Chemiluminescence detection of telomere DNA in human cells on a membrane by using fluorescein-5-isothiocyanate-labeled primers

Telomere DNA is related to cell aging and cancer genesis because the telomeric region of DNA sequences at chromosome ends are shortened with cell divisions. Therefore, a sensitive and specific detection method is required for the telomere DNA. Here we propose a chemiluminescence (CL)-based method for the sensitive detection of telomere DNA in human cells. In this study, the telomere DNA was amplified by polymerase chain reaction (PCR) using special forward and reverse primers labeled with fluorescein-5-isothiocyanate (FITC) at the 5′ end, and then the FITC-containing PCR products were detected by CL reaction with 3,4,5-trimethoxyphenylglyoxal (TMPG) after electrophoresis followed by Southern blot onto a nylon membrane. The TMPG reagent specifically reacted with guanine moiety in DNA at room temperature and provided CL intensities. The CL intensities from the PCR products could be enhanced approximately 10-fold using FITC-labeled primers as compared with those using nonlabeled primers. The detection limit of the PCR products with the proposed method was 0.3 ng on the membrane. The developed CL method could quantitatively determine the telomere DNA in a small number of human cells (∼350) and gave approximately 10 times higher sensitivity than a conventional fluorescence-based method.     

Annexin A5 inhibits engulfment through internalization of PS-expressing cell membrane patches

Apoptosis and subsequent clearance of apoptotic cells are important for the prevention of diseases. Therefore, it is essential to understand the mechanisms underlying the biology of phagocytic clearance of apoptotic cells. The best characterized "eat me" signal on the surface of apoptotic cells is phosphatidylserine (PS). Recently, we demonstrated that annexin A5 mediates the internalization of PS-expressing membrane patches and down regulates surface expression of tissue factor. Here, we investigated the role of PS in the phagocytosis of apoptotic cells using annexin A5. Using a novel flow cytometric-based phagocytosis assay, we observed that engulfment was inhibited with 20% if annexin A5 was added to PS-expressing cells that had completed apoptosis. The inhibition increased to more than 50% if annexin A5 was added during the apoptotic process. This inhibition is specific for annexin A5, since the mutant M23 and annexin A1 did not further increase the inhibition of phagocytosis when added during the apoptotic process. Interestingly, cells with internalized annexin A5 still express PS at their surface. We conclude that other ligands within the PS-expressing membrane patch act together with PS as an "eat me" signal.     

Macrophage recognition of externalized phosphatidylserine and phagocytosis of apoptotic Jurkat cells--existence of a threshold

Phosphatidylserine (PS) is predominantly confined to the inner leaflet of plasma membrane in cells, but it is externalized on the cell surface during apoptosis. This externalized PS is required for effective phagocytosis of apoptotic cells by macrophages. Because PS trans-bilayer asymmetry is not absolute in different types of nonapoptotic cells, we hypothesized that the amounts of externalized PS may be critical for macrophage discrimination between apoptotic and nonapoptotic cells. We developed a sensitive electron paramagnetic resonance method to quantify the amounts of externalized PS based on specific binding of paramagnetic annexin V-microbead conjugates with PS on cell surfaces. Using this technique, we found that nonapoptotic Jurkat cells externalize 0.9 pmol of endogenous PS/10(6) Jurkat cells. For cells with different amounts of integrated exogenous PS on their surface, no phagocytic response was observed at PS levels <5 pmol/10(6) Jurkat cells; at higher PS concentrations, phagocytosis increased in a concentration-dependent manner. Apoptosis in Jurkat cells caused externalization of approximately 240 pmol PS/10(6) Jurkat cells; these amounts of externalized PS are manyfold higher than the threshold amounts of PS required for phagocytosis. Thus, macrophages have a sensitivity threshold for PS externalized on the cell surface that provides for reliable recognition and distinction between normal cells with low contents of externalized PS and apoptotic cells with remarkably elevated PS levels.     

Optimization of expression of an annexin V-hirudin chimeric protein in Escherichia coli

A human Annexin V-Hirudin chimeric protein, Annexin V-Hirudin C, was expressed in Escherichia coli. A broad range of parameters such as plasmid stability during propogation and expression, expression capacity stability, the culture media, the growth time before induction and the induction duration were examined and optimized. Recombinant Annexin V-Hirudin C was purified from the cell lysate supernatants by ethanol precipitation, DEAE-cellulose chromatography and Sephadex G-75 chromatography, and the purified protein showed dose-dependent thrombin inhibitory activity. The overall production of purified Annexin V-Hirudin C protein is 10 mg/l/OD600.     

