Epidermal Growth Factor Receptor Activation Remodels the Plasma Membrane Lipid Environment To Induce Nanocluster Formation

Signal transduction is regulated by the lateral segregation of proteins into nanodomains on the plasma membrane. However, the molecular mechanisms that regulate the lateral segregation of cell surface receptors, such as receptor tyrosine kinases, upon ligand binding are unresolved. Here we used high-resolution spatial mapping to investigate the plasma membrane nanoscale organization of the epidermal growth factor (EGF) receptor (EGFR). Our data demonstrate that in serum-starved cells, the EGFR exists in preformed, cholesterol-dependent, actin-independent nanoclusters. Following stimulation with EGF, the number and size of EGFR nanoclusters increase in a time-dependent manner. Our data show that the formation of EGFR nanoclusters requires receptor tyrosine kinase activity. Critically, we show for the first time that production of phosphatidic acid by phospholipase D2 (PLD2) is essential for ligand-induced EGFR nanocluster formation. In accordance with its crucial role in regulating EGFR nanocluster formation, we demonstrate that modulating PLD2 activity tunes the degree of EGFR nanocluster formation and mitogen-activated protein kinase signal output. Together, these data show that EGFR activation drives the formation of signaling domains by regulating the production of critical second-messenger lipids and modifying the local membrane lipid environment.The epidermal growth factor (EGF) receptor (EGFR) is a single transmembrane domain protein that possesses intrinsic tyrosine kinase (TK) activity. Ligand binding to the extracellular domain induces conformational changes that promote activation of the intracellular TK domain. The kinase domain then autophosphorylates a number of tyrosine residues in the C-terminal region of the protein, creating docking sites for adapter and effector proteins. Thus, the active form of the EGFR could reasonably be expected to be a dimer. However, recent studies using single-molecule imaging, image correlation spectroscopy (ICS), fluorescence correlation spectroscopy (FCS), and immunoelectron microscopy (immuno-EM) show that the EGFR is, in fact, nonrandomly organized into oligomers on the plasma membrane (6, 7, 16, 34, 44). ICS measurements estimate that, in the absence of ligand, there are, on average, 2.2 EGFRs per cluster, which increases to 3.7 receptors per cluster upon stimulation (7). Single-molecule tracking experiments also suggest that unliganded EGFRs continually fluctuate between monomers and dimers that are primed for activation (5). Furthermore, the organization of the EGFR is dynamic and clustering of the EGFR increases over time after EGF stimulation (7, 16). However, neither the precise role of EGFR oligomerization in signal transduction nor the mechanisms driving oligomer formation have been resolved.The organization of the EGFR into oligomers is dependent upon cellular cholesterol. Saffarian et al., using FCS, estimated that 70% of EGFRs exist as monomers, 20% as dimers, and 10% as oligomers (34). However, depletion of cholesterol decreases the percentage of monomeric receptors and increases the proportion of oligomeric receptors. Cholesterol depletion and actin depolymerization also alter the diffusion coefficient of the EGFR and the confinement area size (22). The finding that EGFR membrane organization is dependent upon cholesterol is of particular interest because a number of studies have demonstrated that EGFR activity is negatively regulated by cholesterol (4, 23, 28, 32).Phospholipase D2 (PLD2) hydrolyzes phosphatidylcholine (PC) to produce choline and phosphatidic acid (PA). PLD2 is localized to the plasma membrane (10), associates with the EGFR (39), and is rapidly activated upon EGF stimulation, leading to increased production of PA (15, 38, 39). A number of lines of evidence suggest that PA is an important mediator of EGFR action. First, exogenous PA is mitogenic when incubated with cells (17, 19, 42, 45). Second, direct interaction with membrane PA regulates the activity of a number of components downstream of the EGFR, including Sos (47) and Raf (12, 13, 30, 31).In the current study, we used high-resolution spatial analysis techniques to investigate EGFR plasma membrane organization. Using these approaches, we identified PA as the key molecular component responsible for driving EGFR nanocluster formation in response to EGF binding and demonstrated that the level of PLD2 activity regulates the duration of mitogen-activated protein kinase (MAPK) signal output.     

