Marcelo Ehrlich

Endocytosis by Random Initiation and Stabilization of Clathrin-Coated Pits

Clathrin-coated vesicles carry traffic from the plasma membrane to endosomes. We report here the real-time visualization of cargo sorting and endocytosis by clathrin-coated pits in living cells. We have detected the formation of coats by monitoring incorporation of fluorescently tagged clathrin or its adaptor AP-2; we have also followed clathrin-mediated uptake of transferrin and of single LDL or reovirus particles. The intensity of a cargo-loaded clathrin cluster grows steadily during its lifetime, and the time required to complete assembly is proportional to the size of the cargo particle. These results are consistent with a nucleation-growth mechanism and an approximately constant growth rate. There are no strongly preferred nucleation sites. A proportion of the nucleation events are weak and short lived. Cargo incorporation occurs primarily or exclusively in a newly formed coated pit. Our data lead to a model in which coated pits initiate randomly but collapse unless stabilized, perhaps by cargo capture.

The Mode of Bone Morphogenetic Protein (BMP) Receptor Oligomerization Determines Different BMP-2 Signaling Pathways

Bone morphogenetic proteins (BMPs) are multifunctional proteins regulating cell growth, differentiation, and apoptosis. BMP-2 signals via two types of receptors (BRI and BRII) that are expressed at the cell surface as homomeric as well as heteromeric complexes. Prior to ligand binding, a low but measurable level of BMP-receptors is found in preformed hetero-oligomeric complexes. The major fraction of the receptors is recruited into hetero-oligomeric complexes only after ligand addition. For this, BMP-2 binds first to the high affinity receptor BRI and then recruits BRII into the signaling complex. However, ligand binding to the preformed complex composed of BRII and BRI is still required for signaling, suggesting that it may mediate activating conformational changes. Using several approaches we have addressed the following questions: (i) Are preformed complexes incompetent of signaling in the absence of BMP-2? (ii) Which domains of the BRII receptors are essential for this complex formation? (iii) Are there differences in signals sent from BMP-inducedversus preformed receptor complexes? By measuring the activation of Smads, of p38 MAPK and of alkaline phosphatase, we show that the ability of kinase-deficient BRII receptor mutants to inhibit BMP signaling depends on their ability to form heteromeric complexes with BRI. Importantly, a BRII mutant that is incapable in forming preassembled receptor complexes but recruits into a BMP-induced receptor complex does not interfere with the Smad pathway but does inhibit the induction of alkaline phosphatase as well as p38 phosphorylation. These results indicate that signals induced by binding of BMP-2 to preformed receptor complexes activate the Smad pathway, whereas BMP-2-induced recruitment of receptors activates a different, Smad-independent pathway resulting in the induction of alkaline phosphatase activity via p38 MAPK.

Initiation of Smad-dependent and Smad-independent signaling via distinct BMP-receptor complexes.

Background: BMP-2 (bone morphogenetic protein-2) signals via two types of transmembrane serine/threonine kinase receptors (BRI and BRII), which form heteromeric complexes prior to and after ligand binding. Within a BMP-bound receptor complex, BRII transphosphorylates and activates BRI-a for further signaling. We investigated which signaling pathway is initiated by BMP-2 via preformed receptor complexes versus BMP-2-induced signaling receptor complexes.Methods: Immunofluorescence co-patching was used to study the oligomerization of receptors at the surface of live cells. Binding and chemical cross-linking of iodinated BMP-2 followed by immunoprecipitation was used to show association of receptors in the presence of ligand. Western blots with use of anti-phospho-Smad1 antibodies and reporter gene assays with use of SBE-lux were employed to show activation of the Smad pathway. Phosphorylation of p38-MAPK was shown by Western blots. Induction of alkaline phosphatase was determined by staining the cells. The cluster density of receptors was determined with use of image correlation spectroscopy.Results and Conclusion: We showed that the Smad pathway is induced by preformed receptor complexes, whereas BMP-2-induced signaling complexes result in the activation of p38-MAPK. We also found evidence that the clustering of BRI-a at the membrane is altered in the presence of BRII, suggesting that it associates with existing clusters of BRII to initiate efficient Smad signaling. These data clearly demonstrate that it is critical to fully understand receptor oligomerization in order to estimate signaling outcome for distinct receptor and ligand mutants.Clinical Relevance: The development of BMP-2 antagonists is of special importance for a number of human disorders caused by several members of the BMP/TGF-&bgr; (transforming growth factor-beta) superfamily. Since manipulation of BMP-signaling is complex, it is important to understand what influence it might have during the initiation of signaling—:i.e., the oligomerization of BMP receptors to form a signaling receptor complex. There might be cases where either the Smad or the p38 pathway should be targeted.

