Sic persistence suggested the loss of Nox2 had resulted in a reduction in the cells ability to turn and explore their environment. Pankov et al [22] demonstrated that total Rac1 activity was important in determining whether random cell migration followed a more intrinsic random or directionally persistent pattern of motion. The data here suggests that, at least in part, some of these regulatory functions of Rac1 could be through Nox2. In contrast to random motion, directional migration moves cells rapidly between points. When challenged with a gradient, loss of Nox2 in BMM resulted in a complete loss of chemotaxis towards CSF-1 and a loss of cell migration and directional persistence. The BMM were able to sense and respond to CSF-1 stimulation as observed by the increase in the speed of WT and Nox2KO BMM, although Nox2KO BMMs were significantly slower than WT cells. Thus the loss of chemotaxis suggested a critical role for Nox2 further downstream from the cell sensing of the external signal and/or in cellular polarisation. A possible mechanism by which Nox2 could affect the directionality of the cell could be by the redox modulation of the intracellular signalling gradients established by phophoinositides. The phosphoinositides PtdIns(3,4,5)P3 (PIP3) and PtdIns(3,4)P2[PI(3,4)P2] along with PI3K and PTENS are key signaling molecules in this process [23,24]. This process involves both localized accumulation and activation of PI3Ks, which generate PIP3/PI(3,4)P2, and the phosphatase PTEN, which removes them [25]. Cells with altered PI3K or PTEN activity can usually show chemokinesis but exhibit a significantly reduced chemotaxis [24,26]. Many of these 
signaling molecules have been shown to be redox sensitive. Leslie et al [27] demonstrated that oxidative stress with H2O2 resulted 
11967625 in the inactivation of PTEN. PTEN is a member of the Protein Tyrosine Phosphatase family which can be physiologically regulated through reversible oxidation resulting in their inactivation [28,29]. The inactivation of PTEN results in an increase in cellular phosphoinositides and thus the loss of any gradient established by the phosphoinositides to a chemoattractant signal. Also phosphoinositides, PtdIns(3,4,5)P3 (PIP3) and PtdIns(3,4)P2[PI(3,4)P2], have been shown, by way of their Phox domains for subunits p40phox and p47phox, to be involved in the recruitment and activation of these Nox regulatory proteins, [30,31] thus establishing a means for the redox modulation of these downstream signalling molecules. Cellular polarisation is equally important for directed cellular migration in which the small GTPase are important in this process and in particular Cdc42. Cdc42 is a AZ-876 manufacturer master regulator of cell polarity by being active towards the front of migrating cells [32] and by restricting where lamellipodia forms [33]. Its importance isNox2 and ChemotaxisNox2 and ChemotaxisFigure 3. Nox2KO BMMs have reduced cell displacement. WT and Nox2KO BMMs were seeded on plastic, CSF-1 TA02 biological activity deprived then stimulated with CSF-1 and imaged as described in material and Methods. A and B) cell tracks of WT and Nox2KO BMM respectively. The tracks have been re-set to co-ordinate (0,0). C and D) The number of cells reaching a set circular horizon was monitored and found to be significantly (p = 0.02) more in the WT than Nox2KO BMM following CSF-1 stimulation. (E and F) mean cell migration speed and mean persistence of direction were calculated from cell tracks above (see material and methods for de.Sic persistence suggested the loss of Nox2 had resulted in a reduction in the cells ability to turn and explore their environment. Pankov et al [22] demonstrated that total Rac1 activity was important in determining whether random cell migration followed a more intrinsic random or directionally persistent pattern of motion. The data here suggests that, at least in part, some of these regulatory functions of Rac1 could be through Nox2. In contrast to random motion, directional migration moves cells rapidly between points. When challenged with a gradient, loss of Nox2 in BMM resulted in a complete loss of chemotaxis towards CSF-1 and a loss of cell migration and directional persistence. The BMM were able to sense and respond to CSF-1 stimulation as observed by the increase in the speed of WT and Nox2KO BMM, although Nox2KO BMMs were significantly slower than WT cells. Thus the loss of chemotaxis suggested a critical role for Nox2 further downstream from the cell sensing of the external signal and/or in cellular polarisation. A possible mechanism by which Nox2 could affect the directionality of the cell could be by the redox modulation of the intracellular signalling gradients established by phophoinositides. The phosphoinositides PtdIns(3,4,5)P3 (PIP3) and PtdIns(3,4)P2[PI(3,4)P2] along with PI3K and PTENS are key signaling molecules in this process [23,24]. This process involves both localized accumulation and activation of PI3Ks, which generate PIP3/PI(3,4)P2, and the phosphatase PTEN, which removes them [25]. Cells with altered PI3K or PTEN activity can usually show chemokinesis but exhibit a significantly reduced chemotaxis [24,26]. Many of these signaling molecules have been shown to be redox sensitive. Leslie et al [27] demonstrated that oxidative stress with H2O2 resulted 11967625 in the inactivation of PTEN. PTEN is a member of the Protein Tyrosine Phosphatase family which can be physiologically regulated through reversible oxidation resulting in their inactivation [28,29]. The inactivation of PTEN results in an increase in cellular phosphoinositides and thus the loss of any gradient established by the phosphoinositides to a chemoattractant signal. Also phosphoinositides, PtdIns(3,4,5)P3 (PIP3) and PtdIns(3,4)P2[PI(3,4)P2], have been shown, by way of their Phox domains for subunits p40phox and p47phox, to be involved in the recruitment and activation of these Nox regulatory proteins, [30,31] thus establishing a means for the redox modulation of these downstream signalling molecules. Cellular polarisation is equally important for directed cellular migration in which the small GTPase are important in this process and in particular Cdc42. Cdc42 is a master regulator of cell polarity by being active towards the front of migrating cells [32] and by restricting where lamellipodia forms [33]. Its importance isNox2 and ChemotaxisNox2 and ChemotaxisFigure 3. Nox2KO BMMs have reduced cell displacement. WT and Nox2KO BMMs were seeded on plastic, CSF-1 deprived then stimulated with CSF-1 and imaged as described in material and Methods. A and B) cell tracks of WT and Nox2KO BMM respectively. The tracks have been re-set to co-ordinate (0,0). C and D) The number of cells reaching a set circular horizon was monitored and found to be significantly (p = 0.02) more in the WT than Nox2KO BMM following CSF-1 stimulation. (E and F) mean cell migration speed and mean persistence of direction were calculated from cell tracks above (see material and methods for de.
