Sylvain V. Costes

The Transformation Suppressor Pdcd4 Is a Novel Eukaryotic Translation Initiation Factor 4A Binding Protein That Inhibits Translation

ABSTRACT Pdcd4 is a novel transformation suppressor that inhibits tumor promoter-induced neoplastic transformation and the activation of AP-1-dependent transcription required for transformation. A yeast two-hybrid analysis revealed that Pdcd4 associates with the eukaryotic translation initiation factors eIF4AI and eIF4AII. Immunofluorescent confocal microscopy showed that Pdcd4 colocalizes with eIF4A in the cytoplasm. eIF4A is an ATP-dependent RNA helicase needed to unwind 5′ mRNA secondary structure. Recombinant Pdcd4 specifically inhibited the helicase activity of eIF4A and eIF4F. In vivo translation assays showed that Pdcd4 inhibited cap-dependent but not internal ribosome entry site (IRES)-dependent translation. In contrast, Pdcd4D418A, a mutant inactivated for binding to eIF4A, failed to inhibit cap-dependent or IRES-dependent translation or AP-1 transactivation. Recombinant Pdcd4 prevented eIF4A from binding to the C-terminal region of eIF4G (amino acids 1040 to 1560) but not to the middle region of eIF4G(amino acids 635 to 1039). In addition, both Pdcd4 and Pdcd4D418A bound to the middle region of eIF4G. The mechanism by which Pdcd4 inhibits translation thus appears to involve inhibition of eIF4A helicase, interference with eIF4A association-dissociation from eIF4G, and inhibition of eIF4A binding to the C-terminal domain of eIF4G. Pdcd4 binding to eIF4A is linked to its transformation-suppressing activity, as Pdcd4-eIF4A binding and consequent inhibition of translation are required for Pdcd4 transrepression of AP-1.

Inhibition of Metastatic Outgrowth from Single Dormant Tumor Cells by Targeting the Cytoskeleton

Metastatic breast cancer may emerge from latent tumor cells that remain dormant at disseminated sites for many years. Identifying mechanisms regulating the switch from dormancy to proliferative metastatic growth has been elusive due to the lack of experimental models of tumor cell dormancy. We characterized the in vitro growth characteristics of cells that exhibit either dormant (D2.0R, MCF-7, and K7M2AS1.46) or proliferative (D2A1, MDA-MB-231, and K7M2) metastatic behavior in vivo. Although these cells proliferate readily in two-dimensional culture, we show that when grown in three-dimensional matrix, distinct growth properties of the cells were revealed that correlate to their dormant or proliferative behavior at metastatic sites in vivo. In three-dimensional culture, cells with dormant behavior in vivo remained cell cycle arrested with elevated nuclear expression of p16 and p27. The transition from quiescence to proliferation of D2A1 cells was dependent on fibronectin production and signaling through integrin beta1, leading to cytoskeletal reorganization with filamentous actin (F-actin) stress fiber formation. We show that phosphorylation of myosin light chain (MLC) by MLC kinase (MLCK) through integrin beta1 is required for actin stress fiber formation and proliferative growth. Inhibition of integrin beta1 or MLCK prevents transition from a quiescent to proliferative state in vitro. Inhibition of MLCK significantly reduces metastatic outgrowth in vivo. These studies show that the switch from dormancy to metastatic growth may be regulated, in part, through epigenetic signaling from the microenvironment, leading to changes in the cytoskeletal architecture of dormant cells. Targeting this process may provide therapeutic strategies for inhibition of the dormant-to-proliferative metastatic switch.

Human CD8+ T Cells Store RANTES in a Unique Secretory Compartment and Release It Rapidly after TcR Stimulation

The chemokine RANTES is secreted rapidly after activation of human CD8+ T cells, with a cycloheximide-resistant burst during the first hour. This pattern was observed in purified memory and effector phenotype CD8+ cells from blood as well as in blasts. In contrast, secretion of other chemokines and interferon-gamma by these cells was sensitive to cycloheximide and detectable only after a lag. Immunofluorescence microscopy of CD8+ memory and effector cells and blasts showed RANTES present in intracellular vesicles that do not significantly colocalize with cytotoxic granule markers or other markers of defined cytoplasmic compartments. Immunoelectron microscopy confirmed that RANTES is stored in small vesicles distinct from the lysosomal secretory granules. RANTES+ vesicles polarize rapidly in response to TcR engagement and are more rapidly depleted from the cytoplasm. These results show that CD8+ T cells have two distinct TcR-regulated secretory compartments characterized by different mobilization kinetics, effector molecules, and biological function.

