Two-dimensional molecular condensation in cell signaling and mechanosensing

Abstract

Membraneless organelles (MLO) regulate diverse biological processes in a spatiotemporally controlled manner spanning from inside to outside of the cells. The plasma membrane (PM) at the cell surface serves as a central platform for forming multi-component signaling hubs that sense mechanical and chemical cues during physiological and pathological conditions. During signal transduction, the assembly and formation of membrane-bound MLO are dynamically tunable depending on the physicochemical properties of the surrounding environment and partitioning biomolecules. Biomechanical properties of MLO-associated membrane structures can control the microenvironment for biomolecular interactions and assembly. Lipid-protein complex interactions determine the catalytic region’s assembly pattern and assembly rate and, thereby, the amplitude of activities. In this review, we will focus on how cell surface microenvironments, including membrane curvature, surface topology and tension, lipid-phase separation, and adhesion force, guide the assembly of PM-associated MLO for cell signal transductions.

Introduction

Membraneless organelles (MLOs) initiate biochemical reactions in a biological system through nucleating local multi-component biomolecular assembly via boundary-spanning networking (percolation or gelation) or density transition (phase separation), generating different biomolecular assembly statuses and resulting in corresponding functions. The biocatalytic core of macromolecule condensates is generally assembled from functional structured domains via the lock-and-key model. In combination, besides the ‘spacer’ effect of intrinsically disordered regions (IDRs), the flexible interactions using the interactive motifs within the IDRs will tune the assembly states of the biocatalytic core by initiating homo- or hetero-oligomerization and bypassing the threshold of percolation or phase separation [ 1– 4] . To trigger a transition to activate, inhibit, or amplify biochemical reactions, critical percolation/gelation/phase separation concentrations are essential factors to initiate the on-site responses [ 5, 6] . Local microenvironments are crucial to change the aforementioned critical concentrations by providing a different threshold to pass the energy barrier for a higher-order assembly. The influential microenvironments are complex and often mixed with multiple factors. These include the molecular grammar within phase separating constituents, chemical composition, and surrounding mechanical environments, such as adhesion and associated topology.

Protein MLO formation on a lipid bilayer refers to the process by which specific proteins can separate into distinct regions or phases within the membrane. The proteins that can undergo this process often have a high degree of intrinsic disorder, such as intrinsically disordered proteins (IDPs) or low complexity domains (LCDs). Bilayer or monolayer membrane structures spatially organizes biocatalysis in an area spanning tens to hundreds of square micrometers. In contrast, the biomolecular-based biochemical reactions could be triggered within the MLOs sized from tens to thousands of square nanometers. These MLOs can perform a wide range of functions within the cell, such as regulation of gene expression, signaling, and metabolic processes. The biochemical activities in the confined spaces within MLOs depend on the biophysical properties within the condensates, such as the viscoelastic properties, surface tension, and interactive networking of the constituents. These features influence the effective density, dwell time, constituents stoichiometry, and conformational flexibility of partitioning components [7]. For example, compared with three-dimensional assembly in cytoplasm or nucleoplasm, phase separation on a two-dimensional surface, such as lipid bilayer or filamentous cytoskeleton, that has limited dimensionality in molecular movements, requires a much lower critical concentration to initiate nanometer-sized clustering.

Membrane-bound MLOs are often de novo assembled from resting state molecules on demand during signal transductions. Depending on the interaction motifs, affinity strength, conformational flexibility, and networking pattern, the assembled membrane-bound MLOs exhibit different connectivity and fluidity, enabling tunable biocatalysis for signal transduction. Due to complex environmental cues and additional spatiotemporal regulations in signal transduction, the biomolecular assembly is dynamically tuned, allowing transition into a different phase for either activation or suppression of downstream signal transduction cascades.

Here, we will discuss the emerging roles of membrane-bound MLO in regulating PM-based signal transduction, with an additional focused discussion on mechanoregulation in biomolecular condensation at the two-dimensional interface between the cell and the surrounding physicochemical environment.

