Changes in membrane lipid composition cause alterations in epithelial cell–cell adhesion structures in renal papillary collecting duct cells

In epithelial tissues, adherens junctions (AJ) mediate cell–cell adhesion by using proteins called E-cadherins, which span the plasma membrane, contact E-cadherin on other cells and connect with the actin cytoskeleton inside the cell. Although AJ protein complexes are inserted in detergent-resistant membrane microdomains, the influence of membrane lipid composition in the preservation of AJ structures has not been extensively addressed. In the present work, we studied the contribution of membrane lipids to the preservation of renal epithelial cell–cell adhesion structures. We biochemically characterized the lipid composition of membranes containing AJ complexes. By using lipid membrane-affecting agents, we found that such agents induced the formation of new AJ protein-containing domains of different lipid composition. By using both biochemical approaches and fluorescence microscopy we demonstrated that the membrane phospholipid composition plays an essential role in the in vivo maintenance of AJ structures involved in cell–cell adhesion structures in renal papillary collecting duct cells.     

Effects of curvature and cell–cell interaction on cell adhesion in microvessels

It has been found that both circulating blood cells and tumor cells are more easily adherent to curved microvessels than straight ones. This motivated us to investigate numerically the effect of the curvature of the curved vessel on cell adhesion. In this study, the fluid dynamics was carried out by the lattice Boltzmann method (LBM), and the cell dynamics was governed by the Newton’s law of translation and rotation. The adhesive dynamics model involved the effect of receptor-ligand bonds between circulating cells and endothelial cells (ECs). It is found that the curved vessel would increase the simultaneous bond number, and the probability of cell adhesion is increased consequently. The interaction between traveling cells would also affect the cell adhesion significantly. For two-cell case, the simultaneous bond number of the rear cell is increased significantly, and the curvature of microvessel further enhances the probability of cell adhesion.     

How does the plasma membrane participate in cellular signaling by receptors for immunoglobulin E?

Accumulating evidence strongly supports the view that the plasma membrane participates in transmembrane signaling by IgE-receptors (IgE-Fc epsilon RI) through the formation of lipid-based domains, also known as rafts. Ongoing biochemical and biophysical experiments investigate the composition, structure, and dynamics of the corresponding membrane components and how these are related to functional coupling between Fc epsilon RI and Lyn tyrosine kinase to initiate signaling in mast cells.     

The carboxy-terminus of the formin FMNL1ɣ bundles actin to potentiate adenocarcinoma migration

The formin family of proteins contributes to spatiotemporal control of actin cytoskeletal rearrangements during motile cell activities. The FMNL subfamily exhibits multiple mechanisms of linear actin filament formation and organization. Here we report novel actin-modifying functions of FMNL1 in breast adenocarcinoma migration models. FMNL1 is required for efficient cell migration and its three isoforms exhibit distinct localization. Suppression of FMNL1 protein expression results in a significant impairment of cell adhesion, migration, and invasion. Overexpression of FMNL1ɣ, but not FMNL1β or FMNL1α, enhances cell adhesion independent of the FH2 domain and FMNL1ɣ rescues migration in cells depleted of all three endogenous isoforms. While FMNL1ɣ inhibits actin assembly in vitro, it facilitates bundling of filamentous actin independent of the FH2 domain. The unique interactions of FMNL1ɣ with filamentous actin provide a new understanding of formin domain functions and its effect on motility of diverse cell types suggest a broader role than previously realized.     

The missing link: do cortical microtubules define plasma membrane nanodomains that modulate cellulose biosynthesis?

Cellulose production is a crucial aspect of plant growth and development. It is functionally linked to cortical microtubules, which self-organize into highly ordered arrays often situated in close proximity to plasma membrane-bound cellulose synthase complexes (CSCs). Although most models put forward to explain the microtubule-cellulose relationship have considered mechanisms by which cortical microtubule arrays influence the orientation of cellulose microfibrils, little attention has been paid to how microtubules affect the physicochemical properties of cellulose. A recent study using the model system Arabidopsis, however, indicates that microtubules can modulate the crystalline and amorphous content of cellulose microfibrils. Microtubules are required during rapid growth for reducing crystalline content, which is predicted to increase the degree to which cellulose is tethered by hemicellulosic polysaccharides. Such tethering is, in turn, critical for maintaining unidirectional cell expansion. In this article, we hypothesize that cortical microtubules influence the crystalline content of cellulose either by controlling plasma membrane fluidity or by modulating the deposition of noncellulosic wall components in the vicinity of the CSCs. We discuss the current limitations of imaging technology to address these hypotheses and identify the image acquisition and processing strategies that will integrate live imaging with super resolution three-dimensional information.     

The missing link: do cortical microtubules define plasma membrane nanodomains that modulate cellulose biosynthesis?