Phosphatidylserine directly and positively regulates fusion of myoblasts into myotubes

Cell membrane consists of various lipids such as phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Among them, PS is a molecular marker of apoptosis, because it is located to the inner leaflet of plasma membrane generally but it is moved to the outer leaflet during programmed cell death. The process of apoptosis has been implicated in the fusion of muscle progenitor cells, myoblasts, into myotubes. However, it remained unclear whether PS regulates muscle cell differentiation directly. In this paper, localization of PS to the outer leaflet of plasma membrane in proliferating primary myoblasts and during fusion of these myoblasts into myotubes is validated using Annexin V. Moreover, we show the presence of PS clusters at the cell–cell contact points, suggesting the importance of membrane ruffling and PS exposure for the myogenic cell fusion. Confirming this conclusion, experimentally constructed PS, but not PC liposomes dramatically enhance the formation of myotubes from myoblasts, thus demonstrating a direct positive effect of PS on the muscle cell fusion. In contrast, myoblasts exposed to PC liposomes produce long myotubes with low numbers of myonuclei. Moreover, pharmacological masking of PS on the myoblast surface inhibits fusion of these cells into myotubes in a dose-dependent manner.     

A novel aminophospholipid transporter exclusively expressed in spermatozoa is required for membrane lipid asymmetry and normal fertilization

Through the use of a functionally unbiased signal peptide trap screen, we have discovered an ATP-dependent aminophospholipid transporter that is exclusively expressed in the acrosomal region of spermatozoa; it is about 62% similar to the flippase, FIC1. We disrupted the transporter gene and found that the size of litters from male null mice was slightly smaller than found with wild-type males. Sperm morphology and motility were the same between null and wild-type littermates, but agents (merocyanine and annexin) that measure phospholipid packing or phosphatidylserine (PS) in the outer membrane leaflet showed that PS already existed in the outer leaflet of null spermatozoa before sperm capacitation. Fertilization rates were normal when null spermatozoa were added to zona pellucida-free eggs, but in the presence of the extracellular matrix, fewer transporter(-/-) spermatozoa bound tightly or penetrated the zona pellucida (ZP), and fewer underwent acrosome reactions. In vitro fertilization was compromised, especially at early time points or at low sperm concentrations after mixing null spermatozoa and eggs. Thus, a new aminophospholipid transporter expressed exclusively in spermatozoa is critical for normal phospholipid distribution in the bilayer, and for normal binding, penetration, and signaling by the zona pellucida.     

Ca(2+) and membrane binding to annexin 3 modulate the structure and dynamics of its N terminus and domain III

Annexin 3 (ANX A3) represents approximately 1% of the total protein of human neutrophils and promotes tight contact between membranes of isolated specific granules in vitro leading to their aggregation. Like for other annexins, the primary molecular events of the action of this protein is likely its binding to negatively charged phospholipid membranes in a Ca(2+)-dependent manner, via Ca(2+)-binding sites located on the convex side of the highly conserved core of the molecule. The conformation and dynamics of domain III can be affected by this process, as it was shown for other members of the family. The 20 amino-acid, N-terminal segment of the protein also could be affected and also might play a role in the modulation of its binding to the membranes. The structure and dynamics of these two regions were investigated by fluorescence of the two tryptophan residues of the protein (respectively, W190 in domain III and W5 in the N-terminal segment) in the wild type and in single-tryptophan mutants. By contrast to ANX A5, which shows a closed conformation and a buried W187 residue in the absence of Ca(2+), domain III of ANX A3 exhibits an open conformation and a widely solvent-accessible W190 residue in the same conditions. This is in agreement with the three-dimensional structure of the ANX A3-E231A mutant lacking the bidentate Ca(2+) ligand in domain III. Ca(2+) in the millimolar concentration range provokes nevertheless a large mobility increase of the W190 residue, while interaction with the membranes reduces it slightly. In the N-terminal region, the W5 residue, inserted in the central pore of the protein, is weakly accessible to the solvent and less mobile than W190. Its amplitude of rotation increases upon binding of Ca(2+) and returns to its original value when interacting with membranes. Ca(2+) concentration for half binding of the W5A mutant to negatively charged membranes is approximately 0.5 mM while it increases to approximately 1 mM for the ANX A3 wild type and to approximately 3 mM for the W190 ANX A3 mutant. In addition to the expected perturbation of the W190 environment at the contact surface between the protein and the membrane bilayer, binding of the protein to Ca(2+) and to membranes modulates the flexibility of the ANX A3 hinge region at the opposite of this interface and might affect its membrane permeabilizing properties.     

