Cortical Polarity of the RING Protein PAR-2 Is Maintained by Exchange Rate Kinetics at the Cortical-Cytoplasmic Boundary

Arata, Yukinobu; Hiroshima, Michio; Pack, Chan‐Gi; Ramanujam, Ravikrishna; Motegi, Fumio; Nakazato, Kenichi; Shindo, Yoichi; Wiseman, Paul W.; Sawa, Hitoshi; Kobayashi, Tetsuya; Brandão, Hugo B.; Shibata, Tatsuo; Sako, Yasushi

doi:10.1016/j.celrep.2016.07.047

Cited by 25 publications

(29 citation statements)

References 36 publications

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“…The reaction-diffusion dynamics of MEX-5 and PIE-1 are analogous in some ways to the reaction-diffusion dynamics of the PAR proteins. PAR-3, PAR-6, and PAR-2 continuously exchange between a symmetrically distributed, fast-diffusing cytoplasmic pool and an asymmetrically distributed cortical pool (27)(28)(29)(30)(31). The kinetics at which the PAR proteins bind to and dissociate from the cortex differs significantly along the A/P axis, leading to their concentration in the appropriate cortical domains.…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, whereas the anterior PAR domain appears to be maintained through the dynamic clustering and dispersion of the aPAR/PAR-6 at the cell cortex (25,(31)(32)(33), MEX-5 and PIE-1 appear to bind to distributed sites within the cytoplasm, suggesting that their gradients do not form through clustering and dispersion. Clustering is thought to increase the avidity of the PAR proteins for the cortex, which may explain why the association of the PAR proteins with the cortex is significantly more stable [k off < 0.008 s −1 for PAR-6 and PAR-2 (27,28,30)] than the association of MEX-5 and PIE-1 with their cytoplasmic binding partner/s (k off ∼ 0.1-0.2 s −1 ).…”

Section: Discussionmentioning

confidence: 99%

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Rapid diffusion-state switching underlies stable cytoplasmic gradients in the Caenorhabditis elegans zygote

Han

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Protein concentration gradients organize cells and tissues and commonly form through diffusion away from a local source of protein. Interestingly, during the asymmetric division of the zygote, the RNA-binding proteins MEX-5 and PIE-1 form opposing concentration gradients in the absence of a local source. In this study, we use near-total internal reflection fluorescence (TIRF) imaging and single-particle tracking to characterize the reaction/diffusion dynamics that maintain the MEX-5 and PIE-1 gradients. Our findings suggest that both proteins interconvert between fast-diffusing and slow-diffusing states on timescales that are much shorter (seconds) than the timescale of gradient formation (minutes). The kinetics of diffusion-state switching are strongly polarized along the anterior/posterior (A/P) axis by the PAR polarity system such that fast-diffusing MEX-5 and PIE-1 particles are approximately symmetrically distributed, whereas slow-diffusing particles are highly enriched in the anterior and posterior cytoplasm, respectively. Using mathematical modeling, we show that local differences in the kinetics of diffusion-state switching can rapidly generate stable concentration gradients over a broad range of spatial and temporal scales.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Rapid diffusion-state switching underlies stable cytoplasmic gradients in the Caenorhabditis elegans zygote

Han

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…Biophysical studies, using fluorescence recovery after photobleaching (FRAP) (Cheeks et al, 2004;Goehring et al, 2011a;Nakayama et al, 2009) or single-molecule imaging (Arata et al, 2016;Robin et al, 2014;Sailer et al, 2015), have shown that PAR proteins exchange dynamically between the cytoplasm and the cell surface, where they move locally by diffusion or transport. At the same time, genetic and biochemical studies have revealed a complex web of local interactions through which PAR proteins modulate the recruitment or dissociation of one another (see below).…”

