5430 research outputs found
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The Bookplate Collection
No full textFew vintage bookplates from the collection at Markus Library
Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/objects-tell-stories/1019/thumbnail.jp
Structure and Mechanism of PCAT1, a Polypeptide Processing and Secretion Transporter
Full text linkATP-binding cassette (ABC) transporters utilize the energy from ATP to transport substrates across biological membranes. Various ABC transporters perform diverse biological functions across all forms of life ranging from importing essential nutrients to exporting toxic drugs (Ford and Beis 2019; ter Beek, Guskov, and Slotboom 2014). Bacterial cells utilize a class of ABC transporters for exporting proteins or peptides. Unlike the Sec translocon machinery, the ABC peptide exporters are dedicated to specific peptides (Fath and Kolter 1993). These peptides function as quorum sensing peptides, biofilms, or antimicrobial peptides. Among these ABC peptide exporters are Peptidase Containing ABC Transporters (PCATs) that perform dual functions of peptide maturation through proteolytic cleavage and peptide export (Gebhard 2012). As the name connotes, these transporters contain an accessory cysteine protease domain that interacts with the core ABC transporter. This structural feature is essential for the function and is unique among ABC transporters, making it a biologically interesting target for investigation. Although PCATs, which were first described 20 years ago, are essential to prokaryotic life, structural and functional studies of these proteins have been lacking. Until recently, only soluble parts of the proteins have been crystallized. The first full-length structure of PCATs was described in 2015 (Lin, Huang, and Chen 2015). However, structures of PCATs in complex with their substrates are needed to understand how PCATs recognize and transport their peptide substrates. To understand how PCATs work at the atomic level, I mainly took a structural approach. To this end, I decided to use cryo-electron microscopy (cryo-EM) and single-particle reconstruction techniques to obtain high-resolution structures of PCATs in various conformational states in complex with their substrates. These structures, together with the biochemical evidence, give us a clearer mechanistic picture of PCATs. First, to understand how PCATs bind substrate, I determined the structure of a PCAT from Clostridium thermocellum (abbreviated as PCAT1) in the substrate-bound inward-facing conformation. I have identified structural features that enable the substrate to bind, translocate into the transmembrane cavity, and orient properly for cleavage. Next, to understand how substrate binding coordinates with ATP binding, I have determined three structures of PCAT1 in the active-turnover condition, where PCAT1 is allowed to transition freely through the transport cycle. In addition, I have determined the structure of a clear outward-facing structure PCAT1 trapped in the Mg2+ condition that elucidates how the behavior of the core transporter affects the accessory peptidase domain. These structures together enable us to propose a mechanism of how the ATP binding and hydrolysis cycle is synchronized with substrate binding and processing, a unique feature crucial for strict coupling of cleavage and translocation. In addition to the cryo-EM work, I collaborated with Dr. Paul Dominic Olinares in Professor Brian T. Chait\u27s laboratory to study the stoichiometry of the PCAT1-substrate complex and intermediates of PCAT1 in the transport cycle. This work allows us to delineate the steps along the transport cycles that are short-lived and cannot be captured using structural study
Transcriptomic and Proteomic Studies of Intercellular Communication Between Melanocytes and Keratinocytes in Human Skin
Full text linkThe epidermis is a stratified epithelium composed of multiple cell types. Keratinocytes are the most prevalent cells in the epidermis and provide the barrier function of the skin. Interspersed amongst the keratinocytes in the basal layer of the epidermis are melanocytes, the pigment producing cells of the epidermis. Melanocytes produce melanin pigment which resides in melanosomes, organelles that are transferred to keratinocytes. In my thesis work, I focused on identifying downstream results of intercellular communication between melanocytes and keratinocytes for each of these cell types. I characterized the transcriptomes of human melanocytes and keratinocytes that were freshly derived from human tissue in Chapter 2. These data served as a reference for comparison and validation for the work that followed. In Chapter 3, I studied the effect of the presence of melanocytes on keratinocyte gene expression. I identified Neuronal Cell Adhesion Molecule (NRCAM) as being upregulated in keratinocytes in the presence of melanocytes, which is the first suggestion of a role for NRCAM in melanocyte-keratinocyte interactions. In Chapter 4, I studied the effects of endothelin-1 (ET-1), a signaling molecule produced by keratinocytes, on melanocyte gene expression. I found that genes involved in cell morphology, neurite growth, and cytoskeletal organization were upregulated in melanocytes in response to ET-1. I specifically identified microtubule associated protein 2 (MAP2) as being upregulated in response to ET-1, which suggests a mechanism for how ET-1 induces changes in melanocyte morphology. I also found that the nerve growth factor receptor (NGFR) is upregulated in melanocytes in response to ET-1. This reveals a new locus of interaction between two cell-cell signaling pathways in melanocytes and keratinocytes. In Chapter 5, I used a proteomic approach to study keratinocyte phagosomes. This is the first such proteomic characterization of phagosomes from an epithelial cell. In the final chapter of my thesis, I outlined the immediate future directions of this work, and discussed how these basic biological findings may be applied to our understanding of disease states
