Modeling in molecular genetics allows students to make connections between biological scales.

Students often see college courses as the presentation of disconnected facts, especially in the life sciences. Student-created Structure Mechanism/Relationship Function (SMRF) models were analyzed to understand students' abilities to make connections between genotype, phenotype, and evolution. Students were divided into two sections; one section received instructions that included a specific gene as an example related to larger issues like human disease or the environment. The other section was only given generic examples, like gene X and phenotype Y. Coding of exam models and a comprehensive (extensive) model reveled students were able to make links and work within and between biological scales of organization. Modeling provided a way to show and allow students to practice and demonstrate the ability to build step-by-step causal relationships that link ideas together. We also observed a small differing with students receiving the specific prompt performing better than students receiving generic prompt at the point in the semester where linking across many biological scales was required to be successful.

Online discussions in biochemistry to increase peer interactions and student interest.

The online environment poses a number of challenges, including creating opportunities for students to interact and get to know each other. I propose adding online discussions that can allow students to explore the real-world implications of biochemistry to provide course context, build the classroom community, diversify student assessment, and practice communication and information literacy skills. Utilizing current events and elements of students' daily lives can help students interconnect what they are learning and increase student interest in the course. These discussions ask students to explore a topic, reflect on what they would like to learn, find information, and report back on what they have learned. Herein I provide an example of a discussion prompt, a table of other potential discussion topics that are aligned with common biochemistry course topics, and results from student survey supporting discussion format.

Modeling in the Classroom: Making Relationships and Systems Visible.

As an instructional tool, models can transform the student experience from the static to the dynamic, the flat to the 3D, and the siloed to the integrated. Few practical resources exist to help instructors transition toward model-based classroom practices. The Modeling in the Classroom evidence-based teaching guide provides instructors with a tool kit for incorporating models and modeling into their classrooms (https://lse.ascb.org/evidence-based-teaching-guides/modeling-in-the-classroom). The guide discusses the underpinnings of modeling as a core scientific practice, one that can enable student development of systems thinking skills and understanding of biological concepts. The guide describes a variety of model types, including phylogenetic trees, simulations, animations, diagrams, conceptual models, concept maps, and tactile models supported by summaries of and links to articles and resources. In this paper, we will introduce key findings describing why and how to use models in the classroom. We also describe open research questions needed to address classroom implementation, instructional design, and development of students' knowledge and skills. It is our hope that the guide will provide a suitable combination of research-based findings and practical suggestions that instructors will be supported and encouraged to thoughtfully incorporate modeling to support learning goals.

Protein phosphatase 2A is crucial for sarcomere organization in Caenorhabditis elegans striated muscle.

Protein phosphatase 2A (PP2A) is a heterotrimer composed of single catalytic and scaffolding subunits and one of several possible regulatory subunits. We identified PPTR-2, a regulatory subunit of PP2A, as a binding partner for the giant muscle protein UNC-89 (obscurin) in Caenorhabditis elegans. PPTR-2 is required for sarcomere organization when its paralogue, PPTR-1, is deficient. PPTR-2 localizes to the sarcomere at dense bodies and M-lines, colocalizing with UNC-89 at M-lines. PP2A components in C. elegans include one catalytic subunit LET-92, one scaffolding subunit (PAA-1), and five regulatory subunits (SUR-6, PPTR-1, PPTR-2, RSA-1, and CASH-1). In adult muscle, loss of function in any of these subunits results in sarcomere disorganization. rsa-1 mutants show an interesting phenotype: one of the two myosin heavy chains, MHC A, localizes as closely spaced double lines rather than single lines. This "double line" phenotype is found in rare missense mutants of the head domain of MHC B myosin, such as unc-54(s74). Analysis of phosphoproteins in the unc-54(s74) mutant revealed two additional phosphoserines in the nonhelical tailpiece of MHC A. Antibodies localize PPTR-1, PAA-1, and SUR-6 to I-bands and RSA-1 to M-lines and I-bands. Therefore, PP2A localizes to sarcomeres and functions in the assembly or maintenance of sarcomeres.

