[ANXA8 Regulates Proliferation of Human Non-Small Lung Cancer Cells A549 via EGFR-AKT-mTOR Signaling Pathway].

Annexin A8 (ANXA8) is a member of the annexin family, which had been reported to regulate multiple cancer cellular processes including proliferation, metastasis and inflammation. However, the specific role of ANXA8 in lung cancer cell biology remains unknown. Our previous transcriptome study revealed that ANXA8 mRNA was downregulated in curcumin analog (MHMD) -treated human non-small lung cancer cells (A549 cell line). Here, we continued to study the ANXA8 expression in A549 cells using reverse transcription-quantitative PCR and Western blotting, compared with that in human normal bronchial epithelium cells (BE-AS-2B cell line). Overexpression of ANXA8 via transfection of pEGFP-ANXA8 recombinant vector contributed to the proliferation and migration of A549 cells. Moreover, the cell cycle protein cyclin E1 was upregulated in ANXA8-transfected A549 cells. Knockdown of ANXA8 using an RNA interference technique decreased A549 cell viability and restrained their migration in vitro. The expression levels of multiple cellular factors, including EGFR, PI3K, Akt, mTOR, p70S6K and 4EBP1, in the epidermal growth factor receptor (EGFR) signaling pathway were also altered by ANXA8 knockdown or overexpression in A549 cells, which confirmed the activation of the EGFR/Akt/mTOR signaling pathway by ANXA8. The present results provided evidence to support further investigation of the functional identification of ANXA8 in lung cancer cells in the future.

Annexins and Membrane Repair Dysfunctions in Muscular Dystrophies.

Muscular dystrophies constitute a group of genetic disorders that cause weakness and progressive loss of skeletal muscle mass. Among them, Miyoshi muscular dystrophy 1 (MMD1), limb girdle muscular dystrophy type R2 (LGMDR2/2B), and LGMDR12 (2L) are characterized by mutation in gene encoding key membrane-repair protein, which leads to severe dysfunctions in sarcolemma repair. Cell membrane disruption is a physiological event induced by mechanical stress, such as muscle contraction and stretching. Like many eukaryotic cells, muscle fibers possess a protein machinery ensuring fast resealing of damaged plasma membrane. Members of the annexins A (ANXA) family belong to this protein machinery. ANXA are small soluble proteins, twelve in number in humans, which share the property of binding to membranes exposing negatively-charged phospholipids in the presence of calcium (Ca2+). Many ANXA have been reported to participate in membrane repair of varied cell types and species, including human skeletal muscle cells in which they may play a collective role in protection and repair of the sarcolemma. Here, we discuss the participation of ANXA in membrane repair of healthy skeletal muscle cells and how dysregulation of ANXA expression may impact the clinical severity of muscular dystrophies.

Roles of Annexin A protein family in autophagy regulation and therapy.

Annexin A is a kind of calcium-dependent phospholipid-binding proteins, which contributes to the formation of the cell membranes and cytoskeleton and played a part as a membrane skeleton to stabilize lipid bilayer. Autophagy is one of the most important programmed cell death mechanisms. And recently some reports suggest that annexin A family protein is associated with autophagy for annexin A can regulate the formation of vesicular lipid membranes and promote cell exocytosis. In this review, we summarized the roles of annexin A protein family in autophagy regulation and targeted medical treatment for better diagnoses and therapies.

Annexin A8 regulates Wnt signaling to maintain the phenotypic plasticity of retinal pigment epithelial cells.

Wnt signalling mediates complex cell-cellinteractions during development and proliferation. Annexin A8 (AnxA8), a calcium-dependent phospholipid-binding protein, and canonical Wnt signalling mechanisms have both been implicated in retinal pigment epithelial (RPE) cell differentiation. The aim here was to examine the possibility of cross-talk between AnxA8 and Wnt signalling, as both are down-regulated upon fenretinide (FR)-mediated RPE transdifferentiation. AnxA8 suppression in RPE cells via siRNA or administration of FR induced neuronal-like cell transdifferentiation and reduced expression of Wnt-related genes, as measured by real-time PCR and western blotting. AnxA8 gene expression, on the other hand, remained unaltered upon manipulating Wnt signalling, suggesting Wnt-related genes to be downstream effectors of AnxA8. Co-immunoprecipitation revealed an interaction between AnxA8 and ß-catenin, which was reduced in the presence of activated TGF-ß1. TGF-ß1 signalling also reversed the AnxA8 loss-induced cell morphology changes, and induced ß-catenin translocation and GSK-3ß phosphorylation in the absence of AnxA8. Ectopic over-expression of AnxA8 led to an increase in active ß-catenin and GSK-3ß phosphorylation. These data demonstrate an important role for AnxA8 as a regulator of Wnt signalling and a determinant of RPE phenotype, with implications for regenerative medicine approaches that utilise stem cell-derived RPE cells to treat conditions such as age-related macular degeneration.

