Role for the molecular chaperones Zuo1 and Ssz1 in quorum sensing via activation of the transcription factor Pdr1.

Zuo1 functions as a J-protein cochaperone of its partner Hsp70. In addition, the C terminus of Zuo1 and the N terminus of Ssz1, with which Zuo1 forms a heterodimer, can independently activate the Saccharomyces cerevisiae transcription factor pleiotropic drug resistance 1 (Pdr1). Here we report that activation of Pdr1 by Zuo1 or Ssz1 causes premature growth arrest of cells during the diauxic shift, as they adapt to the changing environmental conditions. Conversely, cells lacking Zuo1 or Ssz1 overgrow, arresting at a higher cell density, an effect overcome by activation of Pdr1. Cells lacking the genes encoding plasma membrane transporters Pdr5 and Snq2, two targets of Pdr1, also overgrow at the diauxic shift. Adding conditioned medium harvested from cultures of wild-type cells attenuated the overgrowth of both zuo1Δssz1Δ and pdr5Δsnq2Δ cells, suggesting the extracellular presence of molecules that signal growth arrest. In addition, our yeast two-hybrid analysis revealed an interaction between Pdr1 and both Zuo1 and Ssz1. Together, our results support a model in which (i) membrane transporters, encoded by Pdr1 target genes act to promote cell-cell communication by exporting quorum sensing molecules, in addition to playing a role in pleiotropic drug resistance; and (ii) molecular chaperones function at promoters to regulate this intercellular communication through their activation of the transcription factor Pdr1.

The nuclear envelope: form and reformation.

The membrane system that encloses genomic DNA is referred to as the nuclear envelope. However, with emerging roles in signaling and gene expression, these membranes clearly serve as more than just a physical barrier separating the nucleus and cytoplasm. Recent progress in our understanding of nuclear envelope architecture and composition has also revealed an intriguing connection between constituents of the nuclear envelope and human disease, providing further impetus to decipher this cellular structure and the dramatic remodeling process it undergoes with each cell division.

Using personas and the ADKAR framework to evaluate a network designed to facilitate sustained change toward active learning in the undergraduate classroom.

One promising practice for increasing active learning in undergraduate science education is the use of a mentoring network. The Promoting Active Learning and Mentoring (PALM) Network was launched with practitioners from several professional societies and disciplines to make changes in their teaching based on evidence-based practices and to encourage the members to reflect deeply on their teaching experiences. Members of the Network interviewed seven previous Fellows, 1 to 6 years after completing their fellowship, to better understand the value of the Network and how these interactions impacted their ability to sustain change toward more active teaching practices. The interviews resulted in the creation of three personas that reflect the kinds of educators who engaged with the Network: Neil the Novice, Issa the Isolated, and Etta the Expert. Key themes emerged from the interviews about how interactions with the PALM Network sustained change toward evidence-based teaching practices allowing the members to readily adapt to the online learning environment during the COVID-19 pandemic. Understanding how the personas intersect with the ADKAR model contributes to a better understanding of how mentoring networks facilitate transformative change toward active learning and can inform additional professional development programs. Supplementary Information: The online version contains supplementary material available at 10.1007/s44217-022-00023-w.

The COPI complex functions in nuclear envelope breakdown and is recruited by the nucleoporin Nup153.

Nuclear envelope breakdown is a critical step in the cell cycle of higher eukaryotes. Although integral membrane proteins associated with the nuclear membrane have been observed to disperse into the endoplasmic reticulum at mitosis, the mechanisms involved in this reorganization remain to be fully elucidated. Here, using Xenopus extracts, we report a role for the COPI coatomer complex in nuclear envelope breakdown, implicating vesiculation as an important step. We have found that a nuclear pore protein, Nup153, plays a critical role in directing COPI to the nuclear membrane at mitosis and that this event provides feedback to other aspects of nuclear disassembly. These results provide insight into how key steps in nuclear division are orchestrated.

Nuclear envelope breakdown is coordinated by both Nup358/RanBP2 and Nup153, two nucleoporins with zinc finger modules.

When higher eukaryotic cells transition into mitosis, the nuclear envelope, nuclear pore complexes, and nuclear lamina are coordinately disassembled. The COPI coatomer complex, which plays a major role in membrane remodeling at the Golgi, has been implicated in the process of nuclear envelope breakdown and requires interactions at the nuclear pore complex for recruitment to this new site of action at mitosis. Nup153, a resident of the nuclear pore basket, was found to be involved in COPI recruitment, but the molecular nature of the interface between COPI and the nuclear pore has not been fully elucidated. To better understand what occurs at the nuclear pore at this juncture, we have probed the role of the nucleoporin Nup358/RanBP2. Nup358 contains a repetitive zinc finger domain with overall organization similar to a region within Nup153 that is critical to COPI association, yet inspection of these two zinc finger domains reveals features that also clearly distinguish them. Here, we found that the Nup358 zinc finger domain, but not a zinc finger domain from an unrelated protein, binds to COPI and dominantly inhibits progression of nuclear envelope breakdown in an assay that robustly recapitulates this process in vitro. Moreover, the Nup358 zinc finger domain interferes with COPI recruitment to the nuclear rim. Consistent with a role for this pore protein in coordinating nuclear envelope breakdown, Nup358-specific antibodies impair nuclear disassembly. Significantly, targeting either Nup153 or Nup358 for inhibition perturbs nuclear envelope breakdown, supporting a model in which these nucleoporins play nonredundant roles, perhaps contributing to COPI recruitment platforms on both the nuclear and cytoplasmic faces of the pore. We found that an individual zinc finger is the minimal interface for COPI association, although tandem zinc fingers are optimal. These results provide new information about the critical components of nuclear membrane remodeling and lay the foundation for a better understanding of how this process is regulated.