Helping Practitioners and Researchers Identify and Use Education Research Literature.

Evidence-based teaching practices are being encouraged to increase student skills and understanding in the sciences. Finding, interpreting, and applying education literature to a specific context are barriers to adopting these evidence-based practices. Here, we introduce a new feature, Evidence-Based Teaching Guides This feature identifies literature associated with specific pedagogies, which we distill to practical recommendations for teaching. The goals of the feature are: to provide instructors with tools to make research-supported choices to implement the pedagogy in question, to articulate the reasons for their choices, and to develop increased awareness of biology education research. We think these guides may also be useful for biology education researchers in identifying critical components, adaptations, and contextual features that could be investigated for a given pedagogy. Each guide consists of a website with a visual map of instructional choices associated with the topic and linked pages that summarize findings from the literature and provide additional links to and summaries of key articles. Each guide will include an instructor checklist of recommendations consolidated from the entire guide in order to provide instructors with a snapshot of instructional choices and actionable advice.

Group Work.

Science, technology, engineering, and mathematics faculty are increasingly incorporating both formal and informal group work in their courses. Implementing group work can be improved by an understanding of the extensive body of educational research studies on this topic. This essay describes an online, evidence-based teaching guide published by CBE-Life Sciences Education (LSE). The guide provides a tour of research studies and resources related to group work (including many articles from LSE). Instructors who are new to group work, as well as instructors who have experienced difficulties in implementing group work, may value the condensed summaries of key research findings. These summaries are organized by teaching challenges, and actionable advice is provided in a checklist for instructors. Education researchers may value the inclusion of empirical studies, key reviews, and meta-analyses of group-work studies. In addition to describing key features of the guide, this essay also identifies areas in which further empirical studies are warranted.

Scientific Process Flowchart Assessment (SPFA): A Method for Evaluating Changes in Understanding and Visualization of the Scientific Process in a Multidisciplinary Student Population.

The scientific process is nonlinear, unpredictable, and ongoing. Assessing the nature of science is difficult with methods that rely on Likert-scale or multiple-choice questions. This study evaluated conceptions about the scientific process using student-created visual representations that we term "flowcharts." The methodology, Scientific Process Flowchart Assessment (SPFA), consisted of a prompt and rubric that was designed to assess students' understanding of the scientific process. Forty flowcharts representing a multidisciplinary group without intervention and 26 flowcharts representing pre- and postinstruction were evaluated over five dimensions: connections, experimental design, reasons for doing science, nature of science, and interconnectivity. Pre to post flowcharts showed a statistically significant improvement in the number of items and ratings for the dimensions. Comparison of the terms used and connections between terms on student flowcharts revealed an enhanced and more nuanced understanding of the scientific process, especially in the areas of application to society and communication within the scientific community. We propose that SPFA can be used in a variety of circumstances, including in the determination of what curricula or interventions would be useful in a course or program, in the assessment of curriculum, or in the evaluation of students performing research projects.

The SH3 domain of UNC-89 (obscurin) interacts with paramyosin, a coiled-coil protein, in Caenorhabditis elegans muscle.

UNC-89 is a giant polypeptide located at the sarcomeric M-line of Caenorhabditis elegans muscle. The human homologue is obscurin. To understand how UNC-89 is localized and functions, we have been identifying its binding partners. Screening a yeast two-hybrid library revealed that UNC-89 interacts with paramyosin. Paramyosin is an invertebrate-specific coiled-coil dimer protein that is homologous to the rod portion of myosin heavy chains and resides in thick filament cores. Minimally, this interaction requires UNC-89's SH3 domain and residues 294-376 of paramyosin and has a KD of ∼1.1 µM. In unc-89 loss-of-function mutants that lack the SH3 domain, paramyosin is found in accumulations. When the SH3 domain is overexpressed, paramyosin is mislocalized. SH3 domains usually interact with a proline-rich consensus sequence, but the region of paramyosin that interacts with UNC-89's SH3 is α-helical and lacks prolines. Homology modeling of UNC-89's SH3 suggests structural features that might be responsible for this interaction. The SH3-binding region of paramyosin contains a "skip residue," which is likely to locally unwind the coiled-coil and perhaps contributes to the binding specificity.