Secretome of the carcinogenic helminth Spirocerca lupi reveals specific parasite proteins associated with its different life stages.

Spirocerca lupi is a parasitic and carcinogenic nematode of canids distributed in tropical and subtropical regions around the world. The excretion-secretion proteins (PES) of S. lupi have been suggested to play a role in the pathogenesis of its infection. We aimed to identify the PES of different stages of S. lupi and search for proteins that would be useful for diagnostic, therapeutic and vaccination purposes as well as understand their functions. A nano-UPLC mass spectrometry de novo analysis was performed on proteins collected from cultures of S. lupi L3 larvae, L4 females, adult females and adult males from naturally infected hosts. A total of 211 proteins were identified in all cultures. Accordingly, 117, 130, 99 and 116 proteins were detected in L3 larva, L4 females, adult females and adult males, respectively, with a strong correlation in the biological replicates (Pearson coefficients > 0.73). Fourty-four proteins were detected in all developmental stages, 64 were stage-specific and 49 were exclusively identified in L4 females. Cell compartment enrichment analysis revealed that proteins common to all stages were cytoplasmatic (p < 9.x10-6), whereas L4 unique proteins were in collagen trimers, and macromolecular complexes (p < 0.00001). Functional enrichment analysis of proteins showed significant enrichment in lipid metabolism in L3-unique proteins (p<0.00005), in mannose metabolism and protein de-glycosylation for L4-unique proteins (p < 0.00004), and in phosphorus metabolism in proteins shared by all stages (p <  2.1 x10-9). Interestingly, annexin 6, associated with cancer in humans, was detected in all life stages, but in a larger abundance in L4 females and adults. These findings indicate that S. lupi establishes complex interactions with its hosts by an arsenal of proteins expressed in different patterns in each life stage which influence the pathogenesis and oncogenesis of S. lupi and may be used as potential targets for diagnostic assays, drug targets or vaccine candidates.

The involvement of spinal annexin A10/NF-κB/MMP-9 pathway in the development of neuropathic pain in rats.

BACKGROUND: Neuropathic pain (NP) is a prevalent disease, which badly impairs the life quality of patients. The underlying mechanism of NP is still not fully understood. It has been reported that spinal Annexin A10 (ANXA10) contributes to NP. This study aims at exploring the underlying mechanisms of spinal ANXA10 in regulating NP in rats. METHODS: Spinal nerve ligation (SNL) was adopted to establish a NP model in rats. After SNL, paw withdrawal threshold and paw withdrawal latency were recorded to measure pain behaviors, RT-PCR was used to check the change of the expression of spinal ANXA10 mRNA, western blot analysis was used to detect the change of the protein level of ANXA10, nuclear factor kappa B (NF-κB), and maisrix metalloproteinase-9 (MMP-9) in the spinal cord. The levels of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukine-1ß (IL-1ß), and interleukine-6 (IL-6), were explored by ELISA kits. The effects of both knockdown of spinal ANXA10 and inhibition of NF-κB on pain behaviors and the expression of MMP-9 and proinflammatory cytokines were investigated. RESULTS: Our present findings highlighted that SNL caused pain hypersensitivity and increased the expression of spinal ANXA10/pNF-κB, TNF-α, IL-1ß, and IL-6 both in the early and late phase of NP in rats, while spinal MMP-9 was only slightly increased in the early phase of NP. Knockdown of ANXA10 at the spinal cord level suppressed the SNL-induced hyperalgesia and blocked the activation of NF-κB, TNF-α and IL-1ß both in the early and late phase of NP. Spinal ANXA10 knockdown could prevent the upregulation of spinal MMP-9 in the early phase and inhibit IL-6 expression in the late phase of SNL-induced NP. CONCLUSIONS: In conclusion, spinal ANXA10/NF-κB/MMP-9 pathway, along with the activation of proinflammatory cytokines, was involved in the SNL-induced NP. MMP-9 may act as the downstream target of ANXA10/NF-κB pathway in the development rather than the maintenance of NP.

