Wetting of phase-separated droplets on plant vacuole membranes leads to a competition between tonoplast budding and nanotube formation

Abstract

Seeds of dicotyledonous plants store proteins in dedicated membrane-bounded organelles called protein storage vacuoles (PSVs). Formed during seed development through morphological and functional reconfiguration of lytic vacuoles in embryos [M. Feeney et al., Plant Physiol. 177, 241–254 (2018)], PSVs undergo division during the later stages of seed maturation. Here, we study the biophysical mechanism of PSV morphogenesis in vivo, discovering that micrometer-sized liquid droplets containing storage proteins form within the vacuolar lumen through phase separation and wet the tonoplast (vacuolar membrane). We identify distinct tonoplast shapes that arise in response to membrane wetting by droplets and derive a simple theoretical model that conceptualizes these geometries. Conditions of low membrane spontaneous curvature and moderate contact angle (i.e., wettability) favor droplet-induced membrane budding, thereby likely serving to generate multiple, physically separated PSVs in seeds. In contrast, high membrane spontaneous curvature and strong wettability promote an intricate and previously unreported membrane nanotube network that forms at the droplet interface, allowing molecule exchange between droplets and the vacuolar interior. Furthermore, our model predicts that with decreasing wettability, this nanotube structure transitions to a regime with bud and nanotube coexistence, which we confirmed in vitro. As such, we identify intracellular wetting [J. Agudo-Canalejo et al., Nature 591, 142–146 (2021)] as the mechanism underlying PSV morphogenesis and provide evidence suggesting that interconvertible membrane wetting morphologies play a role in the organization of liquid phases in cells.

Intracellular wetting mediates contacts between liquid compartments and membrane-bound organelles

“…Further, it implies that elastocapillarity (the interplay of inherent material attributes of both the subcompartment and the vacuole membrane, respectively droplet surface tension and membrane elasticity) governs protein storage vacuole formation. To distinguish this droplet-mediated mechanism of cellular membrane reshaping from conventional membrane remodeling complexes that form membrane coats such as clathrin ( Avinoam et al, 2015 ), we refer to this novel form of droplet-mediated membrane remodeling as “liquid scaffolding.” Liquid scaffolding during vacuole remodeling has been confirmed recently ( Kusumaatmaja et al, 2021 ).…”

“…To distinguish this droplet-mediated mechanism of cellular membrane reshaping from conventional membrane remodeling complexes that form membrane coats such as clathrin (Avinoam et al, 2015), we refer to this novel form of droplet-mediated membrane remodeling as "liquid scaffolding." Liquid scaffolding during vacuole remodeling has been confirmed recently (Kusumaatmaja et al, 2021).…”

“…While the effect of cytosol–droplet interactions on cellular organization and physiology has been established (reviewed in Alberti et al, 2019 ; Banani et al, 2017 ; Shin and Brangwynne, 2017 ), the mechanism underlying droplet–membrane interactions remains poorly understood. The functional importance of droplet–membrane interactions has only very recently been shown, for example in T cell receptor signal transduction ( Su et al, 2016 ), pre- and postsynaptic densities ( Milovanovic et al, 2018 ; Zeng et al, 2018 ), the development of tight junctions that control paracellular transport ( Beutel et al, 2019 ), droplet transport by hitchhiking on moving lysosomes ( Liao et al, 2019 ), the assembly of membranes implicated in autophagy ( Fujioka et al, 2020 ; Agudo-Canalejo et al, 2021 ), endoplasmic reticulum–mediated droplet splitting ( Lee et al, 2020 ), and formation of protein storage vacuoles ( Kusumaatmaja et al, 2021 ). These examples demonstrate that referring to droplets as “membrane-less organelles” is misleading as, in fact, droplets frequently interact with several distinct classes of membrane: the plasma membrane and membrane-bound organelles as well as small vesicular compartments and membrane sheets.…”

Spontaneous phase separation of ternary fluid mixtures

“…q t (ru a ) + q b (ru a u b ) = Àq b P ab + q b Z(q b u a + q a u b ). (8) In this work, for simplicity, we have assumed all components have the same density, such that the total density r = C 1 + C 2 + C 3 = 1 is constant. The pressure tensor is related to the chemical potential via…”

“…1,2 More recently, it has also been shown to play fundamental roles in volcanic eruptions 3,4 and in the organisation of cellular matters, leading to the formation of the so-called biomolecular condensates or membraneless organelles. [5][6][7][8] In industry, better understanding of the phase separation process is necessary, among others, for tuning the formation of fractures in alloys in the field of metallurgy, 9,10 for manipulating the structure of polymer blends which in turn affect their mechanical and electrical properties, [11][12][13] and for controlling the morphology of complex emulsions for applications in drug delivery [14][15][16] and in the food industry. [17][18][19] Extensive theoretical and experimental studies on phase separation have been carried out in the case where the fluid mixtures separate into two distinct, immiscible fluid components.…”