Analysis of Microbial Water Contamination, Soil Microbial Community Structure, and Soil Respiration in a Collaborative First-Year Students as Scholars Program (SAS).

The persistence of college students in STEM majors after their first-year of college is approximately 50%, with underrepresented populations displaying even higher rates of departure. For many undergraduates, their first-year in college is defined by large class sizes, poor access to research faculty, and minimal standing in communities of scholars. Pepperdine University and Whittier College, funded by a National Science Foundation award to Improve Undergraduate Stem Education (NSF IUSE), partnered in the development of first-year classes specifically geared to improve student persistence in STEM and academic success. This Students as Scholars Program (SAS) engaged first-year undergraduates in scholarly efforts during their first semester in college with a careful approach to original research design and mentoring by both faculty and upperclassmen experienced in research. Courses began by introducing hypothesis formulation and experimental design partnered with the scientific focus of each course (ecological, biochemical, microbiological). Students split into research teams, explored the primary literature, designed research projects, and executed experiments over a 6-7 week period, collecting, analyzing, and interpreting data. Microbiology-specific projects included partnerships with local park managers to assess water quality and microbial coliform contamination at specified locations in a coastal watershed. In addition, students explored the impact of soil salinity on microbial community structure. Analysis of these samples included next-generation sequencing and microbiome compositional analysis via collaboration with students from an upper division microbiology course. This cross-course collaboration facilitated additional student mentoring opportunities between upperclassmen and first-year students. This approach provided first-year students an introduction to the analysis of complex data sets using bioinformatics and statistically reliable gas-exchange replicates. Assessment of the impact of this program revealed students to view the research as challenging, but confidence building as they take their first steps as biology majors. In addition, the direct mentorship of first-year students by upperclassmen and faculty was viewed positively by students. Ongoing assessments have revealed SAS participants to display a 15% increased persistence rate in STEM fields when compared to non-SAS biology majors.

Moderate endoplasmic reticulum stress activates a PERK and p38-dependent apoptosis.

The endoplasmic reticulum (ER) has the ability to signal organelle dysfunction via a complex signaling network known as the unfolded protein response (UPR). In this work, hamster fibroblast cells exhibiting moderate levels of ER stress were compared to those exhibiting severe ER stress. Inhibition of N-linked glycosylation was accomplished via a temperature-sensitive mutation in the Dad1 subunit of the oligosaccharyltransferase (OST) complex or by direct inhibition with tunicamycin (Tm). Temperature shift (TS) treatment generated weak activation of ER stress signaling when compared to doses of Tm that are typically used in ER stress studies (500-1000 nM). A dose-response analysis of key ER stress signaling mediators, inositol-requiring enzyme 1 (IRE1) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), revealed 20-40 nM of Tm to generate activation intensity similar to TS treatment. In parental BHK21 cells, moderate (20-40 nM) and high doses (200-1000 nM) of Tm were compared to identify physiological and signaling-based differences in stress response. Inhibition of ER Ca2+ release via ITPR activity with 2-aminoethoxydiphenyl borate (2-APB) or Xestospongin C (XeC) was sufficient to protect against apoptosis induced by moderate but not higher doses of Tm. Analysis of kinase activation over a range of Tm exposures revealed the p38 stress-activated protein kinase (SAPK) to display increasing activation with Tm dosage. Interestingly, Tm induced the extracellular regulated kinases (Erk1/2) only at moderate doses of Tm. Inhibition of ER transmembrane stress sensors (IRE1, PERK) or cytosolic signaling mediators (p38, Jnk1, Erk1/2) was used to evaluate pathways involved in apoptosis activation during ER stress. Inhibition of either PERK or p38 was sufficient to reduce cell death and apoptosis induced by moderate, but not high, doses of Tm. During ER stress, cells exhibited a rapid decline in anti-apoptotic Mcl-1 and survivin proteins. Inhibition of PERK was sufficient to block this affect. This work reveals moderate doses of ER stress to generate patterns of stress signaling that are distinct from higher doses and that apoptosis activation at moderate levels of stress are dependent upon PERK and p38 signaling. Studies exploring ER stress signaling should recognize that this signaling acts as a rheostat rather than a simple switch, behaving distinctively in a dose-dependent manner.

