Diego Krapf, Ph.D. - US grants

In this project the PIs will study the regulation of Kv2.1 channel clusters that may play several important biological roles in the brain. Currently, the mechanism by which clusters Kv2.1 are regulated and maintained is unknown. The project focuses on the biophysics of Kv2.1 voltage-gated K+ channel cell surface dynamics with particular emphasis on cytoskeleton-membrane interactions in live cells. The overall goal of the research is to improve our understanding of the mechanism by which the cortical cytoskeleton functionally forms a diffusion limiting fence that selectively corrals a sub-population of Kv2.1 channels. The specific research aims are: (1) characterize the dynamics of clustered and non-clustered channels within various surface regions of cultured neurons, (2) measure the influence of the cortical cytoskeleton and raft microdomains on Kv2.1 channel dynamics, (3) build a microscope to implement high-speed particle tracking and optical tweezers, and (4) determine the mechanism that forms Kv2.1 clusters on the cell surface. This experimental work will lead to unique insights in the molecular mechanism of membrane protein dynamics. The project focuses on investigating Kv2.1 clustering regulation, dynamics and interactions with the cytoskeleton which may lead to improved treatments for acute ischemic stroke through enhanced neuro-protective approaches. The research program will be integrated with an educational component by building an optical tweezers setup for educational purposes. Under-represented students will be recruited at minority meetings and through a Women & Minorities in Engineering Program at Colorado State University.

The project is jointly sponsored by the Physics and the Molecular Cell Biology Divisions at NSF.

Endoplasmic reticulum/plasma membrane (ER/PM) junctions are known to be sites of calcium ion (Ca2+) influx. Recently, the PI discovered that these junctions function as trafficking hubs for insertion and removal of plasma membrane proteins. Furthermore, the PI has found that the voltage gated potassium channel Kv2.1 interacts with the endoplasmic reticulum, dramatically increasing ER/PM junction surface area and structurally changing the junction morphology. The PI's findings show that the Kv2.1 potassium channel remodels to cortical ER, which is likely within 30 nm of the plasma membrane. Kv2.1 is playing a structural role similar to that of Orai, for the PI proposes that Kv2.1 is binding an ER membrane protein. Thus, Kv2.1-mediated ER enrichment on the cell surface is a novel specialized organelle with specific functions in protein transport vital to cell signaling.

The current project focuses on understanding the biology of ER/PM junctions with particular emphasis on the regulation of the ER/PM junction structure and its function in the modulation of membrane protein trafficking. The PI will answer the following questions: What is the role of Kv2.1 in protein trafficking at ER/PM junctions? How are ER/PM junctions dynamically regulated by Kv2.1? What are the relationships between the cortical cytoskeleton, ER, and Kv2.1? Which theoretical framework can be used to describe the assembly and maintenance of these domains? How does large-scale membrane behavior emerge from the interactions between Kv2.1 and ER? The fusion of multicolor single-molecule tracking in living cells and advanced stochastic process analysis, which are integral to the project, will provide answer to these questions. This research will offer excellent opportunities for graduate and undergraduate student participation in interdisciplinary research through the collaboration between two laboratories with very different backgrounds. The research program will be integrated with an outreach component by developing a microscopy laboratory for students at a local elementary school. The goal of the outreach program is to foster scientific enquiry and to motivate students to appreciate science from an early age. This lab presents a unique opportunity to leverage integration of education and research, giving students access to hands-on practical learning.

This project is being jointly supported by the Physics of Living Systems program in the Division of Physics and the Cellular Dynamics and Function Program in the Division of Molecular and Cellular Biosciences.

An award is made to Colorado State University (CSU) to acquire a microscopy system that combines multiple techniques in a single platform. This instrument will be housed in a facility that enables secure 24-hour access for users and will be maintained by a staff scientist. The instrument will be included in a new module for an undergraduate lab course that serves about 100 students annually. Students in a graduate course on microscopy will also be introduced to the instrument. The instrument will be made available to a broad user base both inside and outside of CSU, by including it in a Microscopy Imaging Network (MIN). This network has a pilot grant program for new instrument users. These pilot grants support user training and preliminary data acquisition on this and other microscopy instruments at CSU.

The instrument will combine scanning probe and optical techniques. The scanning probe techniques enable imaging of the topographical features and mechanical properties of samples at very high resolution. The optical techniques include spinning disc confocal microscopy, fluorescence recovery after photobleaching (FRAP), and photoactivation (PA). The instrument will be configured to perform imaging of wet samples including live cells. Combining these fundamentally different yet complementary techniques on a single platform enables interrogation of multiple sample properties simultaneously and in the exact same field of view. Therefore specific measurements made with one technique (e.g. mechanical properties) can be correlated with features (e.g. location of chemical functional groups or biological structures) measured by another technique. Changes in these correlations and spatial organization can be monitored over time to observe biological phenomena. The initial user group consists of 19 investigators in ten academic departments from three universities (CSU, University of Colorado at Denver, and University of Denver). These investigators will use the instrument for interdisciplinary and collaborative research projects including biochemical and biophysical studies and for the development of new materials for biomedical and tissue engineering applications.

