensity signals of complexes confirms furthermore that αvβ3-uPAR complexes are comprised of one integrin and two uPAR proteins. Remarkably, formation of comparable hetero-protein complexes is largely suppressed in a cholesterol-free lipid composition, highlighting the importance of cholesterol in αvβ3-uPAR complex formation. Our results are intriguing in light of the already established significance of cholesterol in integrin function in cellular membranes.

Mapping Ceacam 1-4L's Nanoscale Spatial Distribution, Structure, Dynamics, and Association with Lipid Ordered Domains by Super-Resolution Microscopy

Super-resolution microscopy has provided novel quantitative insights into the nanoscale spatial distribution of membrane proteins, including the observation that some systems form micro- and nano-sized clusters. However, details of the protein's self-association state (monomer / dimer / oligomer) within the different cluster types are often missing. Since it has been shown that a protein's oligomeric form might be a crucial determinant of its function, it is important to have a technique capable of measuring both spatial distribution and association state. To that end, we have applied a correlated dSTORM/homo-FRET approach to characterize CEACAM 1-4L, the progenitor isoform of the carcinoembryonic antigen-related cellular adhesion molecule (CEACAMs) family. CEACAMs are cell surface glycoproteins involved in homo- and hetero-philic intercellular interactions that control cellular growth, differentiation, tumourigenesis, inflammation and infection. While CEACAM1-4L is known to exist as a monomer or oligomer with the former being hypothesized to be involved in activatory intercellular interactions, the basis for its spatial distribution and association state as a function of distribution remain poorly understood. Our correlated approach involved live cell homo-FRET studies of eYFP-CEACAM1-4L in HeLa cells followed by fixed cell dSTORM imaging performed using nanobody labeling with Alexa Fluor 647 (AF647) conjugated to an anti-eYFP single domain antibody. Cluster analysis of the dSTORM datasets using Voronoi tessellation revealed that the CEACAM1-4L nanoclusters were preferentially comprised of monomeric CEACAM1-4L while microclusters contained a heterogeneous distribution of CEACAM1-4L monomers and oligomers. To further characterize these clusters, super-resolution imaging using Alexa-647 labelled cholera toxin was performed to examine the co-localization of lipid ordered domains, which are known to drive membrane protein lateral distribution and clustering, with CEACAM1-4L. Finally, live-cell single particle tracking (SPT) of CEACAM1-4L was performed to investigate the putative confined, obstructed, free diffusion, and directed motion dynamics of CEACAM1-4L.

Structure and in Situ Organization of ATP Sythase and Respiratory Chain Complexes

Mitochondria are the powerhouses of eukaryotic cells and the main site of ATP synthesis in cells performing aerobic respiration. Located on the cristae of the inner mitochondrial membrane, the ATP synthase uses the energy stored in a transmembrane gradient of protons to power the synthesis of ATP while the respiratory chain complexes replenish the gradient. Using the technique of electron cryo-tomography and subtomogram averaging, we are characterizing how the structure and spatial distribution of membrane proteins influence mitochondrial function. So far we have discovered: 1) the structure of the ATP synthase varies dramatically between different eukaryotic lineages, 2) that in all species studied so far, the ATP synthase form rows of dimers along the highly curved edges of the cristae membranes, 3) that the structure of the rows varies between protozoan and multicellular organisms, and 4) the purpose of the rows appears to be the maintenance of organized inner membrane invaginations. In parallel, we have shown that the proteins of the respiratory chain are located in the flat membrane regions of cristae and that they form species specific supercomplexes. Alongside the cryo-ET studies, we have determined a 6.2A structure of the mitochondrial ATP synthase of Polytomella sp. using single particle cryo-EM. Our structure revealed four highly tilted membrane-intrinsic helices (∼20° to the membrane plane) adjacent to the rotor ring which we assign to the elusive a-subunit. Based on this structure, we present a model describing how protons move through the ATP synthase.

The shape of the transmembrane domain is a novel endocytosis signal for single-spanning membrane proteins.

Endocytosis is crucial for all cells as it allows them to incorporate material from the extracellular space and control the availability of transmembrane proteins at the plasma membrane. In yeast, endocytosis followed by recycling to the plasma membrane results in a polarised distribution of membrane proteins by a kinetic mechanism. Here, we report that increasing the volume of residues that constitute the exoplasmic half of the transmembrane domain (TMD) in the yeast SNARE Sso1, a type II membrane protein, results in its polarised distribution at the plasma membrane. Expression of this chimera in strains affected in either endocytosis or recycling revealed that this polarisation is achieved by endocytic cycling. A bioinformatics search of the Saccharomyces cerevisiae proteome identified several proteins with high-volume exoplasmic hemi-TMDs. Our experiments indicate that TMDs from these proteins can confer a polarised distribution to the Sso1 cytoplasmic domain, indicating that the shape of the TMD can act as a novel endocytosis and polarity signal in yeast. Additionally, a high-volume exoplasmic hemi-TMD can act as an endocytosis signal in a mammalian cell line.