Widespread hyperplasia induced by transgenic TGFalpha in ApcMin mice is associated with only regional effects on tumorigenesis.

Using a mouse predisposed to neoplasia by a germ line mutation in Apc (Apc(Min)), we tested whether induced hyperplasia is sufficient to increase intestinal tumor multiplicity or size in the intestine. We found that hyperplasia in the jejunum correlated with a significant increase in tumor multiplicity. However, tumor multiplicity was unchanged in the hyperplastic colon. This result indicates that even an intestine predisposed to neoplasia can, in certain regions including the colon, accommodate net increased cell growth without developing more neoplasms. Where hyperplasia correlated with increased tumor multiplicity, it did not increase the size or net growth of established tumors. This result suggests that the event linking hyperplasia and neoplasia in the jejunum is tumor establishment. Two novel observations arose in our study: the multiple intestinal neoplasia (Min) mutation partially suppressed both mitosis and transforming growth factor alpha-induced hyperplasia throughout the intestine; and zinc treatment alone increased tumor multiplicity in the duodenum of Min mice.

A Randomized Crossover Design to Assess Learning Impact and Student Preference for Active and Passive Online Learning Modules.

Medical education increasingly involves online learning experiences to facilitate the standardization of curriculum across time and space. In class, delivering material by lecture is less effective at promoting student learning than engaging students in active learning experience and it is unclear whether this difference also exists online. We sought to evaluate medical student preferences for online lecture or online active learning formats and the impact of format on short- and long-term learning gains. Students participated online in either lecture or constructivist learning activities in a first year neurologic sciences course at a US medical school. In 2012, students selected which format to complete and in 2013, students were randomly assigned in a crossover fashion to the modules. In the first iteration, students strongly preferred the lecture modules and valued being told "what they need to know" rather than figuring it out independently. In the crossover iteration, learning gains and knowledge retention were found to be equivalent regardless of format, and students uniformly demonstrated a strong preference for the lecture format, which also on average took less time to complete. When given a choice for online modules, students prefer passive lecture rather than completing constructivist activities, and in the time-limited environment of medical school, this choice results in similar performance on multiple-choice examinations with less time invested. Instructors need to look more carefully at whether assessments and learning strategies are helping students to obtain self-directed learning skills and to consider strategies to help students learn to value active learning in an online environment.

Improving medical students' knowledge of genetic disease: a review of current and emerging pedagogical practices.

Genetics is an essential subject to be mastered by health professional students of all types. However, technological advances in genomics and recent pedagogical research have changed the way in which many medical training programs teach genetics to their students. These advances favor a more experience-based education focused primarily on developing student's critical thinking skills. In this review, we examine the current state of genetics education at both the preclinical and clinical levels and the ways in which medical and pedagogical research have guided reforms to current and emerging teaching practices in genetics. We discover exciting trends taking place in which genetics is integrated with other scientific disciplines both horizontally and vertically across medical curricula to emphasize training in scientific critical thinking skills among students via the evaluation of clinical evidence and consultation of online databases. These trends will produce future health professionals with the skills and confidence necessary to embrace the new tools of medical practice that have emerged from scientific advances in genetics, genomics, and bioinformatics.

Experiences of mentors training underrepresented undergraduates in the research laboratory.

Successfully recruiting students from underrepresented groups to pursue biomedical science research careers continues to be a challenge. Early exposure to scientific research is often cited as a powerful means to attract research scholars with the research mentor being critical in facilitating the development of an individual's science identity and career; however, most mentors in the biological sciences have had little formal training in working with research mentees. To better understand mentors' experiences working with undergraduates in the laboratory, we conducted semistructured interviews with 15 research mentors at a public university in the Midwest. The interviewed mentors were part of a program designed to increase the number of American Indians pursuing biomedical/biobehavioral research careers and represented a broad array of perspectives, including equal representation of male and female mentors, mentors from underrepresented groups, mentors at different levels of their careers, and mentors from undergraduate and professional school departments. The mentors identified benefits and challenges in being an effective mentor. We also explored what the term underrepresented means to the mentors and discovered that most of the mentors had an incomplete understanding about how differences in culture could contribute to underrepresented students' experience in the laboratory. Our interviews identify issues relevant to designing programs and courses focused on undergraduate student research.

