An unbiased classification approach reveals multiple fusion categories of VAMP2-mediated exocytosis
Presented by Professor Stephanie Gupton
Department of Cell Biology and Physiology, School of Medicine
University of North Carolina at Chapel Hill
Exocytosis is a fundamental process that secretes cargo into the extracellular space and potentially inserts lipids and proteins into the plasma membrane. A pH-sensitive variant of GFP (pHluorin) fused to the luminal end of a vesicle-SNARE protein, such as VAMP2, provides a fluorescent intensity readout of the fusion pore opening during exocytosis and subsequent fate of the v-SNARE. We previously reported an automated analysis platform that identifies such exocytic events, and records several parameters of their fusion, such as the frequency, half-life fluorescence decay, and spatio-temporal distribution. This led us to hypothesize there were discrete types of exocytic events based on the behavior of the v-SNARE after fusion pore opening. Here, we introduce a novel machine-learning method to automatically classify TIRF images of VAMP2-pHluorin exocytic events in developing murine cortical neurons. We used multiple classifiers, including hierarchical agglomerative clustering, dynamic time warping, and feature selection followed by dimensionality reduction to categorize exocytic events in an unsupervised way. Using a majority-rule committee of 28 indices, run with each of the classifiers individually, we determined the most likely number of classes. The committee always selected four discrete classes of fusion, with each classifier similarly grouping exocytic events. This was surprising, as classically, two modes of fusion are recognized. During full-vesicle-fusion (FVF), the fusion pore dilates after opening and the vesicle collapses into the membrane. During kiss-and-run (KNR) exocytosis a transient fusion pore closes after cargo secretion. All four classes were tetanus sensitive, indicating they were bona fide VAMP2-mediated fusion events, but exhibited unique fluorescence intensity profiles after fusion pore opening. To determine if any of these differences were pH sensitive, we buffered the media with HEPES. This increased the half-life of VAMP2-pHluorin fluorescence in two classes, indicating vesicle re-acidification, consistent with KNR fusion. One KNR class exhibited an immediate decay of fluorescence after fusion pore opening, whereas the other demonstrated a delay in the onset of decay, consistent with the fusion pore remaining open. The two classes that were HEPES-insensitive exhibited fluorescence decay consistent with VAMP2-pHluorin diffusing in the plasma membrane, consistent with FVF. One of these classes also exhibited immediate fluorescence decay, whereas the other class demonstrated a delay in the onset of fluorescence decay. Simultaneous imaging of VAMP2-phluorin with VAMP2-TagRFP to reveal the fate of the vesicle further supports the existence of the four classes. This validated novel unsupervised classification of exocytic modes now allows us to answer important previously unapproachable biological questions. We will provide examples of manipulations that alter the proportion of events and their distribution in neurons.
Functional Biomaterials for Regenerative Medicine
Presented by Professor Treena Livingston Arinzeh
Department of Biomedical Engineering, New Jersey Institute of Technology
Stem cells are a promising cell source in the tissue engineering and regenerative medicine fields. Intense studies have been focused at the cell and molecular biology levels on understanding the relationship between stem cell growth and differentiation in an effort to control these processes. Recent discoveries have shown that the microenvironment can influence stem cell self-renewal and differentiation, which has had a tremendous impact on identifying potential strategies for using these cells effectively in the body. This presentation will describe studies examining the influence of biomaterials on stem cell behavior with an emphasis on identifying biomaterial designs and chemistries that impart appropriate cues to stem cells to affect their behavior both in vitro and in vivo. Recent results using biomimetic materials, specifically piezoelectric polymers, that provide electromechanical cues to stem cells and novel glycosaminoglycan mimetics that prolong the bioactivity of growth factors and induce differentiation will be discussed.
Treena Livingston Arinzeh, PhD is a Professor of Biomedical Engineering at the New Jersey Institute of Technology (NJIT). Dr. Arinzeh received her B.S. from Rutgers University in Mechanical Engineering, her M.S.E. in Biomedical Engineering from Johns Hopkins University, and her Ph.D. in Bioengineering from the University of Pennsylvania. She worked for several years as a project manager at a stem cell technology company, Osiris Therapeutics, Inc. Dr. Arinzeh joined the faculty of NJIT as one of the founding faculty members of the department of Biomedical Engineering and served as interim chairperson and graduate director. Her most notable or cited work to date has been in the use of allogeneic mesenchymal stem cells (MSCs) with bioactive ceramics to induce bone formation in a large bone defect without the use of immunosuppressive therapy. This study served the basis for FDA approval to pursue clinical trials using allogeneic MSCs for various applications. Dr. Arinzeh has been recognized with numerous awards, including the National Science Foundation (NSF) CAREER Award and the Presidential Early Career Award for Scientists and Engineers (PECASE). She was nominated by the Governor of Connecticut to the Connecticut Stem Cell Research Advisory Committee. She is a fellow of the American Institute for Medical and Biological Engineering (AIMBE) and the Biomedical Engineering Society (BMES). She recently served as the chairperson for the National Institutes of Health (NIH) Musculoskeletal Tissue Engineering (MTE) Study Section. She is currently a co-PI of the NSF Science and Technology Center on Engineering Mechanobiology, which is a multi-institutional center with the University of Pennsylvania and Washington University in Saint Louis. She has also made a significant impact in the recruitment and mentoring of underrepresented minorities and women in biomedical engineering and other STEM fields.
