Shigella enters epithlial cells via internalization into a vacuole. Subsequent vacuolar membrane rupture allows bacterial escape into the cytosol for replication and cell-to-cell spread. Bacterial effectors such as IpgD, a PI(4,5)P2 phosphatase that generates PI(5)P and alters host actin, facilitate this internalization. Here, we identify host proteins involved in Shigella uptake and vacuolar membrane rupture by high-content siRNA screening and subsequently focus on Rab11, a constituent of the recycling compartment. Rab11-positive vesicles are recruited to the invasion site before vacuolar rupture, and Rab11 knockdown dramatically decreases vacuolar membrane rupture. Additionally, Rab11 recruitment is absent and vacuolar rupture is delayed in the ipgD mutant that does not dephosphorylate PI(4,5)P2 into PI(5)P. Ultrastructural analyses of Rab11-positive vesicles further reveal that ipgD mutant-containing vacuoles become confined in actin structures that likely contribute to delayed vacular rupture. These findings provide insight into the underlying molecular mechanism of vacuole progression and rupture during Shigella invasion.
Protein kinase inhibitors have emerged as new drugs in various therapeutic areas including leishmaniasis, an important parasitic disease. Members of the Leishmania Casein Kinase 1 family represent promising therapeutic targets. The Leishmania casein kinase 1 isoform 2 (CK1.2) has been identified as an exokinase capable of phosphorylating host proteins thus exerting a potential immune-suppressive action on infected host cells. Moreover its inhibition reduces promastigote growth. Despite these important properties, its requirement for intracellular infection and its chemical validation as therapeutic target in the disease-relevant amastigote stage remain to be established. In this report, we used a multi-disciplinary approach combining bio-informatics, biochemical, and pharmacological analyses with macrophage infection assay to characterize and define Leishmania CK1.2 as a valid drug target. We showed that recombinant and transgenic Leishmania CK1.2 (i) can phosphorylate CK1-specific substrates, (ii) are sensitive to temperature, and (iii) are susceptible to CK1-specific inhibitors. CK1.2 is constitutively expressed at both the promastigote insect and the vertebrate amastigote stages. We further demonstrated that reduction of CK1 activity by specific inhibitors such as D4476 blocks promastigote growth, strongly compromises axenic amastigote viability and decreases the number of intracellular L. donovani and L. amazonensis amastigotes in infected macrophages. These results underline the potential role of CK1 kinases in intracellular survival. The identification of differences in structure and inhibition profiles, compared to mammalian CK1 kinases, opens new opportunities for Leishmania CK1.2 anti-leishmanial drug development. Our report provides the first chemical validation of Leishmania CK1 protein kinases as therapeutic targets, required for amastigote intracellular survival.
The Notch signaling pathway is involved in liver development and regeneration. Here, we investigate the role of the 4 mammalian Notch paralogs in the regulation of hepatoblast proliferation and hepatocytic differentiation. Our model is based on bipotential mouse embryonic liver (BMEL) progenitors that can differentiate into hepatocytes or cholangiocytes in vitro and in vivo. BMEL cells were subjected to Notch antagonists or agonists. Blocking Notch activation with a ?-secretase inhibitor, at 50 ?M for 48 h, reduced cell growth by 50%. S-phase entry was impaired, but no apoptosis was induced. A systematic paralog-specific strategy was set using lentiviral transduction with constitutively active forms of each Notch receptor along with inhibition of endogenous Notch signaling. This assay demonstrates that proliferation of BMEL cells requires Notch2 and Notch4 activity, resulting in significant down-regulation of p27(Kip1) and p57(Kip2) cyclin-dependent kinase inhibitors. Conversely, Notch3-expressing cells proliferate less and express 3-fold higher levels of p57(Kip2). The Notch3 cells present a hepatocyte-like morphology, enhanced multinucleation, and a ploidy shift. Moreover, Notch3 activity is conducive to hepatocytic differentiation in vitro, while its paralogs impede this fate. Our study provides the first evidence of a functional diversity among the mammalian Notch homologues in the proliferation and hepatocytic-lineage commitment of liver progenitors.-Ortica, S., Tarantino, N., Aulner, N., Israël, A., Gupta-Rossi, N. The 4 Notch receptors play distinct and antagonistic roles in the proliferation and hepatocytic differentiation of liver progenitors.
Human leishmaniases are parasitic diseases causing severe morbidity and mortality. No vaccine is available and numerous factors limit the use of current therapies. There is thus an urgent need for innovative initiatives to identify new chemotypes displaying selective activity against intracellular Leishmania amastigotes that develop and proliferate inside macrophages, thereby causing the pathology of leishmaniasis.