Sensitive Detection of Cell Surface Membrane Proteins in Living Breast Cancer Cells Using Multicolor Fluorescence Microscopy with a Plasmonic Chip.

A plasmonic chip was applied to live cancer cell imaging. The epithelial cell adhesion molecule (EpCAM) is a surface marker that can be used to classify breast cancer cell lines into distinct differentiation states. EpCAM and the nuclei of two kinds of living breast cancer cells, MDA-MB231 and MCF-7, were stained with allophycocyanin (APC)-labeled anti-EpCAM antibody and 4',6-diamidino-2-phenylindole (DAPI), respectively, and the cells were scattered on either a plasmonic chip (metal-coated wavelength-scale grating substrate) or a control glass slide. Multicolor fluorescence microscopic imaging allowed fluorescence images of APC-EpCAM to be obtained on the plasmonic chip that were more than 10 times brighter compared with those on the glass slide. In contrast, in the fluorescence images of DAPI-stained nuclei, no difference in brightness was observed between substrates. The fluorescence enhancement of APC-EpCAM in the cell membrane in contact with the plasmonic chip is thought to be due to the excitation of APC molecules localized within the surface plasmon field. Analysis of the cross section of a fluorescence image revealed a distribution of EpCAM at a higher level of fluorescence in the center of the cell image because of contact between the cell membrane and the plasmonic chip. In contrast, fluorescence images of APC-EpCAM taken on a glass slide were so dark that only the outline of the cell was characterized. The plasmonic chip thus constitutes a simple and powerful tool for analyzing the distribution and kinetics of surface marker proteins in cell membranes contacting the chip.

Inhomogeneity Based Characterization of Distribution Patterns on the Plasma Membrane.

Cell surface protein and lipid molecules are organized in various patterns: randomly, along gradients, or clustered when segregated into discrete micro- and nano-domains. Their distribution is tightly coupled to events such as polarization, endocytosis, and intracellular signaling, but challenging to quantify using traditional techniques. Here we present a novel approach to quantify the distribution of plasma membrane proteins and lipids. This approach describes spatial patterns in degrees of inhomogeneity and incorporates an intensity-based correction to analyze images with a wide range of resolutions; we have termed it Quantitative Analysis of the Spatial distributions in Images using Mosaic segmentation and Dual parameter Optimization in Histograms (QuASIMoDOH). We tested its applicability using simulated microscopy images and images acquired by widefield microscopy, total internal reflection microscopy, structured illumination microscopy, and photoactivated localization microscopy. We validated QuASIMoDOH, successfully quantifying the distribution of protein and lipid molecules detected with several labeling techniques, in different cell model systems. We also used this method to characterize the reorganization of cell surface lipids in response to disrupted endosomal trafficking and to detect dynamic changes in the global and local organization of epidermal growth factor receptors across the cell surface. Our findings demonstrate that QuASIMoDOH can be used to assess protein and lipid patterns, quantifying distribution changes and spatial reorganization at the cell surface. An ImageJ/Fiji plugin of this analysis tool is provided.

Single-point single-molecule FRAP distinguishes inner and outer nuclear membrane protein distribution.

The normal distribution of nuclear envelope transmembrane proteins (NETs) is disrupted in several human diseases. NETs are synthesized on the endoplasmic reticulum and then transported from the outer nuclear membrane (ONM) to the inner nuclear membrane (INM). Quantitative determination of the distribution of NETs on the ONM and INM is limited in available approaches, which moreover provide no information about translocation rates in the two membranes. Here we demonstrate a single-point single-molecule FRAP microscopy technique that enables determination of distribution and translocation rates for NETs in vivo.