Cellulose production is a crucial aspect of plant growth and development. It is functionally linked to cortical microtubules, which self-organize into highly ordered arrays often situated in close proximity to plasma membrane-bound cellulose synthase complexes (CSCs). Although most models put forward to explain the microtubule–cellulose relationship have considered mechanisms by which cortical microtubule arrays influence the orientation of cellulose microfibrils, little attention has been paid to how microtubules affect the physicochemical properties of cellulose. A recent study using the model system Arabidopsis, however, indicates that microtubules can modulate the crystalline and amorphous content of cellulose microfibrils. Microtubules are required during rapid growth for reducing crystalline content, which is predicted to increase the degree to which cellulose is tethered by hemicellulosic polysaccharides. Such tethering is, in turn, critical for maintaining unidirectional cell expansion. In this article, we hypothesize that cortical microtubules influence the crystalline content of cellulose either by controlling plasma membrane fluidity or by modulating the deposition of noncellulosic wall components in the vicinity of the CSCs. We discuss the current limitations of imaging technology to address these hypotheses and identify the image acquisition and processing strategies that will integrate live imaging with super resolution three-dimensional information.     

The membrane scaffold CD82 regulates cell adhesion by altering α4 integrin stability and molecular density

Hematopoietic stem/progenitor cell (HSPC) interactions with the bone marrow microenvironment are important for maintaining HSPC self-renewal and differentiation. In recent work, we identified the tetraspanin protein, CD82, as a regulator of HPSC adhesion and homing to the bone marrow, although the mechanism by which CD82 mediated adhesion was unclear. In the present study, we determine that CD82 expression alters cell–matrix adhesion, as well as integrin surface expression. By combining the superresolution microscopy imaging technique, direct stochastic optical reconstruction microscopy, with protein clustering algorithms, we identify a critical role for CD82 in regulating the membrane organization of α4 integrin subunits. Our data demonstrate that CD82 overexpression increases the molecular density of α4 within membrane clusters, thereby increasing cellular adhesion. Furthermore, we find that the tight packing of α4 into membrane clusters depend on CD82 palmitoylation and the presence of α4 integrin ligands. In combination, these results provide unique quantifiable evidence of CD82’s contribution to the spatial arrangement of integrins within the plasma membrane and suggest that regulation of integrin density by tetraspanins is a critical component of cell adhesion.     

Host–pathogen interactions: Impenetrable barriers or entry portals? The role of cell–cell adhesion during infection

Cell-cell adhesion plays a fundamental role in cell polarity and organogenesis. It also contributes to the formation and establishment of physical barriers against microbial infections. However, a large number of pathogens, from viruses to bacteria and parasites, have developed countless strategies to specifically target cell adhesion molecules in order to adhere to and invade epithelial cells, disrupt epithelial integrity, and access deeper tissues for dissemination. The study of all these processes has contributed to the characterization of molecular machineries at the junctions of eukaryotic cells that have been better understood by using pathogens as probes.     

The CAR group of Ig cell adhesion proteins–Regulators of gap junctions?

Members of the CAR group of Ig-like type I transmembrane proteins mediate homotypic cell adhesion, share a common overall extracellular domain structure and are closely related at the amino acid sequence level. CAR proteins are often found at tight junctions and interact with intracellular scaffolding proteins, suggesting that they might modulate tight junction assembly or function. However, impairment of tight junction integrity has not been reported in mouse knockout models or zebrafish mutants of CAR members. In contrast, in the same knockout models deficits in gap junction communication were detected in several organ systems, including the atrioventricular node of the heart, smooth muscle cells of the intestine and the ureter and in Sertoli cells of the testes. Possible interactions between BT-IgSF and connexin41.8 on the disturbed pattern of pigment stripes found in zebrafish mutants and between ESAM and connexin43 during hematopoiesis in the mouse are also discussed. On the basis of the combined data and phenotypic similarities between CAR member mutants and connexin mutants I hypothesize that they primarily play a role in the organization of gap junction communication. Also see the video abstract here: https://youtu.be/i0yq2KhuDAE .     

Down-regulation of Pkd2 by siRNAs suppresses cell–cell adhesion in the mouse melanoma cells

The Pkd2 gene encodes an integral protein (~130 kDa), named polycystin-2 (PC-2). PC-2 is mainly involved in autosomal dominant polycystic kidney disease. Recently, polycystin-1/polycystin-2 complex has been shown to act as an adhesion complex mediating or regulating cell–cell or cell–matrix adhesion, suggesting that PC-2 may play a role in cell–cell/cell–matrix interactions. Here, we knocked down the expression of Pkd2 gene with small interfering RNAs (siRNAs) in the mouse melanoma cells (B16 cells), indicating that the cells transfected with the targeted siRNAs significantly suppressed cell–cell adhesion, but not cell–matrix adhesion, compared to the cells transfected with non-targeted control (NC) siRNA. This study provides the first directly functional evidence that PC-2 mediates cell–cell adhesion. Furthermore, we demonstrated that PC-2 modulated cell–cell adhesion may be, at least partially, associated with E-cadherin. Collectively, these findings for the first time showed that PC-2 may mediate cell–cell adhesion, at least partially, through E-cadherin.     