Spatial-Temporal Regulation of Molecular Condensation and Function

To avoid undesired intermolecular interactions but still enable efficient reactions for diverse cellular activities, the spatial distribution of most biomolecules by membrane compartmentalization is evolved to separate different biomolecules by membrane systems. However, non-membrane-gated functional biomolecules, despite their lack of membrane boundary, are still able to be integrated into the functional macromolecular complex by being condensed and separated from surrounding environments, namely membranelles organelle (MLO) [7]. Dynamic multi-component assembly process and the exchange between the diluted and dense phase are susceptible to physicochemical environments that allow the spatial-temporally regulated molecular assembly to initiate biochemical activities on demand during signal transductions. During signaling events, phase-separating clients could be actively recruited by scaffolding molecules to form signaling condensates in distinct cellular spaces, including cytoplasm [ 8‒ 10] , nucleoplasm [ 11‒ 15] , or in a membrane-associated manner [ 16‒ 28] , in responses to intracellular or extracellular stimuli.

Cytoplasm condensates

Since a large portion of the cytoplasm volume is occupied by different macromolecular, it has been a mystery for centuries to understand the complex mechanisms by which the cell maintains order in this crowded and chaotic environment. With the past ten years of intense research, a large number of membraneless compartments were reported that are commonly believed to create micro-environments that allow biochemical interactions to take place during physiological or pathological conditions. For example, P granules in C. elegans embryos are involved in germ line segregation and modulation of post-transcriptional gene expression, depending on the constitutive component PGL-1/3 [ 16, 29] . In yeast, the Spa2-mediated polarisome complex forms phase-separating condensates at the bud tip to facilitate actin cable polymerization during polarized cell growth and condense in the cytoplasm for stress adaptation [30].

Nucleoplasm condensates

The formation of nuclear condensate has also emerged as a central paradigm for transcriptional reprogramming during diverse signaling [31]. Transcriptional condensates could control RNA production by recruiting and enriching transformation factors (TFs) and coactivators, which subsequently modulate the distribution of enhancers and super-enhancers [32]. In return, RNA can also provide dynamic feedback on transcription through stoichiometry changes [14]. In addition, the assembly of nucleoli has been shown to enhance ribosome biogenesis [ 12, 33] . In plant circadian clock regulation, photobodies in Arabidopsis are formed by CRY2 with transcription factors, responding to blue light [34]. During plant biotic stress signaling, defense-activated condensates can be triggered in the nucleus to recruit RNA polymerase II and transcriptional coactivators of the Mediator complex to reprogram plant defense gene expression profile for resistance. Interestingly, chromatin crosslinking can become mechanical environments that provide a regulatory framework for condensates’ formation and function in the nucleus [35].

Cell surface- and intracellular membranes

Unlike a three-dimensional physiochemical environment, the surface of intracellular membrane structures and plasma membrane (PM) provides a unique two-dimensional platform, allowing different bond strengths and availability of associated MLO compared to 3D-MLO. The lipid bilayer surface facilitates the nucleation of protein clusters and the formation of the dense phase through a reduction in diffusion and restriction in coarsening. Still, it requires a much lower critical concentration [ 21, 36, 37] . Emerging evidence shows that membrane-associated MLOs regulate diverse intracellular activities, such as mediating autophagy [ 38, 39] , activating immune signaling pathways [ 17, 18, 20, 22, 25, 40‒ 43] , cytoskeleton and cell adhesion [ 17, 24, 42, 44‒ 46] , and synaptic development and signaling [ 47‒ 51] .

