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Simon Flexner as a young boy
No full textSimon Flexner as a young boy, circa 1871
Courtesy of the American Philosophical Society Library
The son of an immigrant Jewish peddler, Simon Flexner had been growing up in Louisville, Kentucky. When he was ten, his delinquency so worried his parents that his father arranged a private tour of the town jail as a warning to his son of where he would end up if he didn’t change his way. Simon Flexner dropped out of school in the eighth grade, and until he fell victim, at sixteen, to typhoid fever, he had drifted from one menial job to another.
His nearly fatal illness and long convalescence transformed the indolent adolescent. As he himself expressed it, “I appear to have become wide awake almost at once”. From a job as a drugstore apprentice he went on to earn a degree, with highest grades, at the Louisville College of Pharmacy, and was soon sharing ownership of a drugstore with one of his brothers.
In 1890, he moved to the Johns Hopkins University to study under William Henry Welch. Within two years, Flexner was named associate professor and became Welch’s first assistant.https://digitalcommons.rockefeller.edu/jem-the-beginnings/1022/thumbnail.jp
Flexner family
No full textFlexner family, circa 1870s
Courtesy of the American Philosophical Society Library
Simon Flexner was the fourth son of seven in a large family of nine children: Jacob Flexner, Henry, and Isadore; then Simon, followed by Bernard Flexner, Abraham Flexner, and Washington. The two sisters, Mary and Gertrude, were the youngest. Jacob became a pharmacist and physician; Bernard became a Zionist leader, and Abraham became an educator, eventually influencing the direction of medical education in the United States.https://digitalcommons.rockefeller.edu/jem-the-beginnings/1025/thumbnail.jp
Details of the Exhibit
No full textDetails of the exhibit JEM: The Beginnings
Idea, design - Olga Nilova, Special Collections Librarian
Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/jem-the-beginnings/1031/thumbnail.jp
Details of the Exhibit
No full textDetails of the exhibit JEM: The Beginnings
William H. Welch. Public health in theory and practice. New Haven, 1925
Idea, design - Olga Nilova, Special Collections Librarian
Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/jem-the-beginnings/1035/thumbnail.jp
Single-Molecule Investigation of Chromatin-Associated Factors in Genome Organization and Epigenetic Maintenance
Get PDFThe central dogma of biology has laid the foundation for understanding gene expression through the mechanisms of transcription and translation. However, another layer of eukaryotic gene regulation lies in the complex structure of chromatin. This scaffold of structural proteins and enzymatic regulators determines what genes are expressed at what times, leading to cell differentiation, cell fate, and often disease. Currently, the field of chromatin biology has relied on basic biochemistry and cellular assays to identify key epigenetic regulators and their role in genomic maintenance. For this thesis work, I have developed a biophysical platform to study chromatin-associated factors at the single-molecule level (Chapter 2). This methodology allows us to extract key mechanistic details often obscured by standard bulk methodologies. Using this platform, we posed the question of how epigenetic factor, Polycomb repressive complex 2 (PRC2) engages with chromatin (Chapter 3). PRC2 is a major epigenetic machinery that maintains transcriptionally silent heterochromatin in the nucleus and plays critical roles in embryonic development and oncogenesis. It is generally thought that PRC2 propagates repressive histone marks by modifying neighboring nucleosomes in a strictly linear progression. However, the behavior of PRC2 on native-like chromatin substrates remains incompletely characterized, making the precise mechanism of PRC2-mediated heterochromatin maintenance elusive. Our understanding of this process was limited by the resolution of structural techniques that fail to identify PRC2-binding modes on long chromatin substrates. In short, we found direct evidence that PRC2 can simultaneously engage nonadjacent nucleosome pairs. The demonstration of PRC2\u27s ability to bridge noncontiguous chromosomal segments furthers our understanding of how Polycomb complexes spread epigenetic modifications and compact chromatin. In addition to this single-molecule chromatin binding technology, I also created a singlemolecule platform harnessing correlative force and fluorescence microscopy to assay the material properties of phase separated condensates (Chapter 2). This assay combined methodology to visualize condensate formation at the single-molecule level, in addition to optical trapping of individual droplets to investigate their material properties. Utilizing this technology, we interrogated the role of linker histone H1 (Chapter 4). The linker histones are the most abundant group of chromatin-binding proteins that bind and organize eukaryotic chromatin. However, roles for the diverse and largely unstructured H1 proteins beyond chromatin compaction remain unclear. We used correlative single-molecule force and fluorescence microscopy to directly visualize the behavior of H1 on DNA under different tensions. Unexpectedly, our results show that H1 preferentially coalesces around nascent, relaxed singlestranded DNA. In vitro bulk assays confirmed that H1 has a higher propensity to form phaseseparated condensates with single-stranded DNA than with double-stranded DNA. Furthermore, we dissected the material properties of different H1:DNA condensates by controlled droplet fusion with optical tweezers, and found that increased DNA length and GC content result in more viscous, gel-like H1 condensates. Overall, our findings suggest a potential role for linker histones to sense and coacervate single-stranded nucleic acids in the nucleus, forming reaction hubs for genome maintenance. This work also provides a new perspective to understand how various H1 subtypes and disease-associated mutations affect chromatin structure and function. In summary, we have gained a greater understanding of the biophysical basis for chromatin regulation by both PRC2 and histone H1. Both of the biophysical platforms created for these studies can be applied to various new targets in chromatin biology. They will enable the investigation of a multiplicity of binding interactions, regulatory mechanisms, and material properties of protein-nucleic acid complexes (Chapters 5 & 6). I believe single-molecule techniques will become a major toolset to study chromatin biology, identifying the intricacies and interactions between epigenetic factors and our genome