Plasma membrane proteomics in the maize primary root growth zone: novel insights into root growth adaptation to water stress

Previous work on maize (Zea mays L.) primary root growth under water stress showed that cell elongation is maintained in the apical region of the growth zone but progressively inhibited further from the apex. These responses involve spatially differential and coordinated regulation of osmotic adjustment, modification of cell wall extensibility, and other cellular growth processes that are required for root growth under water-stressed conditions. As the interface between the cytoplasm and the apoplast (including the cell wall), the plasma membrane likely plays critical roles in these responses. Using a simplified method for enrichment of plasma membrane proteins, the developmental distribution of plasma membrane proteins was analysed in the growth zone of well-watered and water-stressed maize primary roots. The results identified 432 proteins with differential abundances in well-watered and water-stressed roots. The majority of changes involved region-specific patterns of response, and the identities of the water stress-responsive proteins suggest involvement in diverse biological processes including modification of sugar and nutrient transport, ion homeostasis, lipid metabolism, and cell wall composition. Integration of the distinct, region-specific plasma membrane protein abundance patterns with results from previous physiological, transcriptomic and cell wall proteomic studies reveals novel insights into root growth adaptation to water stress.

Intermediate Filaments and Polarization in the Intestinal Epithelium.

The cytoplasmic intermediate filament cytoskeleton provides a tissue-specific three-dimensional scaffolding with unique context-dependent organizational features. This is particularly apparent in the intestinal epithelium, in which the intermediate filament network is localized below the apical terminal web region and is anchored to the apical junction complex. This arrangement is conserved from the nematode Caenorhabditis elegans to humans. The review summarizes compositional, morphological and functional features of the polarized intermediate filament cytoskeleton in intestinal cells of nematodes and mammals. We emphasize the cross talk of intermediate filaments with the actin- and tubulin-based cytoskeleton. Possible links of the intermediate filament system to the distribution of apical membrane proteins and the cell polarity complex are highlighted. Finally, we discuss how these properties relate to the establishment and maintenance of polarity in the intestine.

Measuring Exocytosis Rate Using Corrected Fluorescence Recovery After Photoconversion.

Exocytosis plays crucial roles in regulating the distribution and function of plasma membrane (PM) and extracellular matrix proteins. However, measuring the exocytosis rate of a specific protein by conventional methods is very difficult because of exocytosis-independent trafficking such as endocytosis, which also affects membrane protein distribution. Here, we describe a novel method, corrected fluorescence recovery after photoconversion, in which exocytosis-dependent and -independent trafficking events are measured simultaneously to accurately determine exocytosis rate. In this method, the protein-of-interest is tagged with Dendra2, a green-to-red photoconvertible fluorescent protein. Following the photoconversion of PM-localized Dendra2, both the recovery of the green signal and the changes in the photoconverted red signal are measured, and the rate of exocytosis is calculated from the changing rates of these two signals.

Probing Role of Cholesterol in Integrin Crosstalk and Complexation of Integrins with GPI-Anchored Urokinase Receptors and Gangliosides using Model Lipid Mixtures

It is now widely recognized that lipid composition may have a profound influence on membrane protein distribution and function. However, due to the complexity of plasma membranes, the underlying biophysical mechanisms of such a lipid-mediated regulation of membrane protein functionality remain elusive. Exemplary, there is still limited understanding about the role of cholesterol in the formation of functional complexes of membrane proteins with other membrane constituents, such as different membrane proteins and glycolipids. To address this potentially important topic, here we explore the influence of cholesterol content on the interaction behavior of different integrins (αvβ3 and α5β1), also known as integrin crosstalk, as well as the complexation of integrins with GPI-anchored urokinase plasminogen activator receptor (uPAR) and ganglioside GM3, respectively, using model lipid mixtures of well-defined compositions. Membrane protein complexation and distribution in the planar model membrane is analyzed using complementary confocal detection methods, including fluorescence correlation spectroscopy (FCS), confocal fluorescence intensity analysis, and photon counting histogram (PCH) method. Experimental results are presented, which not only confirm α5β1-αvβ3, αvβ3-uPAR, and α5β1-GM3 complexations in the fluid lipid bilayer, but also demonstrate the important role of cholesterol therein. They highlight the potential significance of cholesterol in the regulation of membrane protein complexes in biological membranes.

Polarized Localization Microscopy Detects Membrane Curvature and Reveals the Interplay between Membrane Orientation and Protein Dynamics

