M. Dean Chamberlain

Direct positive regulation of PTEN by the p85 subunit of phosphatidylinositol 3-kinase

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is deregulated in many human diseases including cancer, diabetes, obesity, and autoimmunity. PI3K consists of a p110 catalytic protein and a p85α regulatory protein, required for the stabilization and localization of p110-PI3K activity. The p110-PI3K enzyme generates the key signaling lipid phosphatidylinositol 3,4,5-trisphosphate, which is dephosphorylated by the PI3-phosphatase PTEN. Here we show another function for the p85α regulatory protein: it binds directly to and enhances PTEN lipid phosphatase activity. We demonstrate that ectopically expressed FLAG-tagged p85 coimmunoprecipitates endogenous PTEN in an epidermal growth factor dependent manner. We also show epidermal growth factor dependent coimmunoprecipitation of endogenous p85 and PTEN proteins in HeLa cells. Thus p85 regulates both p110-PI3K and PTEN-phosphatase enzymes through direct interaction. This finding underscores the need for caution in analyzing PI3K activity because anti-p85 immunoprecipitations may contain both p85:p110-PI3K and p85:PTEN-phosphatase enzymes and thus measure net PI3K activity. We identify the N-terminal SH3-BH region of p85α, absent in the smaller p55α and p50α isoforms, as the region that mediates PTEN binding and regulation. Cellular expression of p85ΔSH3-BH results in substantially increased magnitude and duration of pAkt levels in response to growth factor stimulation. The ability of p85 to bind and directly regulate both p110-PI3K and PTEN-PI3-phosphatase allows us to explain the paradoxical insulin signaling phenotypes observed in mice with reduced PI3K or PTEN proteins. This discovery will impact ongoing studies using therapeutics targeting the PI3K/PTEN/Akt pathway.

Biodegradable scaffold with built-in vasculature for organ-on-a-chip engineering and direct surgical anastomosis

We report the fabrication of a scaffold (hereafter referred to as AngioChip) that supports the assembly of parenchymal cells on a mechanically tunable matrix surrounding a perfusable, branched, three-dimensional microchannel network coated with endothelial cells. The design of AngioChip decouples the material choices for the engineered vessel network and for cell seeding in the parenchyma, enabling extensive remodelling while maintaining an open-vessel lumen. The incorporation of nanopores and micro-holes in the vessel walls enhances permeability, and permits intercellular crosstalk and extravasation of monocytes and endothelial cells on biomolecular stimulation. We also show that vascularized hepatic tissues and cardiac tissues engineered by using AngioChips process clinically relevant drugs delivered through the vasculature, and that millimeter-thick cardiac tissues can be engineered in a scalable manner. Moreover, we demonstrate that AngioChip cardiac tissues implanted via direct surgical anastomosis to the femoral vessels of rat hindlimbs establish immediate blood perfusion.

Expression of the ExeAB complex of Aeromonas hydrophila is required for the localization and assembly of the ExeD secretion port multimer

Aeromonas hydrophila secretes protein toxins via the type II pathway, involving the products of at least two operons, exeAB (gspAB) and exeC‐N (gspC‐N). In the studies reported here, aerolysin secretion was restored to C5.84, an exeA::Tn5–751 mutant, by overexpression of exeD alone in trans. Expression studies indicated that these results did not reflect a role of ExeAB in the regulation of the exeC‐N operon. Instead, immunoblot analysis showed that ExeD did not multimerize in C5.84, and fractionation of the membranes showed that the monomeric ExeD remained in the inner membrane. Expression of ExeAB, but not either protein alone, from a plasmid in C5.84 resulted in increases in the amount of multimeric ExeD, which correlated with increases in aerolysin secretion. Pulse‐chase analysis also sug‐gested that the induction of ExeAB allowed multimerization of previously accumulated monomer ExeD. In C5.84 cells overproducing ExeD, it multimerized even in the absence of ExeAB and, although most remained in the inner membrane, an amount similar to that in wild‐type outer membranes fractionated with the outer membrane of the overproducing cells. These results indicate that the secretion defect of exeAB mutants is a result of an inability to assemble the ExeD secretin in the outer membrane. The localization and multimerization of overproduced ExeD in these mutants further suggests that the ExeAB complex plays either a direct or indirect role in the transport of ExeD into the outer membrane.

Disrupted RabGAP function of the p85 subunit of phosphatidylinositol 3-kinase results in cell transformation.

