Formation Of Membrane Domains Research Articles

Extracting Curvature Preferences of Lipids Assembled in Flat Bilayers Shows Possible Kinetic Windows for Genesis of Bilayer Asymmetry and Domain Formation in Biological Membranes

Studies on the assembly of pure lipid components allow mechanistic insights toward understanding the structural and functional aspects of biological membranes. Molecular dynamic (MD) simulations on membrane systems provide molecular details on membrane dynamics that are difficult to obtain experimentally. A large number of MD studies have remained somewhat disconnected from a key concept of amphipathic assembly resulting in membrane structures--shape parameters of lipid molecules in those structures in aqueous environments. This is because most of the MD studies have been done on flat lipid membranes. With the above in view, we analyzed MD simulations of 26 pure lipid patches as a function of (1) lipid type(s) and (2) time of MD simulations along with 35-40 ns trajectories of five pure lipids. We report, for the first time, extraction of curvature preferences of lipids from MD simulations done on flat bilayers. Our results may lead to mechanistic insights into the possible origins of bilayer asymmetries and domain formation in biological membranes.

MITOL Regulates Endoplasmic Reticulum-Mitochondria Contacts via Mitofusin2

The mitochondrial ubiquitin ligase MITOL regulates mitochondrial dynamics. We report here that MITOL regulates mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) domain formation through mitofusin2 (Mfn2). MITOL interacts with and ubiquitinates mitochondrial Mfn2, but not ER-associated Mfn2. Mutation analysis identified a specific interaction between MITOL C-terminal domain and Mfn2 HR1 domain. MITOL mediated lysine-63-linked polyubiquitin chain addition to Mfn2, but not its proteasomal degradation. MITOL knockdown inhibited Mfn2 complex formation and caused Mfn2 mislocalization and MAM dysfunction. Sucrose-density gradient centrifugation and blue native PAGE retardation assay demonstrated that MITOL is required for GTP-dependent Mfn2 oligomerization. MITOL knockdown reduced Mfn2 GTP binding, resulting in reduced GTP hydrolysis. We identified K192 in the GTPase domain of Mfn2 as a major ubiquitination site for MITOL. A K192R mutation blocked oligomerization even in the presence of GTP. Taken together, these results suggested that MITOL regulates ER tethering to mitochondria by activating Mfn2 via K192 ubiquitination.

The Role of Lipid Domains in Bacterial Cell Processes

Membranes are vital structures for cellular life forms. As thin, hydrophobic films, they provide a physical barrier separating the aqueous cytoplasm from the outside world or from the interiors of other cellular compartments. They maintain a selective permeability for the import and export of water-soluble compounds, enabling the living cell to maintain a stable chemical environment for biological processes. Cell membranes are primarily composed of two crucial substances, lipids and proteins. Bacterial membranes can sense environmental changes or communication signals from other cells and they support different cell processes, including cell division, differentiation, protein secretion and supplementary protein functions. The original fluid mosaic model of membrane structure has been recently revised because it has become apparent that domains of different lipid composition are present in both eukaryotic and prokaryotic cell membranes. In this review, we summarize different aspects of phospholipid domain formation in bacterial membranes, mainly in Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. We describe the role of these lipid domains in membrane dynamics and the localization of specific proteins and protein complexes in relation to the regulation of cellular function.

Effective Charge and Membrane Binding, Mixing, and Permeation of Lipopeptides Characterized by Zeta Potential Measurements

We demonstrate the use of zeta potential measurements of liposomes to address membrane binding of peptides and surfactants, membrane-induced protonation and counterion binding effects, membrane asymmetry and permeation, and membrane domain formation. Instead of estimating membrane binding from the surface charge density by guessing the effective charge per molecule, we used what we refer to as an equi-activity evaluation to correct for binding and, hence, measure the effective charge. To this end, zeta potentials were recorded for an array of different lipid and peptide concentrations. It turns that the effective charge of a membrane-bound peptide is not straightforward to be guessed, because it may depend sensitively on membrane-induced (de)protonation and counterion-specific neutralization effects. The importance of the effective charge for trans-membrane flip-flop and interactions with other membrane components underlines the value of its direct measurement as explained here. Another interesting feature of the zeta potential is that it specifically reflects the charge density in the outer leaflet of the liposome. This allows for addressing the asymmetric binding of a peptide and detecting its threshold for transmembrane equilibration due to bilayer asymmetry stress or pore formation. Finally, composition-dependent changes of the apparent charge already at low membrane content may indicate the formation of peptide-rich domains. These approaches are demonstrated for the Bacillus lipopeptides surfactin and fengycin, as well as for SDS in different buffers.