Section: The Molecular Basis For Complementary Par Domainsmentioning

confidence: 99%

“…Finally, a RING domain stabilizes PAR-2 binding to the cortex and mediates positive feedback in which membrane-localized PAR-2 facilitates recruitment of cytoplasmic PAR-2 (Hao et al, 2006;Motegi et al, 2011). Recent single molecule imaging studies indicate that PAR-2 forms oligomers at the membrane that appear to undergo size-dependent dissociation (Arata et al, 2016). Because oligomerization is common to many other RING domains (Deshaies and Joazeiro, 2009), RING domainmediated oligomerization of PAR-2 may both stabilize PAR-2 binding and potentiate recruitment of cytoplasmic PAR-2.…”

Section: The Hierarchical Recruitment Of Posterior Parsmentioning

confidence: 99%

The PAR proteins: from molecular circuits to dynamic self-stabilizing cell polarity

2017

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PAR proteins constitute a highly conserved network of scaffolding proteins, adaptors and enzymes that form and stabilize cortical asymmetries in response to diverse inputs. They function throughout development and across the metazoa to regulate cell polarity. In recent years, traditional approaches to identifying and characterizing molecular players and interactions in the PAR network have begun to merge with biophysical, theoretical and computational efforts to understand the network as a pattern-forming biochemical circuit. Here, we summarize recent progress in the field, focusing on recent studies that have characterized the core molecular circuitry, circuit design and spatiotemporal dynamics. We also consider some of the ways in which the PAR network has evolved to polarize cells in different contexts and in response to different cues and functional constraints.

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“…S4B), indicating that the particles attached to the plasma membrane were Halo–RalGDS molecules. The detection of the transient appearance of single particles on the plasma membrane allows us to analyze the interaction kinetics between cytoplasmic proteins and membrane components [27,28]. The fraction of monomeric Halo–RalGDS among the particles was estimated to be about 40% (Supplementary Table S1), based on the fluorescence intensity distribution (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Single-molecule fluorescence imaging of RalGDS on cell surfaces during signal transduction from Ras to Ral

Yoshizawa

Umeki²,

Yanagawa³

et al. 2017

BIOPHYSICS

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RalGDS is one of the Ras effectors and functions as a guanine nucleotide exchange factor for the small G-protein, Ral, which regulates membrane trafficking and cytoskeletal remodeling. The translocation of RalGDS from the cytoplasm to the plasma membrane is required for Ral activation. In this study, to understand the mechanism of Ras–Ral signaling we performed a single-molecule fluorescence analysis of RalGDS and its functional domains (RBD and REMCDC) on the plasma membranes of living HeLa cells. Increased molecular density of RalGDS and RBD, but not REMCDC, was observed on the plasma membrane after EGF stimulation of the cells to induce Ras activation, suggesting that the translocation of RalGDS involves an interaction between the GTP-bound active form of Ras and the RBD of RalGDS. Whereas the RBD played an important role in increasing the association rate constant between RalGDS and the plasma membrane, the REMCDC domain affected the dissociation rate constant from the membrane, which decreased after Ras activation or the hyperexpression of Ral. The Y64 residue of Ras and clusters of RalGDS molecules were involved in this reduction. From these findings, we infer that Ras activation not merely increases the cell-surface density of RalGDS, but actively stimulates the RalGDS–Ral interaction through a structural change in RalGDS and/or the accumulation of Ral, as well as the GTP–Ras/RalGDS clusters, to induce the full activation of Ral.

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Cortical Polarity of the RING Protein PAR-2 Is Maintained by Exchange Rate Kinetics at the Cortical-Cytoplasmic Boundary

Cited by 25 publications

References 36 publications

Rapid diffusion-state switching underlies stable cytoplasmic gradients in the Caenorhabditis elegans zygote

Rapid diffusion-state switching underlies stable cytoplasmic gradients in the Caenorhabditis elegans zygote

The PAR proteins: from molecular circuits to dynamic self-stabilizing cell polarity

Single-molecule fluorescence imaging of RalGDS on cell surfaces during signal transduction from Ras to Ral

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