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Multi-Modal Regualtion of Actin Networks
Full text linkEukaryotic cells employ the actin cytoskeleton to maintain cell shape, support motility, and sense and respond to external mechanical stimuli. An intricate network of actin filaments constitutes the foundation of tissue architecture by forming key cellular structures including the muscle fiber, the filopodium, the lamellipodium, and the cell cortex. Cellular and tissue level studies have shown that more than 150 actin-binding proteins regulate almost every single aspect of actin physiology, such as actin polymerization, actin severing, and actin crosslinking, but at the molecular level, the structural mechanisms by which different actin binding proteins bind, assemble, and regulate different actin networks remain largely elusive. It is also known that the actin cytoskeleton mediates mechanical coupling between eukaryotic cells and their tissue microenvironments. Although cellular and tissue level studies suggest the architecture and composition of actin networks are modulated by force, it is still unclear how interactions between actin filaments and associated proteins are mechanically regulated. Force-sensitive actin binding interactions are fundamental for cells to sense and respond to mechanical stimuli by converting mechanical cues into biochemical signals, a key process known as cellular mechanotransduction. In this thesis, single molecule biophysical techniques including simultaneous optical trapping and confocal microscopy assays and in vitro reconstituted myosin motor-based total internal reflection fluorescence microscopy assays were employed to study how mechanical forces applied on actin filaments can regulate actin binding. A case study on a homologous pair of essential adhesion actin-binding proteins, α-catenin and vinculin, reveals α-catenin directly senses force on actin, while vinculin does not. Near atomic-resolution cryo-electron microscopy structures of both proteins bound to Factin, structure-guided protein engineering, and ongoing nuclear magnetic resonance and force reconstitution cryo-electron microscopy studies demonstrate that α-catenin\u27s C-terminus is a modular detector of F-actin tension and suggest a force-sensing mechanism. Cryo-electron microscopy was also employed to study how adhesion actinbinding proteins and the calponin-homology domain actin-binding proteins assemble cellular actin networks by binding and crosslinking actin filaments. Actin binding by another essential adhesion actin-binding protein talin, and actin bundling by both α- catenin and vinculin were additionally structurally characterized. Using a unique calponin-homology domain actin-binding protein, T-plastin, as an example, the thesis established the structural mechanism by which T-plastin crosslinks actin filaments. In summary, with biochemical, biophysical, and structural methods, this thesis systematically studied the molecular mechanisms for the multi-modal regulation of actin networks by actin binding proteins
Sign-Inverting Vectors Underlie a Coordinate Transformation in the Drosophila Central Brain
Full text linkOur coherent sense of orientation is built from combining sensory experiences across different modalities. How animal brains compute and remember the directions of motion of the body, limbs, objects or landmarks in a shared reference frame is only now becoming clear.This dissertation focuses on a system of neurons that signals wind direction in the Drosophila central brain. We found that the activity of these neurons, called PFNa, can be thought of as a set of vectors whose summed activity yields the direction of wind in an allocentric reference frame — that is, relative to external landmarks like the sun, as opposed to relative to the animal\u27s head. We also discovered that PFNa neurons alternate signaling modes to convey the forward and backward wind directions. Wind from the front is signaled in the canonical way, by raising the membrane potential and thus the spike rate of the PFNa neurons whose activity is aligned with a master heading signal. Wind from the back, however, is signaled with hyperpolarization of the PFNa neurons, which induces a non-canonical, prominent, 2-6 Hz membrane potential oscillation, reminiscent of mammalian delta rhythms. These membrane potential oscillations drive a calcium response in the PFNa cells that represent angles 180° away from the master heading signal. The ability to induce quantitatively precise calcium signals via both depolarization and hyperpolarization of the membrane potential is a new mechanism for implementing positive and negative signs in vector computations in the fly brain
Mechanistic Understanding of the Role of TRF1 in Telomere Replication