Ankyrin-Based Trafficking and Scaffolding of Membrane Proteins: Implications for Plasma Membrane Stability, Formation, and Specialization

Through the collaborative actions of b-spectrin and ankyrin, a cytoskeletal adaptor protein, integral and peripheral membrane proteins find order and stability in the relatively fluid environment of the plasma membrane. Not only is the ankyrin/β-spectrin complex responsible for the proper targeting and retention of membrane proteins but it facilitates the formation of multi-protein complexes to maximize local signaling between membrane and effector proteins. Dysfunction in ankyrin or β-spectrin causes deficiencies in fundamental cellular properties such as membrane stability, excitability, and adhesion. This review focuses on the direct effects of ankyrin function on membrane proteins in terms of binding and stability, intracellular transport, membrane targeting and retention, and altered biophysical properties. We propose that ankyrin and β-spectrin are important for the normal progression of many membrane proteins along their biosynthetic pathway from stabilizing the membrane protein to it’s proper trafficking and eventual targeting and retention at membrane domains. The second half of the review addresses how an ankyrin/membrane protein interaction influences the local membrane environment with particular emphasis on membrane stability, membrane domain formation, and membrane domain specialization. We propose that not only are ankyrins necessary for erythroid membrane stability but they are required in some cells types for membrane domain formation and integral for the formation of specialized membrane domains in myocytes.

Curvature Stimulates Assembly of Gag Shell through Distinct Fluid-Like Intermediate

HIV particles assemble on the plasma membrane of a host cell. While the plasma membrane provides an integrative platform for the virus assembly, it also constitutes a mechanical barrier for viral budding and fission. Gag, the polyprotein forming a shell lining the envelope membrane of HIV, is sufficient to overcome this barrier: self-assembly of Gag into small clusters on the membrane surface leads to membrane bending and, ultimately, to production of virus-like membrane particles. The mechanisms behind membrane curvature creation by Gag remain controversial. Here we reveal that Gag adsorption and polymerization is modulated by membrane curvature. Negative curvatures stimulate formation of distinct fluid-like membrane domains tightly packed with Gag molecules, which further polymerizes into a stable protein shell. The nucleation of these domains happens at physiologically relevant high curvatures and Gag polymerization leads to stabilization of these highly bent membrane configurations. Our findings indicate a novel mechanism of negative curvature creation based upon curvature-driven polymerization of Gag and involving curvature-polymerization feedback.

Biophysical and Biological Behaviour of Ciprofloxacin and Ciprofloxacin Derivatives: A Route to Counteract Bacteria Resistance?

Studies of bacterial membranes are fundamental in the understanding and counteracting of bacterial resistance. Direct attack on bacterial membranes is thought to be a way to counteract bacterial resistance mechanisms, which are mostly based on intracellular adaptations. Therefore, a current trend in antibiotic research is specifically targeting bacterial membranes, leading to permeation of the membrane, inducing the formation of membrane domains and ultimately leading to cell death. The fact that such effects are not observed in the interaction with mammalian cells illustrates the capacity that such antibiotics have to discriminate between bacterial and mammalian cells, most likely due to the differences in the lipid composition of the two types of cell membranes.In this work we present fluorescence spectroscopy studies of the interaction of ciprofloxacin and phenanthroline/copper/ciprofloxacin complex with several lipidic mimetic systems. POPE/POPG (0.75:0.25), POPE/POPG/Cardiolipin (0.67:0.23:0.10), E. coli total lipid extract and DMPC liposomes were used and the results obtained point out to a very different behavior between ciprofloxacin and its copper complex. Preliminary results of the interaction of these compounds with OmpF proteoliposomes will also be presented. These results, together with biological data, suggest that the ciprofloxacin complex can be an alternative to counteract bacterial resistance to these antibiotics.Acknowledgements: The authors are grateful to Fundacao para a Ciencia e Tecnologia (FCT, Portugal) for funding through project PTDC/SAU-FAR/111414/2009

