Cell Biology and Metabolism Branch, National Institutes of Health, Bethesda
Saturday, 10 September, 17:45 - 18:30
Emerging visualization technologies are playing an increasingly important role in the study of numerous aspects of cell biology, capturing processes at the level of whole organisms down to single molecules. While developments in probes and microscopes are dramatically expanding the areas of productive imaging, there are still significant roadblocks. Primary challenges include 1) fluorophore bleed-through, which limits the number of fluorophores that can be simultaneously imagined, 2) imaging speeds that are too slow, and 3) labeling densities that are too low for deciphering fine subcellular architecture. Here, I will discuss new imaging methods that can overcome these roadblocks, focusing on their potential for clarifying subcellular organelle dynamics. To surmount fluorophore bleed-through, we combined excitation-based spectral unmixing and lattice light sheet microscopy to visualize up to six organelles (i.e., ER, Golgi, mitochondria, lysosomes, peroxisomes and lipid droplets) simultaneously within cells. This allowed us to track these organelles through time and analyze their inter-organelle contacts. To increase temporal resolution during imaging, we employed total internal reflection fluorescence combined with structured illumination microscopy to visualize organelle dynamics at very high temporal-spatial resolution. Examining the ER, we observed that many peripheral ER sheets seen using diffraction-limited imaging are actually tight matrices of tubules connected by three-way junctions. Interconversion between the loose ER polygonal arrays and dense matrices could occur on the order of ~250 msec and was energy-dependent. Viewed at higher resolution using lattice light sheet microscopy combined with point accumulation for nanoscale topology (PAINT), the peripheral ER sheets represented a complex meshwork of tightly cross-linked ER tubules. I discuss possible roles this complex ER structural organization has for diverse cellular functions.
Jennifer Lippincott-Schwartz is Section Chief of the Cell Biology and Metabolism Branch, NICHD, NIH and NIH Distinguished Investigator. She received her BA from Swarthmore College, MS in Biology from Stanford University and PhD in Biochemistry from the Johns Hopkins University. Her research uses live cell imaging approaches to analyze the spatio-temporal behavior and dynamic interactions of molecules and organelles in cells. Her group has pioneered the use of green fluorescent protein (GFP) technology for quantitative analysis and modeling of intracellular protein traffic and organelle biogenesis in live cells and embryos, providing novel insights into cell compartmentalization, protein trafficking and organelle inheritance. Most recently, her research has focused on the development and use of photoactivatable fluorescent proteins, including the development of photoactivated localization microscopy, (i.e., PALM), a superresolution imaging technique that enables visualization of molecule distributions at high density at the nano-scale.
Her work has been recognized with election to the National Academy of Sciences and the National Institute of Medicine, and with the Royal Microscopy Society Pearse Prize and the Society of Histochemistry Feulgen Prize. She is President of the American Society of Cell Biology for 2014. She serves on the scientific advisory boards of the Howard Hughes Medical Institute, the Weizmann Institute of Sciences, the Searle Scholar Program, and the Salk Institute.
Royal Institute of Technology (KTH), Stockholm
Monday, 12 September, 09:00 - 09:45
We have classified all the protein coding genes in humans using a combination of genomics, transcriptomics, proteomics and antibody-based profiling. We have used this data to study the global protein expression patterns in human cells, tissues and organs as well as a discovery tool to find potential biomarkers and drug targets for disease. As part of this effort, we have developed bacterial cell factories for production of human protein fragments and CHO cell factories for production of human full-length proteins with a native fold and glycosylation. The bacterial cell factory has been used to generate 55,000 human protein fragments called PRESTs that has been used primarily as antigens for antibody generation and secondly as standards in mass spectrometry based targeted proteomics.
Selected own references: Uhlen et al (2015) Science 347: 394 Uhlen et al (2016) Mol Systems Biol. 12: 862Martinoglu et al (2014) Nature Communication, 5:3038.Wein et al (2014) Nature Medicine 20: 992-1000Kampf et al (2014), FASEB J 28(7): 2901-2914.Lee et al (2016) Cell Metabolism, 12;24(1):172-84 Forsström et al (2014) Mol Cell Proteomics 13: 1585- 1597Edfors et al (2014) Mol Cell Proteomics 13: 1611-1624.
Dr Uhlén received his PhD in chemistry at the Royal Institute of Technology (KTH), Stockholm, Sweden. After a post-doc period at the EMBL in Heidelberg, Germany, he became professor in microbiology at KTH in 1998. Dr Uhlen has more than 350 publications in bioscience with the focus on the development and use of affinity reagents in biotechnology and biomedicine.
In the eighties, he was the first to describe the use of affinity tags for purification of proteins, a principle now widely used in bioscience. In the 90's, his group described a new strategy for DNA analysis called Pyrosequencing, a method that was further developed by a US company (454/Roche) into the first of a new generation of next generation sequencing methods. His group also developed a new affinity reagent called Affibodies, based on combinatorial principles and, in addition, developed alkali-stable variations of protein A, now commercially available for purification of antibodies (MabSelectSure). In the early 2000, his group started an international effort, with groups in Sweden, India, China and South Korea, for the creation of a Human Protein Atlas (www.proteinatlas.org) with the aim to systematically map the human proteome with antibodies.
Dr Uhlén is member of the Royal Swedish Academy of Engineering Science (IVA), the Royal Swedish Academy of Science (KVA), EMBO and member of the Human Proteome Organization (HUPO) council. He was Vice-President of the Royal Institute of Technology (KTH), responsible for external relations, from 1999 to 2001. Recently, he became the Director of a new center Science for Life Laboratory Stockholm for high-throughput bioscience (www.scilifelab.org). He has received numerous awards, including the Göran Gustavsson prize, the Gold Medal of the Royal Swedish Academy of Engineering Sciences, the Akzo Noble Award, the HUPO Distinguished Award, the KTH Great Prize, the ABRF award, the Scheele prize and H.M. the King’s Medal with the ribbon of the Order of Seraphim.