Full text linkMammalian telomeres are hard to replicate, and the shelterin subunit TRF1 is important for facilitating the replication of telomeric DNA. Deletion of TRF1 results in several phenotypes that are thought to be associated with telomere replication stress, including ATR signaling, fork stalling, sister telomere associations, and fragile telomeres. Fragile telomeres are structures that resemble common fragile sites (CFSs), but how they are formed is not known. TRF1 functions in part by recruiting the BLM helicase, which can resolve G-quadruplexes on the lagging-strand template. Deletion of BLM leads to lagging strand specific fragile telomere formation. In this thesis, I report that analogous to CFSs, fragile telomeres in BLM-deficient cells involve double-strand break (DSB) formation, in this case by the SLX4/SLX1 nuclease. The DSBs are repaired by POLD3/POLD4-dependent break-induced replication (BIR), resulting in fragile telomeres containing conservatively replicated DNA. BIR also promotes fragile telomere formation in cells with FokI-induced telomeric DSBs and in alternative lengthening of telomeres (ALT) cells, which have spontaneous telomeric damage. BIR of telomeric DSBs competes with PARP1-, LIG3-, and XPF-dependent alternative non-homologous end joining (alt-NHEJ). Collectively, these findings indicate that fragile telomeres can arise from BIR-mediated repair of telomeric DSBs. Even though BLM loss can induce BIR-dependent telomere fragility on the lagging strand, it fails to explain all other phenotypes caused by TRF1 loss, including leading strand fragile telomeres, ATR signaling, fork stalling, and sister telomere associations. Even the frequency of lagging strand fragile telomeres observed in BLM-deleted MEFs is not comparable to that in TRF1-deleted cells. Therefore, it is likely that TRF1 has functions other than recruiting BLM to prevent replication stress at telomeres. In this thesis, I report two newly-identified functional modules in TRF1, one in the TRFH domain and the other one, surprisingly, in the Myb domain. Mutating each module leads to extensive telomere fragility and telomere replication stress. Interestingly, the general transcription factor IIH (TFIIH) complex is also important for facilitating telomere replication. Deleting components of the TFIIH complex causes extensive fragile telomere formation and telomere replication stress. Lastly, I have determined the nature of sister telomere associations caused by TRF1 loss. They are alt-NHEJ-mediated sister telomere fusions caused by the partial loss of TRF2, indirectly as a result of TRF1 deletion. Altogether, this work provides detailed mechanistic insights into the role of TRF1 during telomere replication and the consequences of the loss of TRF1 functions
Vintage Laboratory Glassware
No full textVintage laboratory glassware (circa 1910) and residence of the Schermerhorn family in the background
Idea, design: Olga Nilova, Special Collections Librarian
Photo by Lubosh Stepanek
See also The Evolving Campushttps://digitalcommons.rockefeller.edu/objects-tell-stories/1001/thumbnail.jp
Fragment Of the Old Library Card Catalog
No full textFragment of the old library card catalog
Before the widespread use of typewritten cards, cards for the catalogs were handwritten in the approved style. According to Melvil Dewey, the Director of the New York State Library, “The fact remains that nothing pays the candidate for a library position better for the time it costs than to be able to write a satisfactory library hand.” In the late 1960s, two developments changed the future of cataloging. The Library of Congress created the MARC format, enabling the machine readability of bibliographic records. The Online Computer Library Center (OCLC) was developed in Dublin, Ohio, and started providing cataloging information via cable and terminal to all its member libraries. These two developments paved the way for the creation of Online Public Access Catalogs (OPACs). Because of the considerable amount of cost savings, most libraries converted to online catalogs and froze and discarded their card catalogs.https://digitalcommons.rockefeller.edu/objects-tell-stories/1017/thumbnail.jp
Louise Pearce
No full textLouise Pierce, ca. 1920 and John Hopkins University
Louise Pearce was a physician-pathologist who in 1913 became the first woman appointed to a research position at Rockefeller. She trained at Stanford University (A.B., 1907) and Johns Hopkins University (M.D., 1912). After working for many years at the Rockefeller Institute, she became president of Women\u27s Medical College in Philadelphia, 1946-1951. Pearce received honorary doctorates from Wilson College (1947), Beaver College (1948), Bucknell University (1950), Skidmore (1950), and Women\u27s Medical College in Philadelphia (1952).
