Identification of endosomal epidermal growth factor receptor signaling targets by functional organelle proteomics

Epidermal growth factor (EGF) receptor (EGFR) signal transduction is organized by scaffold and adaptor proteins, which have specific subcellular distribution. On a way from the plasma membrane to the lysosome EGFRs are still in their active state and can signal from distinct subcellular locations. To identify organelle-specific targets of EGF receptor signaling on endosomes a combination of subcellular fractionation, two-dimensional DIGE, fluorescence labeling of phosphoproteins, and MALDI-TOF/TOF mass spectrometry was applied. All together 23 EGF-regulated (phospho)proteins were identified as being differentially associated with endosomal fractions by functional organelle proteomics; among them were proteins known to be involved in endosomal trafficking and cytoskeleton rearrangement (Alix, myosin-9, myosin regulatory light chain, Trap1, moesin, cytokeratin 8, septins 2 and 11, and CapZbeta). Interestingly R-Ras, a small GTPase of the Ras family that regulates cell survival and integrin activity, was associated with endosomes in a ligand-dependent manner. EGF-dependent association of R-Ras with late endosomes was confirmed by confocal laser scanning immunofluorescence microscopy and Western blotting of endosomal fractions. EGFR tyrosine kinase inhibitor gefitinib was used to confirm EGF-dependent regulation of all identified proteins. EGF-dependent association of signaling molecules, such as R-Ras, with late endosomes suggests signaling specification through intracellular organelles.     

Cartography of an organelle

Peptide counting in mass spectrometry allows researchers to draw a quantitative proteomic map of the ER and Golgi.     

The receptosome: an intermediate organelle of receptor mediated endocytosis in cultured fibroblasts

Receptor-mediated endocytosis of specific ligands is mediated through clustering of receptor-ligand complexes in coated pits on the cell surface. Following this clustering event, the ligand is internalized into a noncoated intracellular vesicle, the receptosome, which selectively avoids fusion with lysosomes and moves toward the Golgi region of the cell by saltatory motion. Using alpha 2-macroglobulin as the ligand and electron microscopic cytochemical methods, we have shown the unusual appearance of this previously undescribed organelle and have followed the labeled ligand in these vesicles in the cytoplasm. To accomplish this, cells were incubated with immunolabeled alpha 2-macroglobulin at 4C under conditions where ligand-receptor complexes cluster into coated pits on the cell surface. Formation of the receptosome occurs between 2 and 5 min after raising the temperature of cells to 37C. These labeled receptosomes were seen to associate with many small vesicular elements in the cytoplasm, and were often found near the Golgi or GERL region after 15 min. Between 15 and 30 min a significant transfer of labeled ligand occurred from the receptosomal population to a population of small uniform lysosomes. By 60 min, all of the label was contained in these small lysosomes. Immunocytochemical studies showed that the receptosomes were not associated with clathrin, actin, myosin or tubulin. This unique, short-lived, specialized organelle selectively delivers the products of receptor-mediated endocytosis to intracellular sites.     

Identification of an endogenous peptide-ligand for the benzodiazepine receptor

An inhibitor of ³H-diazepam binding with characteristics that distinguish it from the other endogenous ligands reported for the benzodiazepine receptor was obtained from bovine brain. After isolation by gel filtration and ion exchange chromatography, the inhibitory factor was found to be a weakly charged molecule of approximately 3000 daltons. Although heat stable, the activity of the factor can be destroyed by treatment with papain. This factor, presumably peptide in nature, inhibited ³H-diazepam binding competitively and in a concentration dependent fashion.     

Identification and characterization of a sphere organelle protein

Sphere organelles are nuclear structures in amphibian oocytes that are easily visible by light microscopy. These structures are up to 10 microns in diameter and have been described morphologically for decades, yet their function remains obscure. The present study defines a protein component of the sphere organelle, named SPH-1, which is recognized by a mAb raised against purified Xenopus laevis oocyte nucleoplasm. SPH-1 is an 80-kD protein which is localized specifically to spheres and is undetectable elsewhere on lampbrush chromosomes or in nucleoli. We show using confocal microscopy that SPH-1 is localized to the cortex of sphere organelles. Furthermore, we have isolated a cDNA that can encode SPH-1. When epitope-tagged forms of SPH-1 are expressed in X. laevis oocytes the protein specifically localizes to spheres, demonstrating that the cloned cDNA encodes the sphere antigen. Comparison of the predicted amino acid sequence with sequence databases shows SPH-1 is related to p80-coilin, a protein associated with coiled bodies; coiled bodies are nuclear structures found in plant and animal cells. The sphere-specific mAb stains X. laevis tissue culture cells in a punctate nuclear pattern, showing that spheres or sphere antigens are present in somatic cells as well as germ cells and suggesting a general and essential function for spheres in all nuclei.     

