dissipative-microgravimetry-to-study-binding-dynamics-phospholipid

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

University of Münster

TRPV5 and TRPV6 constitute the Ca(2+) influx pathway in a variety of epithelial cells. Here, we identified S100A10 as the first auxiliary protein of these epithelial Ca(2+) channels using yeast two-hybrid and GST pull-down assays. This S100 protein forms a heterotetrameric complex with annexin 2 and associates specifically with the conserved sequence VATTV located in the C-terminal tail of TRPV5 and TRPV6. Of these five amino acids, the first threonine plays a crucial role since the corresponding mutants (TRPV5 T599A and TRPV6 T600A) exhibited a diminished capacity to bind S100A10, were redistributed to a subplasma membrane area and did not display channel activity. Using GST pull-down and co-immunoprecipitation assays we demonstrated that annexin 2 is part of the TRPV5-S100A10 complex. Furthermore, the S100A10-annexin 2 pair colocalizes with the Ca(2+) channels in TRPV5-expressing renal tubules and TRPV6-expressing duodenal cells. Importantly, downregulation of annexin 2 using annexin 2-specific small interfering RNA inhibited TRPV5 and TRPV6-mediated currents in transfected HEK293 cells. In conclusion, the S100A10-annexin 2 complex plays a crucial role in routing of TRPV5 and TRPV6 to plasma membrane.

Functional expression of the epithelial Ca(2+) channels (TRPV5 and TRPV6) requires association of the S100A10-annexin 2 complex.

Annexin A9 is a novel member of the annexin family of Ca(2+) and phospholipid binding proteins which has so far only been identified in EST data bases and whose deduced protein sequence shows mutations in residues considered crucial for Ca(2+) coordination in other annexins. To elucidate whether the annexin A9 protein is expressed as such and to characterize its biochemical properties we probed cell extracts with specific anti-annexin A9 antibodies and developed a recombinant expression system. We show that the protein is found in HepG2 hepatoma cell lysates and that a green fluorescent protein-tagged form is abundantly expressed in the cytosol of HeLa cells. Recombinant expression in bacteria yields a soluble protein that can be enriched by conventional chromatographic procedures. The protein is capable of binding phosphatidylserine containing liposomes albeit only at Ca(2+) concentrations exceeding 2 mM. Moreover and in contrast to other annexins this binding appears to be irreversible as the liposome-bound annexin A9 cannot be released by Ca(2+) chelation. These results indicate that annexin A9 is a unique member of the annexin family whose intracellular activity is not subject to Ca(2+) regulation.

Atypical properties displayed by annexin A9, a novel member of the annexin family of Ca(2+) and lipid binding proteins.

Annexins are a well-known multigene family of Ca(2+)-regulated phospholipid-binding and membrane-binding proteins. Recent work employing annexin-knockdown or - knockout models has provided new insights into the biological functions of different annexin proteins. Transient annexin depletion by RNA interference and the expression of dominant-negative mutant proteins has revealed roles for the proteins in membrane processes ranging from the control of membrane structure to certain membrane transport phenomena. Although such functions correlate well with the ability of annexins to interact with cellular membranes in a reversible and regulated manner, some activities are membrane independent, probably because annexins can also engage in specific protein-protein interactions. Among other things, this is evident in annexin A1- and A2-knockout mice, which show impaired regulation of neutrophil extravasation and defects in plasmin generation, respectively.

Annexins--unique membrane binding proteins with diverse functions.

Phosphorylation of the Ca2+ and membrane-binding protein annexin 1 by epidermal growth factor (EGF) receptor tyrosine kinase has been thought to be involved in regulation of the EGF receptor trafficking. To elucidate the interaction of annexin 1 during EGF receptor internalization, we followed the distribution of annexin 1-GFP fusion proteins at sites of internalizing EGF receptors. The observed association of annexin 1 with EGF receptors was confirmed by immunoprecipitation. We found that this interaction was independent of a functional phosphorylation site in the annexin 1 N-terminal domain but mediated through the Ca2+ binding core domain.

