D. Scott Witherow

A Novel Kind of G Protein Heterodimer: The Gβ5-RGS Complex

The fifth member of the G protein g the subunit family, G g 5, has been shown to bind exclusively to a subfamily of regulators of G protein signaling (RGS) including RGS6, RGS7, RGS9, and RGS11. This interaction occurs through a G protein gamma-like (GGL) domain present in members of this RGS subfamily and is the only reported instance in which a G g subunit is not bound to a G n subunit. The G g 5-RGS interaction has been demonstrated both in vitro and in vivo and has been shown to stabilize the dimer against proteolytic degradation. GTPase activating protein (GAP) assays suggest that G g 5-RGS7 acts specifically on G f o, however in cell-based assays it also inhibited G f i- and G f q-mediated signaling. The role of the dimer in signaling and the function of G g 5 moiety within the complex are poorly understood. This review summarizes the information about the assembly and function of G g 5-RGS dimers, as well as their posttranslational modifications and localization.

Expression levels of RGS7 and RGS4 proteins determine the mode of regulation of the G protein-activated K+ channel and control regulation of RGS7 by Gβ5

Regulators of G protein signaling RGS4 and RGS7 accelerate the kinetics of K+ channels (GIRKs) in the Xenopus oocyte system. Here, via quantitative analysis of RGS expression, we reveal biphasic effects of RGSs on GIRK regulation. At low concentrations, RGS4 inhibited basal GIRK activity, but stimulated it at high concentrations. RGS7, which is associated with the G protein subunit Gβ5, is regulated by Gβ5 by two distinct mechanisms. First, Gβ5 augments RGS7 activity, and second, it increases its expression. These dual effects resolve previous controversies regarding RGS4 and RGS7 function and indicate that they modulate signaling by mechanisms supplementary to their GTPase‐activating protein activity.

Spinal cord injury induces expression of RGS7 in microglia/macrophages in rats.

RGS proteins regulate G protein‐mediated signalling pathways through direct interaction with the Gα subunits and facilitation of GTP hydrolysis. An RGS subfamily consisting of RGS 6, 7, 9, and 11 also interacts with the G protein β subunit Gβ5 via a characteristic Gγ‐like domain. Thus far, these complexes were found only in neurons, with RGS7 being the most widely distributed in the brain. Here we confirm the expression of RGS7 in spinal neurons and show as a novel finding that following an experimental spinal cord injury in rats, expression of RGS7 is induced in a subpopulation of other cells. Immunofluorescent confocal microscopy using a series of cell specific antibodies identified these RGS7 positive cells as activated microglia and/or invading peripheral macrophages. To rule out interference from the adjacent neurons and confirm the presence of RGS7‐Gβ5 complex in inflammatory cells, we performed immunocytochemistry, RT‐PCR, Western blot, and immunoprecipitation using microglial (BV2) and peripheral macrophage (RAW) cell lines. Expression of RGS7 mRNA and protein are nearly undetectable in non‐stimulated BV2 and RAW cells, but remarkably increased after stimulation with LPS or TNF‐α In addition, RGS7‐positive cells were also found in the perinodular rim in the rat spleen. Our findings show that RGS7‐Gβ5 complex is expressed in immunocompetent cells such as resident microglia and peripheral macrophages following spinal cord injury. This expression might contribute to the post‐traumatic inflammatory responses in the central nervous system.

A laboratory-intensive course on RNA interference and model organisms.

RNA interference (RNAi) is a powerful method to silence gene expression in a variety of organisms and is generating interest not only as a useful tool for research scientists but also as a novel class of therapeutics in clinical trials. Here, we report that undergraduate and graduate students with a basic molecular biology background were able to demonstrate conceptual knowledge and technical skills for using RNAi as a research tool upon completion of an intensive 8-wk RNAi course with a 2-h lecture and 5-h laboratory per week. Students were instructed on design of RNAi experiments in model organisms and perform multiweek laboratory sessions based on journal articles read and discussed in class. Using Nicotiana benthamiana, Caenorhabditis elegans, and mammalian cell culture, students analyzed the extent of silencing using both qualitative assessment of phenotypic variations and quantitative measurements of RNA levels or protein levels. We evaluated the course over two semesters, each with a separate instructor. In both semesters, we show students met expected learning outcomes as demonstrated by successful laboratory experiment results, as well as positive instructor assessments of exams and lab reports. Student self-assessments revealed increased confidence in conceptual knowledge and practical skills. Our data also suggest that the course is adaptable to different instructors with varying expertise.