Songbird Brain Organization and its Molecular Convergence with Humans for Vocal Imitation Learning
Full text linkThe rich diversity of neural cell populations and their specialized connections give rise to a suite of complex behaviors across vertebrates. The presence of common neural cell types may be a product of either shared ancestry through homology or parallel evolution through convergence. However, the extent as to which these two processes are utilized by diverse nervous systems is poorly understood. Understanding the evolutionary relationships of these neural populations, both within and across vertebrate species, is critical for the study of complex trait evolution. Here, I investigate these two evolutionary processes within the adult avian brain and their impact on our understanding of the evolution of vocal imitation learning across species. Much is still unknown about the number and organization of unique neural cell populations in the avian brain. The avian dorsal and ventral pallium, separated by a vestigial ventricle divide, each contain brain circuits governing complex behaviors like navigation and vocal imitation. However, it is not known whether the cell types in these regions are molecularly distinct or represent homologous populations. In Chapter 1, I tested these competing hypotheses of avian brain organization by investigating potential homology between neural cell populations above and below the ventricle. Using RNA sequencing, I laser capture microdissected (LCM) neural subdivisions and performed differential gene expression and network co-expression analyses. We found that each avian subdivision in the dorsal pallium exhibited remarkable molecular similarity to a region in the ventral pallium. Interestingly, each matching population was defined by the same co-expression networks specialized in anatomical structure development, with such gene regulation similarity being most parsimonious with homologous origins. This work settles a debate of avian brain organization that lasted more than two decades, and it offers important insights into neural cell type diversity and function. With a better understanding of avian neural cell population diversity and functions, I can more accurately assess their homologous or convergent relationships to mammalian neural cell types. In Chapter 2, I investigated the analogous neural cell types involved in vocal imitation between songbirds and humans. Convergent gene expression patterns and connectivity exist between primary motor regions in the human (laryngeal motor cortex, LMC), and songbird (robust nucleus of the arcopallium, RA) controlling the vocal organs. However, it is unknown if the premotor control of these primary motor regions is convergent as well. In songbirds, HVC and LMAN are two premotor regions each critical for proper production and imitation of sounds respectively. In humans, prominent premotor regions activated during speech include the LMC, Broca\u27s area, and the Supplementary Motor Area (SMA). Using LCM and RNA-Seq, I generated vocal brain region specific gene sets for each of the four principal song nuclei in the zebra finch (Area X, HVC, LMAN, RA) and compared these with genes sets from human brain regions active during speech using gene set enrichment analysis. I found a weak correlation (10 genes) with the genes specialized in zebra finch HVC and human Broca\u27s area. Similarly, songbird LMAN most closely matched the human SMA (12 genes), with genes enriched for specialized connectivity. In contrast, songbird HVC exhibited the strongest genetic correlation (\u3e200 genes) with human LMC, followed closely by motor RA. Strikingly, utilizing single cell RNA-Seq data from human primary motor cortex (PMC), I found that songbird HVC cells were more like the intratelencephalic neurons in the PMC superficial layers, while songbird RA cells were more like long-range projection neurons in the PMC deep layers. These results offer strong evidence of an analogous microcircuit between two spatially distinct regions in songbirds and two layers of a cortical column in humans for vocal imitation. We molecularly profiled the song system of the budgerigar parakeet to test for further convergence in the parrot lineage, but were limited by sequence gaps and overall quality of the reference genome assembly. To overcome this limitation, I generated a haplotypephased, chromosome-scale budgerigar reference genome assembly that is an order of magnitude more contiguous than the current