<Emphasis Type="Italic">TRAF1/C5</Emphasis>polymorphism is not associated with increased mortality in rheumatoid arthritis: two large longitudinal studies

Introduction  

Recently an association between a genetic variation in TRAF1/C5 and mortality from sepsis or cancer was found in rheumatoid arthritis (RA). The most prevalent cause of death, cardiovascular disease, may have been missed in that study, since patients were enrolled at an advanced disease stage. Therefore, we used an inception cohort of RA patients to investigate the association between TRAF1/C5 and cardiovascular mortality, and replicate the findings on all-cause mortality. As TRAF1/C5 associated mortality may not be restricted to RA, we also studied a large cohort of non-RA patients.     

Accelerated hand bone mineral density loss is associated with progressive joint damage in hands and feet in recent-onset rheumatoid arthritis

Introduction

To investigate whether accelerated hand bone mineral density (BMD) loss is associated with progressive joint damage in hands and feet in the first year of rheumatoid arthritis (RA) and whether it is an independent predictor of subsequent progressive total joint damage after 4 years.

Methods

In 256 recent-onset RA patients, baseline and 1-year hand BMD was measured in metacarpals 2-4 by digital X-ray radiogrammetry. Joint damage in hands and feet were scored in random order according to the Sharp-van der Heijde method at baseline and yearly up to 4 years.

Results

68% of the patients had accelerated hand BMD loss (>-0.003 g/cm²) in the first year of RA. Hand BMD loss was associated with progressive joint damage after 1 year both in hands and feet with odds ratios (OR) (95% confidence intervals [CI]) of 5.3 (1.3-20.9) and 3.1 (1.0-9.7). In univariate analysis, hand BMD loss in the first year was a predictor of subsequent progressive total joint damage after 4 years with an OR (95% CI) of 3.1 (1.3-7.6). Multivariate analysis showed that only progressive joint damage in the first year and anti-citrullinated protein antibody positivity were independent predictors of long-term progressive joint damage.

Conclusions

In the first year of RA, accelerated hand BMD loss is associated with progressive joint damage in both hands and feet. Hand BMD loss in the first year of recent-onset RA predicts subsequent progressive total joint damage, however not independent of progressive joint damage in the first year.     

How the sea squirt nucleus tells mesoderm not to be endoderm

Sea squirts are simple invertebrate chordates. In this issue of Developmental Cell, Takatori et?al. show nuclear migration within ascidian mesendodermal cells enables polarized localization of Not mRNA, which encodes a homeobox protein that distinguishes mesoderm from endoderm fates. The link between nuclear migration and mRNA localization suggests exciting parallels with protostomes.     

Cell and molecular biology of SAE, a cell line from the spiny dogfish shark, Squalus acanthias

Cartilaginous fish, primarily sharks, rays and skates (elasmobranchs), appeared 450 million years ago. They are the most primitive vertebrates, exhibiting jaws and teeth, adaptive immunity, a pressurized circulatory system, thymus, spleen, and a liver comparable to that of humans. The most used elasmobranch in biomedical research is the spiny dogfish shark, Squalus acanthias. Comparative genomic analysis of the dogfish shark, the little skate (Leucoraja erincea), and other elasmobranchs have yielded insights into conserved functional domains of genes associated with human liver function, multidrug resistance, cystic fibrosis, and other biomedically relevant processes. While genomic information from these animals is informative in an evolutionary framework, experimental verification of functions of genomic sequences depends heavily on cell culture approaches. We have derived the first multipassage, continuously proliferating cell line of a cartilaginous fish. The line was initiated from embryos of the spiny dogfish shark. The cells were maintained in a medium modified for fish species and supplemented with cell type-specific hormones, other proteins and sera, and plated on a collagen substrate. SAE cells have been cultured continuously for three years. These cells can be transfected by plasmids and have been cryopreserved. Expressed Sequence Tags generated from a normalized SAE cDNA library included a number of markers for cartilage and muscle, as well as proteins influencing tissue differentiation and development, suggesting that SAE cells may be of mesenchymal stem cell origin. Examination of SAE EST sequences also revealed a cartilaginous fish-specific repetitive sequence that may be evidence of an ancient mobile genetic element that most likely was introduced into the cartilaginous fish lineage after divergence from the lineage leading to teleosts.     

