1. Wound Healing Research
Wound healing serves an important function in biology, as it enables the tissue maintenance that maintains compartmentalization of cell types that drives various processes, including the barrier function of skin and other epithelial tissues. There is significant data to support the theory that the wound healing capability of cells is frequently highjack during cancer progression, where tumors migrate through an invasive migration process from one tissue to another, termed听metastasis.听 Other recent data has shown that viral infection leads to a collective migration in human skin cells, which suggests that activation of wound healing is a normal physiological response to viral infection. Although wound healing is conclusively responsible for a wide array of physiological and pathophysiological response in human biology, several key questions remain to be answered: 1. How do cells dtect a wound? 2. How do cells detect that wound has been healed?听
听Live Cell Imaging of the Activity of "Sheddase" TACE
Chapnick DA, Bunker E, Liu X.
Science Signaling. 2015 Feb 24听365(8):rs1.
Abstract: Ligand shedding has gained increased attention as a major posttranslational modification mechanism used by cells to respond to diverse environmental conditions. The protease TACE [tumor necrosis factor 伪 (TNF伪)-converting enzyme, encoded by ADAM17] is a critical mediator of such ligand shedding, regulating the maturation and release of an impressive range of extracellular substrates that induce diverse cellular responses. Exactly how this protease is itself activated is unclear, in part because of the lack of available tools to measure TACE activity with temporal and spatial resolution in live cells. We have developed a FRET (fluorescence resonance energy transfer)-based biosensor called TSen that measures TACE activation kinetics in live cells with a high degree of specificity. In combination with chemical biology approaches, we used TSen to probe the dependence of various means of TACE activation on the induction of the kinases p38 and ERK. Using TSen, we also identified a previously unknown connection between actin cytoskeletal disruption and TACE activation. Cytoskeletal disruption led to rapid and robust TACE activation in some cell types and the accumulation of TACE at the plasma membrane, enabling increased cleavage of endogenous substrates. Our study highlights both the versatility of TSen as a tool to unravel the mechanisms of TACE activation in live cells and the importance of actin cytoskeletal integrity as a modulator of TACE activity.
Quantifying Cellular Motility
In the following publication, we demonstrate the capabilities of our motility analysis software, named听Pathfinder, which is intended to aid researchers in complex quantification of cellular motility听without the need for complex programming skills.
Chapnick DA, Jacobsen J, Liu X.
PLoS One. 2013 Dec 27;8(12):e82444. doi: 10.1371/journal.pone.0082444.听
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Abstract:听Understanding how cells migrate individually and collectively during development and cancer metastasis can be significantly aided by a computation tool to accurately measure not only cellular migration speed, but also migration direction and changes in migration direction in a temporal and spatial manner. We have developed such a tool for cell migration researchers, named Pathfinder, which is capable of simultaneously measuring the migration speed, migration direction, and changes in migration directions of thousands of cells both instantaneously and over long periods of time from fluorescence microscopy data. Additionally, we demonstrate how the Pathfinder software can be used to quantify collective cell migration. The novel capability of the Pathfinder software to measure the changes in migration direction of large populations of cells in a spatiotemporal manner will aid cellular migration research by providing a robust method for determining the mechanisms of cellular guidance during individual and collective cell migration.
Highlights:听The Pathfinder program can transform time-lapse microscopy videos into single cell or population data that quantifies cellular Speed, Persistence, and Direction:
Understanding Context Dependent Biochemical Activities and Collective Behavior During Wound Healing
The following publication highlights three main principles. First, TGF尾 induces wound directed collective migration in human keratinocytes. Second, this ligand dependent behavior is driven by local activation of MAPK towards the leading edge of groups of cells. Third, this spatially constrained MAPK activity is functionally different than global activation of MAPK, which leads to stochastic collective migration (not solely directed towards the wound).
Chapnick DA, Liu X.
Mol Biol Cell. 2014 May 15;25(10):1586-93. doi: 10.1091/mbc.E14-01-0697.
Abstract:听During wound healing and cancer metastasis, cells are frequently observed to migrate in collective groups. This mode of migration relies on the cooperative guidance of leader and follower cells throughout the collective group. The upstream determinants and molecular mechanisms behind such cellular guidance remain poorly understood. We use live-cell imaging to track the behavior of epithelial sheets of keratinocytes in response to transforming growth factor 尾 (TGF尾), which stimulates collective migration primarily through extracellular regulated kinase 1/2 (Erk1/2) activation. TGF尾-treated sheets display a spatial pattern of Erk1/2 activation in which the highest levels of Erk1/2 activity are concentrated toward the leading edge of a sheet. We show that Erk1/2 activity is modulated by cellular density and that this functional relationship drives the formation of patterns of Erk1/2 activity throughout sheets. In addition, we determine that a spatially constrained pattern of Erk1/2 activity results in collective migration that is primarily wound directed. Conversely, global elevation of Erk1/2 throughout sheets leads to stochastically directed collective migration throughout sheets. Our study highlights how the spatial patterning of leader cells (cells with elevated Erk1/2 activity) can influence the guidance of a collective group of cells during wound healing.
Highlights:听TGF尾听Induces Wound Directed Collective Migration:
TGF尾听Induces Spatially Constrained MAPK, in a Cell Density Dependent Manner:
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2. Systems biology of TGF-尾 signaling in normal and cancer cells
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We aim to understand the role of transforming growth factor-尾 (TGF-尾) signaling in normal and cancer cells. TGF-尾 is a multi-functional cytokine responsible for regulating growth and differentiation of a wide variety of cell types and tissues. It is a potent inhibitor of normal epithelial cell proliferation and possesses tumor suppressing activity. The majority of human tumors are of epithelial origin and their proliferation is no longer inhibited by TGF-尾. This usually arises either from a loss of key signaling molecules in the TGF-尾 signal transduction pathway (e.g., TGF-尾 receptors), or from activation of oncogenes. TGF-尾 can positively regulate expression of many tumor suppressor genes which cause the cell to stop proliferation and yet negatively regulate expression of many proto-oncogenes/oncogenes which promote cell cycle progression. The action of TGF-尾 in normal epithelial cells is to downshift the engine that drives cell proliferation by shifting the balance of the activity of the oncogene and tumor suppressor gene.
TGF-尾 signals via two receptor Ser/Thr protein kinases, termed type I and type II TGF-尾 receptors. The type II receptor phosphorylates and activates the type I receptor, which then phosphorylates Smad2 or Smad3 within their C-terminal Ser-Ser-X-Ser motifs. Once activated, Smad2 or Smad3 associate with the shared signaling molecule Smad4, translocate to the nucleus and in concert with additional transcription factors alters the transcription of a large repertoire of genes. Although several key downstream targets that transduce the TGF-尾 signals from the cell surface to the nucleus have been identified, it remains to be determined how TGF-尾 can mediate myriad cellular responses and regulate so many important physiological processes. To address how TGF-尾 signaling could mediate such diverse effects, we use the two following approaches: 1) Find as yet unidentified molecules that may mediate diverse functions, and 2) understand how the TGF-尾 network behaves as a system, which may identify how known network components interact to produce unexpected emergent behavior. The principal methods used to achieve these aims include cDNA library screening for genes that mediate TGF-尾 resistance (Erickson et al., MBC 2009), purification and identification by mass spectrometry of protein complexes associated with well-established signaling transducers (Knuesel et al., 2003, Zhu et al., 2007) and computational modeling of TGF- 尾/Smad signaling dynamics (Clarke et al., 2006; Clarke et al. 2008; Clarke et al. 2009; Zi et al. 2011).