UNC-89 (obscurin) binds to MEL-26, a BTB-domain protein, and affects the function of MEI-1 (katanin) in striated muscle of Caenorhabditis elegans.

The ubiquitin proteasome system is involved in degradation of old or damaged sarcomeric proteins. Most E3 ubiquitin ligases are associated with cullins, which function as scaffolds for assembly of the protein degradation machinery. Cullin 3 uses an adaptor to link to substrates; in Caenorhabditis elegans, one of these adaptors is the BTB-domain protein MEL-26 (maternal effect lethal). Here we show that MEL-26 interacts with the giant sarcomeric protein UNC-89 (obscurin). MEL-26 and UNC-89 partially colocalize at sarcomeric M-lines. Loss of function or gain of function of mel-26 results in disorganization of myosin thick filaments similar to that found in unc-89 mutants. It had been reported that in early C. elegans embryos, a target of the CUL-3/MEL-26 ubiquitylation complex is the microtubule-severing enzyme katanin (MEI-1). Loss of function or gain of function of mei-1 also results in disorganization of thick filaments similar to unc-89 mutants. Genetic data indicate that at least some of the mel-26 loss-of-function phenotype in muscle can be attributed to increased microtubule-severing activity of MEI-1. The level of MEI-1 protein is reduced in an unc-89 mutant, suggesting that the normal role of UNC-89 is to inhibit the CUL-3/MEL-26 complex toward MEI-1.

EGFR ligands exhibit functional differences in models of paracrine and autocrine signaling.

Epidermal growth factor (EGF) family peptides are ligands for the EGF receptor (EGFR). Here, we elucidate functional differences among EGFR ligands and mechanisms underlying these distinctions. In 32D/EGFR myeloid and MCF10A breast cells, soluble amphiregulin (AR), transforming growth factor alpha (TGFα), neuregulin 2 beta, and epigen stimulate greater EGFR coupling to cell proliferation and DNA synthesis than do EGF, betacellulin, heparin-binding EGF-like growth factor, and epiregulin. EGF competitively antagonizes AR, indicating that its functional differences reflect dissimilar intrinsic activity at EGFR. EGF stimulates much greater phosphorylation of EGFR Tyr1045 than does AR. Moreover, the EGFR Y1045F mutation and z-cbl dominant-negative mutant of the c-cbl ubiquitin ligase potentiate the effect of EGF but not of AR. Both EGF and AR stimulate phosphorylation of EGFR Tyr992. However, the EGFR Y992F mutation and phospholipase C gamma inhibitor U73122 reduce the effect of AR much more than that of EGF. Expression of TGFα in 32D/EGFR cells causes greater EGFR coupling to cell proliferation than does expression of EGF. Moreover, expression of EGF in 32D/EGFR cells causes these cells to be largely refractory to stimulation with soluble EGF. Thus, EGFR ligands are functionally distinct in models of paracrine and autocrine signaling and EGFR coupling to biological responses may be specified by competition among functionally distinct EGFR ligands.

The Q43L mutant of neuregulin 2ß is a pan-ErbB receptor antagonist.

