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Open Access Publications from the University of California

Scholarly Works (3 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2012)

Epithelial remodeling, in which apical-basal polarized cells switch to a migratory phenotype, plays essential roles in development and disease of multicellular organisms. How cytoskeleton dynamics, especially microtubule dynamics, are controlled and contribute to epithelial remodeling in a more physiological three-dimensional (3D) environment is not understood. We optimize confocal live-cell imaging to analyze microtubule function and dynamics during 3D epithelial remodeling of polarized Madin-Darby kidney epithelial cells that undergo a partial epithelial-to-mesenchymal transition (pEMT) in response to hepatocyte growth factor (HGF). We found that cellular extensions at the basal surface of HGF-treated cysts are densely packed with microtubules. Computational tracking of EB1-2xEGFP showed large numbers of microtubules growing persistently from the apical domain into these extensions and an increase in microtubule growth rate in response to HGF. We tested the role of microtubule plus-end-tracking protein (+TIP) complexes in 3D epithelial remodeling by depleting cells of EB1, an adaptor protein that mediates recruitment of other +TIP proteins to growing microtubule plus ends. In EB1-depleted cells, microtubules displayed rapid lateral and retrograde movements demonstrating that EB1 is required to stabilize and organize microtubules in HGF-induced extensions. EB1-depleted cysts formed shorter, more branched, extensions suggesting that EB1 is required for productive HGF-induced extension outgrowth. Analysis of cell-matrix interactions and F-actin dynamics revealed that control extensions progressively pulled on and deformed the extracellular matrix (ECM) typically with one F-actin-rich protrusion near the cell tip with cell-matrix adhesions that turned over in a coordinated fashion. In contrast, EB1-depleted cells produced multiple highly dynamic protrusions with nascent adhesions that were uncoordinated, mislocalized and did not productively engage the matrix. As a result EB1-depleted extensions rapidly protruded, retracted and changed direction. Finally we show that trafficking of VAMP3-positive vesicles to the protrusion tip is disrupted in EB1-depleted cells. Together these data suggest that EB1-mediated organization of the MT cytoskeleton and associated vesicle delivery to the tip of HGF-induced extensions are likely required to coordinate cell-matrix adhesion and protrusion dynamics during 3D epithelial remodeling. It will be important in the future to investigate a broader role for EB1-mediated +TIP complexes in other normal and disease states of epithelial remodeling.

    Cover page: Function and dynamics of microtubules and plus-end-tracking proteins during 3D epithelial remodeling
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2009)
    Polarity of the microtubule (MT) cytoskeleton is essential for many cell functions. Cytoplasmic linker-associated proteins (CLASPs) are MT-associated proteins thought to organize intracellular MTs and display a unique spatiotemporal regulation. In migrating epithelial cells, CLASPs track MT plus ends in the cell body but bind along MTs in the lamella. In this study, we demonstrate that glycogen synthase kinase 3beta (GSK3beta) directly phosphorylates CLASPs at multiple sites in the domain required for MT plus end tracking. Although complete phosphorylation disrupts both plus end tracking and association along lamella MTs, we show that partial phosphorylation of the identified GSK3beta motifs determines whether CLASPs track plus ends or associate along MTs. In addition, we find that expression of constitutively active GSK3beta destabilizes lamella MTs by disrupting lateral MT interactions with the cell cortex. GSK3beta-induced lamella MT destabilization was partially rescued by expression of CLASP2 with mutated phosphorylation sites. This indicates that CLASP-mediated stabilization of peripheral MTs, which likely occurs in the vicinity of focal adhesions, may be regulated by local GSK3beta inactivation.
      Cover page: GSK3β phosphorylation modulates CLASP–microtubule association and lamella microtubule attachment
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2023)
      BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic disease characterised by aberrant fibroblast/myofibroblast accumulation and excessive collagen matrix deposition in the alveolar areas of lungs. As the first approved IPF medication, pirfenidone (PFD) significantly decelerates lung function decline while its underlying anti-fibrotic mechanism remains elusive. METHODS: We performed transcriptomic and immunofluorescence analyses of primary human IPF tissues. RESULTS: We showed that myocardin-related transcription factor (MRTF) signalling is activated in myofibroblasts accumulated in IPF lungs. Furthermore, we showed that PFD inhibits MRTF activation in primary human lung fibroblasts at clinically achievable concentrations (half-maximal inhibitory concentration 50-150 µM, maximal inhibition >90%, maximal concentration of PFD in patients <100 µM). Mechanistically, PFD appears to exert its inhibitory effects by promoting the interaction between MRTF and actin indirectly. Finally, PFD-treated IPF lungs exhibit significantly less MRTF activation in fibroblast foci areas than naïve IPF lungs. CONCLUSIONS: Our results suggest MRTF signalling as a direct target for PFD and implicate that some of the anti-fibrotic effects of PFD may be due to MRTF inhibition in lung fibroblasts.
        Cover page: Inhibition of MRTF activation as a clinically achievable anti-fibrotic mechanism for pirfenidone.
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