Specific association of annexin 1 with plasma membrane-resident and internalized EGF receptors mediated through the protein core domain.

Annexin A8 is a poorly characterized member of the annexin family of Ca2+-regulated membrane binding proteins. Initially only identified at the cDNA level it had been tentatively linked to acute promyelocytic leukaemia (APL) due to its high and regulated expression in APL-derived cells. Here we identify unique properties of the annexin A8 protein. We show that it binds Ca2+-dependently and with high specificity to phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2) and is also capable of interacting with F-actin. In line with these characteristics annexin A8 is recruited to F-actin-associated PtdIns(4,5)P2-rich membrane domains formed in HeLa cells upon infection with non-invading enteropathogenic Escherichia coli. These properties suggest a role of annexin A8 in the organization of certain actin-associated membrane domains.

Annexin A8 displays unique phospholipid and F-actin binding properties.

S100A10, also known as p11 or annexin 2 light chain, is a member of the S100 family of small, dimeric EF hand-type Ca(2+)-binding proteins that generally modulate cellular target proteins in response to intracellular Ca(2+) signals. In contrast to all other S100 proteins, S100A10 is Ca(2+) insensitive because of amino acid replacements in its Ca(2+)-binding loops that lock the protein in a permanently active state. Within cells, the majority of S100A10 resides in a tight heterotetrameric complex with the peripheral membrane-binding protein annexin A2 that directs the complex to specific target membranes, in particular the plasma membrane and the membrane of early endosomes. Several other Ca(2+)-independent interaction partners of S100A10 have been described in the recent past. Many of these interactions, which have been shown to be of functional significance for the respective partner, involve plasma membrane-resident proteins. In most of these cases, S100A10, probably residing in a complex with annexin A2, appears to regulate the intracellular trafficking of the respective target protein and thus its functional expression at the cell surface. In this paper, we review the current information on S100A10 protein interactions placing a particular emphasis on data that contribute to an understanding of the mechanistic basis of the S100A10 action. Based on these data, we propose that S100A10 functions as a linker tethering certain transmembrane proteins to annexin A2 thereby assisting their traffic to the plasma membrane and/or their firm anchorage at certain membrane sites.

S100A10/p11: family, friends and functions.

The epithelial Ca(2+) channel TRPV5 plays an essential role in transcellular Ca(2+) transport and is one of the most Ca(2+)-selective members of the transient receptor potential superfamily. Regulation of the abundance of TRPV5 at the cell surface is critical in body Ca(2+) homeostasis. However, little is known about the mechanisms underlying TRPV5 endo- and exocytosis. Here, we show that TRPV5 is constitutively internalized in a dynamin- and clathrin-dependent manner. Internalized TRPV5 first appears in small vesicular structures and then localizes to perinuclear structures positive for Rab11a. TRPV5 has a half-life of more than 8 h and is stable even after internalization from the cell surface for more than 3 h. Disruption of cell surface delivery of newly synthesized TRPV5 by brefeldin A does not reduce TRPV5-mediated Ca(2+) influx in cells, suggesting the presence of a stable intracellular pool of the channel capable of recycling back to the surface. Furthermore, the endocytic recycling kinetics is decreased upon treatment with Ca(2+) chelator BAPTA-AM, indicating that the channel's trafficking pathways are dynamically controlled by Ca(2+).

TRPV5 is internalized via clathrin-dependent endocytosis to enter a Ca2+-controlled recycling pathway.

Cell adhesion and motility require a dynamic remodelling of the membrane-associated actin cytoskeleton in response to extracellular stimuli that are primarily transmitted through receptor tyrosine kinases. In a cellular model system for tyrosine phosphorylation-based growth factor signaling, we observed that annexin A2 is tyrosine-phosphorylated upon insulin receptor activation. The phosphorylation precedes peripheral actin accumulations and subsequent cell detachment. These morphological changes are inhibited by annexin A2 depletion and require Rho/ROCK signaling downstream of tyrosine-phosphorylated annexin A2. A phospho-mimicking annexin A2 mutant is sufficient to drive peripheral actin accumulation and the resulting cell detachment in the absence of insulin stimulation. Thus, a tyrosine phosphorylation switch in annexin A2 is an important event in triggering Rho/ROCK-dependent and actin-mediated changes in cell morphology associated with the control of cell adhesion.

