Faculty Bibliography

Mosaic mutant analysis, the study of cellular defects in scattered mutant cells in a wild-type environment, is a powerful approach for identifying critical functions of genes and has been applied extensively to invertebrate model organisms. A highly versatile technique has been developed in mouse: MASTR (mosaic mutant analysis with spatial and temporal control of recombination), which utilizes the increasing number of floxed alleles and simultaneously combines conditional gene mutagenesis and cell marking for fate analysis. A targeted allele (R26(MASTR)) was engineered; the allele expresses a GFPcre fusion protein following FLP-mediated recombination, which serves the dual function of deleting floxed alleles and marking mutant cells with GFP. Within 24 hr of tamoxifen administration to R26(MASTR) mice carrying an inducible FlpoER transgene and a floxed allele, nearly all GFP-expressing cells have a mutant allele. The fate of single cells lacking FGF8 or SHH signaling in the developing hindbrain was analyzed using MASTR, and it was revealed that there is only a short time window when neural progenitors require FGFR1 for viability and that granule cell precursors differentiate rapidly when SMO is lost. MASTR is a powerful tool that provides cell-type-specific (spatial) and temporal marking of mosaic mutant cells and is broadly applicable to developmental, cancer, and adult stem cell studies.

The transition of chronic pancreatic fibroinflammatory disease to neoplasia is a primary example of the paradigm linking inflammation to carcinogenesis. However, the cellular and molecular mediators bridging these entities are not well understood. Because TLR4 ligation can exacerbate pancreatic inflammation, we postulated that TLR4 activation drives pancreatic carcinogenesis. In this study, we show that lipopolysaccharide accelerates pancreatic tumorigenesis, whereas TLR4 inhibition is protective. Furthermore, blockade of the MyD88-independent TRIF pathway is protective against pancreatic cancer, whereas blockade of the MyD88-dependent pathway surprisingly exacerbates pancreatic inflammation and malignant progression. The protumorigenic and fibroinflammatory effects of MyD88 inhibition are mediated by dendritic cells (DCs), which induce pancreatic antigen-restricted Th2-deviated CD4(+) T cells and promote the transition from pancreatitis to carcinoma. Our data implicate a primary role for DCs in pancreatic carcinogenesis and illustrate divergent pathways in which blockade of TLR4 signaling via TRIF is protective against pancreatic cancer and, conversely, MyD88 inhibition exacerbates pancreatic inflammation and neoplastic transformation by augmenting the DC-Th2 axis.

Neuron electrical properties are critical to function and generally subtype specific, as are patterns of axonal and dendritic projections. Specification of motoneuron morphology and axon pathfinding has been studied extensively, implicating the combinatorial action of Lim-homeodomain transcription factors. However, the specification of electrical properties is not understood. Here, we address the key issues of whether the same transcription factors that specify morphology also determine subtype specific electrical properties. We show that Drosophila motoneuron subtypes express different K(+) currents and that these are regulated by the conserved Lim-homeodomain transcription factor Islet. Specifically, Islet is sufficient to repress a Shaker-mediated A-type K(+) current, most likely due to a direct transcriptional effect. A reduction in Shaker increases the frequency of action potential firing. Our results demonstrate the deterministic role of Islet on the excitability patterns characteristic of motoneuron subtypes.

The poor prognosis of glioblastoma (GBM) routinely treated with ionizing radiation (IR) has been attributed to the relative radioresistance of glioma-initiating cells (GIC). Other studies indicate that although GIC are sensitive, the response is mediated by undefined factors in the microenvironment. GBM produce abundant transforming growth factor-beta (TGF-beta), a pleotropic cytokine that promotes effective DNA damage response. Consistent with this, radiation sensitivity, as measured by clonogenic assay of cultured murine (GL261) and human (U251, U87MG) glioma cell lines, increased by approximately 25% when treated with LY364947, a small-molecule inhibitor of TGF-beta type I receptor kinase, before irradiation. Mice bearing GL261 flank tumors treated with 1D11, a pan-isoform TGF-beta neutralizing antibody, exhibited significantly increased tumor growth delay following IR. GL261 neurosphere cultures were used to evaluate GIC. LY364947 had no effect on the primary or secondary neurosphere-forming capacity. IR decreased primary neurosphere formation by 28%, but did not reduce secondary neurosphere formation. In contrast, LY364947 treatment before IR decreased primary neurosphere formation by 75% and secondary neurosphere formation by 68%. Notably, GL261 neurospheres produced 3.7-fold more TGF-beta per cell compared with conventional culture, suggesting that TGF-beta production by GIC promotes effective DNA damage response and self-renewal, which creates microenvironment-mediated resistance. Consistent with this, LY364947 treatment in irradiated GL261 neurosphere-derived cells decreased DNA damage responses, H2AX and p53 phosphorylation, and induction of self-renewal signals, Notch1 and CXCR4. These data motivate the use of TGF-beta inhibitors with radiation to improve therapeutic response in patients with GBM. Cancer Res; 72(16); 4119-29. (c)2012 AACR.