Improved apoptosis detection in ovine neutrophils by annexin V and carboxyfluorescein diacetate staining

Neutrophil apoptosis is critical for final resolution of the inflammation in the tissues and for maintenance of neutrophil homeostasis under normal condition. An early hallmark of apoptotic cells is translocation of phosphatidylserine (PS) residues, normally located in the inner leaflet of cellular membrane, to the external cell surface; exposed PS is recognized by specific PS receptors on disposing cells. Here we report an improved procedure to detect neutrophil apoptosis by simultaneous staining for exposed PS with Cy3-labeled annexin V (Cy3) and for membrane integrity with the vital dye 6-carboxyfluorescein diacetate (6-CFDA) based on the APOAC apoptosis detection kit (Sigma). Spontaneous apoptosis was evaluated in ovine neutrophils cultured ex vivo for 18 h. We investigated the multiple parameters involved in the assay, i.e. the type of fixative (methanol, paraformaldehyde, or no fixation) and the type of slide (coated with Vectabond, polylysine or Parafilm^®). Results indicated that both the adhesion to the slide and the fixation can modify neutrophil functional status and morphology, which result in misleading apoptosis detection. In order to minimize these artifacts, we have developed an improved APOAC assay procedure, staining cells while in suspension and using Parafilm^® coated slides.     

Identification of novel binding partners (annexins) for the cell death signal phosphatidylserine and definition of their recognition motif

Identification and clearance of apoptotic cells prevents the release of harmful cell contents thereby suppressing inflammation and autoimmune reactions. Highly conserved annexins may modulate the phagocytic cell removal by acting as bridging molecules to phosphatidylserine, a characteristic phagocytosis signal of dying cells. In this study five members of the structurally and functionally related annexin family were characterized for their capacity to interact with phosphatidylserine and dying cells. The results showed that AnxA3, AnxA4, AnxA13, and the already described interaction partner AnxA5 can bind to phosphatidylserine and apoptotic cells, whereas AnxA8 lacks this ability. Sequence alignment experiments located the essential amino residues for the recognition of surface exposed phosphatidylserine within the calcium binding motifs common to all annexins. These amino acid residues were missing in the evolutionary young AnxA8 and when they were reintroduced by site directed mutagenesis AnxA8 gains the capability to interact with phosphatidylserine containing liposomes and apoptotic cells. By defining the evolutionary conserved amino acid residues mediating phosphatidylserine binding of annexins we show that the recognition of dying cells represent a common feature of most annexins. Hence, the individual annexin repertoire bound to the cell surface of dying cells may fulfil opsonin-like function in cell death recognition.     

Determination of molecular groups involved in the interaction of annexin A5 with lipid membrane models at the air-water interface

Annexin A5 (AnxA5) is a member of a family of homologous proteins sharing the ability to bind to negatively charged phospholipid membranes in a Ca²⁺-dependent manner. In this paper, we used polarization-modulated infrared reflection absorption spectroscopy (PMIRRAS), Brewster angle microscopy (BAM), and ellipsometry to investigate changes both in the structure of AnxA5 and phospholipid head groups associated with membrane binding. We found that the secondary structure of AnxA5 in the AnxA5/Ca²⁺/lipid ternary complex is conserved, mainly in α-helices and the average orientation of the α-helices of the protein is slightly tilted with respect to the normal to the phospholipid monolayer. Upon interaction between AnxA5 and phospholipids, a shift of the ν_as PO₂⁻ band is observed by PMIRRAS. This reveals that the phosphate group is the main group involved in the binding of AnxA5 to phospholipids via Ca²⁺ ions, even when some carboxylate groups are accessible (PS). PMIRRAS spectra also indicate a change of carboxylate orientation in the aspartate and glutamate residues implicated in the association of the AnxA5, which could be linked to the 2D crystallization of protein under the phospholipid monolayer. Finally, we demonstrated that the interaction of AnxA5 with pure carboxylate groups of an oleic acid monolayer is possible, but the orientation of the protein under the lipid is completely different.     

