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Course-based undergraduate research experiences (CUREs) provide a way for students to gain research experience in a classroom setting. Few examples of cell culture CUREs or online CUREs exist in the literature. The Cell Biology Education Consortium (CBEC) provides a network and resources for instructors working to incorporate cell-culture based research into the classroom. In this article, we provide examples from six instructors from the CBEC network on how they structure their cell-culture CUREs and how they transitioned the labs to online in the spring semester of 2020. We intend for these examples to provide instructors with ideas for strategies to set up cell culture CUREs, how to change that design mid-term, and for creating online CUREs in the future.

American Society for Microbiology (ASM) Curriculum Guidelines highlight the importance of instruction about informational flow in organisms, including regulation of gene expression. However, foundational central dogma concepts and more advanced gene regulatory mechanisms are challenging for undergraduate biology students. To increase student comprehension of these principles, we designed an activity for upper-level biology students centered on construction and analysis of physical models of bacterial riboswitches. Students manipulate an inexpensive bag of supplies (beads, pipe cleaners) to model two conformations of a riboswitch in a bacterial transcript. After initial pilot testing, we implemented the activity in three upper-level classes at one research-intensive and two primarily undergraduate institutions. To assess student perceptions of learning gains, we utilized a pre/post-activity 5-point Likert-type survey instrument to characterize student perceptions of confidence in both their understanding of riboswitches and their ability to apply the central dogma to riboswitches. Median post-test ranks were significantly higher than median pre-test ranks ( p < 0.0001) when compared by the Wilcoxon signed-rank test ( n = 31). Next, we assessed post-activity knowledge via use of a rubric to score student responses on exam questions. More than 80% of students could correctly describe and diagram examples of riboswitches; data from initial iterations were used to enhance curriculum materials for subsequent implementations. We conclude that this riboswitch activity leads to both student-reported increases in confidence in the ASM curriculum dimension of gene regulation, including central dogma concepts, and demonstrated student ability to diagram riboswitches, predict outcomes of riboswitches, and connect riboswitches to evolutionary roles.

We developed an interactive laboratory that allows students to identify and grade tissue samples from human breast biopsies, using techniques similar to those used by actual pathologists. This unique lab develops a practical and intellectual understanding of basic tissue structures that make up living systems, utilizing technology to bring together pathology, cancer biology, genetics, and bioethics in a relevant and engaging way that leaves a lasting impression on students. The activities described are appropriate for students at all levels of high school and college, especially those with an interest in health care careers.

The adaptor protein Amot130 scaffolds components of the Hippo pathway to promote the inhibition of cell growth. This study describes how Amot130 through binding and activating the ubiquitin ligase AIP4/Itch achieves these effects. AIP4 is found to bind and ubiquitinate Amot130 at residue Lys-481. This both stabilizes Amot130 and promotes its residence at the plasma membrane. Furthermore, Amot130 is shown to scaffold a complex containing overexpressed AIP4 and the transcriptional co-activator Yes-associated protein (YAP). Consequently, Amot130 promotes the ubiquitination of YAP by AIP4 and prevents AIP4 from binding to large tumor suppressor 1. Amot130 is found to reduce YAP stability. Importantly, Amot130 inhibition of YAP dependent transcription is reversed by AIP4 silencing, whereas Amot130 and AIP4 expression interdependently suppress cell growth. Thus, Amot130 repurposes AIP4 from its previously described role in degrading large tumor suppressor 1 to the inhibition of YAP and cell growth.

Large tumor suppressor (LATS)1/2 protein kinases transmit Hippo signaling in response to intercellular contacts and serum levels to limit cell growth via the inhibition of Yes-associated protein (YAP). Here low serum and high LATS1 activity are found to enhance the levels of the 130-kDa isoform of angiomotin (Amot130) through phosphorylation by LATS1/2 at serine 175, which then forms a binding site for 14-3-3. Such phosphorylation, in turn, enables the ubiquitin ligase atrophin-1 interacting protein (AIP)4 to bind, ubiquitinate, and stabilize Amot130. Consistently, the Amot130 (S175A) mutant, which lacks LATS phosphorylation, bound AIP4 poorly under all conditions and showed reduced stability. Amot130 and AIP4 also promoted the ubiquitination and degradation of YAP in response to serum starvation, unlike Amot130 (S175A). Moreover, silencing Amot130 expression blocked LATS1 from inhibiting the expression of connective tissue growth factor, a YAP-regulated gene. Concordant with phosphorylated Amot130 specifically mediating these effects, wild-type Amot130 selectively induced YAP phosphorylation and reduced transcription of connective tissue growth factor in an AIP4-dependent manner versus Amot130 (S175A). Further, Amot130 but not Amot130 (S175A) strongly inhibited the growth of MDA-MB-468 breast cancer cells. The dominant-negative effects of Amot130 (S175A) on YAP signaling also support that phosphorylated Amot130 transduces Hippo signaling. Likewise, Amot130 expression provoked premature growth arrest during mammary cell acini formation, whereas Amot130 (S175A)-expressing cells formed enlarged and poorly differentiated acini. Taken together, the phosphorylation of Amot130 by LATS is found to be a key feature that enables it to inhibit YAP-dependent signaling and cell growth.

Green fluorescent protein (GFP) and variants have become powerful tools to study protein localization, interactions, and dynamics. We present here a mass spectrometry-based proteomics strategy to examine protein-protein interactions using anti-GFP single-chain antibody V(H)H in a combination with a novel stable isotopic labeling reagent, isotope tag on amino groups (iTAG). We demonstrate that the single-chain V(H)H (GFP nanotrap) allows us to identify interacting partners of the Syk protein-tyrosine kinase bearing a GFP epitope tag with high efficiency and high specificity. Interacting proteins identified include CrkL, BLNK, α- and β-tubulin, Csk, RanBP5 and DJ-1. The iTAG reagents were prepared with simple procedures and characterized with high accuracy in the determination of peptides in model peptide mixtures and as well as in complex mixture. Applications of the iTAG method and GFP nanotrap to an analysis of the nucleocytoplasmic trafficking of Syk led to the identification of location-specific associations between Syk and multiple proteins. While the results reveal that the new quantitative proteomic strategy is generally applicable to integrate protein interaction data with subcellular localization, extra caution should be taken in evaluating the results obtained by such affinity purification strategies as many interactions appear to occur following cell lysis.