TMPRSS2/ERG Promotes Epithelial to Mesenchymal Transition through the ZEB1/ZEB2 Axis in a Prostate Cancer Model

Prostate cancer is the most common non-dermatologic malignancy in men in the Western world. Recently, a frequent chromosomal aberration fusing androgen regulated TMPRSS2 promoter and the ERG gene (TMPRSS2/ERG) was discovered in prostate cancer. Several studies demonstrated cooperation between TMPRSS2/ERG and other defective pathways in cancer progression. However, the unveiling of more specific pathways in which TMPRSS2/ERG takes part, requires further investigation. Using immortalized prostate epithelial cells we were able to show that TMPRSS2/ERG over-expressing cells undergo an Epithelial to Mesenchymal Transition (EMT), manifested by acquisition of mesenchymal morphology and markers as well as migration and invasion capabilities. These findings were corroborated in vivo, where the control cells gave rise to discrete nodules while the TMPRSS2/ERG-expressing cells formed malignant tumors, which expressed EMT markers. To further investigate the general transcription scheme induced by TMPRSS2/ERG, cells were subjected to a microarray analysis that revealed a distinct EMT expression program, including up-regulation of the EMT facilitators, ZEB1 and ZEB2, and down-regulation of the epithelial marker CDH1(E-Cadherin). A chromatin immunoprecipitation assay revealed direct binding of TMPRSS2/ERG to the promoter of ZEB1 but not ZEB2. However, TMPRSS2/ERG was able to bind the promoters of the ZEB2 modulators, IL1R2 and SPINT1. This set of experiments further illuminates the mechanism by which the TMPRSS2/ERG fusion affects prostate cancer progression and might assist in targeting TMPRSS2/ERG and its downstream targets in future drug design efforts.

The δ Region of Outer-Capsid Protein μ1 Undergoes Conformational Change and Release from Reovirus Particles during Cell Entry

ABSTRACT Cell entry by reoviruses requires a large, transcriptionally active subvirion particle to gain access to the cytoplasm. The features of this particle have been the subject of debate, but three primary candidates—the infectious subvirion particle (ISVP), ISVP*, and core particle forms—that differ in whether putative membrane penetration protein μ1 and adhesin σ1 remain particle bound have been identified. Experiments with antibody reagents in this study yielded new information about the steps in particle disassembly during cell entry. Monoclonal antibodies specific for the δ region of μ1 provided evidence for a conformational change inμ 1 and for release of the δ proteolytic fragment from entering particles. Antiserum raised against cores provided evidence for entry-related changes in particle structure and identified entering particles that largely lack the δ fragment inside cells. Antibodies specific for σ1 showed that it is also largely shed from entering particles. Limited coimmunostaining with markers for late endosomes and lysosomes indicated the particles lacking δ andσ 1 did not localize to those subcellular compartments, and other observations suggested that both the particles and free δ were released into the cytoplasm. Essentially equivalent findings were obtained with native ISVPs and highly infectious recoated particles containing wild-type proteins. Poorly infectious recoated particles containing a hyperstable mutant form of μ1, however, showed no evidence for the in vitro and intracellular changes in particle structure normally detected by antibodies, and these particles instead accumulated in late endosomes or lysosomes. Recoated particles with hyperstable μ1 were also ineffective at mediating erythrocyte lysis in vitro and promoting α-sarcin coentry and intoxication of cells in cultures. Based on these and other findings, we propose that ISVP* is a transient intermediate in cell entry which mediates membrane penetration and is then further uncoated in the cytoplasm to yield particles, resembling cores, that largely lack the δ fragment ofμ 1.

A Legionella effector acquired from protozoa is involved in sphingolipids metabolism and is targeted to the host cell mitochondria

Legionella pneumophila infects alveolar macrophages and protozoa through establishment of an intracellular replication niche. This process is mediated by bacterial effectors translocated into the host cell via the Icm/Dot type IV secretion system. Most of the effectors identified so far are unique to L. pneumophila; however, some of the effectors are homologous to eukaryotic proteins. We performed a distribution analysis of many known L. pneumophila effectors and found that several of them, mostly eukaryotic homologous proteins, are present in different Legionella species. In‐depth analysis of LegS2, a L. pneumophila homologue of the highly conserved eukaryotic enzyme sphingosine‐1‐phosphate lyase (SPL), revealed that it was most likely acquired from a protozoan organism early during Legionella evolution. The LegS2 protein was found to translocate into host cells using a C‐terminal translocation domain absent in its eukaryotic homologues. LegS2 was found to complement the sphingosine‐sensitive phenotype of a Saccharomyces serevisia SPL‐null mutant and this complementation depended on evolutionary conserved residues in the LegS2 catalytic domain. Interestingly, unlike the eukaryotic SPL that localizes to the endoplasmic reticulum, LegS2 was found to be targeted mainly to host cell mitochondria. Collectively, our results demonstrate the remarkable adaptations of a eukaryotic protein to the L. pneumophila pathogenesis system.

