Deutz, L. N. | Search bioRxiv

bioRxiv2023

Atypical Protein Kinase C Promotes its own Asymmetric Localisation by Phosphorylating Cdc42 in Polarising Cells

Atypical protein kinase C (aPKC) is a major regulator of cell polarity. Acting in conjunction with Par6, Par3 and the small GTPase Cdc42, aPKC becomes asymmetrically localised and drives the polarisation of cells. aPKC activity is crucial for its own asymmetric localisation, suggesting a hitherto unknown feedback mechanism contributing to polarisation. Here we show in the C. elegans zygote that the feedback relies on aPKC phosphorylation of Cdc42 at serine 71. The turnover of CDC-42 phosphorylation ensures optimal aPKC asymmetry and activity throughout polarisation by tuning Par6/aPKC association with Par3 and Cdc42. Moreover, turnover of Cdc42 phosphorylation regulates actomyosin cortex dynamics that are known to drive aPKC asymmetry. Given the widespread role of aPKC and Cdc42 in cell polarity, this form of self-regulation of aPKC may be vital for the robust control of polarisation in many cell types. Key findings/graphical abstract- Phosphorylation of CDC-42 by aPKC accelerates aPKC dissociation from CDC-42, limiting aPKC activity - CDC-42/aPKC dissociation promotes aPKC association with PAR-3 and, thereby, aPKC asymmetry due to actomyosin flow - Cycling of CDC-42 phosphorylation fuels the exchange of aPKC between anteriorly transported PAR-3 and aPKC-active CDC-42 complexes - Turnover of CDC-42 phosphorylation alternates its association with effectors, aPKC and MRCK-1, ensuring proper actomyosin dynamics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/563985v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@e0ec65org.highwire.dtl.DTLVardef@c00b8dorg.highwire.dtl.DTLVardef@36565dorg.highwire.dtl.DTLVardef@160149a_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Membrane extraction in native lipid nanodiscs reveals dynamic regulation of Cdc42 complexes during cell polarization

AbstractEmbryonic development requires the establishment of cell polarity to enable cell fate segregation and tissue morphogenesis. This process is regulated by Par complex proteins, which partition into polarized membrane domains and direct downstream polarized cell behaviors. The kinase aPKC (along with its cofactor Par6) is a key member of this network and can be recruited to the plasma membrane by either the small GTPase Cdc42 or the scaffolding protein Par3. Although in vitro interactions among these proteins are well established, much is still unknown about the complexes they form during development. Here, to enable the study of membrane-associated complexes in vivo, we used a maleic acid copolymer to rapidly isolate membrane proteins from single C. elegans zygotes into lipid nanodiscs. We show that native lipid nanodisc formation enables detection of endogenous complexes involving Cdc42, which are undetectable when cells are lysed in detergent. We found that Cdc42 interacts more strongly with aPKC/Par6 during polarity maintenance than polarity establishment, two developmental stages that are separated by only a few minutes. We further show that Cdc42 and Par3 do not bind aPKC/Par6 simultaneously, confirming recent in vitro findings in an in vivo context. Our findings establish a new tool for studying membrane-associated signaling complexes and reveal an unexpected mode of polarity regulation via Cdc42.

cell biology↗