Proper epithelial structure requires adherens junction (AJ) assembly. In the early Drosophila embryo, AJ assembly depends on Bazooka (Baz; PAR-3), but it is unclear how Baz affects AJ assembly and what precursors are involved. To understand this process at the molecular level, we counted the number of core AJ proteins and Baz proteins at an average spot AJ (SAJ) and determined their dynamics with fluorescence recovery after photobleaching experiments. These data reveal that SAJs are subdivided into Baz clusters and cadherin–catenin clusters with independent protein numbers and dynamics. This independence suggests that precursory cadherin–catenin clusters might form before SAJ assembly. We identify cadherin–catenin clusters forming between apical microvilli. Further analyses show that they form independently of Baz and that Baz functions in repositioning them to apicolateral sites for full SAJ assembly. Our data implicate cell protrusions in initial cadherin–catenin clustering in the Drosophila melanogaster embryo. Then, independent Baz clusters appear to engage the cadherin–catenin clusters to assemble SAJs.

SummaryEpithelial cell polarity is essential for animal development. The scaffold protein Bazooka (Baz/PAR-3) forms apical polarity landmarks to organize epithelial cells. However, it is unclear how Baz is recruited to the plasma membrane and how this is coupled with downstream effects. Baz contains an oligomerization domain, three PDZ domains, and binding regions for the protein kinase aPKC and phosphoinositide lipids. With a structure-function approach, we dissected the roles of these domains in the localization and function of Baz in the Drosophila embryonic ectoderm. We found that a multifaceted membrane association mechanism localizes Baz to the apical circumference. Although none of the Baz protein domains are essential for cortical localization, we determined that each contributes to cortical anchorage in a specific manner. We propose that the redundancies involved might provide plasticity and robustness to Baz polarity landmarks. We also identified specific downstream effects, including the promotion of epithelial structure, a positive-feedback loop that recruits aPKC, PAR-6 and Crumbs, and a negative-feedback loop that regulates Baz.

To form regulated barriers between body compartments, epithelial cells polarize into apical and basolateral domains and assemble adherens junctions (AJs). Despite close links with polarity networks that generate single polarized domains, AJs distribute isotropically around the cell circumference for adhesion with all neighboring cells [1-3]. How AJs avoid the influence of polarity networks to maintain their isotropy has been unclear. In established epithelia, trans cadherin interactions could maintain AJ isotropy [4], but AJs are dynamic during epithelial development and remodeling [5, 6], and thus specific mechanisms may control their isotropy. In Drosophila, aPKC prevents hyper-polarization of junctions as epithelia develop from cellularization to gastrulation [7]. Here, we show that aPKC does so by inhibiting a positive feedback loop between Bazooka (Baz)/Par-3, a junctional organizer [5, 8-10], and centrosomes. Without aPKC, Baz and centrosomes lose their isotropic distributions and recruit each other to single plasma membrane (PM) domains. Surprisingly, our loss- and gain-of-function analyses show that the Baz-centrosome positive feedback loop is driven by Par-1, a kinase known to phosphorylate Baz and inhibit its basolateral localization [8, 11, 12]. We find that Par-1 promotes the positive feedback loop through both centrosome microtubule effects and Baz phosphorylation. Normally, aPKC attenuates the circuit by expelling Par-1 from the apical domain at gastrulation. The combination of local activation and global inhibition is a common polarization strategy [13-16]. Par-1 seems to couple both effects for a potent Baz polarization mechanism that is regulated for the isotropy of Baz and AJs around the cell circumference.

Bazooka/PAR-3 apicolateral polarity landmarks are established by the combination of two basolateral displacement activities in the Drosophila embryo. Basolateral PAR-1 activity acts redundantly with a basal-to-apical transport mechanism. With disruption of either mechanism alone Bazooka can polarize, but disruption of both blocks polarization.

Live imaging is critical for understanding the structure and activities of protein interaction networks in cells. By tagging proteins of interest with fluorescent proteins, such as green fluorescent protein (GFP), their localization in cells can be determined and correlated with cellular activities. This can be extended into developmental systems such as Drosophila to understand the molecular and cellular bases of development. In this chapter, we review sample preparation techniques and basic imaging considerations for Drosophila embryos. We then discuss how these techniques can be extended to count absolute protein numbers at specific subcellular locations, and determine their dynamics using fluorescence recovery after photobleaching (FRAP). These techniques can help reveal the structure and dynamics of protein complexes in live cells.