Calcium,leukocyte cell death and the use of annexin V: fatal encounters

One hallmark of programmed cell death (PCD) is redistribution of phosphatidylserine (PS) to the plasma membrane’s outer leaflet. Annexin V is widely used in cell death research due to its calcium-dependent ability to bind phosphatidylserine, thus marking apoptotic cells. However, calcium is invariably used at high concentrations in annexin V staining, at doses that can induce cell death. We used flow cytometric annexin V staining, together with propidium iodide and TMRM for determination of dissipation of mitochondrial potential, with a variety of calcium concentrations, cell media, and incubation times, to identify a possible bias in PCD determination of human primary leukocytes. Here we show that measurements of PCD in human monocytes, polymorphonuclear cells, and monocyte-derived dendritic cells using annexin V may be dramatically affected by calcium concentration, time of incubation on ice, and media choice. We propose a method that enables accurate and unbiased annexin V staining, without affecting results. Uriel Trahtemberg and Mizhir Atallah contributed equally to this publication.     

Eukaryotic expression and secretion of EGFP-labeled annexin A5

The Ca²⁺-dependent binding of annexin A5 to phosphatidylserine on cell surfaces is a reliable marker for apoptosis that is widely used in flow cytometry based apoptosis assays. In this approach, annexin A5 must be coupled to a fluorescent dye, but standard dyes such as fluorescein are photolabile, and the heterogeneous chemical linkage partially inhibits binding to phosphatidylserine. Recombinant fusions comprising annexin A5 and fluorescent proteins are available for prokaryotic expression, but can be purified only at low concentrations due to their low solubility in the cytoplasm. Here we describe a eukaryotic expression system for the secretion of functional recombinant annexin A5, with and without fluorescent protein fusions, in different formats. Metal affinity purification yielded up to 18 μg of histidine-tagged annexin A5 fusions per ml processed cell culture supernatants. Furthermore the supernatant itself was sufficient for direct use in apoptosis assays. The availability of such fusion proteins offers new and more economical opportunities for the development and application of this widely utilized apoptosis assay.     

Differential expression of annexin V during spermatogenesis in the newt Cynops pyrrhogaster

 In order to isolate genes whose expression is up-regulated after the initiation of meiosis, we screened a cDNA expression library of newt testes with antiserum against homogenates of testes derived from the spermatogonial and spermatocyte stages. We report the isolation of spermatocyte-specific cDNA clones encoding a newt homologue of the calcium-dependent phospholipid-binding protein, annexin V. Northern blot analysis showed that newt annexin V mRNA was 1.7 kb in length and was expressed strongly in testes, but weakly in other organs. In situ hybridization revealed that the expression of newt annexin mRNA was barely observed in spermatogonia, but increased significantly in leptotene-zygotene primary spermatocytes and reached a maximum level in pachytene spermatocytes and round spermatids. The newt annexin V cDNA predicted a 323-amino acid protein and had a 68% homology to human annexin V. The predicted amino acid sequence contained a conserved 4-fold internal repeat of approximately 70 residues like other annexin proteins. Immunoblot analysis using the monoclonal antibody against newt annexin V showed that the protein was expressed scarcely in spermatogonia but was abundantly expressed in stages from primary spermatocytes to spermatids; this pattern was consistent to that of the mRNA. Immunohistochemical analysis revealed that newt annexin V was localized in the cytoplasm of the spermatogenic cells, but not in somatic cells such as Sertoli cells or pericystic cells. These results indicate that the expression of newt annexin V is up-regulated in the spermatogenic cells after the initiation of meiosis and suggest that newt annexin V plays an important role in spermatogenesis. Received: 8 December 1995 / Accepted: 12 February 1996 Edited by H. Shimada/D. Tautz     

A novel retinoid binding property of human annexin A6

Vitamin A (all-trans retinol) and all-trans retinoid acid (ATRA) interacted with human annexin A6 (AnxA6) as evidenced by AnxA6-induced blue shift of retinoid absorption maxima, by AnxA6-Trp fluorescence quenching and by a fluorescence resonance energy transfer from a Trp residue of AnxA6 to retinol. In addition, both retinoids stimulated the calcium-dependent binding of AnxA6 to liposomes, accompanied by oligomerization of AnxA6. Up to our knowledge, it is a first report supporting the hypothesis of a direct implication of AnxA6 in vitamin A-dependent tissue mineralization.     