Nanoscale membrane curvature is a necessary component of countless cellular processes. However, the understanding of these processes is limited by the experimental techniques and its inability to detect and resolve nanoscale membrane curvature. Here we present Polarized Localization Microscopy (PLM), a novel optical microscopy technique that detects and resolves membrane curvature with 15 nm resolution. PLM combines the advantages of polarized total internal reflection fluorescence microscopy (TIRF) and fluorescence localization microscopy to reveal single-fluorophore locations and orientations without sacrificing the localization precision of the point spread function. We demonstrate PLM capability of resolving membrane orientations by draping a supported lipid bilayer over polystyrene nanoparticles of varying sizes on a glass coverslip, thus creating a model membrane with coexisting flat and curved regions and membrane radii of curvature as small as 20 nm. Further, we introduce theoretical analysis that enables mapping out the membrane topology from the density of detected localizations in fluorescence excitation polarization. Using this approach, the interplay between membrane topology and cholera toxin subunit B (CTxB) dynamics was addressed. CTxB partitioned to curved membrane over the nanoparticle and further drove formation of lipid tubules. PLM detected the binding of an exocytosis protein complex to nanoscale membrane curvature. PLM was utilized to perform high throughput single particle tracking to reveal the correlated effects of membrane curvature, dynamics, and protein distribution. By providing super-resolution of membrane topology, PLM is able to reveal the critical components in various cellular processes such as endocytosis/exocytosis, viral infections, and neurotransmission.

Loss of the BBSome perturbs endocytic trafficking and disrupts virulence of Trypanosoma brucei

Cilia (eukaryotic flagella) are present in diverse eukaryotic lineages and have essential motility and sensory functions. The cilium's capacity to sense and transduce extracellular signals depends on dynamic trafficking of ciliary membrane proteins. This trafficking is often mediated by the Bardet-Biedl Syndrome complex (BBSome), a protein complex for which the precise subcellular distribution and mechanisms of action are unclear. In humans, BBSome defects perturb ciliary membrane protein distribution and manifest clinically as Bardet-Biedl Syndrome. Cilia are also important in several parasites that cause tremendous human suffering worldwide, yet biology of the parasite BBSome remains largely unexplored. We examined BBSome functions in Trypanosoma brucei, a flagellated protozoan parasite that causes African sleeping sickness in humans. We report that T. brucei BBS proteins assemble into a BBSome that interacts with clathrin and is localized to membranes of the flagellar pocket and adjacent cytoplasmic vesicles. Using BBS gene knockouts and a mouse infection model, we show the T. brucei BBSome is dispensable for flagellar assembly, motility, bulk endocytosis, and cell viability but required for parasite virulence. Quantitative proteomics reveal alterations in the parasite surface proteome of BBSome mutants, suggesting that virulence defects are caused by failure to maintain fidelity of the host-parasite interface. Interestingly, among proteins altered are those with ubiquitination-dependent localization, and we find that the BBSome interacts with ubiquitin. Collectively, our data indicate that the BBSome facilitates endocytic sorting of select membrane proteins at the base of the cilium, illuminating BBSome roles at a critical host-pathogen interface and offering insights into BBSome molecular mechanisms.

Model of Growth Cone Membrane Polarization via Microtubule Length Regulation

We present a mathematical model of membrane polarization in growth cones. We proceed by coupling an active transport model of cytosolic proteins along a two-dimensional microtubule (MT) network with a modified Dogterom-Leibler model of MT growth. In particular, we consider a Rac1-stathmin-MT pathway in which the growth and catastrophe rates of MTs are regulated by cytosolic stathmin, while the stathmin is regulated by Rac1 at the membrane. We use regular perturbation theory and numerical simulations to determine the steady-state stathmin concentration, the mean MT length distribution, and the resulting distribution of membrane-bound proteins. We thus show how a nonuniform Rac1 distribution on the membrane generates a polarized distribution of membrane proteins. The mean MT length distribution and hence the degree of membrane polarization are sensitive to the precise form of the Rac1 distribution and parameters such as the catastrophe-promoting constant and tubulin association rate. This is a consequence of the fact that the lateral diffusion of stathmin tends to weaken the effects of Rac1 on the distribution of mean MT lengths.

Artifacts in single-molecule localization microscopy.

Single-molecule localization microscopy provides subdiffraction resolution images with virtually molecular resolution. Through the availability of commercial instruments and open-source reconstruction software, achieving super resolution is now public domain. However, despite its conceptual simplicity, localization microscopy remains prone to user errors. Using direct stochastic optical reconstruction microscopy, we investigate the impact of irradiation intensity, label density and photoswitching behavior on the distribution of membrane proteins in reconstructed super-resolution images. We demonstrate that high emitter densities in combination with inappropriate photoswitching rates give rise to the appearance of artificial membrane clusters. Especially, two-dimensional imaging of intrinsically three-dimensional membrane structures like microvilli, filopodia, overlapping membranes and vesicles with high local emitter densities is prone to generate artifacts. To judge the quality and reliability of super-resolution images, the single-molecule movies recorded to reconstruct the images have to be carefully investigated especially when investigating membrane organization and cluster analysis.