Multivalent interactions drive the Toxoplasma AC9:AC10:ERK7 complex to concentrate ERK7 in the apical cap

The Toxoplasma inner membrane complex (IMC) is a specialized organelle that is crucial for the parasite to establish an intracellular lifestyle and ultimately cause disease. The IMC is composed of both membrane and cytoskeletal components, further delineated into the apical cap, body, and basal subcompartments. The apical cap cytoskeleton was recently demonstrated to govern the stability of the apical complex, which controls parasite motility, invasion, and egress. While this role was determined by individually assessing the apical cap proteins AC9, AC10, and the MAP kinase ERK7, how the three proteins collaborate to stabilize the apical complex is unknown. In this study, we use a combination of deletion analyses and yeast-2-hybrid experiments to establish that these proteins form an essential complex in the apical cap. We show that AC10 is a foundational component of the AC10:AC9:ERK7 complex and demonstrate that the interactions among them are critical to maintain the apical complex. Importantly, we identify multiple independent regions of pairwise interaction between each of the three proteins, suggesting that the AC9:AC10:ERK7 complex is organized by multivalent interactions. Together, these data support a model in which multiple interacting domains enable the oligomerization of the AC9:AC10:ERK7 complex and its assembly into the cytoskeletal IMC, which serves as a structural scaffold that concentrates ERK7 kinase activity in the apical cap.

Multivalent Interactions between Molecular Components Involved in Clathrin Independent Endocytosis Drive Protein Phase Separation

Endocytosis of transmembrane receptors initiates via molecular interactions between the activated receptor and the endocytic machinery. A specific group of receptors, including the β1-adrenergic receptor (β1-AR), is internalized through a non-clathrin pathway known as Fast Endophilin Mediated Endocytosis (FEME). A key question is: how does the endocytic machinery assemble and how is it modulated by activated receptors during FEME. Here we show that endophilin, a major regulator of FEME, undergoes a phase transition into liquid-like condensates, which facilitates the formation of multi-protein assemblies by enabling the phase partitioning of endophilin binding proteins. The phase transition can be triggered by specific multivalent binding partners of endophilin in the FEME pathway such as the third intracellular loop (TIL) of the β1-AR, and the proline-rich-motifs of lamellipodin (LPD-PRMs). Other endocytic accessory proteins can either partition into, or target interfacial regions of, these condensate droplets. On the membrane, TIL promotes protein clustering in the presence of endophilin and LPD-PRMs. Our results demonstrate how the multivalent interactions between endophilin, LPD-PRMs and TIL regulate protein assembly formation on the membrane, providing mechanistic insights into the priming and initiation steps of FEME.

Multivalent interactions make adherens junction–cytoskeletal linkage robust during morphogenesis

Embryogenesis requires cells to change shape and move without disrupting epithelial integrity. This requires robust, responsive linkage between adherens junctions and the actomyosin cytoskeleton. Using Drosophila morphogenesis, we define molecular mechanisms mediating junction–cytoskeletal linkage and explore the role of mechanosensing. We focus on the junction–cytoskeletal linker Canoe, a multidomain protein. We engineered the canoe locus to define how its domains mediate its mechanism of action. To our surprise, the PDZ and FAB domains, which we thought connected junctions and F-actin, are not required for viability or mechanosensitive recruitment to junctions under tension. The FAB domain stabilizes junctions experiencing elevated force, but in its absence, most cells recover, suggesting redundant interactions. In contrast, the Rap1-binding RA domains are critical for all Cno functions and enrichment at junctions under tension. This supports a model in which junctional robustness derives from a large protein network assembled via multivalent interactions, with proteins at network nodes and some node connections more critical than others.

The length scale of multivalent interactions is evolutionarily conserved in fungal and vertebrate phase-separating proteins

One key feature of proteins that form liquid droplets by phase separation inside a cell is multivalency—the presence of multiple sites that mediate interactions with other proteins. We know little about the variation of multivalency on evolutionary time scales. Here, we investigated the long-term evolution (∼600 million years) of multivalency in fungal mRNA decapping subunit 2 protein (Dcp2), and in the FET protein family. We found that multivalency varies substantially among the orthologs of these proteins. However, evolution has maintained the length scale at which sequence motifs that enable protein-protein interactions occur. That is, the total number of such motifs per hundred amino acids is higher and less variable than expected by neutral evolution. To help explain this evolutionary conservation, we developed a conformation classifier using machine-learning algorithms. This classifier demonstrates that disordered segments in Dcp2 and FET proteins tend to adopt compact conformations, which is necessary for phase separation. Thus, the evolutionary conservation we detected may help proteins preserve the ability to undergo phase separation. Altogether, our study reveals that the length scale of multivalent interactions is an evolutionarily conserved feature of two classes of phase-separating proteins in fungi and vertebrates.

Multivalent interactions facilitate motor-dependent protein accumulation at growing microtubule plus ends

Growing microtubule ends provide platforms for the accumulation of plus-end tracking proteins that organize into comets of mixed protein composition. Using a reconstituted fission yeast system consisting of end-binding protein Mal3, kinesin Tea2 and cargo Tip1, we found that these proteins can be driven into liquid phase droplets both in solution and at microtubule ends under crowding conditions. In the absence of crowding agents, cryo-electron tomography revealed that motor-dependent comets consist of disordered networks where multivalent interactions appear to facilitate the non-stoichiometric accumulation of cargo Tip1. We dissected the contribution of two disordered protein regions in Mal3 and found that both are required for the ability to form droplets and Tip1 accumulation, while autonomous Mal3 comet formation only requires one of them. Using theoretical modeling, we explore possible mechanisms by which motor activity and multivalent interactions may lead to the observed enrichment of Tip1 at microtubule ends.