How does plasma membrane surface area accommodate alterations in guard cell volume during stomatal closing?

During stomatal movement, guard cells undergo considerable and repetitive variations in cell volume and consequently surface area over a period of minutes. Due to limited stretching capability of the plasma membrane, alterations in the surface area must accommodate the volume changes through membrane turnover. Using fluorescence imaging and electrophysiology techniques, extensive studies imply that endocytosis may be a critical mechanism for the plasma membrane turnover. In contrast to the conventional studies, using transmission electronic microscope in combination with laser confocal microscope so that the membrane turnover can be detected without a resolution limitation, our works, recently published in the Journal of Experimental Botany, has provided strong evidences that excretion and folding of plasma membrane are critical for the accommodation of the cell volume alterations in intact guard cells in Vicia faba L. These results have opened a new perspective on the mechanism for the membrane turnover during stomatal movement. In this addendum, we further discuss some key issues about the mechanisms for the accommodation of the cell volume alterations during stomatal movements.Key words: stomata, guard cell, plasma membrane, surface area, endocytosis, excretion, accommodationGuard cells control stomatal movement thereby regulating gas exchange in plants. During stomatal movement, guard cells undergo considerable and repetitive variations in cell volume and consequently surface area over a period of minutes. It was proposed that the alterations of the plasma membrane surface area could be up to 40%,¹ whereas the maximum possible stretching of membranes was limited to only about 2%.² Furthermore, due to the presence of a turgor pressure, it has been commonly thought that membrane infoldings should not occur in the guard cells.³ Therefore, it is reasonable to propose that alterations in the surface area must be accomplished by addition and removal of membrane material to and from the plasma membrane.⁴ While many studies imply that endocytosis most likely functions to accommodate the alterations in guard cell volume, many crucial questions about this mechanism deserve to be argued.     

The role of cell adhesion molecules in neurite outgrowth on Müller cells

The roles of neural cell adhesion molecule (NCAM), L1, N-cadherin, and integrin in neurite outgrowth on various substrates were studied. Antibodies against these cell surface molecules were added to explants of chick retina and the neurites from retinal ganglion cells were examined for effects of the antibodies on neurite length and fasciculation. On laminin, an anti-integrin antibody completely inhibited neurite outgrowth. The same antibody did not inhibit neurite outgrowth on polylysine or Müller cells. Antibodies to NCAM, L1, and N-cadherin did not significantly inhibit neurite outgrowth on laminin but produced significant inhibition on Müller cells. The inhibition of neurite outgrowth on glia by anti-L1 antibodies supports the hypothesis that L1 is capable of acting in a heterophilic binding mechanism. On laminin, both anti-N-cadherin and anti-L1 caused defasciculation of neurites from retinal ganglion cells, while anti-NCAM did not. None of these antibodies produced defasciculation on Müller cells. The results indicate that these three cell adhesion molecules may be very important in interactions with glia as axons grow from the retina to the tectum and may be less important in axon-axon interactions along this pathway. No evidence was found supporting the role of integrins in axon growth on Müller cells.     

The Na-K-ATPase α₁β₁ heterodimer as a cell adhesion molecule in epithelia

The ion gradients generated by the Na-K-ATPase play a critical role in epithelia by driving transepithelial transport of various solutes. The efficiency of this Na-K-ATPase-driven vectorial transport depends on the integrity of epithelial junctions that maintain polar distribution of membrane transporters, including the basolateral sodium pump, and restrict paracellular diffusion of solutes. The review summarizes the data showing that, in addition to pumping ions, the Na-K-ATPase located at the sites of cell-cell junction acts as a cell adhesion molecule by interacting with the Na-K-ATPase of the adjacent cell in the intercellular space accompanied by anchoring to the cytoskeleton in the cytoplasm. The review also discusses the experimental evidence on the importance of a specific amino acid region in the extracellular domain of the Na-K-ATPase β(1) subunit for the Na-K-ATPase trans-dimerization and intercellular adhesion. Furthermore, a possible role of N-glycans linked to the Na-K-ATPase β(1) subunit in regulation of epithelial junctions by modulating β(1)-β(1) interactions is discussed.     