Student Learning Outcomes and Attitudes When Biotechnology Lab Partners Are of Different Academic Levels

The North Carolina State University Biotechnology Program offers laboratory-intensive courses to both undergraduate and graduate students. In “Manipulation and Expression of Recombinant DNA,” students are separated into undergraduate and graduate sections for the laboratory, but not the lecture, component. Evidence has shown that students prefer pairing with someone of the same academic level. However, retention of main ideas in peer learning environments has been shown to be greater when partners have dissimilar abilities. Therefore, we tested the hypothesis that there will be enhanced student learning when lab partners are of different academic levels. We found that learning outcomes were met by both levels of student, regardless of pairing. Average undergraduate grades on every assessment method increased when undergraduates were paired with graduate students. Many of the average graduate student grades also increased modestly when graduate students were paired with undergraduates. Attitudes toward working with partners dramatically shifted toward favoring working with students of different academic levels. This work suggests that offering dual-level courses in which different-level partnerships are created does not inhibit learning by students of different academic levels. This format is useful for institutions that wish to offer “boutique” courses in which student enrollment may be low, but specialized equipment and faculty expertise are needed.

Biochemical Purification and Functional Analysis of Complexes between the G-Protein Subunit Gβ5 and RGS Proteins

Regulator of G-protein signaling (RGS) proteins of the R7 subfamily (RGS6, 7, 9, and 11) contain a unique Ggamma-like (GGL) domain that enables their association with the G-protein beta subunit Gbeta5. The existence of these complexes was demonstrated by their purification from native tissues as well as by reconstitution in vitro. According to pulse-chase analysis, Gbeta5 and RGS7 monomers undergo rapid proteolytic degradation in cells, whereas the dimer is stable. Studies of the functional role of Gbeta5-RGS dimers using GTPase activity, ion channel, and calcium mobilization assays showed that, similarly to other RGS proteins, they can negatively regulate G-protein-mediated signal transduction. Protein-protein interactions involving the Gbeta5-RGS7 complex can be studied in cells using fluorescence resonance energy transfer utilizing Gbeta5, RGS, and Galpha subunits fused to the cyan and yellow versions of green fluorescent protein.

Assessment of a novel group-centered testing schema in an upper-level undergraduate molecular biotechnology course

Providing students with assignments that focus on critical thinking is an important part of their scientific and intellectual development. However, as class sizes increase, so does the grading burden, prohibiting many faculty from incorporating critical thinking assignments in the classroom. In an effort to continue to provide our students with meaningful critical thinking exercises, we implemented a novel group‐centered, problem‐based testing scheme. We wanted to assess how performing critical thinking problem sets as group work compares to performing the sets as individual work, in terms of student attitudes and learning outcomes. During two semesters of our recombinant DNA course, students had the same lecture material and similar assessments. In the Fall semester, student learning was assessed by two collaborative take‐home exams, followed immediately by individual, closed‐book in‐class exams on the same content, as well as a final cumulative exam. Student teams on the take‐home exams were instructor‐assigned, and each team turned in one collaborative exam. In the Spring semester, the control group of students were required to turn in their own individual take‐home exams, followed by the in‐class exams and final cumulative exam. For the majority of students, learning outcomes were met, regardless of whether they worked in teams. In addition, collaborative learning was favorably received by students and grading was reduced for instructors. These data suggest that group‐centered, problem‐based learning is a useful model for achievement of student learning outcomes in courses where it would be infeasible to provide feedback on individual critical thinking assignments due to grading volume.

Lab Session 11 – Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot: Part 1

Abstract Today you will stain your membrane with Ponceau Red to confirm the transfer of your proteins to the nitrocellulose and complete the western blot that was started in the last laboratory session.