Tyrosine phosphorylation of annexin A2 regulates Rho-mediated actin rearrangement and cell adhesion.

Endocytosis of activated growth factor receptors regulates spatio-temporal cellular signaling. In the case of the EGF receptor, sorting into multivesicular bodies (MVBs) controls signal termination and subsequently leads to receptor degradation in lysosomes. Annexin A1, a Ca(2+)-regulated membrane binding protein often deregulated in human cancers, interacts with the EGF receptor and is phosphorylated by internalized EGF receptor on endosomes. Most relevant for EGF receptor signal termination, annexin A1 is required for the formation of internal vesicles in MVBs that sequester ligand-bound EGF receptor away from the limiting membrane. To elucidate the mechanism underlying annexin A1-dependent EGF receptor trafficking we employed an N-terminally truncated annexin A1 mutant that lacks the EGF receptor phosphorylation site and the site for interaction with its protein ligand S100A11. Overexpression of this dominant-negative mutant induces a delay in EGF-induced EGF receptor transport to the LAMP1-positive late endosomal/lysosomal compartment and impairs ligand-induced EGF receptor degradation. Consistent with these findings, EGF-stimulated EGF receptor and MAP kinase pathway signaling is prolonged. Importantly, depletion of S100A11 also results in a delayed EGF receptor transport and prolonged MAP kinase signaling comparable to the trafficking defect observed in cells expressing the N-terminally truncated annexin A1 mutant. These results strongly suggest that the function of annexin A1 as a regulator of EGF receptor trafficking, degradation and signaling is critically mediated through an N-terminal interaction with S100A11 in the endosomal compartment. This interaction appears to be essential for lysosomal targeting of the EGF receptor, possibly by providing a physical scaffold supporting inward vesiculation in MVBs. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Disruption of the annexin A1/S100A11 complex increases the migration and clonogenic growth by dysregulating epithelial growth factor (EGF) signaling.

Annexin A10 is the latest identified member of the annexin family of Ca(2+)- and phospholipid-binding proteins. In previous studies, downregulation of annexin A10 was correlated with dedifferentiation, invasion, and tumor progression, pointing to a possible tumor suppressor role. However, the biochemical characteristics and functions of annexin A10 remain unknown. We show that annexin A10 displays biochemical characteristics atypical for an annexin, indicating a Ca(2+)- and membrane-binding-independent function. Annexin A10 co-localizes with the mRNA-binding proteins SFPQ and PSPC1 at paraspeckles, an only recently discovered nuclear body, and decreases paraspeckle numbers when overexpressed in HeLa cells. In addition, annexin A10 relocates to dark perinucleolar caps upon transcriptional inhibition of RNA polymerase II. We mapped the cap-binding function of annexin A10 to the proximal part of the core domain, which is missing in the short isoform of annexin A10, and show its independence from the remaining functional type II Ca(2+)-binding site. In contrast to this, paraspeckle recruitment required additional core regions and was negatively affected by the mutation of the last type II Ca(2+)-binding site. Additionally, we show that overexpression of annexin A10 in HeLa cells increases their sensitivity to apoptosis and reduces colony formation. The identification of unique nuclear and biochemical characteristics of annexin A10 points towards its membrane-independent role in paraspeckle-associated mRNA regulation or processing.

The tumor suppressor annexin A10 is a novel component of nuclear paraspeckles.