Rethinking the dispersal of Homo sapiens out of Africa

Current fossil, genetic, and archeological data indicate that Homo sapiens originated in Africa in the late Middle Pleistocene. By the end of the Late Pleistocene, our species was distributed across every continent except Antarctica, setting the foundations for the subsequent demographic and cultural changes of the Holocene. The intervening processes remain intensely debated and a key theme in hominin evolutionary studies. We review archeological, fossil, environmental, and genetic data to evaluate the current state of knowledge on the dispersal of Homo sapiens out of Africa. The emerging picture of the dispersal process suggests dynamic behavioral variability, complex interactions between populations, and an intricate genetic and cultural legacy. This evolutionary and historical complexity challenges simple narratives and suggests that hybrid models and the testing of explicit hypotheses are required to understand the expansion of Homo sapiens into Eurasia.     

Increased incidence of pregnancy complications in women who later develop scleroderma: a case control study

Introduction  

Studies have shown that fetal progenitor cells persist in maternal blood or bone marrow for more than 30 years after delivery. Increased trafficking of fetal cells occurs during pregnancy complications, such as hypertension, preeclampsia, miscarriage and intra-uterine growth restriction (IUGR). Women with these pregnancy complications are significantly more often HLA-class II compatible with their spouses. Women who later develop scleroderma also give birth to an HLA-class II child more often. From these prior studies we hypothesized that preeclampsia and other pregnancy complications could be associated with increased levels of fetal cell trafficking, and later be involved in the development of scleroderma.     

RETRACTED ARTICLE: Mast cells are the main interleukin 17-positive cells in anticitrullinated protein antibody-positive and -negative rheumatoid arthritis and osteoarthritis synovium

Introduction  

Mast cells have been implicated to play a functional role in arthritis, especially in autoantibody-positive disease. Among the cytokines involved in rheumatoid arthritis (RA), IL-17 is an important inflammatory mediator. Recent data suggest that the synovial mast cell is a main producer of IL-17, although T cells have also been implicated as prominent IL-17 producers as well. We aimed to identify IL-17 expression by mast cells and T cells in synovium of arthritis patients.     

The relation between cartilage biomarkers (C2C,C1,2C,CS846, and CPII) and the long-term outcome of rheumatoid arthritis patients within the CAMERA trial

Introduction  

The aim of this study was to investigate whether serum biomarker levels of C2C, C1,2C, CS846, and CPII can predict the long-term course of disease activity and radiographic progression early in the disease course of rheumatoid arthritis (RA).     

Pore-forming toxins induce multiple cellular responses promoting survival

Pore-forming toxins (PFTs) are secreted proteins that contribute to the virulence of a great variety of bacterial pathogens. They inflict one of the more disastrous damages a target cell can be exposed to: disruption of plasma membrane integrity. Since this is an ancient form of attack, which bears similarities to mechanical membrane damage, cells have evolved response pathways to these perturbations. Here, it is reported that PFTs trigger very diverse yet specific response pathways. Many are triggered by the decrease in cytoplasmic potassium, which thus emerges as a central regulator. Upon plasma membrane damage, cells activate signalling pathways aimed at restoring plasma membrane integrity and ion homeostasis. Interestingly these pathways do not require protein synthesis. Cells also trigger signalling cascades that allow them to enter a quiescent-like state, where minimal energy is consumed while waiting for plasma membrane damage to be repaired. More specifically, protein synthesis is arrested, cytosolic constituents are recycled by autophagy and energy is stored in lipid droplets.