Identification of novel proteins in Neospora caninum using an organelle purification and monoclonal antibody approach

Neospora caninum is an important veterinary pathogen that causes abortion in cattle and neuromuscular disease in dogs. Neospora has also generated substantial interest because it is an extremely close relative of the human pathogen Toxoplasma gondii, yet does not appear to infect humans. While for Toxoplasma there are a wide array of molecular tools and reagents available for experimental investigation, relatively few reagents exist for Neospora. To investigate the unique biological features of this parasite and exploit the recent sequencing of its genome, we have used an organelle isolation and monoclonal antibody approach to identify novel organellar proteins and develop a wide array of probes for subcellular localization. We raised a panel of forty-six monoclonal antibodies that detect proteins from the rhoptries, micronemes, dense granules, inner membrane complex, apicoplast, mitochondrion and parasite surface. A subset of the proteins was identified by immunoprecipitation and mass spectrometry and reveal that we have identified and localized many of the key proteins involved in invasion and host interaction in Neospora. In addition, we identified novel secretory proteins not previously studied in any apicomplexan parasite. Thus, this organellar monoclonal antibody approach not only greatly enhances the tools available for Neospora cell biology, but also identifies novel components of the unique biological characteristics of this important veterinary pathogen.     

Identification of an organelle-specific myosin V receptor

Class V myosins are widely distributed among diverse organisms and move cargo along actin filaments. Some myosin Vs move multiple types of cargo, where the timing of movement and the destinations of selected cargoes are unique. Here, we report the discovery of an organelle-specific myosin V receptor. Vac17p, a novel protein, is a component of the vacuole-specific receptor for Myo2p, a Saccharomyces cerevisiae myosin V. Vac17p interacts with the Myo2p cargo-binding domain, but not with vacuole inheritance-defective myo2 mutants that have single amino acid changes within this region. Moreover, a region of the Myo2p tail required specifically for secretory vesicle transport is neither required for vacuole inheritance nor for Vac17p-Myo2p interactions. Vac17p is localized on the vacuole membrane, and vacuole-associated Myo2p increases in proportion with an increase in Vac17p. Furthermore, Vac17p is not required for movement of other cargo moved by Myo2p. These findings demonstrate that Vac17p is a component of a vacuole-specific receptor for Myo2p. Organelle-specific receptors such as Vac17p provide a mechanism whereby a single type of myosin V can move diverse cargoes to distinct destinations at different times.     

Identification of TLT2 as an engulfment receptor for apoptotic cells

Clearance of apoptotic cells (efferocytosis) is critical to the homeostasis of the immune system by restraining inflammation and autoimmune response to intracellular Ags released from dying cells. TLRs-mediated innate immunity plays an important role in pathogen clearance and in regulation of the adaptive immune response. However, the regulation of efferocytosis by activation of TLRs has not been well characterized. In this study, we found that activation of TLR3 or TLR9, but not of TLR2, enhances engulfment of apoptotic cells by macrophages. We found that the activation of TLR3 upregulates the expression of triggering receptor expressed on myeloid cells (TREM)-like protein 2 (TLT2), a member of the TREM receptor family, on the surface of macrophages. Blocking TLT2 on the macrophage surface by either specific anti-TLT2 Ab or soluble TLT2 extracellular domain attenuated the enhanced ability of macrophages with TLR3 activation to engulf apoptotic cells. To the contrary, overexpression of TLT2 increased the phagocytosis of apoptotic cells. We found that TLT2 specifically binds to phosphatidylserine, a major "eat me" signal that is exposed on the surface of apoptotic cells. Furthermore, we found that TLT2 mediates phagocytosis of apoptotic cells in vivo. Thus, our studies identified TLT2 as an engulfment receptor for apoptotic cells. Our data also suggest a novel mechanism by which TREM receptors regulate inflammation and autoimmune response.     

Lis1 is an initiation factor for dynein-driven organelle transport

The molecular motor cytoplasmic dynein is responsible for most minus-end-directed, microtubule-based transport in eukaryotic cells. It is especially important in neurons, where defects in microtubule-based motility have been linked to neurological diseases. For example, lissencephaly is caused by mutations in the dynein-associated protein Lis1. In this paper, using the long, highly polarized hyphae of the filamentous fungus Aspergillus nidulans, we show that three morphologically and functionally distinct dynein cargos showed transport defects in the genetic absence of Lis1/nudF, raising the possibility that Lis1 is ubiquitously used for dynein-based transport. Surprisingly, both dynein and its cargo moved at normal speeds in the absence of Lis1 but with reduced frequency. Moreover, Lis1, unlike dynein and dynactin, was absent from moving dynein cargos, further suggesting that Lis1 is not required for dynein-based cargo motility once it has commenced. Based on these observations, we propose that Lis1 has a general role in initiating dynein-driven motility.     