Direct visualization of superselective colloid-surface binding mediated by multivalent interactions

Reliably distinguishing between cells based on minute differences in receptor density is crucial for cell–cell or virus–cell recognition, the initiation of signal transduction, and selective targeting in directed drug delivery. Such sharp differentiation between different surfaces based on their receptor density can only be achieved by multivalent interactions. Several theoretical and experimental works have contributed to our understanding of this “superselectivity.” However, a versatile, controlled experimental model system that allows quantitative measurements on the ligand–receptor level is still missing. Here, we present a multivalent model system based on colloidal particles equipped with surface-mobile DNA linkers that can superselectively target a surface functionalized with the complementary mobile DNA-linkers. Using a combined approach of light microscopy and Foerster resonance energy transfer (FRET), we can directly observe the binding and recruitment of the ligand–receptor pairs in the contact area. We find a nonlinear transition in colloid-surface binding probability with increasing ligand or receptor concentration. In addition, we observe an increased sensitivity with weaker ligand–receptor interactions, and we confirm that the timescale of binding reversibility of individual linkers has a strong influence on superselectivity. These unprecedented insights on the ligand–receptor level provide dynamic information into the multivalent interaction between two fluidic membranes mediated by both mobile receptors and ligands and will enable future work on the role of spatial–temporal ligand–receptor dynamics on colloid-surface binding.

Recruitment and organization of ESCRT-0 and ubiquitinated cargo via condensation

The general mechanisms by which ESCRTs are specifically recruited to various membranes, and how ESCRT subunits are spatially organized remain central questions in cell biology. At the endosome and lysosomes, ubiquitination of membrane proteins triggers ESCRT-mediated substrate recognition and degradation. Using the yeast lysosome/vacuole, we define the principles by which substrate engagement by ESCRTs occurs at this organelle. We find that multivalent interactions between ESCRT-0 and polyubiquitin is critical for substrate recognition at yeast vacuoles, with a lower-valency requirement for cargo engagement at endosomes. Direct recruitment of ESCRT-0 induces dynamic foci on the vacuole membrane, and forms fluid condensates in vitro with polyubiquitin. We propose that self-assembly of early ESCRTs induces condensation, an initial step in ESCRT-assembly/nucleation at membranes. This property can be tuned specifically at various organelles by modulating the number of binding interactions.One-Sentence SummaryCondensation of multivalent ESCRT-0/polyubiquitin assemblies organizes cargo sorting reactions at lysosomes

Multivalent interactions with RNA drive RNA binding protein recruitment and dynamics in biomolecular condensates in Xenopus oocytes

RNA localization and biomolecular condensate formation are key biological strategies for organizing the cytoplasm and generating cellular and developmental polarity. While enrichment of RNAs and RNA-binding proteins (RBPs) is a hallmark of both processes, the functional and structural roles of RNA-RNA and RNA-protein interactions within condensates remain unclear. Recent work from our laboratory has shown that RNAs required for germ layer patterning in Xenopus oocytes localize in novel biomolecular condensates, termed Localization bodies (L-bodies). L-bodies are composed of a non-dynamic RNA phase enmeshed in a more dynamic protein-containing phase. However, the interactions that drive the biophysical characteristics of L-bodies are not known. Here, we test the role of RNA-protein interactions using an L-body RNA-binding protein, PTBP3, which contains four RNA-binding domains (RBDs). We find that binding of RNA to PTB is required for both RNA and PTBP3 to be enriched in L-bodies in vivo. Importantly, while RNA binding to a single RBD is sufficient to drive PTBP3 localization to L-bodies, interactions between multiple RRMs and RNA tunes the dynamics of PTBP3 within L-bodies. In vitro, recombinant PTBP3 phase separates into non-dynamic structures in an RNA-dependent manner, supporting a role for RNA-protein interactions as a driver of both recruitment of components to L-bodies and the dynamics of the components after enrichment. Our results point to a model where RNA serves as a concentration-dependent, non-dynamic substructure and multivalent interactions with RNA are a key driver of protein dynamics.

Towards Chemotactic Supramolecular Nanoparticles: From Autonomous Surface Motion Following Specific Chemical Gradients to Multivalency-Controlled Disassembly

<p>Nature designs chemotactic supramolecular structures that can selectively bind specific groups present on surfaces, autonomously scan them moving along density gradients, and react once a critical concentration is encountered. While such properties are key in many biological functions, these also offer inspirations for designing artificial systems capable of similar bioinspired autonomous behaviors. One approach is to use soft molecular units that self-assemble in aqueous solution generating nanoparticles (NPs) that display specific chemical groups on their surface, enabling for multivalent interactions with complementarily functionalized surfaces. However, a first challenge is to explore the behavior of these assemblies at sufficiently high-resolution to gain insights on the molecular factors controlling their behaviors. Here we show that, coupling coarse-grained molecular models and advanced simulation approaches, it is possible to study the (autonomous or driven) motion of self-assembled NPs on a receptor-grafted surface at submolecular resolution. As an example, we focus on self-assembled NPs composed of facially amphiphilic oligomers. We observe how tuning the multivalent interactions between the NP and the surface allows to control NP binding, its diffusion along chemical surface gradients, and ultimately, the NP reactivity at determined surface group densities. <i>In silico</i> experiments provide physical-chemical insights on key molecular features in the self-assembling units which determine the dynamic behavior and fate of the NPs on the surface: from adhesion, to diffusion, and disassembly. This offers a privileged point of view into the chemotactic properties of supramolecular assemblies, improving our knowledge on how to design new types of materials with bioinspired autonomous behaviors.</p>