Idea, design: Olga Nilova
Photograph by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/five-rockefeller-trailblazers/1001/thumbnail.jp
Exhibit Detail
No full textExhibit details: Rebecca C. Lancefield. Specific Relationship of Cell Composition to Biological Activity of Hemolytic Streptococci. Harvey Lecture delivered May 15th, 1941
Idea, design - Olga Nilova
Photograph by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/five-rockefeller-trailblazers/1007/thumbnail.jp
Exhibit Detail
No full textExhibit detail: Gertrude Erica Perlmann
Gertrude Erika Perlmann (1912 – 1974) was an Austro-Hungarian Empire-born U.S. biochemist and structural biologist. She is known for her work in protein chemistry, particularly her discoveries on the biology of phosphoproteins and the structure and action of pepsin and pepsinogen.https://digitalcommons.rockefeller.edu/five-rockefeller-trailblazers/1020/thumbnail.jp
DNA Methylation and DNA Methyltransferases in the Clonal Raider Ant, Ooceraea biroi
Full text linkDNA methylation and DNA methyltransferase enzymes (DNMTs) are ubiquitous and predate the origin of eukaryotes. In animals, DNA methylation primarily is carried out by DNMT1, which targets hemi – methylated DNA and maintains methylation patterns through cycles of cell replication, and DNMT3, the de novo methyltransferase. These genes are essential to mammalian development, and mutations lead to embryonic (DNMT1 and DNMT3b) or post – natal (DNMT3a) lethality. Studies of DNA methylation and DNMTs in invertebrates have been limited to non – traditional model organisms because Caenorhabditis elegans and Drosophila melanogaster lack the DNMT enzymes, and their DNA has no detectable methylation. In other invertebrates, global DNA methylation levels are generally much lower than those of mammals and largely concentrated in exons of protein coding genes. In social insects, some studies have argued that DNA methylation regulates social behavior or caste differentiation, but others have challenges this idea and it remains controversial. To further understand the DNMT enzymes, we used the clonal raider ant, Ooceraea biroi, a tractable model organism with robust DNA methylation. We utilized CRISPR/Cas9 to mutate the DNMT genes (DNMT1 and DNMT3) and generate four unique mutants (two targeting different regions of DNMT1, one targeting DNMT3 and a DNMT1/DNMT3 double mutant). In the DNMT1 catalytic domain mutant we observed a drastic drop in global levels of DNA methylation as well as reproductive sterility and increased mortality. We did not observe any reproductive or DNA methylation phenotypes in the other DNMT1 mutant or the DNMT3 mutant. Furthermore, in both of these mutants, we observed faithful transcription of the frameshift mutations in aligned mRNA reads, but did not observe any differential gene expression compared to wildtypes. Recently, studies have come to light regarding CRISPR/Cas9 mutagenesis inducing mechanisms of genomic plasticity, such as alternative splicing or translation reinitiating, which ultimately can rescue gene function. It is possible that we did not detect any phenotypes in these DNMT1 and DNMT3 mutants due to such a mechanism, leading to normal gene function despite successful mutagenesis. The sterility we observed as a result of DNMT1 catalytic domain mutagenesis is consistent with growing work demonstrating that DNMT1 plays an essential, possibly methylation – independent, role during oogenesis in insects. To further evaluate this phenomenon, we characterized DNMT1 mRNA and protein in the ant ovary using fluorescence in situ hybridization and immunohistochemistry. We found DNMT1 present in somatic cells within the ovary, in addition to being maternally provisioned into oocytes early in development. Furthermore, we observed developing oocytes in the ovaries of DNMT1 catalytic domain mutants, indicating that this gene is not essential for the initiation or early stages of oogenesis. Our findings demonstrate that unlike in mammals, normal development after DNMT1 inhibition is possible in insects. However, DNMT1 is essential for longevity and progression of insect oogenesis. Further work to understand the precise mechanism of DNMT1 involvement in oogenesis, and potentially meiosis, may shed light on its evolutionary role and why it has been conserved across so many forms of life
Fall on Campus
No full textFall on campus, 2022
Photo by Juan Rodriquezhttps://digitalcommons.rockefeller.edu/campus/1094/thumbnail.jp