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SEC14-like condensate phase transitions at plasma membranes regulate root growth in Arabidopsis [1]
['Chen Liu', 'Department Of Biology', 'University Of Crete', 'Heraklion', 'Institute Of Molecular Biology', 'Biotechnology', 'Foundation For Research', 'Technology-Hellas', 'Department Of Plant Biology', 'Uppsala Biocenter']
Date: 2023-09
Protein function can be modulated by phase transitions in their material properties, which can range from liquid- to solid-like; yet, the mechanisms that drive these transitions and whether they are important for physiology are still unknown. In the model plant Arabidopsis, we show that developmental robustness is reinforced by phase transitions of the plasma membrane-bound lipid-binding protein SEC14-like. Using imaging, genetics, and in vitro reconstitution experiments, we show that SEC14-like undergoes liquid-like phase separation in the root stem cells. Outside the stem cell niche, SEC14-like associates with the caspase-like protease separase and conserved microtubule motors at unique polar plasma membrane interfaces. In these interfaces, SEC14-like undergoes processing by separase, which promotes its liquid-to-solid transition. This transition is important for root development, as lines expressing an uncleavable SEC14-like variant or mutants of separase and associated microtubule motors show similar developmental phenotypes. Furthermore, the processed and solidified but not the liquid form of SEC14-like interacts with and regulates the polarity of the auxin efflux carrier PINFORMED2. This work demonstrates that robust development can involve liquid-to-solid transitions mediated by proteolysis at unique plasma membrane interfaces.
Funding: Funding for this work was through the Vetenskapsrådet (VR) (298264-2015 to PNM), Svenska Forskningsrådet Formas (MOP-86675 to PNM), Hellenic Foundation for Research & Innovation (HFRI)-Always Strive for Excellence-Theodore Papazoglou (1624 to PNM), Hellenic Foundation of Research and Innovation (HFRI) (06526 to AM), National Secretariat of research and innovation (GR) (Τ2ΕΔΚ-00597 to PNM), H2020 Marie Skłodowska-Curie Actions (RISE 872969 PANTHEON to PNM), Foundation for Research and Technology (FORTH-IMBB) Start-Up Funding (to PNM), and by the Deutsche Forschungsgemeinschaft (SCHA 1274/5-1, 841 Germany’s Excellence Strategy EXC-2070-390732324 PhenoRob to GS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Whether condensates interfacing with membranes can undergo liquid-to-solid transitions like cytoplasmic ones and if these transitions would have any significance is unclear. Here, we discovered that a previously uncharacterized SEC14-like lipid transfer protein that we named SEC FOURTEEN-HOMOLOG8 (SFH8) recruits KISC to the PM. The ESP part of KISC trimmed SFH8 protein removing an IDR, leading to the conversion of SFH8 from a liquid to a more solid filamentous phase that remains attached to the PM, an event that we could also reconstitute in vitro. This liquid-to-solid transition was associated with SFH8 polarization, interaction with PIN2, and robust root development. Remarkably, we showed how spatiotemporally confined proteolysis can yield changes in the material properties of proteins and how these underlie robust development.
In plants, the few known polar plasma membrane proteins provide crucial information for robust development [ 15 – 17 ]. We have previously discovered a link between development and a complex comprising the Arabidopsis caspase-like protease separase (also named EXTRA SPINDLE POLES [ESP]) and 3 Arabidopsis microtubule (MT)-based centromeric protein-E-like Kinesins 7 (KIN7), which belong to the so-called KIN7.3-clade (KIN7.1, KIN7.3, and KIN7.5). This complex (the kinesin-separase complex [KISC]) is recruited to MTs; the most abundant and important kinesin from the KISC is KIN7.3 [ 18 ]. ESP is an evolutionarily conserved protein responsible for sister chromatid separation and membrane fusion in both plants and animals [ 19 , 20 ]. ESP binds to the KIN7.3-clade C termini (the so-called “tails”), inducing conformational changes that expose the MT-avid N-terminal motor domain of KIN7s, thereby increasing KISC binding on MTs. The KISC can also modulate polar domains of the plasma membrane (PM), as the temperature-sensitive radially swollen 4 (rsw4) mutant harbouring a temperature-sensitive ESP variant or KIN7.3-clade mutants display reduced delivery of polar auxin efflux carriers PINFORMED (PINs) at the PM [ 18 ]. Yet, how the KISC acts upon PM polar domains to regulate development remains elusive.
In the model plant, Arabidopsis (Arabidopsis thaliana) LLPS condensates are involved in, for example, the internal chloroplast cargo sorting, transcriptional circuits modulating defence, RNA processing, and temperature sensing [ 6 – 10 ]. Furthermore, plants form conserved condensates like stress granules and processing bodies [ 11 – 13 ]. Recent evidence suggests that like their animal counterparts, plant condensates can interface with membranes. For example, condensates of the TPLATE, a plant-specific complex modulating endocytosis, can likely form on the plasma membrane [ 14 ]. We have also shown that condensates of processing bodies form on membranes in Arabidopsis and can attain polarity (i.e., localizing asymmetrically at the plasma membrane) [ 13 ]. However, the functional significance of condensates at the plasma membrane is unclear.
The past few years have experienced tremendous progress in the evolution of a molecular grammar that underpins LLPS. Molecules such as proteins and RNAs are polymers with attractive groups known as “stickers” that form noncovalent and mainly weak interactions. At certain concentrations, which are determined by various factors (e.g., temperature, redox state, pH), interactions are enabled among intra- or intermolecular stickers. When reaching a system-specific threshold concentration, the whole system containing various proteins and/or RNAs undergoes LLPS. The stickers promote the attraction between charged residues, dipoles, or aromatic groups that are usually provided by the so-called “intrinsically disordered regions” (IDRs) [ 4 ]. Stickers are connected by “spacers” that regulate the density transitions (i.e., liquid-to-solid transitions) by orienting stickers. The IDRs lack a defined structure and thus can easily expose their stickers. Furthermore, IDRs can increase the apparent size known as hydrodynamic radius adopted by the solvated, tumbling protein molecule [ 5 ].
Under certain conditions, biomolecules can separate from their bulk phase through liquid–liquid phase separation (LLPS), thereby attaining liquid-like properties, such as surface tension, which leads to highly circular condensates akin to droplets [ 1 ]. LLPS determines the formation of many evolutionary conserved condensates, such as nucleoli, stress granules, and processing bodies. Starting as liquids, some condensates undergo transitions in their material properties that affect their viscosity, surface tension, and degree of penetrance by other molecules. For example, in Drosophila melanogaster, oskar ribonucleoprotein (RNP) condensates undergo a liquid-to-solid transition, which is important for the polar distribution of some RNAs in the cell [ 2 ]. Whereas oskar RNP liquidity allows RNA sequestration, its solid phase precludes the incorporation of RNA while still allowing protein sequestration. Furthermore, although they are not delimited by membranes, condensates can interface with them or even engulf small vesicles [ 3 ].
Results
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[1] Url:
https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002305
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