Rab proteins regulate vesicle fusion events during the endocytosis, recycling, and degradation of activated receptor tyrosine kinases. The p85α subunit of phosphatidylinositol 3-kinase has GTPase-activating protein activity toward Rab5 and Rab4, an activity severely reduced by a single point mutation (p85-R274A). Expression of p85-R274A resulted in increased platelet-derived growth factor receptor (PDGFR) activation and downstream signaling (Akt and MAPK) and in decreased PDGFR degradation. We now report that the biological consequences of p85-R274A expression cause cellular transformation as determined by the following: aberrant morphological phenotype, loss of contact inhibition, growth in soft agar, and tumor formation in nude mice. Immunohistochemistry shows that the tumors contain activated PDGFR and high levels of activated Akt. Coexpression of a dominant negative Rab5-S34N mutant attenuated these transformed properties. Our results demonstrate that disruption of the RabGAP function of p85α due to a single point mutation (R274A) is sufficient to cause cellular transformation via a phosphatidylinositol 3-kinase-independent mechanism partially reversed by Rab5-S34N expression. This critical new role for p85 in the regulation of Rab function suggests a novel role for p85 in controlling receptor signaling and trafficking through its effects on Rab GTPases.

Digital microfluidic immunocytochemistry in single cells

We report a new technique called Digital microfluidic Immunocytochemistry in Single Cells (DISC). DISC automates protocols for cell culture, stimulation and immunocytochemistry, enabling the interrogation of protein phosphorylation on pulsing with stimulus for as little as 3 s. DISC was used to probe the phosphorylation states of platelet-derived growth factor receptor (PDGFR) and the downstream signalling protein, Akt, to evaluate concentration- and time-dependent effects of stimulation. The high time resolution of the technique allowed for surprising new observations—for example, a 10 s pulse stimulus of a low concentration of PDGF is sufficient to cause >30% of adherent fibroblasts to commit to Akt activation. With the ability to quantitatively probe signalling events with high time resolution at the single-cell level, we propose that DISC may be an important new technique for a wide range of applications, especially for screening signalling responses of a heterogeneous cell population.

The smaller isoforms of ankyrin 3 bind to the p85 subunit of phosphatidylinositol 3'-kinase and enhance platelet-derived growth factor receptor down-regulation

The Src homology 2 (SH2) domains of the p85 subunit of phosphatidylinositol 3′-kinase have been shown to bind to the tyrosine-phosphorylated platelet-derived growth factor receptor (PDGFR). Previously, we have demonstrated that p85 SH2 domains can also bind to the serine/threonine kinase A-Raf via a unique phosphorylation-independent interaction. In this report, we describe a new phosphotyrosine-independent p85 SH2-binding protein, ankyrin 3 (Ank3). In general, ankyrins serve a structural role by binding to both integral membrane proteins at the plasma membrane and spectrin/fodrin proteins of the cytoskeleton. However, smaller isoforms of Ank3 lack the membrane domain and are localized to late endosomes and lysosomes. We found that p85 binds directly to these smaller 120- and 105-kDa Ank3 isoforms. Both the spectrin domain and the regulatory domain of Ank3 are involved in binding to p85. At least two domains of p85 can bind to Ank3, and the interaction involving the p85 C-SH2 domain was found to be phosphotyrosine-independent. Overexpression of the 120- or 105-kDa Ank3 proteins resulted in significantly enhanced PDGFR degradation and a reduced ability to proliferate in response to PDGF. Ank3 overexpression also differentially regulated signaling pathways downstream from the PDGFR. Chloroquine, an inhibitor of lysosomal-mediated degradation pathways, blocked the ability of Ank3 to enhance PDGFR degradation. Immunofluorescence experiments demonstrated that both small Ank3 isoforms colocalized with the lysosomal-associated membrane protein and with p85 and the PDGFR. These results suggest that Ank3 plays an important role in lysosomal-mediated receptor down-regulation, likely through a p85-Ank3 interaction.

Deregulation of Rab5 and Rab4 proteins in p85R274A-expressing cells alters PDGFR trafficking.