Hog1: 20 years of discovery and impact.

The protein kinase Hog1 (high osmolarity glycerol 1) was discovered 20 years ago, being revealed as a central signaling mediator during osmoregulation in the budding yeast Saccharomyces cerevisiae. Homologs of Hog1 exist in all evaluated eukaryotic organisms, and this kinase plays a central role in cellular responses to external stresses and stimuli. Here, we highlight the mechanism by which cells sense changes in extracellular osmolarity, the method by which Hog1 regulates cellular adaptation, and the impacts of the Hog1 pathway upon cellular growth and morphology. Studies that have addressed these issues reveal the influence of the Hog1 signaling pathway on diverse cellular processes.

Endoplasmic reticulum stress or mutation of an EF-hand Ca(2+)-binding domain directs the FKBP65 rotamase to an ERAD-based proteolysis.

FKBP65 is an endoplasmic reticulum (ER)-localized chaperone and rotamase, with cargo proteins that include tropoelastin and collagen. In humans, mutations in FKBP65 have recently been shown to cause a form of osteogenesis imperfecta (OI), a brittle bone disease resulting from deficient secretion of mature type I collagen. In this work, we describe the rapid proteolysis of FKBP65 in response to ER stress signals that activate the release of ER Ca(2+) stores. A large-scale screen for stress-induced cellular changes revealed FKBP65 proteins to decrease within 6-12 h of stress activation. Inhibiting IP(3)R-mediated ER Ca(2+) release blocked this response. No other ER-localized chaperone and folding mediators assessed in the study displayed this phenomenon, indicating that this rapid proteolysis of folding mediator is distinctive. Imaging and cellular fractionation confirmed the localization of FKBP65 (72 kDa glycoprotein) to the ER of untreated cells, a rapid decrease in protein levels following ER stress, and the corresponding appearance of a 30-kDa fragment in the cytosol. Inhibition of the proteasome during ER stress revealed an accumulation of FKBP65 in the cytosol, consistent with retrotranslocation and a proteasome-based proteolysis. To assess the role of Ca(2+)-binding EF-hand domains in FKBP65 stability, a recombinant FKBP65-GFP construct was engineered to ablate Ca(2+) binding at each of two EF-hand domains. Cells transfected with the wild-type construct displayed ER localization of the FKBP65-GFP protein and a proteasome-dependent proteolysis in response to ER stress. Recombinant FKBP65-GFP carrying a defect in the EF1 Ca(2+)-binding domain displayed diminished protein in the ER when compared to wild-type FKBP65-GFP. Proteasome inhibition restored mutant protein to levels similar to that of the wild-type FKBP65-GFP. A similar mutation in EF2 did not confer FKBP65 proteolysis. This work supports a model in which stress-induced changes in ER Ca(2+) stores induce the rapid proteolysis of FKBP65, a chaperone and folding mediator of collagen and tropoelastin. The destruction of this protein may identify a cellular strategy for replacement of protein folding machinery following ER stress. The implications for stress-induced changes in the handling of aggregate-prone proteins in the ER-Golgi secretory pathway are discussed. This work was supported by grants from the National Institutes of Health (R15GM065139) and the National Science Foundation (DBI-0452587).

Caspases indirectly regulate cleavage of the mitochondrial fusion GTPase OPA1 in neurons undergoing apoptosis.

