Abstract
ABSTRACTThe association of molecules within membrane microdomains is critical for the intracellular organization of cells. During polarization of the C. elegans zygote, both polarity proteins and actomyosin regulators associate within dynamic membrane-associated foci. Recently, a novel class of asymmetric membrane-associated structures was described that appeared to be enriched in phosphatidylinositol 4,5-bisphosphate (PIP2), suggesting that PIP2 domains could constitute signaling hubs to promote cell polarization and actin nucleation. Here, we probe the nature of these domains using a variety of membrane- and actin cortex-associated probes. These data demonstrate that these domains are filopodia, which are stimulated transiently during polarity establishment and accumulate in the zygote anterior. The resulting membrane protrusions create local membrane topology that quantitatively accounts for observed local increases in the fluorescence signal of membrane-associated molecules, suggesting molecules are not selectively enriched in these domains relative to bulk membrane and that the PIP2 pool as revealed by PHPLCδ1 simply reflects plasma membrane localization. Given the ubiquity of 3D membrane structures in cells, including filopodia, microvilli and membrane folds, similar caveats are likely to apply to analysis of membrane-associated molecules in a broad range of systems.
Highlights
Micro- to nano-scale heterogeneity in the distribution of proteins and lipids in the plasma membrane has emerged as a fundamental organizing principle of the cell (Simons and Ikonen, 1997; Balla, 2013; Schink et al, 2016; Stone et al, 2017)
Despite being noted over a decade ago, the nature of these domains remains poorly understood. We show that these apparent microdomains are filopodia, which create the illusion of local enrichment of membrane-associated molecules due to induction of changes in local membrane topology
Diverse membrane-associated molecules appear to be coenriched in membrane structures To reveal the nature of these PIP2-enriched domains, we confirmed previous results that polarity-related proteins RHO-1, CDC-42, and CSNK-1 colocalized to a similar class of membrane-associated domains labeled by the PIP2 probe, PHPLCδ1 in C. elegans zygotes
Summary
Micro- to nano-scale heterogeneity in the distribution of proteins and lipids in the plasma membrane has emerged as a fundamental organizing principle of the cell (Simons and Ikonen, 1997; Balla, 2013; Schink et al, 2016; Stone et al, 2017). During polarity establishment in the C. elegans zygote, clustering of a conserved set of PAR proteins (PAR-3, PAR-6 and PKC-3) on. Received 6 February 2019; Accepted 17 June 2019 the membrane is critical for their ability to be segregated into the nascent anterior by actomyosin cortical flows (Rodriguez et al, 2017; Wang et al, 2017; Dickinson et al, 2017), eventually allowing them to be replaced by a second opposing set of PAR proteins (PAR-1, PAR-2, LGL-1 and CHIN-1) on the posterior membrane (Rose and Gonczy, 2014; Goehring, 2014). Asymmetric enrichment of phosphatidylinositol 4,5-bisphosphate (PIP2), has been observed within another class of membraneassociated domains in the anterior of the C. elegans zygote (Nakayama et al, 2009; Wang et al, 2017; Scholze et al, 2018). PIP2-enriched microdomains have been proposed to serve as organizing platforms to coordinate regulation of cortical actin organization, cell polarity and asymmetric division of the zygote (Scholze et al, 2018). Our data argues against local enrichment of PIP2 within the anterior of the embryo or within micron-scale domains
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