@PhdThesis{duepublico_mods_00071244,
  author = 	{Patwardhan, Rutuja},
  title = 	{Role of Rho GTPase networks in Keratinocyte migration},
  year = 	{2020},
  month = 	{Mar},
  day = 	{25},
  keywords = 	{Rho GTPase; Cell migration},
  abstract = 	{Keratinocytes are skin cells, which undergo proliferation, differentiation and migration during normal homeostasis and wound healing. Several groups have shown that the Rho GTPases Cdc42, Rho and Rac play critical role in Keratinocyte maintenance and differentiation. However, little is known about their function in Keratinocyte motility and how their subcellular activity patterns might mediate these effects. Here, a multifaceted approach was used which includes fluorescent live-cell activity sensors, TIRF microscopy, RNAi and overexpression studies to characterize spatio-temporal activity patterns of two major Rho GTPases, Cdc42 and Rho, in Keratinocytes during migration and to understand the underlying mechanisms. The work here confirmed Cdc42 to be an integral part of Keratinocyte migration machinery. First, a live cell sensor for Cdc42 activity was used revealing two major Cdc42 activity patterns during migration. In addition, Cdc42 activity pulses were observed in the lamellum region in the front of the cells as well as a more persistent Cdc42 activity pool at the trailing edge. Interestingly, the latter strongly correlated with the direction of cell migration and overlapped with the retrograde flow of actin and Myosin-II. Next, the potential regulatory mechanisms controlling Cdc42 activity patterns in migratory Keratinocytes were explored. Prior studies have revealed binding of the Cdc42/Rac1 effector PAK to the guanine nucleotide exchange factor (GEF) $\beta$Pix suggesting a positive feedback loop. Using pharmaceutical inhibition and RNAi mediated depletion of PAK2 as well as $\beta$Pix lead to strong decrease of the Cdc42 trailing-edge activity, further strengthening potential existence of such positive feedback loop to control Cdc42 activity patterns in Keratinocytes. Finally, depletion of Cdc42 lead to decrease in cell migration as well as to significant reduction of contraction sites. This confirms that Cdc42 is critical for Keratinocyte migration possibly via controlling actomyosin dynamics. Growth factor stimulation is a major trigger of Keratinocyte cell motility. Interestingly, EGF stimulation did not affect Cdc42 activity patterns suggesting that it is a part of intrinsic cell migration machinery, which does not rely on growth factor stimulation. In contrast to Cdc42, significantly altered Rho activity patterns were seen upon EGF stimulation. However, the changes in subcellular Rho activity signals were very transient. For example, low amplitude activity pulses in the entire cell increased within the first 12 minutes and decreased after an hour. Also, a very prominent, albeit transient, Rho activity increase around the entire cell periphery was observed shortly after EGF addition that was followed by strong contraction of these regions. Such transient global cell contraction may be required to stimulate enhanced migratory behavior, for example, by reorganizing topology of signal networks controlling Rho GTPase activity. The steep and transient rise of Rho activity upon EGF addition may be due to excitability in the system and a focused overexpression screen revealed several Rho activating Lbc type GEFs that enhanced Rho activity waves and thus might be potentially involved in Rho excitability in Keratinocytes. In the last part of the work, a previously established cell system (U2OS) was used to better understand the role of the extracellular matrix and the cellular cortex in the regulation of Rho activity. Rho activity pulses were enhanced by the ECM substrates collagen and fibronectin implicating integrin signaling linked to the pulsatory Rho network dynamics. Furthermore, our findings revealed sub-cellular localization pulses of the ERM proteins ezrin and moesin at the cell cortex that strongly correlated with actin dynamics and Rho activity pulses. Inhibition of Rho-ROCK signaling reduced ezrin pulses suggesting that ERM protein dynamics might be downstream of Rho activity and actomyosin pulses in adherent cells. Together, the work presented here revealed novel insight into the role of the Rho GTPase Cdc42 in Keratinocyte cell migration and advanced knowledge of the molecular mechanisms controlling its sub-cellular activity patterns in this process. Furthermore, our findings link growth factor induced distinct Rho activity patterns to cellular morpho-dynamics and identified potential regulatory GEFs. We are far from understanding the entire complexity of the underlying mechanisms that give rise to distinct local Rho GTPase activity patterns. Future work building on the novel findings presented in this thesis will greatly advance our understanding of Keratinocyte motility and increase the scope of therapeutics for aberrant processes related to wound healing and reepithelization.},
  doi = 	{10.17185/duepublico/71244},
  url = 	{https://duepublico2.uni-due.de/receive/duepublico_mods_00071244},
  url = 	{https://doi.org/10.17185/duepublico/71244},
  file = 	{:https://duepublico2.uni-due.de/servlets/MCRFileNodeServlet/duepublico_derivate_00081249/Diss_Patwardhan.pdf:PDF},
  language = 	{en}
}