A Single-Cell Biochemistry Approach Reveals PAR Complex Dynamics during Cell Polarization

Abstract

Summary Regulated protein-protein interactions are critical for cell signaling, differentiation and development. To study dynamic regulation of protein interactions in vivo, there is a need for techniques that can yield time-resolved information and probe multiple protein binding partners simultaneously, using small amounts of starting material. Here, we describe a single-cell protein interaction assay. Single-cell lysates are generated at defined timepoints and analyzed using single-molecule pull-down, yielding information about dynamic protein complex regulation in vivo. We established the utility of this approach by studying PAR polarity proteins, which mediate polarization of many animal cell types. We uncovered striking regulation of PAR complex composition and stoichiometry during C. elegans zygote polarization, which takes place in less than 20 minutes. PAR complex dynamics are linked to the cell cycle by polo-like kinase 1, and govern movement of PAR proteins to establish polarity. Our results demonstrate an approach to study dynamic biochemical events in vivo.

A Polybasic Domain in aPKC Mediates Par6-Dependent Control of Membrane Targeting and Kinase Activity

“…Mechanisms spatially coupling the aPKC kinase activity with its subcellular localization are essential for aPKC to phosphorylate targets at the right place and right time (Hong, 2018), but molecular details about these mechanism remain largely unknow. Recent studies began to reveal exciting details on how aPKC/Par-6 kinase activity and subcellular localization maybe regulated by the clustering of Par-3(Baz) and diffusive interaction with Cdc42 (Dickinson et al, 2017;Rodriguez et al, 2017), but these studies so far have been limited to the process of anterior-posterior (A-P) polarization in worm one-cell embryos. Here we show that, in Drosophila epithelial cells and cultured mammalian cells, the electrostatic binding of aPKC to PM provides an elegant mechanism enabling Par-6 to play a pivotal role coupling the PM targeting and control of kinase activity of aPKC (Fig.7).…”

“…PM/cortical localization of aPKC/Par-6 complex has been assumed mainly based on protein interactions with other polarity proteins such as Baz (Izumi et al, 1998;Krahn et al, 2010; Morais- de-Sa et al, 2010), Crb (Sotillos et al, 2004), Sdt (Wang et al, 2004), Patj (Hurd et al, 2003) or Cdc42 (Joberty et al, 2000;Lin et al, 2000;Qiu et al, 2000). Recent studies have delineated some detail mechanisms by which Par-3 and Cdc42 coordinates the spatial and temprol control of aPKC kinase activity during the anterior-posterior (A-P) polarization of worm one-cell embryo (Dickinson et al, 2017;Rodriguez et al, 2017), but how aPKC PM localization and kinase activity are regulated during apical-basal (A-B) polarization is much less clear.…”

Apical transport of Crumbs maintains epithelial cell polarity

“…Two general mechanisms for Cdc42-driven positive feedback were identified in yeast: (1) oligomeric clustering of Cdc42 complexes (Altschuler et al, 2008;Bendezu et al, 2015;Irazoqui et al, 2003) and (2) actin cytoskeleton mediated delivery of Cdc42 containing vesicles by a Myosin motor protein (Lechler et al, 2000;Wedlich-Soldner et al, 2003) in S. cerevisiae or microtubule mediated transport of polarizing factors in S. pombe (Martin and Arkowitz, 2014;Martin et al, 2005;Mata and Nurse, 1997;Minc et al, 2009). In fertilized worm oocytes (zygotes), polarization depends on clustering of Cdc42 and PAR-3 complexes (Dickinson et al, 2017;Gotta et al, 2001;Rodriguez et al, 2017;Sailer et al, 2015). The actin cytoskeleton flows to one pole of the worm zygote, pulling Cdc42-containing PAR-3 complexes along with it via bulk fluid 'advection' (Goehring et al, 2011).…”

Planar cell polarity in the C. elegans embryo emerges by differential retention of aPARs at cell-cell contacts

“…Consistent with the idea that aPARs are advected by centripetal flow, we observe that PIV of PAR-3 in ABpl reveals the same directionality as NMY-2 flow, however, with slightly reduced velocities ( Figure 3C). Considering the obvious and measured (Dickinson et al, 2017) differences between PAR-3 and PAR-6 cortical assemblies, it is interesting to note that we cannot detect a significant difference in the kinetics of advection to the medial cortex ( Figure 3D).…”

“…polarity determinants, adhesion or signaling complexes. Polarized activation of cortical flow and transient, avidity-driven interactions have been shown to lead to advection of anterior polarity factors (aPARs) PAR-3, PAR-6, and PKC-3, thereby establishing the anteroposterior axis in C. elegans (Munro et al, 2004;Goehring et al, 2011;Dickinson et al, 2017;Wang et al, 2017;Mittasch et al, 2018). In addition to patterning the anteroposterior axis, where longitudinal cortical flow is required, dorsoventral and left/right (l/r) patterning in C. elegans require rotational cortical flow (Singh and Pohl, 2014;Naganathan et al, 2014;Pohl, 2015;Sugioka and Bowerman, 2018).…”