OsANN3, a calcium-dependent lipid binding annexin is a positive regulator of ABA-dependent stress tolerance in rice.

Annexin is a multigene family that plays critical roles in plant stress responses and various cellular processes. Here, we reported the cloning and functional characterization of a novel rice annexin protein, OsANN3. We found that expression of OsANN3 was induced by polyethylene glycol (PEG) and abscisic acid (ABA) treatments. Overexpression of OsANN3 in rice significantly increased survival rates under drought stress, while knocking down OsANN3 resulted in sensitivity to drought. Meanwhile, OsANN3 overexpression showed enhanced sensitivity to exogenous ABA. Together with its Ca2+ and phospholipid binding activity, we proposed that when plants were subjected to drought stress, OsANN3 might mediate Ca2+ influx by binding to phospholipid to activate ABA signaling pathways. In addition, overexpression OsANN3 showed better growth under drought stress comparing to wild type, such as longer root length and more stomata closure for reducing water loss by regulating ABA-dependent stress response pathways.

When a transmembrane channel isn't, or how biophysics and biochemistry (mis)communicate.

Annexins are a family of soluble proteins that bind to acidic phospholipids such as phosphatidylserine in a calcium-dependent manner. The archetypical member of the annexin family is annexin A5. For many years, its function remained unknown despite the availability of a high-resolution structure. This, combined with the observations of specific ion conductance in annexin-bound membranes, fueled speculations about the possible membrane-spanning forms of annexins that functioned as ion channels. The channel hypothesis remained controversial and did not gather sufficient evidence to become accepted. Yet, it continues to draw attention as a framework for interpreting indirect (e.g., biochemical) data. The goal of the mini-review is to examine the data on annexin-lipid interactions from the last ~30 years from the point of view of the controversy between the two lines of inquiry: the well-characterized peripheral assembly of the annexins at membranes vs. their putative transmembrane insertion. In particular, the potential role of lipid rearrangements induced by annexin binding is highlighted.

The calcium-binding protein EpANN from the lichenized fungus Endocarpon pusillum enhances stress tolerance in yeast and plants.

Annexins are calcium-phospholipid binding proteins that play a significant role in the Ca2+signaling pathway. These proteins are essential for plants to effectively respond to abiotic stresses. However, their functions and mechanisms remain largely unknown in fungi. In this study, an annexin gene, Epann, was cloned from the lichenized fungus Endocarpon pusillum, a drought resistant organism. Our results showed that Epann was induced by several abiotic stresses in E. pusillum. Heterologous expression of the Epann gene enhanced the stress tolerance of Saccharomyces cerevisiae. Under heat-shock conditions, the EpANN proteins were significantly aggregated and the aggregation sites were located on peroxisomes. In heat-shocked cells, Epann reduced the reactive oxygen species level mainly through its intracellular peroxidase activity and regulation of stress-related genes. Transgenic Arabidopsis plants overexpressing Epann exhibited a higher germination rate under oxidative stress and stronger drought tolerance. Our results provide a mechanistic understanding of the role of annexins in abiotic stress responses and suggest that this lichenized fungal gene could be a promising resource to generate stress-tolerant transgenic organisms.

The roles of annexins in vascular endothelium dysfunction accompanying diabetes mellitus type 2.

Impairment in cellular transport, distribution and storage of cholesterol accompanies insulin resistance and diabetes mellitus type 2 as well as other diseases such as obesity, atherosclerosis, and non-alcoholic fatty liver disease. Diabetes mellitus type 2 is a metabolic disorder that is characterized by hyperglycemia in the context of insulin resistance and relative lack of insulin. Type 2 diabetes makes up about 90% of cases of diabetes. Several therapeutic strategies are today being considered to target diabetes mellitus type 2, and the accompanying endothelial dysfunction, but none as yet has proved satisfactory. Accumulating data suggest that annexins, as cholesterol binding proteins that participate in intracellular transport and storage of cholesterol and in the organization of plasma membrane, may participate in development and sustenance of diabetes mellitus type 2 and may serve as predictive markers of this disease.