Eisosomes and Plasma Membrane Organization

A vast body of evidence coming from different microscopy techniques has been instrumental in concluding the 10-year-long debate on whether biological membranes presented lateral segregation of proteins and lipids. Currently, the existence of membrane domains in both eukaryotes and prokaryotes has been common ground. However, the mechanisms that sustain membrane domain formation and maintenance remain largely unknown. Our work is focused on the study of Eisosomes, recently discovered plasma membrane domains in S. cerevisiae. In a first piece of work, we identified new eisosomal components and also showed that eisosomes are involved in sphingolipid metabolism (1). Thereafter, we showed that Pil1 and Lsp1, the major proteinaceous components of eisosomes, are able to form self-assemblies that bind and curve membranes both in vivo and in vitro. We also showed that Lsp1 and Pil1 membrane-sculpting abilities are associated with the generation and organization of membrane domains (2). Thus, our currently published work support the hypothesis that a mechanism for membrane eisosome domain formation is membrane curvature generation directed by Pil1-Lsp1 assemblies. 1. Aguilar PS., et al. (2010) Nature Structural and Molecular Biology 17, 901-908. 2. Olivera-Couto A., et al. (2011) Molecular Biology of the Cell 22, 2360-2372.

Sterol affinity for phospholipid bilayers is influenced by hydrophobic matching between lipids and transmembrane peptides

Lipid self-organization is believed to be essential for shaping the lateral structure of membranes, but it is becoming increasingly clear that also membrane proteins can be involved in the maintenance of membrane architecture. Cholesterol is thought to be important for the lateral organization of eukaryotic cell membranes and has also been implicated to take part in the sorting of cellular transmembrane proteins. Hence, a good starting point for studying the influence of lipid–protein interactions on membrane trafficking is to find out how transmembrane proteins influence the lateral sorting of cholesterol in phospholipid bilayers. By measuring equilibrium partitioning of the fluorescent cholesterol analog cholestatrienol between large unilamellar vesicles and methyl-β-cyclodextrin the effect of hydrophobic matching on the affinity of sterols for phospholipid bilayers was determined. Sterol partitioning was measured in 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers with and without WALP19, WALP23 or WALP27 peptides. The results showed that the affinity of the sterol for the bilayers was affected by hydrophobic matching. An increasing positive hydrophobic mismatch led to stronger sterol binding to the bilayers (except in extreme situations), and a large negative hydrophobic mismatch decreased the affinity of the sterol for the bilayer. In addition, peptide insertion into the phospholipid bilayers was observed to depend on hydrophobic matching. In conclusion, the results showed that hydrophobic matching can affect lipid–protein interactions in a way that may facilitate the formation of lateral domains in cell membranes. This could be of importance in membrane trafficking.

Diffraction studies on natural and model lipid bilayers

In this study we have used neutron diffraction to examine the swelling behaviour and bilayer parameters of membranes reconstituted from polar lipids extracted from B. subtilis and model systems composed of synthetic phospholipids. Evidence for phase separation in the model system (lacking in Lysyl-PG, L-PG) is discussed in relation to its possible contribution to membrane domain formation through lipid-lipid interactions. Comparing these results with those obtained from the bilayers composed of lipids extracted from bacterial cells gives us some indication of the role of L-PG in the B. subtilis plasma membrane.

Limonoid Compounds Inhibit Sphingomyelin Biosynthesis by Preventing CERT Protein-dependent Extraction of Ceramides from the Endoplasmic Reticulum

To identify novel inhibitors of sphingomyelin (SM) metabolism, a new and selective high throughput microscopy-based screening based on the toxicity of the SM-specific toxin, lysenin, was developed. Out of a library of 2011 natural compounds, the limonoid, 3-chloro-8β-hydroxycarapin-3,8-hemiacetal (CHC), rendered cells resistant to lysenin by decreasing cell surface SM. CHC treatment selectively inhibited the de novo biosynthesis of SM without affecting glycolipid and glycerophospholipid biosynthesis. Pretreatment with brefeldin A abolished the limonoid-induced inhibition of SM synthesis suggesting that the transport of ceramide (Cer) from the endoplasmic reticulum to the Golgi apparatus is affected. Unlike the Cer transporter (CERT) inhibitor HPA-12, CHC did not change the transport of a fluorescent short chain Cer analog to the Golgi apparatus or the formation of fluorescent and short chain SM from the corresponding Cer. Nevertheless, CHC inhibited the conversion of de novo synthesized Cer to SM. We show that CHC specifically inhibited the CERT-mediated extraction of Cer from the endoplasmic reticulum membranes in vitro. Subsequent biochemical screening of 21 limonoids revealed that some of them, such as 8β-hydroxycarapin-3,8-hemiacetal and gedunin, which exhibits anti-cancer activity, inhibited SM biosynthesis and CERT-mediated extraction of Cer from membranes. Model membrane studies suggest that 8β-hydroxycarapin-3,8-hemiacetal reduced the miscibility of Cer with membrane lipids and thus induced the formation of Cer-rich membrane domains. Our study shows that certain limonoids are novel inhibitors of SM biosynthesis and suggests that some biological activities of these limonoids are related to their effect on the ceramide metabolism.

