We uncoupled two roles for PI(3)K in regulating neutrophil motility: Rac-mediated protrusion of the leading edge, and polarization of F-actin dynamics that is separable from Rac activation. This two-tiered regulation of motility by PI(3)K can explain why PI(3)K is critical for neutrophil motility within complex tissues in vivo compared to its context-dependent roles in vitro (

Andrew and Insall, 2007;

Chen et al., 2007;

Ferguson et al., 2007;

Heit et al., 2008;

Hoeller and Kay, 2007;

Nishio et al., 2007). A previous report suggests that tail contraction is critical for migration in 3D environments by pushing the nucleus through narrow and constricted spaces (

Lammermann et al., 2008). As another example to support the importance of tail contraction for 3D migration, actomyosin-mediated tail contraction is not essential for motility of

*D. discoideum* over 2D surfaces (

De Lozanne and Spudich, 1987), but is indispensable for locomotion through 3D matrices that offer resistance (

Laevsky and Knecht, 2003). The idea that PI(3)K is important for actomyosin-mediated tail contraction is consistent with the previous report that PI(3)K regulates extravasation from the blood vessel (

Liu et al., 2007) because neutrophils need to be contracted to go through a narrow space between endothelial cells. Although we suggest that PI(3)K-mediated anteroposterior polarity of F-actin dynamics is not through Rac activation at the leading edge, several lines of evidence from in vitro studies suggest that Rac regulates Rho activity or actomyosin contraction (

Pestonjamasp et al., 2006;

Wu et al., 2009). It is intriguing to speculate that Rac may regulate uropod events via PI(3)K activation, through the feedback loop from Rac to PI(3,4,5)P

_{3}-PI(3,4)P

_{2} polarity that has been reported in vitro (

Srinivasan et al., 2003;

Sun et al., 2004;

Weiner et al., 2002) and was demonstrated in vivo in this study.

It remains elusive how the PI(3,4,5)P

_{3}-PI(3,4)P

_{2} gradient regulates polarity of F-actin dynamics, but our data suggest that inhibition of PI(3)K and Rho-ROCK-Myosin pathway leads to similar phenotypes: a rounded tail and loss of stable F-actin polarity. Although spatiotemporal regulation is not clear, PI(3)K was previously demonstrated to suppress basal activity of Rho (

Papakonstanti et al., 2007;

Van Keymeulen et al., 2006) or to regulate ROCK in vitro (

Niggli, 2000). Together with our data, this raises the possibility that PI(3)K, which is mainly active at the leading edge, regulates Rho-ROCK-Myosin-mediated uropod contraction. A possible hypothesis is that PI(3)K might inhibit Rho activity at the leading edge through regulating, for example a Rho GAP and creating gradients of Rho activity from the front to the back. Among Rho GAPs, Arap3, which was screened out as a binding protein to PI(3,4,5)P

_{3}, is a candidate to mediate PI(3)K-dependent Rho regulation from the front to the back (

Krugmann et al., 2002). As another possibility, PI(3,4,5)P

_{3}-PI(3,4)P

_{2} pulse which occasionally appears at the tail as a membrane component () might regulate uropod events directly. Although we cannot rule out this possibility, a role of PI(3,4,5)P

_{3}-PI(3,4)P

_{2} as an instructive cue at the tail is unlikely because the pulse of PI(3,4,5)P

_{3}-PI(3,4)P

_{2} at the tail is much less frequent than PI(3,4,5)P

_{3}-PI(3,4)P

_{2} at the front or stable F-actin at the tail. Thus, we speculate that PI(3,4,5)P

_{3}-PI(3,4)P

_{2} at the tail would have a permissive role together with other instructive cues at the tail if there is a specific function. Alternatively PI(3)K might regulate polarity of F-actin dynamics through Hem-1 (

Weiner et al., 2006) or Pak (

Chung et al., 2001) which were suggested to regulate Rho and/or myosin-mediated tail contraction. It is also possible that PI(3)K might induce a gradient of F-actin dynamics through cofilin activator slingshot, which is activated downstream of PI(3)K in tissue culture systems (

Nishita et al., 2004). Finally, the defects in uropod morphology and F-actin dynamics in PI(3)K-inhibited cells might also be due to altered adhesion at the leading edge. Although we cannot rule out this hypothesis, the scenario of adhesion-mediated consolidation at the front needs to be reconciled with the recent report that leukocytes migrate in the absence of specific adhesive interactions within 3D environments (

Lammermann et al., 2008).

Here, for the first time, we have visualized the dynamics of PI(3)K products PI(3,4,5)P_{3}-PI(3,4)P_{2} during neutrophil migration in intact tissues in vivo. We have shown that PI(3)K is critical for neutrophil polarity and motility in vivo. Cell migration could be directed in vivo with precise spatio-temporal control using light-mediated activation of a novel genetically encoded photoactivatable Rac. This enabled us to demonstrate that Rac activation at the leading edge was sufficient to rescue membrane protrusion but not directed cell migration or polarity of F-actin dynamics in PI(3)K-inhibited cells. With the majority of current data supporting a model that PI(3)K regulates migration by promoting Rac-mediated leading edge formation, our findings that PI(3)K regulates gradients of F-actin dynamics in a pathway that is separable from Rac-mediated protrusion suggest a new paradigm of two-tiered regulation of cell motility by PI(3)K: PI(3)K promotes Rac-mediated actin polymerization at the leading edge while generating anteroposterior polarity of F-actin dynamics ().