Annexin A10 is involved in the development and maintenance of neuropathic pain in mice.

ANXA10 (annexin A10) is a member of the annexin family, and its biological effects are mediated primarily through the calcium-dependent phospholipid-binding and calcium ion binding. We examined the gene expressions of the L5 spinal cord after spinal nerve ligation (SNL)-induced neuropathic pain in mice by gene chip. The results showed that Anxa10 mRNA was the most upregulated gene in annexin family with 73.7-fold increase. Although previous studies have reported that several annexins are involved in nociceptive pain, the role of Anxa10 in pain remains undefined. We aimed to evaluate the role of ANXA10 in mediating injury-induced heat hyperalgesia and mechanical allodynia. We found that SNL induced persistent upregulation of Anxa10 mRNA and protein in the spinal cord of mice. Moreover, ANXA10 was colocalized with the neuronal marker MAP2 and astrocytic marker glial fibrillary acidic protein (GFAP), but not with microglial marker CD11b. Finally, pretreatment with Anxa10 siRNA partially prevented SNL-induced mechanical allodynia and heat hyperalgesia. Posttreatment with Anxa10 siRNA attenuated SNL-induced neuropathic pain. These findings suggest that ANXA10 might be a novel target in the treatment of neuropathic pain.

[Annexins in mitochondria]. / Aneksyny mitochondriów.

Annexins form a family of membrane- and calcium-binding proteins, widely distributed in vertebrates. Their interactions with membranes are regulated by changes of intracellular concentration of calcium ([Ca2+]in.), pH, and the presence of negatively charged phospholipids as well as cholesterol in membranes. As protein participating in membrane fusion and sensors of a [Ca2+]in. Annexins may regulate various signaling pathways including patways involving protein kinase C (PKC isoforms. They also particpate in membrane repair mechanisms (along with actin cytoskeleton and S100 protein), in the vesicular transport (cholesterol enriched domains) as well in in intracellular calcium homeostasis and regulation of mitochondrial function and mitochondrial network structure. The last possibility is a topic of present review commemorating 90th Birthday of Professor Lech Wojtczak.

Annexins are instrumental for efficient plasma membrane repair in cancer cells.

Plasma membrane stress can cause damage to the plasma membrane, both when imposed by the extracellular environment and by enhanced oxidative stress. Cells cope with these injuries by rapidly activating their plasma membrane repair system, which is triggered by Ca(2+) influx at the wound site. The repair system is highly dynamic, depends on both lipid and protein components, and include cytoskeletal reorganization, membrane replacements, and membrane fusion events. Cancer cells experience enhanced membrane stress when navigating through dense extracellular matrix, which increases the frequency of membrane injuries. In addition, increased motility and oxidative stress further increase the risk of plasma membrane lesions. Cancer cells compensate by overexpressing Annexin proteins including Annexin A2 (ANXA2). Annexin family members can facilitate membrane fusion events and wound healing by binding to negatively charged phospholipids in the plasma membrane. Plasma membrane repair in cancer cells depends on ANXA2 protein, which is recruited to the wound site and forms a complex with the Ca(2+)-binding EF-hand protein S100A11. Here they regulate actin accumulation around the wound perimeter, which is required for wound closure. In this review, we will discuss the requirement for Annexins, S100 proteins and actin cytoskeleton in the plasma membrane repair response of cancer cells, which reveals a novel avenue for targeting metastatic cancers.

Defying death: Cellular survival strategies following plasmalemmal injury by bacterial toxins.

The perforation of the plasmalemma by pore-forming toxins causes an influx of Ca(2+) and an efflux of cytoplasmic constituents. In order to ensure survival, the cell needs to identify, plug and remove lesions from its membrane. Quarantined by membrane folds and isolated by membrane fusion, the pores are removed from the plasmalemma and expelled into the extracellular space. Outward vesiculation and microparticle shedding seem to be the strategies of choice to eliminate toxin-perforated membrane regions from the plasmalemma of host cells. Depending on the cell type and the nature of injury, the membrane lesion can also be taken up by endocytosis and degraded internally. Host cells make excellent use of an initial, moderate rise in intracellular [Ca(2+)], which triggers containment of the toxin-inflicted damage and resealing of the damaged plasmalemma. Additional Ca(2+)-dependent defensive cellular actions range from the release of effector molecules in order to warn neighbouring cells, to the activation of caspases for the initiation of apoptosis in order to eliminate heavily damaged, dysregulated cells. Injury to the plasmalemma by bacterial toxins can be prevented by the early sequestration of bacterial toxins. Artificial liposomes can act as a decoy system preferentially binding and neutralizing bacterial toxins.