Cell Memory and Fate in Human Development
Get PDFDevelopment of a single cell, the fertilized egg, into an entire organism is a fascinating example of biological organization that gives rise to many forms of multicellular life. Cell-cell signaling is central to development, particularly in organisms such as vertebrates, where embryonic cells become progressively restricted in their potential through a hierarchy of decision points guided by the signals they receive. All cells contain the same genetic information in the form of DNA, and we now know that the DNA is modified epigenetically to impart distinct functions to each adult cell type. However, we are just beginning to uncover how the epigenome is modified over the course of development and how signaling pathways might direct these modifications. One of the earliest developmental decision points in bilaterian embryos, which includes vertebrates, occurs during gastrulation when cells are directed into one of three primary germ layers. Model systems, including frog, fish, chicken, and mouse, have been very useful for uncovering the identity and function of the key signaling networks driving vertebrate gastrulation. However, how these signals function together in time to modify cellular behavior has been difficult to tease apart in embryos. With the isolation of human embryonic stem cells (hESCs), many aspects of development can now be reconstituted and manipulated in culture, thus allowing us for the first time to investigate the early stages of our own development. We can now also address how mechanisms proposed from model systems function dynamically or in unanticipated ways. In this work, I used hESCs and a stem cell-based model of gastrulation (human gastruloids) to disentangle the function and timing of two key signaling pathways, namely Wnt and Activin/Nodal. From studies in model organisms both pathways are known to be required for initiating gastrulation and for the formation of mesoderm and endoderm, but it has been proposed that the level of Activin/Nodal signaling ultimately determines germ layer identity. In order to test these predictions in a model of human gastrulation, I first developed hESC lines with fluorescent reporters of Activin/Nodal signaling that facilitate quantitative measurements of the cellular response in real-time. With these lines I was able to establish that hESCs display a dose-dependent response to Activin ligands at the level of the intracellular effector Smad2. However, this response is transient, and the cells do not adopt germ layer identities even when the pathway response is transiently saturated. By manipulating the relative timing of Wnt and Activin ligands, I demonstrated that hESCs can record Wnt signals without differentiating and that this signaling memory makes cells competent to form mesoderm and endoderm in response to subsequently supplied Activin. My observations do not eliminate the co-requirement for Wnt and Activin in germ layer differentiation, which has been emphasized by previous studies of mouse and human ESCs, but rather they highlight the fact that instructive Wnt signals occur temporally up-stream of Activin signals to induce complete germ layer patterning. My initial efforts to uncover the mechanism of Wnt signaling memory identified a role for the bromodomain and extra-terminal domain (BET) family of epigenetic readers, which facilitate transcription through interactions with acetylated residues of histones and other proteins and are thought to promote tumor formation in the context of cancer. I showed that inhibition of BET protein function selectively eliminates mesoderm and endoderm in human gastruloids and produces phenotypes in frog embryos consistent with disruption of early Wnt signaling. The concept that Wnt signaling primes cells to differentiate without directing them to commit to a particular fate defines a new aspect to how this signaling pathway functions. These observations also establish an experimental context to further investigate the link between developmental signaling pathways and the epigenetic landscape. As I hope to demonstrate throughout this thesis, hESCs are a powerful means to uncover both conserved and potentially human-specific mechanisms of vertebrate development. In the context of Wnt and Activin signaling my results highlight the importance of the temporal interaction between Wnt and Activin signaling in patterning the vertebrate embryo and suggests an evolutionarily conversed mechanism of Wnt signaling memory that could explain how signals are integrated to direct cellular behavior in both development and disease
B16 and CD1d-Transfected B16 Melanoma
No full textSlide 5-12: B16 and CD1d-Transfected B16 Melanoma are tumorigenic via the subcutaneous routehttps://digitalcommons.rockefeller.edu/nkt-cells/1011/thumbnail.jp
Chemical Biology of Dynein