The photophysics of a Rhodamine head labeled phospholipid in the identification and characterization of membrane lipid phases

The organization of lipids and proteins into domains in cell membranes is currently an established subject within biomembrane research. Fluorescent probes have been used to detect and characterize these membrane lateral heterogeneities. However, a comprehensive understanding of the link between the probes’ fluorescence features and membrane lateral organization can only be achieved if their photophysical properties are thoroughly defined. In this work, a systematic characterization of N-(lyssamine Rhodamine B sulfonyl)-1,2-dioleoyl-sn-3-phosphatidylehanolamine (Rhod-DOPE) absorption and fluorescence behavior in gel, liquid-ordered (lo) and liquid-disordered (ld) model membranes was performed. In agreement with a previous study, it was found that Rhod-DOPE fluorescence lifetimes present a strong sensitivity to lipid phases, becoming significantly shorter in lo membranes as the probe membrane concentration increases. The sensitivity of Rhod-DOPE absorption and fluorescence properties to the membrane phase was further explored. In particular, the fluorescence lifetime sensitivity was shown to be a consequence of the enhanced Rhod-DOPE fluorescence dynamic self-quenching, due to the formation of probe-rich membrane domains in these condensed phases that cannot be considered as typical probe aggregates, as excitonic interaction is not observed. The highly efficient dynamic self-quenching was shown to be specific to lo phases, pointing to an important effect of membrane dipole potential in this process. Altogether, this work establishes how to use Rhod-DOPE fluorescence properties in the study of membrane lipid lateral heterogeneities, in particular cholesterol-enriched lipid rafts.

The Kupershtokh-Medvedev electrostrictive instability as possible mechanism of initiation of phase transitions, domains and pores in lipid membranes and influence of microwave irradiation on cell

One of the possible mechanisms of initiation of local phase transitions and formation of nonuniform structure of biological and model lipid membranes is suggested. It is based on anisotropic electrohydrodynamic instability of Kupershtokh and Medvedev in strong electric field relative to density perturbations. This mechanism may clarify initial stages of formation of membrane domains and pores, some aspects of cell signalization and influence of microwave irradiation of nonthermal intensity on living organisms.

The anti-adhesive mucin podocalyxin may help initiate the transperitoneal metastasis of high grade serous ovarian carcinoma

High grade serous ovarian tumors often metastasize transperitoneally, a process that begins when small tumor nodules de-adhere and are released into the fluid of the abdominal cavity where they float freely to reach new sites on the peritoneal wall. Podocalyxin, a small anti-adhesive sialomucin, has been shown to contribute to non-adhesive membrane domain formation in some epithelia and is overexpressed in a variety of cancers. We therefore assessed podocalyxin expression on a previously characterized tissue microarray and found that 87% (169/194) of high grade serous epithelial ovarian carcinomas were positive for podocalyxin. In addition, cell surface localization of podocalyxin was associated with a significant decrease in disease-free survival in these tumors. When podocalyxin was force-expressed in serous ovarian carcinoma-derived OVCAR-3 cells it was targeted to the cell surface and it decreased the adhesion of these cells to mesothelial monolayers, fibronectin and immobilized β1 integrin-binding antibodies. This decrease in adhesion was associated with a modest decrease in cell surface β1 integrin. In monolayer culture, podocalyxin was targeted to the free, apical domains of OVCAR-3 cells and it appeared to decrease β1 integrin levels on the attached basolateral domains of the same cells. Furthermore, in 3-dimensional basement membrane gel culture, the cells formed small, cohesive nodules and podocalyxin localized to membrane domains at the cell-basement membrane interface. Therefore, podocalyxin's ability to facilitate the formation of non-adhesive membrane domains may contribute to the formation of free-floating high grade serous tumor nodules during the initial steps of transperitoneal metastasis.