Presented by Professor Jennifer A. Lewis
Hansjörg Wyss Professor of Biologically Inspired Engineering , Paulson School of Engineering and Applied Sciences, Harvard University
3D printing enables one to rapidly design and fabricate materials in arbitrary shapes on demand. I will introduce the fundamental principles that underpin both droplet- and filamentary printing methods. I will then describe the development of new functional, structural and biological inks as well as printhead designs that are vastly expanding the capabilities of 3D printing. Finally, I will highlight several examples from our recent work, including the fabrication and characterization of soft electronic, robotic, and shape-morphing architectures.
Jennifer A. Lewis is the Wyss Professor for Biologically Inspired Engineering in the Paulson School of Engineering and Applied Sciences and a core faculty member of the Wyss Institute at Harvard University, where she co-leads the 3D Organ Engineering Initiative. Her research focuses on the directed assembly of functional, structural, and biological materials. She is an elected member of the National Academy of Sciences, National Academy of Engineering, National Academy of Inventors, and the American Academy of Arts and Sciences. She has received numerous awards, including the National Science Foundation Presidential Faculty Fellow Award, the American Chemical Society Langmuir Lecture Award, the Materials Research Society Medal Award, the American Ceramic Society Sosman Award, and, most recently, the Lush Science Prize. Her work on microscale 3D printing was highlighted as one of the “10 Breakthrough Technologies” by the MIT Technology Review, while her bioprinting research was named “one of the top 100 science stories” by Discover Magazine. Her work has enjoyed broad coverage in the popular media. To date, she has co-founded two companies that are commercializing technology from her lab.
Micro Mechanical Methods for Biology (M3B)
Presented by Professor Liwei Lin
University of California, Berkeley
Next-generation autonomous microfluidic components, circuits and systems have been widely investigated for the past decades by researchers from various disciplines, including mechanical engineering, electrical engineering, bioengineering, chemistry and biology. They key focuses have been making microfluidic systems with functions similar to microelectronics with possible applications related to biological/medical problems. This talk will start with the discussions on the background information on the development of microfluidic components and systems. It will then followed with specific progresses from my laboratory in relevant topics, including: (1) the application of optofluidic lithography in the making and demonstration of microfluidic diodes with three different versions: bead-based operations, swing check valves, and spring check valves; (2) micro mechanical platforms for cell mechanobiology to control the cellular functions with two approaches: bi-axial control of substrate stiffness using micropost arrays of varying diameter, and tri-axial stiffness control using microscale springs; and (3) microfluidics based on the 3D printing techniques.
Professor Liwei Lin received PhD degree from the University of California, Berkeley, in 1993. He was an Associate Professor in the Institute of Applied Mechanics, National Taiwan University, Taiwan (1994~1996) and an Assistant Professor in Mechanical Engineering Department, University of Michigan (1996~1999). He joined the University of California at Berkeley in 1999 and is now James Marshall Wells Professor at the Mechanical Engineering Department and Co-Director at Berkeley Sensor and Actuator Center (BSAC), an NSF/Industry/University research cooperative center. His research interests are in design, modeling and fabrication of micro/nano structures, sensors and actuators as well as mechanical issues in micro/nano systems including heat transfer, solid/fluid mechanics and dynamics. Dr. Lin is the recipient of the 1998 NSF CAREER Award for research in MEMS Packaging and the 1999 ASME Journal of Heat Transfer best paper award for his work on micro scale bubble formation. He led the effort to establish the MEMS division in ASME and served as the founding Chairman of the Executive Committee from 2004~2005. He is an ASME Fellow and has 20 issued US patents in the area of MEMS. He was the general co-chair of the 24th international conference on Micro Electro Mechanical Systems at Cancun, Mexico. Currently, he serves as a subject editor for the IEEE/ASME Journal of Microelectromechanical Systems and the North and South America Editor of Sensors and Actuators –A Physical.