Imaging the Impact of Chemically Inducible Proteins on Cellular Dynamics In Vivo

The analysis of dynamic events in the tumor microenvironment during cancer progression is limited by the complexity of current in vivo imaging models. This is coupled with an inability to rapidly modulate and visualize protein activity in real time and to understand the consequence of these perturbations in vivo. We developed an intravital imaging approach that allows the rapid induction and subsequent depletion of target protein levels within human cancer xenografts while assessing the impact on cell behavior and morphology in real time. A conditionally stabilized fluorescent E-cadherin chimera was expressed in metastatic breast cancer cells, and the impact of E-cadherin induction and depletion was visualized using real-time confocal microscopy in a xenograft avian embryo model. We demonstrate the assessment of protein localization, cell morphology and migration in cells undergoing epithelial-mesenchymal and mesenchymal-epithelial transitions in breast tumors. This technique allows for precise control over protein activity in vivo while permitting the temporal analysis of dynamic biophysical parameters

Apurinic/Apyrimidinic Endonuclease 1 Restricts the Internalization of Bacteria Into Human Intestinal Epithelial Cells Through the Inhibition of Rac1.

Pathogenic intestinal bacteria lead to significant disease in humans. Here we investigated the role of the multifunctional protein, Apurinic/apyrimidinic endonuclease 1 (APE1), in regulating the internalization of bacteria into the intestinal epithelium. Intestinal tumor-cell lines and primary human epithelial cells were infected with Salmonella enterica serovar Typhimurium or adherent-invasive Escherichia coli. The effects of APE1 inhibition on bacterial internalization, the regulation of Rho GTPase Rac1 as well as the epithelial cell barrier function were assessed. Increased numbers of bacteria were present in APE1-deficient colonic tumor cell lines and primary epithelial cells. Activation of Rac1 was augmented following infection but negatively regulated by APE1. Pharmacological inhibition of Rac1 reversed the increase in intracellular bacteria in APE1-deficient cells whereas overexpression of constitutively active Rac1 augmented the numbers in APE1-competent cells. Enhanced numbers of intracellular bacteria resulted in the loss of barrier function and a delay in its recovery. Our data demonstrate that APE1 inhibits the internalization of invasive bacteria into human intestinal epithelial cells through its ability to negatively regulate Rac1. This activity also protects epithelial cell barrier function

Apurinic/Apyrimidinic Endonuclease 1 Restricts the Internalization of Bacteria Into Human Intestinal Epithelial Cells Through the Inhibition of Rac1.

Pathogenic intestinal bacteria lead to significant disease in humans. Here we investigated the role of the multifunctional protein, Apurinic/apyrimidinic endonuclease 1 (APE1), in regulating the internalization of bacteria into the intestinal epithelium. Intestinal tumor-cell lines and primary human epithelial cells were infected with Salmonella enterica serovar Typhimurium or adherent-invasive Escherichia coli. The effects of APE1 inhibition on bacterial internalization, the regulation of Rho GTPase Rac1 as well as the epithelial cell barrier function were assessed. Increased numbers of bacteria were present in APE1-deficient colonic tumor cell lines and primary epithelial cells. Activation of Rac1 was augmented following infection but negatively regulated by APE1. Pharmacological inhibition of Rac1 reversed the increase in intracellular bacteria in APE1-deficient cells whereas overexpression of constitutively active Rac1 augmented the numbers in APE1-competent cells. Enhanced numbers of intracellular bacteria resulted in the loss of barrier function and a delay in its recovery. Our data demonstrate that APE1 inhibits the internalization of invasive bacteria into human intestinal epithelial cells through its ability to negatively regulate Rac1. This activity also protects epithelial cell barrier function

Increased Tumor Homing and Tissue Penetration of the Filamentous Plant Viral Nanoparticle <i>Potato virus X</i>

Nanomaterials with elongated architectures have been shown to possess differential tumor homing properties compared to their spherical counterparts. Here, we investigate whether this phenomenon is mirrored by plant viral nanoparticles that are filamentous (<i>Potato virus X</i>) or spherical (<i>Cowpea mosaic virus</i>). Our studies demonstrate that <i>Potato virus X</i> (PVX) and <i>Cowpea mosaic virus</i> (CPMV) show distinct biodistribution profiles and differ in their tumor homing and penetration efficiency. Analogous to what is seen with inorganic nanomaterials, PVX shows enhanced tumor homing and tissue penetration. Human tumor xenografts exhibit higher uptake of PEGylated filamentous PVX compared to CPMV, particularly in the core of the tumor. This is supported by immunohistochemical analysis of the tumor sections, which indicates greater penetration and accumulation of PVX within the tumor tissues. The enhanced tumor homing and retention properties of PVX along with its higher payload carrying capacity make it a potentially superior platform for applications in cancer drug delivery and imaging applications

Regulation of Rac1 and Reactive Oxygen Species Production in Response to Infection of Gastrointestinal Epithelia.