Lateral diffusion in equimolar mixtures of natural sphingomyelins with dioleoylphosphatidylcholine

Cellular membranes of mammals are composed of a complex assembly of diverse phospholipids. Sphingomyelin (SM) and phosphatidylcholine (PC) are important lipids of eukaryotic cellular membranes and neuronal tissues, and presumably participate in the formation of membrane domains, known as “rafts,” through intermolecular interaction and lateral microphase decomposition. In these two-dimensional membrane systems, lateral diffusion of lipids is an essential dynamic factor, which might even be indicative of lipid phase separation process. Here, we used pulsed field gradient nuclear magnetic resonance to study lateral diffusion of lipid components in macroscopically oriented bilayers composed of equimolar mixtures of natural SMs of egg yolk, bovine brain, bovine milk and dipalmitoylphosphatidylcholine (DPPC) with dioleoylphosphatidylcholine (DOPC). In addition, differential scanning calorimetry was used as a complementary technique to characterize the phase state of the lipid bilayers. In fully liquid bilayers, the lateral diffusion coefficients in both DOPC/DPPC and DOPC/SM systems exhibit mean values of the pure bilayers. For DOPC/SM bilayer system, this behavior can be explained by a model where most SM molecules form short-lived lateral domains with preferential SM–SM interactions occurring within them. However, for bilayers in the presence of their low-temperature gel phase, lateral diffusion becomes complicated and cannot simply be understood solely by a simple change in the liquid phase decomposition.

Lipid Raft Formation and Properties are Necessary and Sufficient to Explain the Properties of Membrane Domains in B. Burgdorferi and are Necessary for its Membrane Integrity

In eukaryotic cells liquid ordered (Lo) microdomains (rafts) rich in sphingolipids and cholesterol are believed to have an important role in many membrane functions. Borrelia burgdorferi (B.b), the bacterium that is the causative agent of Lyme disease, contains host-derived free cholesterol and large amounts of cholesteryl glycolipids. We recently found that microdomains large enough to be seen by transmission electron microscopy (TEM) form in its outer membrane (La Rocca et al (2010) Cell Host and Microbe 8, 331-342). In this study, the extent to which B.b. membrane domains have lipid raft properties was investigated. We carried out lipid substitutions in B.b. cells in which cholesterol lipids were partly substituted with various sterols having differing abilities to support ordered domain formation. TEM was used to visualize B.b. membrane domains, while in live, unfixed B.b. cells FRET and fluorescence anisotropy were used to detect domain segregation and measure overall membrane order, respectively. Both in ordinary B.b. cells and in cells after substitution with sterols that promote ordered domain formation in model membranes there was domain segregation and membrane order consistent with liquid ordered/liquid disordered domain co-existence. in contrast, after substitution with sterols that do not support ordered domain formation in model membranes there was no domain segregation and membrane order was more consistent with the liquid disordered state. Association of biotinylated lipids with B.b. domains required that they contain saturated rather than unsaturated acyl chains. Membrane integrity and growth after sterol substitution also required raft-forming sterols. Sterols not supporting ordered domain formation resulted in osmotic lysis of the organisms. These properties demonstrate that B. burgdorferi membrane domains have the properties of lipid rafts.

Determining the Mechanism of Peptide-Induced Dye-Flux from Giant Unilamellar Vesicles

A single-vesicle analysis was employed to compare the dye release mechanisms of several different peptides from phospholipid vesicles. These peptides, which have different amino acid sequences, are similar in that they all form amphipathic α-helices when bound to a phospholipid membrane. Peptides used include δ-lysin, DL-1 (a δ-lysin variant), TPW-3 (a transportan 10 variant), and CE-2 (a cecropin A variant). Previous hypotheses about the mechanisms of these peptides, being all-or-none or graded, were based on ANTS/DPX assay results in populations of large unilamellar vesicles (LUVs). δ-Lysin, CE-2, and TPW-3 were determined to exhibit the graded mechanism of dye release, while DL-1 was determined to exhibit the all-or-none mechanism. Here we use giant unilamellar vesicles (GUVs), which are observable under a microscope, to examine these mechanisms and further test these hypotheses. Preliminary results have shown CE-2 to exhibit graded dye release and DL-1 to exhibit all-or-none dye release, which are consistent with results obtained using LUVs. However, δ-lysin and TPW-3, which were determined to exhibit weaker graded mechanisms using LUVs, displayed characteristics of both the graded and all-or-none mechanisms in GUVs. We have also employed GUVs, coupled with fluorescence microscopy, to investigate two other areas of interest. The first is to determine if peptide accumulation on the membrane is concurrent with dye-flux, and if so, to what extent peptide accumulation is required to cause subsequent dye-flux. The second, is to use GUVs to study peptide-induced domain formation in membranes of various lipid compositions.This research was supported by NIH Grant GM072507.