Identification of Icm protein complexes that play distinct roles in the biogenesis of an organelle permissive for Legionella pneumophila intracellular growth

Legionella pneumophila is a bacterial pathogen that can enter the human lung and grow inside alveolar macrophages. To grow within phagocytic host cells, the bacteria must create a specialized organelle that restricts fusion with lysosomes. Biogenesis of this replicative organelle is controlled by 24 dot and icm genes, which encode a type IV-related transport apparatus. To understand how this transporter functions, isogenic L. pneumophila dot and icm mutants were characterized, and three distinct phenotypic categories were identified. Our data show that, in addition to genes that encode the core Dot/Icm transport apparatus, subsets of genes are required for pore formation and modulation of phagosome trafficking. To understand activities required for virulence at a molecular level, we investigated protein-protein interactions. Specific interactions between different Icm proteins were detected by yeast two-hybrid and gel overlay analysis. These data support a model in which the IcmQ-IcmR complex regulates the formation of a translocation channel that delivers proteins into host cells, and the IcmS-IcmW complex is required for export of virulence determinants that modulate phagosome trafficking.     

Autocrine motility factor receptor is a marker for a distinct membranous tubular organelle

Autocrine motility factor (AMF) is secreted by tumor cells and is capable of stimulating the motility of the secreting cells. In addition to being expressed on the cell surface, its receptor, AMF-R, is found within a Triton X-100 extractable intracellular tubular compartment. AMF-R tubules can be distinguished by double immunofluorescence microscopy from endosomes labeled with the transferrin receptor, lysosomes labeled with LAMP-2, and the Golgi apparatus labeled with beta-COP. AMF-R can also be separated from a LAMP-2 containing lysosomal fraction by differential centrifugation of MDCK cells and is found within a 100,000 g membrane pellet. By electron microscopic immunocytochemistry, AMF-R is localized predominantly to smooth vesicular and tubular membranous organelles as well as to a lesser extent to the plasma membrane and rough endoplasmic reticulum. AMF-R tubules have a variable diameter of 50-250 nm and can acquire an elaborate branched morphology. By immunofluorescence microscopy, AMF-R tubules are clearly distinguished from the calnexin labeled rough endoplasmic reticulum and AMF-R tubule expression is stable to extended cycloheximide treatment. The AMF-R tubule is therefore not a biosynthetic subcompartment of the endoplasmic reticulum. The tubular morphology of the AMF-R tubule is modulated by both the actin and microtubule cytoskeletons. In a similar fashion to that described previously for the tubular lysosome and endoplasmic reticulum, the linear extension and peripheral cellular orientation of the AMF-R tubule are dependent on the integrity of the microtubule cytoskeleton. The AMF-R tubule may thus form part of a family of microtubule- associated tubular organelles.     

Evidence for organelle origin

A selection of evaluations from Faculty of 1000 covering the Methanococcus maripaludis genome; recombination in animal mitochondria; modulating cell cycle progression; gene silencing by microRNA directed methylation; yeast DNA breakage and repair.     

Identification of a tumor cell receptor for VGVAPG, an elastin-derived chemotactic peptide

Extracellular matrix proteins and their proteolytic products have been shown to modulate cell motility. We have found that certain tumor cells display a chemotactic response to degradation products of the matrix protein elastin, and to an elastin-derived peptide, VGVAPG. The hexapeptide VGVAPG is a particularly potent chemotaxin for lung-colonizing Lewis lung carcinoma cells (line M27), with 5 nM VGVAPG eliciting maximal chemotactic response when assayed in 48-microwell chemotaxis chambers. Binding of the elastin-derived peptide to M27 cells was studied using a tyrosinated analog (Y-VGVAPG) to allow iodination. Scatchard analysis of [125I]Y-VGVAPG binding to viable M27 tumor cells at both 37 and 4 degrees C indicates the presence of a single class of high affinity binding sites. The dissociation constant obtained from these studies (2.7 X 10(-9) M) is equivalent to the concentration of VGVAPG required for chemotactic activity. The receptor molecule was identified as an Mr 59,000 species by covalent cross-linking of the radiolabeled ligand to the M27 tumor cell surface and subsequent analysis of the cross-linked material by electrophoresis and size-exclusion high performance liquid chromatography. These results suggest that M27 tumor cell chemotaxis to VGVAPG is initiated by high affinity binding of the peptide to a distinct cell surface receptor.     

Moving mitochondria: establishing distribution of an essential organelle

Mitochondria form a dynamic network responsible for energy production, calcium homeostasis and cell signaling. Appropriate distribution of the mitochondrial network contributes to organelle function and is essential for cell survival. Highly polarized cells, including neurons and budding yeast, are particularly sensitive to defects in mitochondrial movement and have emerged as model systems for studying mechanisms that regulate organelle distribution. Mitochondria in multicellular eukaryotes move along microtubule tracks. Actin, the primary cytoskeletal component used for transport in yeast, has more subtle functions in other organisms. Kinesin, dynein and myosin isoforms drive motor-based movement along cytoskeletal tracks. Milton and syntabulin have recently been identified as potential organelle-specific adaptor molecules for microtubule-based motors. Miro, a conserved GTPase, may function with Milton to regulate transport. In yeast, Mmr1p and Ypt11p, a Rab GTPase, are implicated in myosin V-based mitochondrial movement. These potential adaptors could regulate motor activity and therefore determine individual organelle movements. Anchoring of stationary mitochondria also contributes to organelle retention at specific sites in the cell. Together, movement and anchoring ultimately determine mitochondrial distribution throughout the cell.