Kinetic and Molecular Analysis of Nuclear Export Factor CRM1 Association with Its Cargo In Vivo

ABSTRACT The nucleocytoplasmic transport receptor CRM1 mediates the export of macromolecules from the nucleus to the cytoplasm by forming a ternary complex with a cargo molecule and RanGTP. The in vivo mechanism of CRM1 export complex formation and its mobility throughout the nucleus have not been fully elucidated. More information is required to fully understand complex formation and the dynamics of CRM1-cargo-RanGTP complexes in space and time. We demonstrate true molecular interaction of CRM1 with its Rev cargo in living cells by using fluorescence resonance energy transfer (FRET). Interestingly, we found that the inhibitory effect of leptomycin B on this CRM1-cargo interaction is Ran dependent. Using fluorescence recovery after photobleaching (FRAP), we show that CRM1 moves at rates similar to that of free green fluorescent protein in the nucleoplasm. A slower mobility was detected on the nuclear membrane, consistent with known CRM1 interactions with nuclear pores. Based on these data, we propose an in vivo model in which CRM1 roams through the nucleus in search of high-affinity binding sites. CRM1 is able to bind Rev cargo in the nucleolus, and upon RanGTP binding a functional export complex is produced that is exported to the cytoplasm.

Role of Murine Leukemia Virus Nucleocapsid Protein in Virus Assembly

ABSTRACT The retroviral nucleocapsid protein (NC) originates by cleavage of the Gag polyprotein. It is highly basic and contains one or two zinc fingers. Mutations in either the basic residues or the zinc fingers can affect several events of the virus life cycle. They frequently prevent the specific packaging of the viral RNA, affect reverse transcription, and impair virion assembly. In this work, we explore the role of NC in murine leukemia virus (MLV) particle assembly and release. A panel of NC mutants, including mutants of the zinc finger and of a basic region, as well as truncations of the NC domain of Gag, were studied. Several of these mutations dramatically reduce the release of virus particles. A mutant completely lacking the NC domain is apparently incapable of assembling into particles, although its Gag protein is still targeted to the plasma membrane. By electron microscopy on thin sections of virus-producing cells, we observed that some NC mutants exhibit various stages of budding defects at the plasma membrane and have aberrant particle morphology; electron micrographs of cells expressing some of these mutants are strikingly similar to those of cells expressing “late-domain” mutants. However, the defects of NC mutants with respect to virus release and infectivity could be complemented by an MLV lacking the p12 domain. Therefore, the functions of NC in virus budding and infectivity are completely distinct from viral late-domain function.

FRAP model to determine the bidirectional transport rate of GFP across the nuclear membrane and the mobile fraction in the cytoplasm and nucleus

A mathematical model was developed to predict the bi- directional transport rate of fluorescent proteins across the nuclear membrane during a fluorescence recovery after photobleaching (FRAP) experiment. The model assumes that the total amount of fluorescent protein remains the same in the cell (i.e. no production, loss or exchange with the outside of the cell) and that the cell is in a state of equilibrium; i.e. proteins are leaving and entering the nucleus at an equal rate. The latter assumption has the advantage of not needing to take into account the method of protein transport (e.g. active or passive). The model includes correction for the photobleaching that happens during image acquisition following the deliberate photobleach. In this study, the green fluorescent protein (GFP) was transfected into cells in order to study its free behavior. In the FRAP experiments, either the entire nucleus and part of the cytoplasm or only part of the cytoplasm was photobleached followed by time-series imaging of the fluorescence redistribution. The model was fitted to the curves of intensity loss or recovery after photobleaching using numerical, non-linear methods. In addition, the mobile fractions of free GFP in the cytoplasm and the nucleus could be determined.