Hepatic proteomic analysis revealed altered metabolic pathways in insulin resistant Akt1(+/-)/Akt2(-/-) mice.

OBJECTIVE: The aim of this study was to identify liver proteome changes in a mouse model of severe insulin resistance and markedly decreased leptin levels. METHODS: Two-dimensional differential gel electrophoresis was utilized to identify liver proteome changes in AKT1(+/-)/AKT2(-/-) mice. Proteins with altered levels were identified with tandem mass spectrometry. Ingenuity Pathway Analysis was performed for the interpretation of the biological significance of the observed proteomic changes. RESULTS: 11 proteins were identified from 2 biological replicates to be differentially expressed by a ratio of at least 1.3 between age-matched insulin resistant (Akt1(+/-)/Akt2(-/-)) and wild type mice. Albumin and mitochondrial ornithine aminotransferase were detected from multiple spots, which suggest post-translational modifications. Enzymes of the urea cycle were common members of top regulated pathways. CONCLUSION: Our results help to unveil the regulation of the liver proteome underlying altered metabolism in an animal model of severe insulin resistance.

Annexin5 plays a vital role in Arabidopsis pollen development via Ca2+-dependent membrane trafficking.

The regulation of pollen development and pollen tube growth is a complicated biological process that is crucial for sexual reproduction in flowering plants. Annexins are widely distributed from protists to higher eukaryotes and play multiple roles in numerous cellular events by acting as a putative "linker" between Ca2+ signaling, the actin cytoskeleton and the membrane, which are required for pollen development and pollen tube growth. Our recent report suggested that downregulation of the function of Arabidopsis annexin 5 (Ann5) in transgenic Ann5-RNAi lines caused severely sterile pollen grains. However, little is known about the underlying mechanisms of the function of Ann5 in pollen. This study demonstrated that Ann5 associates with phospholipid membrane and this association is stimulated by Ca2+ in vitro. Brefeldin A (BFA) interferes with endomembrane trafficking and inhibits pollen germination and pollen tube growth. Both pollen germination and pollen tube growth of Ann5-overexpressing plants showed increased resistance to BFA treatment, and this effect was regulated by calcium. Overexpression of Ann5 promoted Ca2+-dependent cytoplasmic streaming in pollen tubes in vivo in response to BFA. Lactrunculin (LatB) significantly prohibited pollen germination and tube growth by binding with high affinity to monomeric actin and preferentially targeting dynamic actin filament arrays and preventing actin polymerization. Overexpression of Ann5 did not affect pollen germination or pollen tube growth in response to LatB compared with wild-type, although Ann5 interacts with actin filaments in a manner similar to some animal annexins. In addition, the sterile pollen phenotype could be only partially rescued by Ann5 mutants at Ca2+-binding sites when compared to the complete recovery by wild-type Ann5. These data demonstrated that Ann5 is involved in pollen development, germination and pollen tube growth through the promotion of endomembrane trafficking modulated by calcium. Our results provide reliable molecular mechanisms that underlie the function of Ann5 in pollen.

Down-regulating annexin gene GhAnn2 inhibits cotton fiber elongation and decreases Ca2+ influx at the cell apex.

Cotton fiber is a single cell that differentiates from the ovule epidermis and undergoes synchronous elongation with high secretion and growth rate. Apart from economic importance, cotton fiber provides an excellent single-celled model for studying mechanisms of cell-growth. Annexins are Ca(2+)- and phospholipid-binding proteins that have been reported to be localized in multiple cellular compartments and involved in control of vesicle secretions. Although several annexins have been found to be highly expressed in elongating cotton fibers, their functional roles in fiber development remain unknown. Here, 14 annexin family members were identified from the fully sequenced diploid G. raimondii (D5 genome), half of which were expressed in fibers of the cultivated tetraploid species G. hirsutum (cv. YZ1). Among them, GhAnn2 from the D genome of the tetraploid species displayed high expression level in elongating fiber. The expression of GhAnn2 could be induced by some phytohormones that play important roles in fiber elongation, such as IAA and GA3. RNAi-mediated down-regulation of GhAnn2 inhibited fiber elongation and secondary cell wall synthesis, resulting in shorter and thinner mature fibers in the transgenic plants. Measurement with non-invasive scanning ion-selective electrode revealed that the rate of Ca(2+) influx from extracellular to intracellular was decreased at the fiber cell apex of GhAnn2 silencing lines, in comparison to that in the wild type. These results indicate that GhAnn2 may regulate fiber development through modulating Ca(2+) fluxes and signaling.