Synergy of Liquid Ordered “Raft Like” Domains and Membrane Curvature in Promoting Sorting of Lipidated Proteins Such As NRas

Cellular membranes define cell boundaries and provide active means for proteins transport and compartmentalization to certain organelles. The prevailing mechanism underlying lipidated protein transport and sorting is based on their selective upconcentration in transient membrane domains of altered fluidity, termed “raft domains”. However the majority of biophysical studies have failed to report increased partition to the lo phase1. We recently illustrated that the farnesylated Gβγ subunit of G protein upconcentrates in highly curved areas and introduced the notion that membrane remodeling may act as a cue for regulating lipidated protein cellular distribution2.Here we capitalized on our developed nanorrays of surface tethered liposomes to quantify the precise effect of membrane curvature and domain formation on the redistribution of the abundant class of signaling proteins, the Ras2-4. Our results demonstrate for the first time that NRas proteins upconcentrate in highly curved areas showing ∼10 fold increased densities as compared to flat bilayers. We furthermore found that NRas does not selectively bind in flat raft domains as compared to ld phases. When however the lo “raft domains” where combined with high curvature a remarkable upconcentration ∼80 fold was documented in the highly curved areas. These findings indicate that lo domains enhance the intrinsic ability of membranes to promote lateral organization of lipidated proteins in highly curved areas.

Giant vesicles as cell models

Tremendous progress has been made in recent years in understanding the working of the living cell, including its micro-anatomy, signalling networks, and regulation of genes. However, an understanding of cellular phenomena using fundamental laws starting from first principles is still very far away. Part of the reason is that a cell is an active and exquisitely complex system where every part is linked to the other. Thus, it is difficult or even impossible to design experiments that selectively and exclusively probe a chosen aspect of the cell. Various kinds of idealised systems and cell models have been used to circumvent this problem. An important example is a giant unilamellar vesicle (GUV, also called giant liposome), which provides a cell-sized confined volume to study biochemical reactions as well as self-assembly processes that occur on the membrane. The GUV membrane can be designed suitably to present selected, correctly-oriented cell-membrane proteins, whose mobility is confined to two dimensions. Here, we present recent advances in GUV design and the use of GUVs as cell models that enable quantitative testing leading to insight into the working of real cells. We briefly recapitulate important classical concepts in membrane biophysics emphasising the advantages and limitations of GUVs. We then present results obtained over the last decades using GUVs, choosing the formation of membrane domains and cell adhesion as examples for in-depth treatment. Insight into cell adhesion obtained using micro-interferometry is treated in detail. We conclude by summarising the open questions and possible future directions.

Increased levels of the cytoplasmic domain of Crumbs repolarise developing Drosophila photoreceptors

Photoreceptor morphogenesis in Drosophila requires remodelling of apico-basal polarity and adherens junctions (AJs), and includes cell shape changes, as well as differentiation and expansion of the apical membrane. The evolutionarily conserved transmembrane protein Crumbs (Crb) organises an apical membrane-associated protein complex that controls photoreceptor morphogenesis. Expression of the small cytoplasmic domain of Crb in crb mutant photoreceptor cells (PRCs) rescues the crb mutant phenotype to the same extent as the full-length protein. Here, we show that overexpression of the membrane-tethered cytoplasmic domain of Crb in otherwise wild-type photoreceptor cells has major effects on polarity and morphogenesis. Whereas early expression causes severe abnormalities in apico-basal polarity and ommatidial integrity, expression at later stages affects the shape and positioning of AJs. This result supports the importance of Crb for junctional remodelling during morphogenetic changes. The most pronounced phenotype observed upon early expression is the formation of ectopic apical membrane domains, which often develop into a complete second apical pole, including ectopic AJs. Induction of this phenotype requires members of the Par protein network. These data point to a close integration of the Crb complex and Par proteins during photoreceptor morphogenesis and underscore the role of Crb as an apical determinant.