The HIV-1 Nef protein is a pathogenic factor modulating the behavior of infected cells. Nef induces actin cytoskeleton changes and impairs cell migration toward chemokines. We further characterized the morphology, cytoskeleton dynamics, and motility of HIV-1-infected lymphocytes. By using scanning electron microscopy, confocal immunofluorescence microscopy, and ImageStream technology, which combines flow cytometry and automated imaging, we report that HIV-1 induces a characteristic remodeling of the actin cytoskeleton. In infected lymphocytes, ruffle formation is inhibited, whereas long, thin filopodium-like protrusions are induced. Cells infected with HIV with nef deleted display a normal phenotype, and Nef expression alone, in the absence of other viral proteins, induces morphological changes. We also used an innovative imaging system to immobilize and visualize living individual cells in suspension. When combined with confocal "axial tomography," this technique greatly enhances three-dimensional optical resolution. With this technique, we confirmed the induction of long filopodium-like structures in unfixed Nef-expressing lymphocytes. The cytoskeleton reorganization induced by Nef is associated with an important impairment of cell movements. The adhesion and spreading of infected cells to fibronectin, their spontaneous motility, and their migration toward chemokines (CXCL12, CCL3, and CCL19) were all significantly decreased. Therefore, Nef induces complex effects on the lymphocyte actin cytoskeleton and cellular morphology, which likely impacts the capacity of infected cells to circulate and to encounter and communicate with bystander cells.
Pathogens use diverse molecular machines to penetrate host cells and manipulate intracellular vesicular trafficking. Viruses employ glycoproteins, functionally and structurally similar to the SNARE proteins, to induce eukaryotic membrane fusion. Intracellular pathogens, on the other hand, need to block fusion of their infectious phagosomes with various endocytic compartments to escape from the degradative pathway. The molecular details concerning the mechanisms underlying this process are lacking. Using both an in vitro liposome fusion assay and a cellular assay, we showed that SNARE-like bacterial proteins block membrane fusion in eukaryotic cells by directly inhibiting SNARE-mediated membrane fusion. More specifically, we showed that IncA and IcmG/DotF, two SNARE-like proteins respectively expressed by Chlamydia and Legionella, inhibit the endocytic SNARE machinery. Furthermore, we identified that the SNARE-like motif present in these bacterial proteins encodes the inhibitory function. This finding suggests that SNARE-like motifs are capable of specifically manipulating membrane fusion in a wide variety of biological environments. Ultimately, this motif may have been selected during evolution because it is an efficient structural motif for modifying eukaryotic membrane fusion and thus contribute to pathogen survival.
To understand signaling pathways in mammalian cells, cell-based assays are relatively new and extremely powerful tools. We have developed a battery of phenotypic assays to study signaling; two of them are described in detail in this chapter. A subset of these assays monitors mitogen-activated protein (MAP) kinase pathways. MAP kinases are principal regulators of fundamental processes in mammalian cells, including growth, cell division, differentiation, stress responses, and neoplastic transformation. Here we describe two cell-based assays querying the function of ERK (extracellular signal regulated kinase), one of the three principal MAP kinases in mammalian cells. We selected human umbilical vein endothelial cells (HUVECs), a primary cell type, because they show a very dynamic response to various activators. Both assays are phenotypic assays and use well-established phosphorylation-specific primary antibodies to study activation. Fluorochrome-coupled secondary antibodies were used to label phosphorylated target proteins; images were captured with the INCell Analyzer 3000 and analyzed with the INCell Analyzer 3000 software. The first of these two assays monitors phosphorylation of ERK1/2, while the second assay monitors activation of the transcription factor CREB (cAMP response element-binding protein). The assays described in this chapter cover major checkpoints of the ERK signaling pathway: (1) MAP kinase activation and (2) subsequent transcription factor activation. Both assays exhibit robust performance and can easily be used for high-throughput screening.
A quinazoline that decreases polyglutamine aggregate burden in a cell-based assay was identified from a high-throughput screen of a chemical-compound library, provided by the NIH Molecular Libraries Small Molecule Repository (MLSMR). A structure and activity study yielded leads with submicromolar potency.
An IKKbeta inhibitor reported to block NF-kappaB transcriptional activities in Jurkat T cells, was found to enhance NF-kappaB translocation in HUVEC cells. These studies suggested a noncanonical NF-kappaB signaling pathway independent of IKKbeta in HUVEC cells.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.