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ABSTRACT: Polarity landmarks guide epithelial development. In the early Drosophila ectoderm, the scaffold protein Bazooka (Drosophila PAR-3) forms apicolateral landmarks to direct adherens junction assembly. However, it is unclear how Bazooka becomes polarized. We report two mechanisms acting in concert to displace Bazooka from the basolateral membrane. As cells form during cellularization, basally localized Bazooka undergoes basal-to-apical transport. Bazooka requires its three PDZ domains to engage the transport mechanism, but with the PDZ domains deleted, basolateral displacement still occurs by gastrulation. Basolateral PAR-1 activity appears to act redundantly with the transport mechanism. Knockdown of PAR-1 sporadically destabilizes cellularization furrows but basolateral displacement of Bazooka still occurs by gastrulation. In contrast, basolateral Bazooka displacement is blocked with disruption of both the transport mechanism and phosphorylation by PAR-1. Thus, Bazooka is polarized through a combination of transport and PAR-1-induced dispersion from basolateral membranes. Our work complements recent findings in C. elegans, and thus suggests the coupling of transport and dispersion is a common protein polarization strategy.
Molecular biology of the cell 09/2012; · 5.98 Impact Factor
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ABSTRACT: Epithelial 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.
Journal of Cell Science 02/2012; 125(Pt 5):1177-90. · 6.11 Impact Factor
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ABSTRACT: 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.
Methods in molecular biology (Clifton, N.J.) 01/2012; 839:1-17.
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ABSTRACT: 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.
The Journal of Cell Biology 07/2009; 185(5):787-96. · 10.26 Impact Factor