The critical processes of mitochondrial fission and fusion are regulated by members of the dynamin family of GTPases. Imbalances in mitochondrial fission and fusion contribute to neuronal cell death. For example, increased fission mediated by the dynamin-related GTPase, Drp1, or decreased fusion resulting from inactivating mutations in the OPA1 GTPase, causes neuronal apoptosis and/or neurodegeneration. Recent studies indicate that post-translational processing regulates OPA1 function in non-neuronal cells and moreover, aberrant processing of OPA1 is induced during apoptosis. To date, the post-translational processing of OPA1 during neuronal apoptosis has not been examined. Here, we show that cerebellar granule neurons (CGNs) or neuroblastoma cells exposed to pro-apoptotic stressors display a novel N-terminal cleavage of OPA1 which is blocked by either pan-caspase or caspase-8 selective inhibitors. OPA1 cleavage occurs concurrently with mitochondrial fragmentation and cytochrome c release in CGNs deprived of depolarizing potassium (5K condition). Although a caspase-8 selective inhibitor prevents both 5K-induced OPA1 cleavage and mitochondrial fragmentation, recombinant caspase-8 fails to cleave OPA1 in vitro. In marked contrast, either caspase-8 or caspase-3 stimulates OPA1 cleavage in digitonin-permeabilized rat brain mitochondria, suggesting that OPA1 is cleaved by an intermembrane space protease which is regulated by active caspases. Finally, the N-terminal truncation of OPA1 induced during neuronal apoptosis removes an essential residue (K301) within the GTPase domain. These data are the first to demonstrate OPA1 cleavage during neuronal apoptosis and they implicate caspases as indirect regulators of OPA1 processing in degenerating neurons.

Cypermethrin blocks a mitochondria-dependent apoptotic signal initiated by deficient N-linked glycosylation within the endoplasmic reticulum.

The endoplasmic reticulum (ER) serves as a critical site of protein synthesis and processing. The temperature-sensitive hamster fibroblast cell line (tsBN7) displays deficient N-linked glycosylation activity at the restrictive temperature and activates cellular apoptosis. Temperature-shifted tsBN7 cells display induction of Grp78 and Gadd153, genes known to be induced by ER stress, and activate apoptosis via the release of cytochrome c from the mitochondria. Cyclosporin A (CsA), a potent blocker of the mitochondrial permeability transition pore (PTP), was sufficient to block cytochrome c release and to rescue tsBN7 cells from apoptosis. CsA-treated cells displayed Grp78 induction at the restrictive temperature, consistent with an ER stress signal being carried to the nucleus, while the apoptosis-associated transcription factor, Gadd153, displayed only a mild induction. Cypermethrin, a type II pyrethroid known to perturb Ca(2+) signaling in neuronal cells, was sufficient to arrest apoptosis under these conditions. This work identifies type II pyrethroids as a valuable new tool in the characterization of cellular stress signaling pathways.

The microarray revolution: Perspectives from educators.

In recent years, microarray analysis has become a key experimental tool, enabling the analysis of genome-wide patterns of gene expression. This review approaches the microarray revolution with a focus upon four topics: 1) the early development of this technology and its application to cancer diagnostics; 2) a primer of microarray research, designed to guide the beginner; 3) a highlight of the Genome Consortium for Active Teaching (GCAT), a worldwide consortium of faculty who are integrating microarrays into the undergraduate teaching laboratory; and 4) the use of microarrays in the biotechnology industry with a look forward to future applications. A central theme within this review is the profound relevance of new, bioinformatics-based, technologies to undergraduate students within the biosciences.

A genetic screen for yeast genes induced by sustained osmotic stress.

The budding yeast, Saccharomyces cerevisiae, responds to changes in external osmolarity through the activation of an osmosensing signal transduction pathway. Using lacZ-reporter gene fusions, clonal cell lines were screened for levels of beta-galactosidase activity in the presence or absence of osmotic stress. A screen of 9,000 transformants displayed 663 (7%) gene fusions that were active in rich medium. Each of the transformants were also assayed for gene activity 24 h following a transfer to high osmolarity medium (0.6 M NaCl) and of the 9,000 clonal cell lines, 86 (1%) displayed a decrease in expression, and seven (0.1%) displayed a reproducible increase in gene expression during primary screening. The chromosomal loci of the lacZ insertions were determined, and the gene(s) associated with that site was examined for osmotically induced expression using RNA blot analysis. Five stress-activated genes were analysed by RNA blot: YDL222C, NMD2, PTC7, FAA4 and YRF1. The genes identified by this screen encompass cellular adaptations to stress including signal transduction, protein myristoylation and fatty acid/sphingolipid content in the cell membrane.