version, enabling genomic studies for parrot vocal learning and beyond. Collectively, this work has important implications for our understanding of neural cell type evolution both within and across species. My work strongly suggests avian neural populations surrounding the ventricle are homologous, and this more holistic understanding of avian neural cell type homology both allows for a deeper understanding of the evolution of complex behaviors within the avian lineage, and also enables scientists to ask fully informed questions of cross-species cell type comparisons. Looking across lineages, the presence of convergent genetic specializations dictating analogous cell types and circuits in highly divergent species offers new insights into the constraints on the genome to generate the cellular phenotypes necessary for vocal imitation. The existence of analogous microcircuitry utilized by songbirds and humans for vocal production learning confirms the value of the songbird as an invaluable model for speech-motor dysfunction. Overall, this work offers important insights into the evolution of the brain and its remarkable potential to converge on specialized cell types necessary for complex behaviors
Novel Optical Tools to Investigate Presenilin Control of Neurotransmission
Full text linkNeurotransmission is a critical function for neurons that underlies the complex processes of the brain. Deficits in synaptic function often manifest in neurodegenerative diseases, motivating the study of control points of neurotransmission. The Endoplasmic Reticulum (ER) has recently been described as one such control point, as ER Ca2+ levels can regulate the magnitude of synaptic vesicle exocytosis. Here we investigate the controversial hypothesis that Presenilins are ER Ca2+ leak channels to determine if a role in ER Ca2+ leak can explain the previously described regulation of neurotransmission by Presenilins. While Presenilins are best known for their involvement in Alzheimer\u27s Disease, this potential secondary role of Presenilins may shed insight into Presenilin involvement in neurodegeneration. To effectively investigate Presenilin control of ER Ca2+, we created a ratiometric version of the low affinity ER-GCaMPs in which we fused the HaloTag protein to allow for direct reporting of ER Ca2+ levels. We coupled this new indicator with in-cell calibrations at physiological temperatures to create a highly robust readout of ER Ca2+ across various cell types. Our analysis revealed a large variation in ER Ca2+ levels in both fibroblasts and hippocampal neurons indicating that ER Ca2+ may be an important control point across cells. The newly designed ratiometric ER-GCaMP provides a tool to study ER Ca2+ biology across various cell types and conditions with increased confidence. The ER-GCaMPs were leveraged to investigate Presenilin control of ER Ca2+. Utilizing Presenilin double knockout mouse embryonic fibroblasts revealed a decrease in resting ER Ca2+ levels, unlike the increase in resting ER Ca2+ levels observed in Presenilin 1 KO Human Embryonic Kidney cells. This result suggests that ER Ca2+ regulation by Presenilins differs across various cell types. Looking in hippocampal neurons conversely showed no effect of Presenilin knockdown or Alzheimer\u27s mutations on ER Ca2+ levels or dynamics, thereby failing to find support for Presenilins as ER Ca2+ leak channels in neurons. The ratiometric ER-GCaMP was a powerful tool to investigate ER Ca2+ regulation by Presenilins across cell types. While Presenilins were not observed to control ER Ca2+ in neurons, they may still have a role in regulating neurotransmission. We utilized vGlut1-pHluorin, a pH sensitive analog of GFP tagged to a synaptic vesicle protein, that allows for reporting of synaptic vesicle cycling. These experiments found evidence that Presenilins do control neurotransmission and that they do so by impacting the fraction of responsive boutons. This result provided insight into the molecular mechanism of Presenilin control of neurotransmission. This thesis investigated Presenilin control of ER Ca2+ across cell types as well as regulation of neurotransmission in hippocampal neurons. Evidence for Presenilin control of ER Ca2+ was observed in non-neuronal cells, but not in hippocampal neurons. However, Presenilins were still found to control neurotransmission. The development of a robust ratiometric probe will also aid future investigations of ER Ca2+ biology and provides a framework for the creation of future ratiometric probes
Florence Sabin
No full textExhibit detail: Florence Sabin
Florence Rena Sabin (1871-1953) was an American anatomist and medical researcher. Her excellent and innovative work on the origins of the lymphatic system, blood cells, and immune system cells, and on the pathology of tuberculosis was well-recognized during her lifetime. She was also a trailblazer for women in science: the first woman to hold a full professorship at Johns Hopkins School of Medicine, the first woman elected to the National Academy of Sciences, and the first woman to head a department at the Rockefeller Institute for Medical Research. In her retirement years, she pursued a second career as a public health activist in Colorado, and in 1951 she received a Lasker Award for this work.