Influenza is caused by influenza A virus (IAV), an enveloped, negative-stranded RNA virus that derives its envelope lipids from the host cell plasma membrane. Here, we examined the functional role of cellular cholesterol in the IAV infection cycle. We show that shifting of cellular cholesterol pools via the Ca(2+)-regulated membrane-binding protein annexin A6 (AnxA6) affects the infectivity of progeny virus particles. Elevated levels of cellular AnxA6, which decrease plasma membrane and increase late endosomal cholesterol levels, impaired IAV replication and propagation, whereas RNA interference-mediated AnxA6 ablation increased viral progeny titers. Pharmacological accumulation of late endosomal cholesterol also diminished IAV virus propagation. Decreased IAV replication caused by upregulated AnxA6 expression could be restored either by exogenous replenishment of host cell cholesterol or by ectopic expression of the late endosomal cholesterol transporter Niemann-Pick C1 (NPC1). Virus released from AnxA6-overexpressing cells displayed significantly reduced cholesterol levels. Our results show that IAV replication depends on maintenance of the cellular cholesterol balance and identify AnxA6 as a critical factor in linking IAV to cellular cholesterol homeostasis.

Annexin A6-balanced late endosomal cholesterol controls influenza A replication and propagation.

To enable leukocyte adhesion to activated endothelium, the leukocyte receptor P-selectin is released from Weibel-Palade bodies (WPB) to the endothelial cell surface where it is stabilized by CD63. Here we report that loss of annexin A8 (anxA8) in human umbilical vein endothelial cells (HUVEC) strongly decreases cell surface presentation of CD63 and P-selectin, with a concomitant reduction in leukocyte rolling and adhesion. We confirm the compromised leukocyte adhesiveness in inflammatory-activated endothelial venules of anxA8-deficient mice. We find that WPB of anxA8-deficient HUVEC contain less CD63, and that this is caused by improper transport of CD63 from late multivesicular endosomes to WPB, with CD63 being retained in intraluminal vesicles. Consequently, reduced CD63 cell surface levels are seen following WPB exocytosis, resulting in enhanced P-selectin re-internalization. Our data support a model in which anxA8 affects leukocyte recruitment to activated endothelial cells by supplying WPB with sufficient amounts of the P-selectin regulator CD63. 

Annexin A8 controls leukocyte recruitment to activated endothelial cells via cell surface delivery of CD63.

The physiological and pathological process of angiogenesis relies on orchestrated endothelial cell (EC) adhesion, migration and formation of new vessels. Here we report that human umbilical vein endothelial cells (HUVECs) deficient in Annexin A8 (AnxA8), a member of the annexin family of Ca- and membrane binding proteins, are strongly deficient in their ability to sprout in response to vascular endothelial growth factor (VEGF)-A, and are strongly impaired in their ability to migrate and adhere to β1 integrin-binding extracellular matrix (ECM) proteins. We find that these cells are defective in the formation of complexes containing the tetraspanin CD63, the main VEGF-A receptor VEGFR2, and the β1 integrin subunit, on the cell surface. We observe that upon VEGF-A activation of AnxA8-depleted HUVECs, VEGFR2 internalization is reduced, phosphorylation of VEGFR2 is increased, and the spatial distribution of Tyr577-phosphorylated focal adhesion kinase (pFAK577) is altered. We conclude that AnxA8 affects CD63/VEGFR2/β1 integrin complex formation, leading to hyperactivation of the VEGF-A signal transduction pathway, and severely disturbed VEGF-A-driven angiogenic sprouting.

Annexin A8 promotes VEGF-A driven endothelial cell sprouting.

Cooperative binding promotes demand-driven recruitment of AnxA8 to cholesterol-containing membranes.

The vertebrate annexin superfamily (AnxA) consists of 12 members of a calcium (Ca) and phospholipid binding protein family which share a high structural homology. In keeping with this hallmark feature, annexins have been implicated in the Ca-controlled regulation of a broad range of membrane events. In this review, we identify and discuss several themes of annexin actions that hold a potential therapeutic value, namely, the regulation of the immune response and the control of tissue homeostasis, and that repeatedly surface in the annexin activity profile. Our aim is to identify and discuss those annexin properties which might be exploited from a translational science and specifically, a clinical point of view.

Annexins in Translational Research: Hidden Treasures to Be Found.

