Amy J. Prunuske

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.

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.

Experiences of Mentors Training Underrepresented Undergraduates in the Research Laboratory

The goal of this research was to better understand the experiences and perspectives of mentors in a program designed to increase the number of American Indian students garnering PhDs. Challenges and benefits associated with mentoring undergraduates were identified through semistructured interviews.

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.

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 Zuo1s C-terminal helical bundle domain results in ribosome dissociation followed by activation of Pdr1 via a direct interaction.

Widespread hyperplasia induced by transgenic TGFα 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.

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.

The Development of an Indigenous Health Curriculum for Medical Students

Indigenous populations experience dramatic health disparities; yet, few medical schools equip students with the skills to address these inequities. At the University of Minnesota Medical School, Duluth campus, a project to develop an Indigenous health curriculum began in September 2013. This project used collaborative and decolonizing methods to gather ideas and opinions from multiple stakeholders, including students, community members, faculty, and administration, to guide the process of adding Indigenous health content to the curriculum to prepare students to work effectively with Indigenous populations. A mixed-methods needs assessment was implemented to inform the instructional design of the curriculum. In June 2014, stakeholders were invited to attend a retreat and complete a survey to understand their opinions of what should be included in the curriculum and in what way. Retreat feedback and survey responses indicated that the most important topics to include were cultural humility, Indigenous culture, social/political/economic determinants of health, and successful tribal health interventions. Stakeholders also emphasized that this content should be taught by tribal members, medical school faculty, and faculty in complementary departments (e.g., American Indian Studies, Education, Social Work) in a way that incorporates experiential learning. Preliminary outcomes include the addition of a seven-hour block of Indigenous content for first-year students taught primarily by Indigenous faculty from several departments. To address the systemic barriers to health and well-being and provider bias that Indigenous patients experience, this project sought to gather data and opinions regarding the training of medical students through a process of Indigenizing research and education.

Community Partnership Designed to Promote Lyme Disease Prevention and Engagement in Citizen Science

The goal of this project is to promote Lyme disease prevention and to cultivate an interest in science through a citizen-science project coordinated by researchers at a public university and teachers at rural high schools. The lesson plan is designed to increase student interest in pursuing a science career through participation in an authentic research experience, utilizing a topic that has implications on the health of the surrounding community. Students are introduced in the classroom to zoonotic diseases transmitted by the Ixodes tick, the health risks of Lyme disease, and disease prevention strategies. Students then participate in a research experience collecting field data and ticks from their community, which are used in university research. To measure changes in student knowledge and attitudes toward Lyme disease and science careers, students completed surveys related to the learning objectives associated with the experience. We found participation in the activity increased student confidence and ability to correctly differentiate a deer tick from a wood tick and to recognize the symptoms of Lyme disease. In addition, students reported increased interest in pursuing a science degree in college or graduate school. Authentic research experience related to a disease relevant to the local community is effective at enhancing high school student engagement in science.