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Open Access Publications from the University of California

Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2016)

Glycogen synthase kinase-3 (GSK3) is a highly conserved multifunctional kinase that regulates many cellular processes in eukaryotes, including cell motility, microtubule function, and cell differentiation (Frame et al., 2001). Dictyostelium GSK3, GskA, has been shown to be essential for chemotaxis (Teo R et al., 2010), although only one GskA substrate, Daydreamer, has been identified so far (Kölsch et al., 2013). Interestingly, the chemotaxis phenotype of Daydreamer null cells was not as severe as the one observed for gskA_ (GskA null) cells, suggesting that there may be more proteins downstream GskA that are regulating chemotaxis. To identify direct downstream effectors of GSK3, a phosphoprotemic assay was performed before and after stimulation with the chemoattractant cAMP in wild-type and gskA- cells (Kölsch et al., 2013). This investigation provided potential direct GSK3 substrate candidates, GxcGG and SogA, based on the phosphorylation of the GSK3 consensus sequence (s/tXXXs/t) in wild-type cells but not in gskA- cells. Our results showed that GxcGG and SogA were phosphorylated in in vitro kinase assays by GSK3. Overall, both sogA- and gxcGG- cells displayed chemotaxis and developmental defects that resulted in incomplete fruiting body development. However they operate at different areas of the GSK3 signaling cascade as visualized by the cell membrane translocation of GFP tagged GxcGG and translocation to the cytosol of GFP tagged SogA during chemotaxis. My investigations suggest that SogA and GxcGG are substrates of GSK3 involved in the cAMP chemotaxis signaling pathway.

    Cover page: Identification of Glycogen Synthase Kinase-3 substrates in Dictyostelium discoideum
    • Thesis
    • Peer Reviewed
    UC San Diego Electronic Theses and Dissertations (2012)

    In a study by Charest et al., 2010 a protein signaling complex was discovered and found to regulate the RasC-TORC-PKB/PKBR1 pathway at the leading edge of chemotaxing Dictyostelium discoideum cells. A novel protein, PHR, is a stable member of this Sca1 protein complex. This present study is an in-depth analysis of phr null strains in the regulation of chemotaxis in Dictyostelium cells.

    Analyses of phr knockout cells show clear alteration of actin and myosin cytoskeleton dynamics as well as altered cell motility. Though it was discovered that PHR is a stable member of the Sca1 protein signaling complex, it does not affect RasC activity. Intriguingly, phr knockout does seem to modulate the downstream TORC2-mediated phosphorylation of PKB and PKBR1 proteins, despite having no observed effect on RasC activity. Thus, additional roles for PHR in regulating chemotaxis outside of the Sca1 complex was proposed. However, due to the fact that the correct topology of the PHR gene is currently unknown, attempts to generate cells expressing epitope-tagged PHR to test for alternative roles in chemotaxis regulation has proven unsuccessful. The exact functions of PHR in the spatiotemporal regulation of chemotaxis remains unclear.

    • 2 supplemental videos
    Cover page: Investigation into the function of a novel protein, PHR, in regulating <i>Dictyostelium discoideum</i> chemotaxis
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2016)
    Streams of migratory cells are initiated by the formation of tandem pairs of cells connected head to tail to which other cells subsequently adhere. The mechanisms regulating the transition from single to streaming cell migration remain elusive, although several molecules have been suggested to be involved. In this work, we investigate the mechanics of the locomotion ofDictyosteliumtandem pairs by analyzing the spatiotemporal evolution of their traction adhesions (TAs). We find that in migrating wild-type tandem pairs, each cell exerts traction forces on stationary sites (∼80% of the time), and the trailing cell reuses the location of the TAs of the leading cell. Both leading and trailing cells form contractile dipoles and synchronize the formation of new frontal TAs with ∼54-s time delay. Cells not expressing the lectin discoidin I or moving on discoidin I-coated substrata form fewer tandems, but the trailing cell still reuses the locations of the TAs of the leading cell, suggesting that discoidin I is not responsible for a possible chemically driven synchronization process. The migration dynamics of the tandems indicate that their TAs' reuse results from the mechanical synchronization of the leading and trailing cells' protrusions and retractions (motility cycles) aided by the cell-cell adhesions.
      Cover page: Cooperative cell motility during tandem locomotion of amoeboid cells.
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2017)
      Cellular traction force microscopy (TFM) requires knowledge of the mechanical properties of the substratum where the cells adhere to calculate cell-generated forces from measurements of substratum deformation. Polymer-based hydrogels are broadly used for TFM due to their linearly elastic behavior in the range of measured deformations. However, the calculated stresses, particularly their spatial patterns, can be highly sensitive to the substratum's Poisson's ratio. We present two-layer elastographic TFM (2LETFM), a method that allows for simultaneously measuring the Poisson's ratio of the substratum while also determining the cell-generated forces. The new method exploits the analytical solution of the elastostatic equation and deformation measurements from two layers of the substratum. We perform an in silico analysis of 2LETFM concluding that this technique is robust with respect to TFM experimental parameters, and remains accurate even for noisy measurement data. We also provide experimental proof of principle of 2LETFM by simultaneously measuring the stresses exerted by migrating Physarum amoeboae on the surface of polyacrylamide substrata, and the Poisson's ratio of the substrata. The 2LETFM method could be generalized to concurrently determine the mechanical properties and cell-generated forces in more physiologically relevant extracellular environments, opening new possibilities to study cell-matrix interactions.
        Cover page: Two-Layer Elastographic 3-D Traction Force Microscopy
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