Analysis of α3 GlyR single particle tracking in the cell membrane

Single particle tracking (SPT) of transmembrane receptors in the plasma membrane often reveals heterogeneous diffusion. A thorough interpretation of the displacements requires an extensive analysis suited for discrimination of different motion types present in the data. Here the diffusion pattern of the homomeric α3-containing glycine receptor (GlyR) is analyzed in the membrane of HEK 293 cells. More specifically, the influence of the α3 RNA splice variants α3K and α3L on lateral membrane diffusion of the receptor is revealed in detail. Using a combination of ensemble and local SPT analysis, free and anomalous diffusion parameters are determined. The GlyR α3 free diffusion coefficient is found to be 0.13 ± 0.01 μm²/s and both receptor variants display confined motion. The confinement probability level and residence time are significantly elevated for the α3L variant compared to the α3K variant. Furthermore, for the α3L GlyR, the presence of directed motion was also established, with a velocity matching that of saltatory vesicular transport. These findings reveal that α3 GlyRs are prone to different types of anomalous diffusion and reinforce the role of RNA splicing in determining lateral membrane trafficking.     

The role of membrane thickness in charged protein–lipid interactions

Charged amino acids are known to be important in controlling the actions of integral and peripheral membrane proteins and cell disrupting peptides. Atomistic molecular dynamics studies have shed much light on the mechanisms of membrane binding and translocation of charged protein groups, yet the impact of the full diversity of membrane physico-chemical properties and topologies has yet to be explored. Here we have performed a systematic study of an arginine (Arg) side chain analog moving across saturated phosphatidylcholine (PC) bilayers of variable hydrocarbon tail length from 10 to 18 carbons. For all bilayers we observe similar ion-induced defects, where Arg draws water molecules and lipid head groups into the bilayers to avoid large dehydration energy costs. The free energy profiles all exhibit sharp climbs with increasing penetration into the hydrocarbon core, with predictable shifts between bilayers of different thickness, leading to barrier reduction from 26 kcal/mol for 18 carbons to 6 kcal/mol for 10 carbons. For lipids of 10 and 12 carbons we observe narrow transmembrane pores and corresponding plateaus in the free energy profiles. Allowing for movements of the protein and side chain snorkeling, we argue that the energetic cost for burying Arg inside a thin bilayer will be small, consistent with recent experiments, also leading to a dramatic reduction in pK_a shifts for Arg. We provide evidence that Arg translocation occurs via an ion-induced defect mechanism, except in thick bilayers (of at least 18 carbons) where solubility-diffusion becomes energetically favored. Our findings shed light on the mechanisms of ion movement through membranes of varying composition, with implications for a range of charged protein–lipid interactions and the actions of cell-perturbing peptides. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Formation of a Bazooka–Stardust complex is essential for plasma membrane polarity in epithelia

Apical–basal polarity in Drosophila melanogaster epithelia depends on several evolutionarily conserved proteins that have been assigned to two distinct protein complexes: the Bazooka (Baz)–PAR-6 (partitioning defective 6)–atypical protein kinase C (aPKC) complex and the Crumbs (Crb)–Stardust (Sdt) complex. These proteins operate in a functional hierarchy, in which Baz is required for the proper subcellular localization of all other proteins. We investigated how these proteins interact and how this interaction is regulated. We show that Baz recruits Sdt to the plasma membrane by direct interaction between the Postsynaptic density 95/Discs large/Zonula occludens 1 (PDZ) domain of Sdt and a region of Baz that contains a phosphorylation site for aPKC. Phosphorylation of Baz causes the dissociation of the Baz–Sdt complex. Overexpression of a nonphosphorylatable version of Baz blocks the dissociation of Sdt from Baz, causing phenotypes very similar to those of crb and sdt mutations. Our findings provide a molecular mechanism for the phosphorylation-dependent interaction between the Baz–PAR-3 and Crb complexes during the establishment of epithelial polarity.     

Ras guanyl nucleotide releasing protein 2 affects cell viability and cell–matrix adhesion in ECV304 endothelial cells

Ras guanyl nucleotide releasing proteins (RasGRPs) are guanine nucleotide exchange factors that activate Ras and Rap. We recently reported that xrasgrp2, which is a homolog of the human rasgrp2, plays a role in vasculogenesis and/or angiogenesis during early development of Xenopus embryos. However, the function of RasGRP2 in human vascular endothelium remains unknown. Therefore we aimed to analyze the function of human RasGRP2 in vascular endothelial cells. RasGRP2 overexpression did not increase Ras activation. However, it slightly increased Ras expression and increased proliferation in ECV304 cells. Furthermore, RasGRP2 overexpression increased Rap1 activation and cell–matrix adhesion in ECV304 cells. These data demonstrate that RasGRP2 increases cell viability and cell–matrix adhesion through increased Ras expression and Rap1 activation, respectively, in endothelial cells.