Platelet‐activating factor interaction with the human erythrocyte membrane

Platelet‐activating factor (PAF) is a soluble signal messenger present in blood at nanomolar concentration. PAF has a wide spectrum of biological activities and is produced by and effective in different cell types. Owing to its important physiological role, we wanted to characterize membrane intercalation and interaction of PAF‐16 (1‐O‐hexadecyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine) by studying its capacity to induce during short‐term incubations at high concentrations cell shape alterations, phosphatidylserine exposure, and hemolysis in human erythrocytes. Our results showed that PAF‐16 at micromolar concentrations rapidly (≤1 min) induces stable but wash‐sensitive echinocytosis and hemolysis, but no substantial phosphatidylserine exposure. In conclusion, our study characterizes PAF‐16 as a highly membrane partitioning non‐permeable molecule accumulating in the outer membrane leaflet. These membrane interacting properties of PAF should, also at physiological concentrations, be important part of its nature as a membrane affector molecule. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:345–348, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20297     

Bisecting GlcNAc mediates the binding of annexin V to Hsp47

The bisecting N-acetylglucosamine (GlcNAc) structure, formed through catalysis by UDP-N-acetylglucosamine : beta-D-mannoside beta-1,4-N-acetylglucosaminyltansferase III (GnT-III), is responsible for a variety of biological functions. We have previously shown that annexin V, a member of the calcium/phospholipid-binding annexin family of proteins, has binding activity toward the bisecting GlcNAc structure. In this study, we reported on a search for potential target glycoproteins for annexin V in a rat hepatoma cell line, M31. Using a glutathione S-transferase (GST)-annexin V immobilized sepharose 4B affinity column to trap interacting proteins produced by the GnT-III-transfected M31 cells, we isolated a 47 kDa protein. It was identified as Hsp47 by an N-terminal sequence analysis. Immunoprecipitation experiments showed that annexin V interacted with Hsp47. The association of annexin V and Hsp47 was abolished by treatment with N-glycosidase F or preincubation with sugar chains containing bisecting GlcNAc, suggesting that the bisecting GlcNAc plays an important role in the interaction. An oligosaccharide analysis of Hsp47 purified from GnT-III-transfected M31 cells was shown to have the bisecting GlcNAc structure, as detected by erythroagglutinating phytohemagglutinin (E4-PHA) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. Surface plasmon resonance analysis showed that annexin V was bound to Hsp47, bearing a bisecting GlcNAc with a Kd of 5.5 microM, whereas no significant binding was observed in the case of Hsp47 without a bisecting GlcNAc. In addition, immunofluorescence microscopy revealed the colocalization of annexin V, Hsp47, and a bisecting GlcNAc sugar chain around the Golgi apparatus. Collectively, these results suggest that the binding of annexin V to Hsp47 is mediated by a bisecting GlcNAc oligosaccharide structure and that Hsp47 is an intracellular ligand glycoprotein for annexin V.     

Measurement of the affinity and cooperativity of annexin V-membrane binding under conditions of low membrane occupancy

We developed a method for measuring the binding affinity of annexin V for phospholipid vesicles and cells at very low levels of membrane occupancy. The annexin V-117 mutant was labeled with fluorescein iodoacetamide on its single N-terminal cysteine residue; binding to phospholipid vesicles containing phosphatidylserine (PS) and 2% rhodamine-phosphatidylethanolamine was measured by fluorescence quenching due to resonance energy transfer; binding to cells with exposed PS was measured by fluorometry after elution of bound protein. The equilibrium constant was calculated as a function of the midpoint of the calcium titration curve, the Hill coefficient, and the concentration of membrane binding sites. Calcium titrations at very low ratios of protein to membrane revealed Hill coefficients of approximately 8 for both vesicles and cells, far higher than previously measured, but as the protein-membrane ratio was increased above 3% of maximum membrane occupancy, the value of the Hill coefficient progressively decreased to a limiting value of about 2. High Hill coefficients were also observed for measurements performed at different ionic strengths and with membrane PS content varied over the range from 20 to 50%. This method allows the accurate determination of the affinity and cooperativity of annexin V-membrane binding and will be useful for the evaluation of modified annexin V derivatives intended for diagnostic and therapeutic applications.     