Generation of reactive oxygen species (ROS) during infection is an immediate host defense leading to microbial killing. APE1 is a multifunctional protein induced by ROS and after induction, protects against ROS-mediated DNA damage. Rac1 and NAPDH oxidase (Nox1) are important contributors of ROS generation following infection and associated with gastrointestinal epithelial injury. The purpose of this study was to determine if APE1 regulates the function of Rac1 and Nox1 during oxidative stress. Gastric or colonic epithelial cells (wild-type or with suppressed APE1) were infected with Helicobacter pylori or Salmonella enterica and assessed for Rac1 and NADPH oxidase-dependent superoxide production. Rac1 and APE1 interactions were measured by co-immunoprecipitation, confocal microscopy and proximity ligation assay (PLA) in cell lines or in biopsy specimens. Significantly greater levels of ROS were produced by APE1-deficient human gastric and colonic cell lines and primary gastric epithelial cells compared to control cells after infection with either gastric or enteric pathogens. H. pylori activated Rac1 and Nox1 in all cell types, but activation was higher in APE1 suppressed cells. APE1 overexpression decreased H. pylori-induced ROS generation, Rac1 activation, and Nox1 expression. We determined that the effects of APE1 were mediated through its N-terminal lysine residues interacting with Rac1, leading to inhibition of Nox1 expression and ROS generation. APE1 is a negative regulator of oxidative stress in the gastrointestinal epithelium during bacterial infection by modulating Rac1 and Nox1. Our results implicate APE1 in novel molecular interactions that regulate early stress responses elicited by microbial infections

A chemically tunable form of E-cadherin for use in intravital imaging.

<p>A) Expression vectors encoding tunable zsGreen (pzsGreen-DD), fluorescent E-cadherin (pE-cadh-zsG) and tunable fluorescent E-cadherin (pE-cadh-zsG-DD). Components include CMV promoter (pCMV), zsGreen fluorescent protein (zsGreen), the Shield-1 binding degradation domain (FKBP-DD), and E-cadherin. B) Schematic of MDA-MB-231-luc-D3H2LN (231LN) cells used to express tunable proteins and the predicted behavior of cells in the presence or absence of Shield-1. 231LN tumor cells were stably transfected with tdTomato and zsGreen alone or as a fusion with E-cadherin. C) Intravital imaging platform (right) with avian embryo imaging chamber (left) to maintain proper temperature (37°C) and humidity (>90%) used to perform <i>in vivo</i> three dimensional time-lapse imaging of micrometastases in the chorioallantoic membrane of the avian embryo.</p

Induction of E-cadherin-zsG-DD protein in 231LN cells by Shield-1 ligand and expression of vimentin.

<p>A) 231LN cells expressing E-cadh-zsG-DD (green) treated with 1.0 µM Shield-1 for 24 hours and immunostained with anti-E-cadherin mAb (red) and Hoechst nuclear stain (blue). Scale bars are 25 µm. B) Western immunoblot analysis of E-cadherin expression in 231LN cells expressing E-cadh-zsG-DD and treated with 1.0 µM Shield-1 using the same mAb as in A). Graph (right) represents analyses performed on three independent induction experiments. Cell lysates of 231LN cells expressing E-cadherin-zsG are shown in the first lane. Lysates of cells expressing E-cadherin-zsG-DD were collected at 0, 4, 8, 12, 16, and 24 hrs after Shield-1 treatment (1.0 µM final), revealing accumulation of Shield-1 stabilized E-cadherin-zsG-DD within cells (∼135 kDa). Far right lane is a positive control of 21PT cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030177#pone.0030177-Souter1" target="_blank">[32]</a> which endogenously express high levels of E-cadherin (∼110 kDa). C) Western immunoblot analysis of markers for epithelial-mesenchymal transition (EMT). Blot (left panels) reveals a decrease in vimentin protein levels when E-cadh-zsG-DD is induced by 1.0 µM Shield-1 treatment. Graph (right) represents analyses performed on three independent induction experiments.</p

Characterization of tunable E-cadherin-zsG-DD protein expression in 231LN cells <i>in vitro</i>.

<p>A) Representative images of 231LN cells expressing fluorescent E-cadherin chimeras. Cell nucleus as stained by Hoechst (blue), E-cadherin-zsGreen (green), and tdTomato to highlight the cytoplasm (red) reveal the changes in cell morphology when E-cadherin is over-expressed (row 2) or induced with Shield-1 for 12 hours (row 4) compared to control (row 1) or un-induced cells (row 3). Arrows (yellow) highlight junctions formed by Shield-1-stabilized E-cadh-zG-DD. Scale bars are 20 µm. Insets show magnified view (250%) of cellular junctions. B) Examples of circularity measurements of representative 231LN cells (left) and 231LN cells expressing E-cadh-zsG-DD treated with 1.0 µM Shield-1 (right). C) Circularity measurements to assess a mesenchymal vs. epithelial morphology in cells described above. N = 70 per group, * denotes p<0.01 between groups, 2-way ANOVA.</p