Activated receptor tyrosine kinases recruit many signaling proteins to activate downstream cell proliferation and survival pathways, including phosphatidylinositol 3-kinase (PI3K) consisting of a p85 regulatory protein and a p110 catalytic protein. We have recently shown the p85alpha protein also has in vitro GTPase activating protein (GAP) activity towards Rab5 and Rab4, small GTPases that regulate vesicle trafficking events for activated receptors. Expression of a GAP-defective mutant, p85R274A, resulted in sustained levels of activated platelet-derived growth factor receptors (PDGFRs) and enhanced downstream signaling. In this report we have characterized Rab5- and Rab4-mediated PDGFR trafficking in cells expressing wild type p85 and GAP-defective mutant p85R274A. Wild type p85 overexpressing cells had slower PDGFR trafficking consistent with enhanced GAP activity deactivating Rab5 and Rab4 to block their vesicle trafficking functions. Mutant p85R274A expression increased the internalization rate of PDGFRs, a Rab5-dependent process, without preventing PDGFR ubiquitination. Immunofluorescence studies further demonstrated that p85R274A-expressing cells showed Rab5 accumulation at intracellular locations. Pull-down and FRAP (fluorescence recovery after photobleaching) experiments indicate this is likely membrane-associated Rab5-GTP, sustained due to decreased p85 GAP activity for the p85R274A mutant. These cells also had substantial amounts of activated PDGFRs in Rab4-positive recycling endosomes, a compartment that usually contains primarily deactivated/dephosphorylated receptors. Our results suggest that the PDGFR-associated GAP activity of p85 regulates both Rab5 and Rab4 functions in cells to influence the movement of activated PDGFR through endosomal compartments. Disruption of this regulation by p85R274A expression impacts PDGFR phosphorylation/dephosphorylation, degradation kinetics and downstream signaling by altering the time receptors spend in specific intracellular endosomal compartments. These results demonstrate that the p85alpha protein is an important regulator of Rab-mediated PDGFR trafficking, which significantly impacts receptor signaling and degradation.

Electrochemiluminescence on digital microfluidics for microRNA analysis.

Electrochemiluminescence (ECL) is a sensitive analytical technique with great promise for biological applications, especially when combined with microfluidics. Here, we report the first integration of ECL with digital microfluidics (DMF). ECL detectors were fabricated into the ITO-coated top plates of DMF devices, allowing for the generation of light from electrically excited luminophores in sample droplets. The new system was characterized by making electrochemical and ECL measurements of soluble mixtures of tris(phenanthroline)ruthenium(II) and tripropylamine (TPA) solutions. The system was then validated by application to an oligonucleotide hybridization assay, using magnetic particles bearing 21-mer, deoxyribose analogues of the complement to microRNA-143 (miRNA-143). The system detects single nucleotide mismatches with high specificity, and has a limit of detection of 1.5 femtomoles. The system is capable of detecting miRNA-143 in cancer cell lysates, allowing for the discrimination between the MCF-7 (less aggressive) and MDA-MB-231 (more aggressive) cell lines. We propose that DMF-ECL represents a valuable new tool in the microfluidics toolbox for a wide variety of applications.

A droplet-based screen for wavelength-dependent lipid production in algae

We report a digital microfluidic system designed for droplet-based long-term culture and analysis of algae. The system includes unique innovations relative to standard devices including an active reservoir structure to maintain homogeneous cell density, a customized device layout capable of controlling a wide range of different droplet volumes, vertical interconnects to collect spent reagents, detection zones compatible with parallel-scale optical measurements using a standard multiwell plate reader, and optimized features for droplet dispensing in parallel. The method allows for automated, multiplexed analysis with significant reductions in human intervention, representing a decrease from 600 pipette steps (for a conventional screen in multiwell plates) to fewer than 20 (for the new microfluidic technique). The system was applied to screen conditions favourable for lipid generation in the widely used algal model for biofuel production, Cyclotella cryptica. A dependence on illumination wavelength was observed, with the best conditions (representing a four-fold increase relative to control) comprising an alternation between yellow (∼580 nm) and blue (∼450 nm) illumination wavelengths. These effects were observed for both micro- and macro-scale cultures, and are consistent with a putative mechanism involving photooxidative stress. We propose that the microfluidic system described here is an attractive new screening tool with potential advantages for applications in renewable energy, biotechnology, materials science, and beyond.

Assay and Stimulation of the Rab5 GTPase by the p85α Subunit of Phosphatidylinositol 3‐Kinase

Rab5 is a small monomeric GTPase involved in regulating vesicle fusion events during receptor-mediated endocytosis. During endocytosis of the activated platelet-derived growth factor receptor, phosphatidylinositol 3-kinase (PI3K) remains associated with the receptor. We have found that the p85 alpha subunit of PI3K binds directly to Rab5 and possesses GTPase-activating protein (GAP) activity toward Rab5. We describe two methods used to characterize the GAP activity of p85 toward the Rab5 protein. The first method is a steady-state GAP assay, used to show that the p85 alpha protein has GAP activity toward Rab5. The second method is a single turnover GAP assay and measures changes in the catalytic rate of Rab5 GTP hydrolysis with or without the p85 alpha protein.