The ErbB4 receptor tyrosine kinase possesses both tumour suppressor and oncogenic activities. Thus pharmacological agents are needed to help elucidate ErbB4 functions. However, limitations of existing ErbB4 agonists and antagonists have led us to seek novel ErbB4 antagonists. The Q43L mutant of the ErbB4 agonist NRG2ß (neuregulin 2ß) stimulates ErbB4 tyrosine phosphorylation, yet fails to stimulate ErbB4 coupling to cell proliferation. Thus in the present paper we hypothesize that NRG2ß/Q43L may be an ErbB4 antagonist. NRG2ß/Q43L competitively antagonizes agonist stimulation of ErbB4 coupling to cell proliferation. NRG2ß/Q43L stimulates less ErbB4 tyrosine phosphorylation than does NRG2ß. In addition, NRG2ß stimulation of cell proliferation requires PI3K (phosphoinositide 3-kinase) activity and NRG2ß stimulates greater Akt phosphorylation than does NRG2ß/Q43L. Moreover, EGFR [EGF (epidermal growth factor) receptor] kinase activity (but not that of ErbB4) is critical for coupling ErbB4 to proliferation. Experiments utilizing ErbB4 splicing isoforms and mutants suggest that NRG2ß and NRG2ß/Q43L may differentially stimulate ErbB4 coupling to the transcriptional co-regulator YAP (Yes-associated protein). Finally, NRG2ß/Q43L competitively antagonizes agonist stimulation of EGFR and ErbB2/ErbB3, indicating that NRG2ß/Q43L is a pan-ErbB antagonist. Thus we postulate that NRG2ß/Q43L and other antagonistic ligands stimulate ErbB tyrosine phosphorylation on a set of residues distinct from that stimulated by agonists, thus suggesting a novel mechanism of ErbB receptor regulation. Moreover, NRG2ß/Q43L and related ligand-based antagonists establish a paradigm for the discovery of anti-ErbB therapeutics.

Immunofluorescent localization of proteins in Caenorhabditis elegans muscle.

Caenorhabditis elegans is a premier model genetic system for discovering new information about the assembly and maintenance of striated muscle. The localization of a protein within a nematode muscle cell can reveal important clues to its function. In C. elegans, proteins can be localized by two different methods at the light microscopy level: GFP tagged proteins and indirect immunofluorescence. Although there are advantages and disadvantages of each method, antibodies can be used to localize proteins expressed at endogenous levels and without tags that might interfere with function. Immunolocalization requires efficient and effective methods of fixation. Here, we describe in detail two different methods for fixation of adult worms, the Nonet method and the Constant Spring method. We also discuss the advantages and the disadvantages of each, and how to choose between them. These methods are also useful for localizing proteins expressed in other cell types.

Functional selectivity of EGF family peptide growth factors: implications for cancer.

Breast, prostate, pancreatic, colorectal, lung, and head and neck cancers exploit deregulated signaling by ErbB family receptors and their ligands, EGF family peptide growth factors. EGF family members that bind the same receptor are able to stimulate divergent biological responses both in cell culture and in vivo. This is analogous to the functional selectivity exhibited by ligands for G-protein coupled receptors. Here we review this literature and propose that this functional selectivity of EGF family members is due to distinctions in the conformation of the liganded receptor and subsequent differences in the sites of receptor tyrosine phosphorylation and receptor coupling to signaling effectors. We also discuss the roles of divergent ligand activity in establishing and maintaining malignant phenotypes. Finally, we discuss the potential of mutant EGF family ligands as cancer chemotherapeutics targeted to ErbB receptors.

Inter-conversion of neuregulin2 full and partial agonists for ErbB4.

The EGF family hormone NRG2beta potently stimulates ErbB4 tyrosine phosphorylation and coupling to IL3 independence. In contrast, the NRG2alpha splicing isoform has lower affinity for ErbB4, does not potently stimulate ErbB4 phosphorylation, and fails to stimulate ErbB4 coupling. Here we investigate these differences. The NRG2beta Q43L mutant potently stimulates ErbB4 phosphorylation but not ErbB4 coupling to IL3 independence. This failure to stimulate ErbB4 coupling is not due to differential ligand purity, glycosylation, or stability. The NRG2alpha K45F mutant potently stimulates ErbB4 phosphorylation but not ErbB4 coupling to IL3 independence. Thus, this failure to stimulate ErbB4 coupling is not due to inadequate affinity for ErbB4. In contrast, the NRG2alpha L43Q/K45F mutant stimulates ErbB4 coupling, even though it does not have greater affinity for ErbB4 than does NRG2alpha/K45F. Collectively, these data indicate that Gln43 of NRG2beta is both necessary and sufficient for NRG2 stimulation of ErbB4 coupling to IL3 independence.