Clustering of Raft-Associated Proteins in the External Membrane Leaflet Modulates Internal Leaflet H-Ras Diffusion and Signaling

ABSTRACT One of the least-explored aspects of cholesterol-enriched domains (rafts) in cells is the coupling between such domains in the external and internal monolayers and its potential to modulate transbilayer signal transduction. Here, we employed fluorescence recovery after photobleaching to study the effects of antibody-mediated patching of influenza hemagglutinin (HA) proteins [raft-resident wild-type HA and glycosylphosphatidylinositol-anchored HA, or the nonraft mutant HA(2A520)] on the lateral diffusion of internal-leaflet raft and nonraft Ras isoforms (H-Ras and K-Ras, respectively). Our studies demonstrate that the clustering of outer-leaflet or transmembrane raft-associated HA proteins (but not their nonraft mutants) retards the lateral diffusion of H-Ras (but not K-Ras), suggesting stabilized interactions of H-Ras with the clusters of raft-associated HA proteins. These modulations were paralleled by specific effects on the activity of H-Ras but not of the nonraft K-Ras. Thus, clustering raft-associated HA proteins facilitated the early step whereby H-Ras is converted to an activated, GTP-loaded state but inhibited the ensuing step of downstream signaling via the Mek/Erk pathway. We propose a model for the modulation of transbilayer signaling by clustering of raft proteins, where external clustering (antibody or ligand mediated) enhances the association of internal-leaflet proteins with the stabilized clusters, promoting either enhancement or inhibition of signaling.

Homomeric and heteromeric complexes among TGF-β and BMP receptors and their roles in signaling.

Transforming growth factor-β (TGF-β) ligands and bone morphogenetic proteins (BMPs) play myriad roles in many biological processes and diseases. Their pluripotent activities are subject to multiple levels of regulation, including receptor oligomerization, endocytosis, association with co-receptors, cellular scaffolds or membrane domains, as well as transcriptional control. In this review, we focus on TGF-β and BMP receptor homomeric and heteromeric complex formation and their modulation by ligand binding, which regulate signaling on a near-immediate timescale. We discuss the current structural, biochemical and biophysical evidence for the oligomerization of these receptors, and the potential roles of distinct oligomeric interactions in signaling.

Disruption of TGF-β growth inhibition by oncogenic ras is linked to p27Kip1 mislocalization

Expression of oncogenic Ras in epithelial tumor cells is linked to the loss of transforming growth factor-β (TGF-β) anti-proliferative activity, and was proposed to involve inhibition of Smad2/3 nuclear translocation. Here we studied several epithelial cell lines expressing oncogenic N-RasK61 and show that TGF-β-induced nuclear translocation of and transcriptional activation by Smad2/3 were unaffected. In contrast, oncogenic Ras mediated nuclear-to-cytoplasmic mislocalization of p27Kip1 (p27) and of the cyclin-dependent kinase (CDK) CDK6, but not CDK2. Concomitantly, oncogenic Ras abrogated the ability of TGF-β to release p27 from CDK6, to enhance its binding to CDK2 and to inhibit CDK2 activity. Inactivation of Ras by a specific antagonist restored the growth inhibitory response to TGF-β with concurrent normalization of p27 and CDK6 localization. Therefore, the disruption of TGF-β-mediated growth inhibition by oncogenic Ras appears to be due to lack of inhibition of CDK2, caused by the sequestration of p27 and CDK2 in different subcellular compartments and by the loss of TGF-β-induced partner switching of p27 from CDK6 to CDK2.

Endosomal signaling of the tomato leucine-rich repeat receptor-like protein LeEix2

Extracellular leucine-rich repeat (LRR) receptor-like proteins (RLPs) represent a unique class of cell-surface receptors, as they lack a functional cytoplasmic domain. Our knowledge of how RLPs that do not contain a kinase or Toll domain function is very limited. The tomato RLP receptor LeEix2 signals to induce defense responses mediated by the fungal protein ethylene-inducing xylanase (EIX). The movement of FYVE-positive endosomes before and after EIX application was examined using spinning disc confocal microscopy. We found that while FYVE-positive endosomes generally observe a random movement pattern, following EIX application a subpopulation of FYVE-positive endosomes follow a directional movement pattern. Further, cellular endosomes travel greater distances at higher speeds following EIX application. Time-course experiments conducted with specific inhibitors demonstrate the involvement of endosomal signaling in EIX-triggered defense responses. Abolishing the existence of endosomes or the endocytic event prevented EIX-induced signaling. Endocytosis/endosome inhibitors, such as Dynasore or 1-butanol, inhibit EIX-induced signaling. Moreover, treatment with Endosidin1, which inhibits an early step in plasma membrane/endosome trafficking, enhances the induction of defense responses by EIX. Our data indicate a distinct endosomal signaling mechanism for induction of defense responses in this RLP system.