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Kipper, Matthew J.

Many applications in medicine and biotechnology involve foreign surfaces that contact blood. Some applications involve long-term exposure to blood, such as heart valve replacements and stents. Other applications require intermediate-term or short-term contact. These include applications such as blood storage and medical procedures like dialysis and blood oxygenation. The materials used in these applications induce undesirable (and potentially catastrophic) blood-material interactions, such as blood clotting and inflammation. In fact, the only known surface that is compatible with flowing whole blood for long-term contact is the inside surfaces of blood vessels. This work will develop new surfaces that have chemical and structural features designed to mimic the inside surfaces of blood vessels. We will also study how the chemistry and structure of these new surfaces can be tuned to control interactions with important blood proteins that regulate blood-material interactions, like clotting. This will enable us to better design materials for blood-contacting applications.

This work will prepare dense polymer brushes containing glycosaminoglycans, which are the polyanionic polysaccharides in the endothelial glycocalyx presented by the cells lining blood vessel walls. The interactions of important blood proteins with these glycocalyx mimics will be investigated by single-molecule fluorescence microscopy experiments. These experiments will be used to test new hypotheses about how blood-compatibility is determined by protein-surface interactions and protein-protein interactions at surfaces. Finally we will demonstrate that glycocalyx mimics result in reduced platelet adhesion and activation, and decrease the propensity for blood to clot.

This work will lead to better understanding of the mechanisms whereby interactions of blood components with blood vessel walls prevent blood clotting. By understanding how the blood vessel wall prevents clotting we can better design blood-contacting surfaces for many applications in cardiovascular medicine and biotechnology. The findings from this research could also lead to advances in handling other complex protein mixtures in applications such as protein separation for the food and biopharmaceutical industries. Our education and outreach activities will include a one-week summer short course for middle school students, a symposium for a middle school students at a charter school that serves low-income and minority students, and contributions to a new textbook being authored by a co-PI.

Transcription is the first step on the path of protein synthesis and consists of copying a DNA sequence into an RNA sequence. In addition to the transcriptional regulation of gene expression, a regulatory layer exists, known as post-transcriptional regulation. In eukaryotes, micro RNAs (miRNAs) are key mediators of this regulatory network. Post-transcriptional regulation is still poorly understood and many fundamental questions remain open. Some features of miRNA-based regulation make it different from other mechanisms of gene regulation: it is stoichiometric and, thus, competition effects emerge, it occurs in the cytoplasm, and it involves transport of both the regulator and targets. The goal of this project is to understand cellular properties that arise from post-transcriptional regulation across spatiotemporal scales, from the motion and interactions of individual molecules to the network level. This project will support an outreach program at Webber Middle School in Fort Collins, CO. Research experiences for students will be developed in coordination with school teachers and integrated within an established exceptional program therein, namely the Webber’s Aerospace Ventures in Education (WAVE), which offers students an engaging, challenging, and rigorous experience simulating how to organize and conduct extra-planetary exploration with the goal of encouraging students to pursue space science research and future careers within the industry. The collaboration with school teachers will enable valuable innovations to the WAVE curriculum. The main activities will focus on testing for the presence of life using biomarkers of RNA and DNA, attending demonstrations of single-molecule RNA tracking, and evaluating the motion RNA in live cells.

The objectives of this project are (i) to dissect the effects of the complex cell environment on post-transcriptional regulation and (ii) to elucidate the crosstalk effects of the post-transcriptional network. The PIs will use optical imaging to track the paths of miRNAs and mRNAs at the single-molecule level in living cells. Experimental trajectories will be analyzed using theoretical and computational approaches that include first passage processes and anomalous diffusion theory for the calculation of kinetic rates coefficients. These coefficients will be employed in mathematical models. The analysis of multiple paths will determine localization patterns and decipher whether the network should be divided into a set of weakly coupled subnetworks. It is posited that a key factor in understanding post-transcriptional regulation lies in its coupling to the transcriptional regulation network. A data-driven mathematical model that combines transcriptional and post-transcriptional regulations as a mixed network of directed (transcription) and bidirectional (post-transcription) links will be built.
This project is being jointly supported by the Physics of Living Systems program in the Division of Physics and the Systems and Synthetic Biology and Genetic Mechanisms Clusters in the Division of Molecular and Cellular Biosciences.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.