To transfer the viral genome into the host cell cytoplasm, internalized influenza A virus (IAV) particles depend on the fusion of the IAV envelope with host endosomal membranes. The antiviral host interferon (IFN) response includes the upregulation of interferon-induced transmembrane protein 3 (IFITM3), which inhibits the release of the viral content into the cytosol. Although IFITM3 induction occurs concomitantly with late endosomal/lysosomal (LE/L) cholesterol accumulation, the functional significance of this process is not well understood. Here we report that LE/L cholesterol accumulation itself plays a pivotal role in the early antiviral defense. We demonstrate that inducing LE/L cholesterol accumulation is antiviral in non-IFN-primed cells, restricting incoming IAV particles and impairing mixing of IAV/endosomal membrane lipids. Our results establish a protective function of LE/L cholesterol accumulation and suggest endosomal cholesterol balance as a possible antiviral target. With annual epidemics occurring in all parts of the world and the risk of global outbreaks, influenza A virus (IAV) infections remain a major threat to public health. Infected host cells detect viral components and mount an interferon (IFN)-mediated response to restrict virus propagation and spread of infection. Identification of cellular factors and underlying mechanisms that establish such an antiviral state can provide novel strategies for the development of antiviral drugs. The contribution of LE/L cholesterol levels, especially in the context of the IFN-induced antiviral response, has remained controversial so far. Here, we report that accumulation of cholesterol in the LE/L compartment contributes to the IFN-induced host cell defense against incoming IAV. Our results establish cholesterol accumulation in LE/L  as a novel antiviral barrier and suggest the endosomal cholesterol balance as a putative druggable host cell factor in IAV infection.

Article	Year
Functional expression of the epithelial Ca(2+) channels (TRPV5 and TRPV6) requires association of the S100A10-annexin 2 complex. The EMBO journal\| PubMed ID: 12660155	2003
Atypical properties displayed by annexin A9, a novel member of the annexin family of Ca(2+) and lipid binding proteins. FEBS letters\| PubMed ID: 12832069	2003
Annexins--unique membrane binding proteins with diverse functions. Journal of cell science\| PubMed ID: 15169834	2004
Specific association of annexin 1 with plasma membrane-resident and internalized EGF receptors mediated through the protein core domain. FEBS letters\| PubMed ID: 15581623	2004
Annexin A8 displays unique phospholipid and F-actin binding properties. FEBS letters\| PubMed ID: 16638567	2006
S100A10/p11: family, friends and functions. Pflugers Archiv : European journal of physiology\| PubMed ID: 17638009	2008
TRPV5 is internalized via clathrin-dependent endocytosis to enter a Ca2+-controlled recycling pathway. The Journal of biological chemistry\| PubMed ID: 18077461	2008
Tyrosine phosphorylation of annexin A2 regulates Rho-mediated actin rearrangement and cell adhesion. Journal of cell science\| PubMed ID: 18565825	2008
Disruption of the annexin A1/S100A11 complex increases the migration and clonogenic growth by dysregulating epithelial growth factor (EGF) signaling. Biochimica et biophysica acta\| PubMed ID: 23246849	2013
The tumor suppressor annexin A10 is a novel component of nuclear paraspeckles. Cellular and molecular life sciences : CMLS\| PubMed ID: 23715859	2014
Annexin A6-balanced late endosomal cholesterol controls influenza A replication and propagation. mBio\| PubMed ID: 24194536	2013
Annexin A8 controls leukocyte recruitment to activated endothelial cells via cell surface delivery of CD63. Nature communications\| PubMed ID: 24769558	2014
Annexin A8 promotes VEGF-A driven endothelial cell sprouting. Cell adhesion & migration\| PubMed ID: 28060564	2017
Cooperative binding promotes demand-driven recruitment of AnxA8 to cholesterol-containing membranes. Biochimica et biophysica acta. Molecular and cell biology of lipids\| PubMed ID: 29306076	2018
Annexins in Translational Research: Hidden Treasures to Be Found. International journal of molecular sciences\| PubMed ID: 29914106	2018
Late Endosomal/Lysosomal Cholesterol Accumulation Is a Host Cell-Protective Mechanism Inhibiting Endosomal Escape of Influenza A Virus. mBio\| PubMed ID: 30042202	2018

Ursula Rescher

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