Endothelial cells line blood vessels and coordinate many aspects of vascular biology. More recent work has shown that endothelial cells provide a key niche in vivo for neural stem cells. In vitro, endothelial cells secrete a factor that expands neural stem cells while inhibiting their differentiation. Here, we show that a transformed mouse endothelial cell line (bEnd.3) maintains human pluripotent stem cells in an undifferentiated state. bEnd.3 cells have a practical advantage over mouse embryonic fibroblasts for pluripotent stem cell maintenance since they can be expanded in vitro and engineered to express genes of interest. We demonstrate this capability by producing fluorescent and drug-resistant feeder cells. Further, we show that bEnd.3 secretes an activity that maintains human embryonic stem cells without direct contact.

Axonal degeneration after traumatic brain injury and nerve compression is considered a common underlying cause of temporary as well as permanent disability. Because a proper functioning of neural network requires phase coherence of all components, even subtle changes in circuitry may lead to network failure. However, it is still not possible to determine which axons will recover or degenerate after injury. Several groups have studied the pressure threshold for axonal injury within a nerve, but difficulty accessing the injured region; insufficient imaging methods and the extremely small dimensions involved have prevented the evaluation of the response of individual axons to injury. We combined microfluidics with atomic force microscopy and in vivo imaging to estimate the threshold force required to 1), uncouple axonal transport without impairing axonal survival, and 2), compromise axonal survival in both individual and bundled axons. We found that rat hippocampal axons completely recover axonal transport with no detectable axonal loss when compressed with pressures up to 65 +/- 30 Pa for 10 min, while dorsal root ganglia axons can resist to pressures up to 540 +/- 220 Pa. We investigated the reasons for the differential susceptibility of hippocampal and DRG axons to mechanical injury and estimated the elasticity of live axons. We found that dorsal root ganglia axons have a 20% lower elastic modulus than hippocampal axons. Our results emphasize the importance of the integrity of the axonal cytoskeleton in deciding the axonal fate after damage and open up new avenues to improve injury diagnosis and to identify ways to protect axons.

Despite their usefulness as fluorophores and synthetic precursors, efficient and reliable routes to coumarin-8-carbaldehydes are lacking. We describe here a high-yielding continuous flow synthesis that requires no manual intermediate purification or work-up, giving access to multigram quantities of the aldehyde product.

Gene expression programs that regulate the abundance of the chaperone BiP adapt the endoplasmic reticulum (ER) to unfolded protein load. However, such programs are slow compared with physiological fluctuations in secreted protein synthesis. While searching for mechanisms that fill this temporal gap in coping with ER stress, we found elevated levels of adenosine diphosphate (ADP)-ribosylated BiP in the inactive pancreas of fasted mice and a rapid decline in this modification in the active fed state. ADP ribosylation mapped to Arg470 and Arg492 in the substrate-binding domain of hamster BiP. Mutations that mimic the negative charge of ADP-ribose destabilized substrate binding and interfered with interdomain allosteric coupling, marking ADP ribosylation as a rapid posttranslational mechanism for reversible inactivation of BiP. A kinetic model showed that buffering fluctuations in unfolded protein load with a recruitable pool of inactive chaperone is an efficient strategy to minimize both aggregation and costly degradation of unfolded proteins.

The sarcoplasmic reticulum calcium pump (SERCA) and its regulator, phospholamban, are essential components of cardiac contractility. Phospholamban modulates contractility by inhibiting SERCA, and this process is dynamically regulated by beta-adrenergic stimulation and phosphorylation of phospholamban. Herein we reveal mechanistic insight into how four hereditary mutants of phospholamban, Arg(9) to Cys, Arg(9) to Leu, Arg(9) to His, and Arg(14) deletion, alter regulation of SERCA. Deletion of Arg(14) disrupts the protein kinase A recognition motif, which abrogates phospholamban phosphorylation and results in constitutive SERCA inhibition. Mutation of Arg(9) causes more complex changes in function, where hydrophobic substitutions such as cysteine and leucine eliminate both SERCA inhibition and phospholamban phosphorylation, whereas an aromatic substitution such as histidine selectively disrupts phosphorylation. We demonstrate that the role of Arg(9) in phospholamban function is multifaceted: it is important for inhibition of SERCA, it increases the efficiency of phosphorylation, and it is critical for protein kinase A recognition in the context of the phospholamban pentamer. Given the synergistic consequences on contractility, it is not surprising that the mutants cause lethal, hereditary dilated cardiomyopathy.