Idea, design: Olga Nilova
Photograph by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/five-rockefeller-trailblazers/1012/thumbnail.jp
Exhibit Detail
No full textExhibit detail: Mary Maynard
Marie Maynard Daly (1921-2003) was an American biochemist. She was the first African-American woman in the United States to earn a Ph.D. in chemistry. Daly made important contributions in four areas of research: the chemistry of histones, protein synthesis, the relationships between cholesterol and hypertension, and creatine\u27s uptake by muscle cells.https://digitalcommons.rockefeller.edu/five-rockefeller-trailblazers/1026/thumbnail.jp
Lillia M. D. Trask, Librarian
No full textLillia M. D. Trask, Librarian,1922
Miss Lillia M.D. Trask was the first professionally trained Librarian of the Rockefeller Institute for Medical Research. She started her library career, at the age of 33, in the Children’s Room at the Orange Free Library, Orange, New Jersey, but a word of the exceptional job that she was doing reached the head of the Children’s Department of the New York Public Library who invited Miss Trask to join the staff. In New York, her work was almost exclusively with children on the Lower East Side, at the Chatham Square and the Seward Park Branch Libraries.
In 1911, she was approached by Dr. Simon Flexner, the director of RIMR, to become its librarian and on August 28, 1911, she entered her second career as a medical librarian.
Courtesy of the Rockefeller Archive Centerhttps://digitalcommons.rockefeller.edu/objects-tell-stories/1004/thumbnail.jp
Glassware and Slides from Günter Blobel Laboratory
No full textGlassware and slides from Günter Blobel laboratory, circa 1990s and the program of a scientific symposium celebrating the life of Günter Blobel with speakers’ signatures, 2019
Günter Blobel (1936-2018) was awarded the Nobel Prize in Physiology and Medicine in 1999 for the discovery that proteins have intrinsic signals that govern their transport and localization in the cell. The Nobel committee cited Blobel’s “elegant biochemical experiments” achieved through the years he spent in the cold room. For those who worked in Blobel’s lab, it was clear that his passion was the pursuit of the experimental test. Dr. Blobel donated all of the Nobel award money to the restoration of Dresden, in particular for the rebuilding of the Frauenkirche (completed in 2005) and the building of a new synagogue.https://digitalcommons.rockefeller.edu/objects-tell-stories/1030/thumbnail.jp
Dr. Merrifield’s Drum Program for Peptide Synthesizer
No full textDr. Merrifield’s drum program for peptide synthesizer, early 1960s
Bruce Merrifield (1921-2006) joined the Rockefeller Institute in 1949 where he started to work as an assistant for Dr. D. W. Woolley. They worked on a dinucleotide growth factor Merrifield discovered in graduate school and on peptide growth factor that Woolley had discovered earlier. These studies led to the need for peptide synthesis and, eventually, to the ideal for solid-phase peptide synthesis in 1959 (in 1984 Bruce Merrifield won the Nobel Prize in Chemistry for this invention). In 1963 Dr. Merrifield was a sole author of a classic paper in the Journal of the American Chemical Society in which the reported a method he called solid-phase peptide synthesis.https://digitalcommons.rockefeller.edu/objects-tell-stories/1028/thumbnail.jp