Annexins - scaffolds modulating PKC localization and signaling.

Spatial and temporal organization of signal transduction is critical to link different extracellular stimuli with distinct cellular responses. A classical example of hormones and growth factors creating functional diversity is illustrated by the multiple signaling pathways activated by the protein kinase C (PKC) family of serine/threonine protein kinases. The molecular requirements for diacylglycerol (DAG) and calcium (Ca(2+)) to promote PKC membrane translocation, the hallmark of PKC activation, have been clarified. However, the underlying mechanisms that establish selectivity of individual PKC family members to facilitate differential substrate phosphorylation and varied signal output are still not fully understood. It is now well believed that the coordinated control and functional diversity of PKC signaling involves the formation of PKC isozyme-specific protein complexes in certain subcellular sites. In particular, interaction of PKC isozymes with compartment and signal-organizing scaffolds, including receptors for activated C-kinase (RACKs), A-kinase-anchoring proteins (AKAPs), 14-3-3, heat shock proteins (HSP), and importins target PKC isozymes to specific cellular locations, thereby delivering PKC isozymes into close proximity of their substrates. In addition, several annexins (Anx), including AnxA1, A2, A5 and A6, display specific and distinct abilities to interact and promote membrane targeting of different PKC isozymes. Together with the ability of annexins to create specific membrane microenvironments, this is likely to enable PKCs to phosphorylate certain substrates and regulate their downstream effector pathways in specific cellular sites. This review aims to summarize the capacity of annexins to modulate the localization and activity of PKC family members and participate in the spatiotemporal regulation of PKC signaling in health and disease.

Annexin A11 in disease.

Ubiquitously expressed in many cell types, annexin A11 (Anxa11) is a member of the multigene family of Ca(2+)-regulated phospholipid-dependent and membrane-binding annexin proteins. Studies have shown that Anxa11 plays an important role in cell division, Ca(2+) signaling, vesicle trafficking and apoptosis. The deregulation and mutation of Anxa11 are involved in systemic autoimmune diseases, sarcoidosis and the development, chemoresistance and recurrence of cancers. Malfunction of Anxa11 may lead to or enhance the metastasis, invasion and drug resistance of cancers through the platelet-derived growth factor receptor (PDGFR) pathway and/or the mitogen-activated protein kinase (MAPK)/p53 pathway. In a variety of diseases, Anxa11 is most commonly reported to function through interactions with apoptosis-linked gene-2 protein (ALG-2) and/or calcyclin (S100A6). Although it has been little studied, Anxa11 is a promising biomarker for the diagnosis, treatment and prognosis of certain diseases. In this review, the associations of Anxa11 with Ca(2+)-regulated exocytosis, cytokinesis, sex differentiation, autoimmune diseases, thrombolysis and cancers are summarized and interpreted.

Annexins and endosomal signaling.

Cell signaling and endocytosis are intimately linked in eukaryotic cells. Signaling receptors at the cell surface enter the endocytic pathway and continue to activate downstream effectors in endosomal compartments. This spatiotemporal regulation of signal transduction provides opportunity for signal diversity and a cell-specific machinery of scaffolding/targeting proteins contributes to establish compartment-specific signaling complexes. Members of the annexin (Anx) protein family, in particular AnxA1, AnxA2, and AnxA6, appear to target their interaction partners to specific membrane microdomains to contribute to the formation of compartment-specific signaling platforms along the endocytic pathway. A major challenge to understand the impact of scaffolding/targeting proteins on spatiotemporal signal transduction along endocytic pathways is the identification, isolation, and functional analysis of low-abundance signal-transducing protein complexes in endocytic compartments. Here, we describe methods to isolate endosomes and to target signaling molecules to endosomes. Applying these methodologies to suitable animal or cell models will enable the dissection of signal transduction in the endocytic compartment in the presence or absence of annexins.