Get PDFCytoplasmic dynein is a AAA (ATPase Associated with various Activities) motor protein that transports cellular cargoes towards the microtubule minus-end. Despite its essential role in intracellular transport, dynein remains the least understood cytoskeletal motor. With speeds \u3e25 μm/min in cells, dynein\u27s cellular functions are challenging to study using genetic approaches, such as CRISPR and RNA interference, as the perturbation timescales far exceed those of dynein action. Fast-acting small molecule probes can be powerful tools to study dynein\u27s many cellular mechanisms, but the design of potent and selective inhibitors of dynein remains challenging. Inhibitors of dynein\u27s motor domain have been reported, such as ciliobrevins, dynapyrazoles and dynarrestin, but the inhibitor-binding site(s) have yet to be elucidated. Though inhibitor-bound dynein structures have been long-sought after, efforts have been curtailed by difficulties in obtaining high-resolution dynein structures and the lack of an inhibitor with the requisite chemical properties, such as compound solubility and stability, for structural studies. The work presented here describes the design and characterization of a class of dynein probes, one of which we use to obtain an inhibitor-bound structure of dynein. The first chapter Chemical inhibitors of AAA proteins provides a brief overview of AAA proteins in general and the inhibitors that have been designed. The chapter emphasizes on the design and characterization of ciliobrevins, dynapyrazoles and dynarrestin, three classes of dynein inhibitors with distinct mechanisms of action. Their limitations as cellular probes are discussed and motivates the need for compounds with improved potency and selectivity. In the second chapter, Dynapyrazoles acutely inhibit intraflagellar transport , I characterize dynapyrazole-A in a cell-based assay that can measure the effect of the compound on intraflagellar transport (IFT). Dynapyrazole-A is an acute reversible inhibitor of dynein 2-dependent retrograde transport and can be a useful probe to study IFT function. However, as dynapyrazole-A inhibits both isoforms of dynein, its use as a chemical probe is limited due to its cytotoxic effects at treatment times \u3e1 hour. This chapter motivates the need for dynein-2 specific inhibitors. Finally, I present a third chapter, Structural insights into the chemical inhibition of dynein , that discusses the design of a dynapyrazole derivative, compound 20, that inhibits the basal ATPase activity of human and S. cerevisiae dynein. I used cryo-EM to obtain a structure of S. cerevisae dynein\u27s motor domain in the presence of the dynapyrazole derivative and find that the compound binds to the regulatory ATPase sites in the AAA3 and AAA4 domains, rather than the main catalytic site in the AAA1 domain. This finding addresses a major gap in our knowledge, as inhibitors of dynein\u27s ATPase activity have been assumed to target the AAA1 domain. Inhibitor design efforts can now be focused on the regulatory ATPase sites to obtain potent and selective small molecule probes of dynein
CRISPR/CASTE: Functional Genomic Studies of the Major Evolutionary Innovations of Ants
Get PDFAnts are social organisms that live in groups and depend intimately on their nestmates for growth and survival. Ants have evolved a number of highly sophisticated, social phenotypes that allow them to form coherent colonies. This thesis explores two particularly derived features of ant biology: complex chemical communication and caste plasticity. To study these features, I had a particular focus on generating and characterizing germ-line mutants. I believe that the study of mutants, and applying molecular biology methods more generally, can lead to insights in ant biology that would not be possible with more traditional methods. I first describe my efforts to develop a CRISPR protocol to make the first germ-line mutant ant lines. I conducted this work using a unique, clonal ant species, Ooceraea biroi, that has many properties making it favorable for laboratory genetics studies. Establishing this protocol required a multi-year optimization process to account for many of the particular features of ant biology, such as egg production and incubation, growing and maintaining lines, and optimizing experimental treatments to produce high mutagenesis rates. I next describe the mutants I generated using these methods, null mutants of a highly conserved insect protein called orco. Orco, or olfactory receptor co-receptor, is required for the function of an important class of chemosensory proteins, the odorant receptors, in insects. I created orco mutant ants and found that they have striking deficiencies in their social behavior and fitness, including an inability to nest with other ants or follow chemical pheromone trails and severely reduced life span and fecundity. These results supported the growing consensus that odorant receptors are key chemosensory proteins for pheromone perception in ants, and provided a new window into ant social behavior and collective organization. Unexpectedly, and unlike orco mutants in other types of insects, I also found that orco mutant ants have severe neuro-anatomical deficiencies, including a loss of most antennal lobe glomeruli and sensory neurons in the antenna. This surprising result implies that orco may play an important role in antennal lobe morphology in ants, and could provide insights into the development and evolution of complex olfactory systems. The following chapter is a critical literature review of the development and evolution of morphological castes, such as workers and queens, in ants. I describe a stereotyped and previously overlooked pattern of morphological variation in ants, and illustrate how this pattern may provide some early insights into the molecular mechanisms of caste plasticity. This chapter provides a falsifiable, mechanistic framework for caste development and suggests a route to start looking for the actual molecules that regulate this interesting process. Finally, I start to realize this promise by characterizing a caste mutant in the laboratory. I discovered a spontaneous \u27winged mutant\u27 that belongs to one of the known clonal lineages of O. biroi and aberrantly expresses queen-like morphology and behavior at worker-like body sizes. These mutants bear a striking resemblance to one class of ant species with derived caste systems, the recurrently evolved workerless social parasites. They could thus provide a window into the mutations that give rise to these unique ants. Overall, this thesis represents the first characterization of mutant lines in ants, and I hope it demonstrates how this approach can be used to generate robust conclusions about ant biology