Effect of bilayer membrane curvature on activity of phosphatidylcholine exchange protein

Effect of bilayer membrane curvature of substrate phosphatidylcholine and inhibitor phosphatidylserine on the activity of phosphatidylcholine exchange protein has been studied by measuring transfer of spin-labeled phosphatidylcholine between vesicles, vesicles and liposomes, and between liposomes. The transfer rate between vesicles was more than 100 times larger than that between vesicles and liposomes. The transfer rate between liposomes was still smaller than that between vesicles and liposomes and nearly the same as that in the absence of exchange protein. The markedly enhanced exchange with vesicles was ascribed to the asymmetric packing of phospholipid molecules in the outer layer of the highly curved bilayer membrane. The inhibitory effect of phosphatidylserine was also greatly dependent on the membrane curvature. The vesicles with diameter of 17 nm showed more than 20 times larger inhibitory activity than those with diameter of 22 nm. The inhibitory effect of liposomes was very small. The size dependence was ascribed to stronger binding of the exchange protein to membranes with higher curvatures. The protein-mediated transfer from vesicles to spiculated erythrocyte ghosts was about four times faster than that to cup-shaped ghosts. This was ascribed to enhanced transfer to the highly curved spiculated membrane sites rather than greater mobility of phosphatidylcholine in the spiculated ghost membrane.     

Biochemical characterization of APPL endosomes: the role of annexin A2 in APPL membrane recruitment

APPL endosomes are a recently identified subpopulation of early endosomes characterized by the presence of two homologous Rab5 effector proteins APPL1 and APPL2. They exhibit only limited colocalization with EEA1, another Rab5 effector and a marker of the canonical early endosomes. Although APPL endosomes appear to play important roles in cargo trafficking and signal transduction, their protein composition and biochemical properties remain largely unknown. Here we employed membrane fractionation methods to characterize APPL endosomes biochemically. We demonstrate that they represent heterogeneous membrane structures which can be discriminated from the canonical EEA1-positive early endosomes by their partly different physical properties and a distinct migration pattern in the continuous density gradients. In search for other potential markers of APPL endosomes we identified Annexin A2 as an interacting partner of both APPL1 and APPL2. Annexin A2 is a Ca(2+) and phosphatidylinositol 4,5-bisphosphate binding protein, previously implicated in several endocytic steps. We show that Annexin A2 co-fractionates and colocalizes with APPL endosomes. Moreover, silencing of its expression causes solubilization of APPL2 from endosomes. Although Annexin A2 is not an exclusive marker of APPL endosomes, our data suggest that it has an important function in membrane recruitment of APPL proteins, acting in parallel to Rab5.     

Cholesterol modulates the membrane binding and intracellular distribution of annexin 6

Annexins are Ca(2+)- and phospholipid-binding proteins that are widely expressed in mammalian tissues and that bind to different cellular membranes. In recent years its role in membrane traffic has emerged as one of its predominant functions, but the regulation of its intracellular distribution still remains unclear. We demonstrated that annexin 6 translocates to the late endocytic compartment in low density lipoprotein-loaded CHO cells. This prompted us to investigate whether cholesterol, one of the major constituents of low density lipoprotein, could influence the membrane binding affinity and intracellular distribution of annexin 6. Treatment of crude membranes or early and late endosomal fractions with digitonin, a cholesterol-sequestering agent, displayed a strong reduction in the binding affinity of a novel EDTA-resistant and cholesterol-sensitive pool of annexin 6 proteins. In addition, U18666A-induced accumulation of cholesterol in the late endosomal compartment resulted in a significant increase of annexin 6 in these vesicles in vivo. This translocation/recruitment correlates with an increased membrane binding affinity of GST-annexin 6 to late endosomes of U18666A-treated cells in vitro. In conclusion, the present study shows that changes in the intracellular distribution and concentration of cholesterol in different subcellular compartments participate in the reorganization of intracellular pools of Ca(2+)-dependent and -independent annexin 6.