Oligomerization and positive feedback on membrane recruitment encode dynamically stable PAR-3 asymmetries in the C. elegans zygote.

Studies of PAR polarity have emphasized a paradigm in which mutually antagonistic PAR proteins form complementary polar domains in response to transient cues. A growing body of work suggests that the oligomeric scaffold PAR-3 can form unipolar asymmetries without mutual antagonism, but how it does so is largely unknown. Here we combine single molecule analysis and modeling to show how the interplay of two positive feedback loops promote dynamically stable unipolar PAR-3 asymmetries in early C. elegans embryos. First, the intrinsic dynamics of PAR-3 membrane binding and oligomerization encode negative feedback on PAR-3 dissociation. Second, membrane-bound PAR-3 promotes its own recruitment through a mechanism that requires the anterior polarity proteins CDC-42, PAR-6 and PKC-3. Using a kinetic model tightly constrained by our experimental measurements, we show that these two feedback loops are individually required and jointly sufficient to encode dynamically stable and locally inducible unipolar PAR-3 asymmetries in the absence of posterior inhibition. Given the central role of PAR-3, and the conservation of PAR-3 membrane-binding, oligomerization, and core interactions with PAR-6/aPKC, these results have widespread implications for PAR-mediated polarity in metazoa.

Regulation of EGF-stimulated activation of the PI-3K/AKT pathway by exocyst-mediated exocytosis.

The phosphoinositide-3 kinase (PI-3K)/AKT cell survival pathway is an important pathway activated by EGFR signaling. Here we show, that in addition to previously described critical components of this pathway, i.e., the docking protein Gab1, the PI-3K/AKT pathway in epithelial cells is regulated by the exocyst complex, which is a vesicle tether that is essential for exocytosis. Using live-cell imaging, we demonstrate that PI(3,4,5)P3 levels fluctuate at the membrane on a minutes time scale and that these fluctuations are associated with local PI(3,4,5)P3 increases at sites where recycling vesicles undergo exocytic fusion. Supporting a role for exocytosis in PI(3,4,5)P3 generation, acute promotion of exocytosis by optogenetically driving exocyst-mediated vesicle tethering up-regulates PI(3,4,5)P3 production and AKT activation. Conversely, acute inhibition of exocytosis using Endosidin2, a small-molecule inhibitor of the exocyst subunit Exo70 (also designated EXOC7), or inhibition of exocyst function by siRNA-mediated knockdown of the exocyst subunit Sec15 (EXOC6), impairs PI(3,4,5)P3 production and AKT activation induced by EGF stimulation of epithelial cells. Moreover, prolonged inhibition of EGF signaling by EGFR tyrosine kinase inhibitors results in spontaneous reactivation of AKT without a concomitant relief of EGFR inhibition. However, this reactivation can be negated by acutely inhibiting the exocyst. These experiments demonstrate that exocyst-mediated exocytosis-by regulating PI(3,4,5)P3 levels at the plasma membrane-subserves activation of the PI-3K/AKT pathway by EGFR in epithelial cells.

An active tethering mechanism controls the fate of vesicles.

Vesicle tethers are thought to underpin the efficiency of intracellular fusion by bridging vesicles to their target membranes. However, the interplay between tethering and fusion has remained enigmatic. Here, through optogenetic control of either a natural tether-the exocyst complex-or an artificial tether, we report that tethering regulates the mode of fusion. We find that vesicles mainly undergo kiss-and-run instead of full fusion in the absence of functional exocyst. Full fusion is rescued by optogenetically restoring exocyst function, in a manner likely dependent on the stoichiometry of tether engagement with the plasma membrane. In contrast, a passive artificial tether produces mostly kissing events, suggesting that kiss-and-run is the default mode of vesicle fusion. Optogenetic control of tethering further shows that fusion mode has physiological relevance since only full fusion could trigger lamellipodial expansion. These findings demonstrate that active coupling between tethering and fusion is critical for robust membrane merger.

Dynamic Opposition of Clustered Proteins Stabilizes Cortical Polarity in the C. elegans Zygote.

Dynamic maintenance of cell polarity is essential for development and physiology. Here we combine experiments and modeling to elucidate mechanisms that maintain cortical polarity in the C. elegans zygote. We show that polarity is dynamically stabilized by two coupled cross-inhibitory feedback loops: one involves the oligomeric scaffold PAR-3 and the kinase PAR-1, and the other involves CDC-42 and its putative GAP CHIN-1. PAR-3 and CDC-42 are both required locally to recruit PAR-6/PKC-3, which inhibits PAR-1 (shown previously) and inhibits local growth/accumulation of CHIN-1 clusters. Conversely, PAR-1 inhibits local accumulation of PAR-3 oligomers, while CHIN-1 inhibits CDC-42 (shown previously), such that either PAR-1 or CHIN-1 can prevent recruitment of PAR-6/PKC-3, but loss of both causes complete loss of polarity. Ultrasensitive dependence of CHIN-1 cluster growth on PAR-6/PKC-3 endows this core circuit with bistable dynamics, while transport of CHIN-1 clusters by cortical flow can stabilize the AP boundary against diffusive spread of PAR-6/PKC-3.