Unfolding of the C-terminal domain of the J-protein Zuo1 releases autoinhibition and activates Pdr1-dependent transcription.

The C-terminal 69 residues of the J-protein Zuo1 are sufficient to activate Pdr1, a transcription factor involved in both pleiotropic drug resistance and growth control. Little is understood about the pathway of activation by this primarily ribosome associated Hsp40 co-chaperone. Here, we report that only the C-terminal 13 residues of Zuo1 are required for activation of Pdr1, with hydrophobic residues being critical for activity. Two-hybrid interaction experiments suggest that the interaction between this 13-residue Zuo1 peptide and Pdr1 is direct, analogous to the activation of Pdr1 by xenobiotics. However, simply dissociation of Zuo1 from the ribosome is not sufficient for induction of Pdr1 transcriptional activity, as the C-terminal 86 residues of Zuo1 fold into an autoinhibitory left-handed four-helix bundle. Hydrophobic residues critical for interaction with Pdr1 are sequestered within the structure of this C-terminal domain (CTD), necessitating unfolding for activation. Thus, although expression of the CTD does not result in activation, alterations that destabilize the structure cause induction of pleiotropic drug resistance. These destabilizing alterations also result in dissociation of the full-length protein from the ribosome. Thus, our results are consistent with an activation pathway in which unfolding of Zuo1's C-terminal helical bundle domain results in ribosome dissociation followed by activation of Pdr1 via a direct interaction.

Using online lectures to make time for active learning.

To make time in class for group activities devoted to critical thinking, we integrated a series of short online lectures into the homework assignments of a large, introductory biology course at a research university. The majority of students viewed the online lectures before coming to class and reported that the online lectures helped them to complete the in-class activity and did not increase the amount of time they devoted to the course. In addition, students who viewed the online lecture performed better on clicker questions designed to test lower-order cognitive skills. The in-class activities then gave the students practice analyzing the information in groups and provided the instructor with feedback about the students' understanding of the material. On the basis of the results of this study, we support creating hybrid course models that allow students to learn the fundamental information outside of class time, thereby creating time during the class period to be dedicated toward the conceptual understanding of the material.

Long-lived Min mice develop advanced intestinal cancers through a genetically conservative pathway.

C57BL/6J mice carrying the Min allele of Adenomatous polyposis coli (Apc) develop numerous adenomas along the entire length of the intestine and consequently die at an early age. This short lifespan would prevent the accumulation of somatic genetic mutations or epigenetic alterations necessary for tumor progression. To overcome this limitation, we generated F(1) Apc(Min/+) hybrids by crossing C57BR/cdcJ and SWR/J females to C57BL/6J Apc(Min/+) males. These hybrids developed few intestinal tumors and often lived longer than 1 year. Many of the tumors (24-87%) were invasive adenocarcinomas, in which neoplastic tissue penetrated through the muscle wall into the mesentery. In a few cases (3%), lesions metastasized by extension to regional lymph nodes. The development of these familial cancers does not require chromosomal gains or losses, a high level of microsatellite instability, or the presence of Helicobacter. To test whether genetic instability might accelerate tumor progression, we generated Apc(Min/+) mice homozygous for the hypomorphic allele of the Nijmegen breakage syndrome gene (Nbs1(DeltaB)) and also treated Apc(Min/+) mice with a strong somatic mutagen. These imposed genetic instabilities did not reduce the time required for cancers to form nor increase the percentage of cancers nor drive progression to the point of distant metastasis. In summary, we have found that the Apc(Min/+) mouse model for familial intestinal cancer can develop frequent invasive cancers in the absence of overt genomic instability. Possible factors that promote invasion include age-dependent epigenetic changes, conservative somatic recombination, or direct effects of alleles in the F(1) hybrid genetic background.

Studying nuclear disassembly in vitro using Xenopus egg extract.

Xenopus egg extract provides an extremely powerful approach in the study of cell cycle regulated aspects of nuclear form and function. Each egg contains enough membrane and protein components to support multiple rounds of cell division. Remarkably, incubation of egg extract with DNA in the presence of an energy regeneration system is sufficient to induce formation of a nuclear envelope around DNA. In addition, these in vitro nuclei contain functional nuclear pore complexes, which form de novo and are capable of supporting nucleocytoplasmic transport. Mitotic entry can be induced by the addition of recombinant cyclin to an interphase extract. This initiates signaling that leads to disassembly of the nuclei. Thus, this cell-free system can be used to decipher events involved in mitotic remodeling of the nuclear envelope such as changes in nuclear pore permeability, dispersal of membrane, and disassembly of the lamina. Both general mechanisms and individual players required for orchestrating these events can be identified via biochemical manipulation of the egg extract. Here, we describe a procedure for the assembly and disassembly of in vitro nuclei, including the production of Xenopus egg extract and sperm chromatin DNA.