MLOs in LAT-based Immune Signaling on the Mammalian Cell PM

Micrometer-sized and submicrometer-sized condensations formed by the components in the T-cell receptor (TCR) pathway are commonly observed on T cell membrane [52]. Three key proteins, linker for activation of T cell (LAT), growth factor receptor-bound protein 2 (Grb2), and Son of Sevenless (Sos) in the TCR pathway, can form tricomponent condensates in solution [53]. Single pMHC ligand: TCR binding events can trigger the condensation of LAT molecules, creating scaffolding signaling hubs downstream of TCR [54]. After T cell activation, SRC Homology 3 (SH3) and SRC Homology 2 (SH2) domains on Grb2 simultaneously bind with multivalent domains on Sos and phosphorylated LAT. Such LAT-centric crosslinking modulates the threshold of complex condensation of LAT, Grb2, and Sos, facilitating downstream signaling transduction [53]. Supported lipid bilayer (SLB) was used to reconstitute the TCR signaling pathway in vitro, and the biochemistry properties, functions, and mechanisms of two-dimensional LAT condensation were studied [55].

LAT condensates are liquid-like and can dynamically move and coarsen with each other [24]. Slow coarsening dynamics and kinetic arrest trap LAT condensation out of equilibrium [26]. Participating proteins are recruited into LAT condensates via percolation and protein-protein interactions, resulting in a higher density and lower mobility of components compared with the surrounding environment [56]. The tyrosine phosphorylation governs LAT condensation on LAT. Decreasing the number of phosphorylated tyrosine sites can weaken the LAT phase separation both on SLB and in live cells [24], which is likely through a reduction in connectivity.

Due to the negative charge of LAT condensates, negatively charged phosphatase CD45 is excluded, while positively charged kinase zeta-chain-associated protein kinase 70 (ZAP70) is recruited and enriched inside the condensate. Thus, the biochemical reactions of TCR signal transduction are positively promoted [24]. The high local component concentration also amplifies the downstream signaling transduction. Actin polymerization is preferentially initiated in LAT condensates with the help of recruited non-catalytic region of tyrosine kinase (Nck), neural Wiskott-Aldrich syndrome protein (N-WASp), and actin-related proteins-2/3 (Arp2/3) [24]. A similar phenomenon occurs in the Nephrin-Nck-N-WASP phase separation on SLB. Because of the condensation, the membrane dwell time of N-WASP and Arp2/3 complex increases, consequently increasing actin assembly [17]. Sos is an important rat sarcoma virus (Ras) guanine nucleotide exchange factor in TCR signaling and can activate Ras once LAT condensates are formed [57]. One-way trafficking of Sos was reported. Upon being recruited by condensation, Sos is trapped on the membrane and continuously activates Ras until being actively removed via endocytosis [58]. The longer molecule dwell time provided in LAT condensates allows kinetic proofreading in the Sos-mediated activation of Ras [41]. In addition to affecting downstream protein behaviors, the formation of LAT condensate on the membrane can also drive lipid phase separation, subsequently driving localization of the Sos substrate and Kirsten rat sarcoma viral oncogene homolog (K-Ras) into the condensates [59]. Ras activation was found to be driven by Ras clustering in a membrane curvature-dependent manner [ 60, 61] . Through the nano-engineering platform, Ras isoforms K-Ras and Harvey rat sarcoma viral oncogene homolog (H-Ras) proteins were found to be highly recruited to the positive curvature area for signaling activation, allowing a mechanical cue and topological cue-mediated Ras signaling. How the topological cues, such as curvature, regulates the activation of oncogenic Ras isoforms in the context of LAT phase separation in an orchestrated manner would require further studies.

MLOs in Mammalian Synaptic Development and Signaling

The process of phase separation plays a critical role in the formation and function of synapses by allowing the proper compartmentalization and localization of proteins, lipids, and signaling molecules. They assemble into small, dynamic, and highly organized MLOs that facilitate the formation of the presynaptic active zones [ 62, 63] , condensed postsynaptic density (PSD) [ 64, 65] , and the clustered reserve pool synaptic vesicles (SVs) [ 66, 67] .

The presynaptic active zone is the site of neurotransmitter release. The active zone is composed of a complex array of proteins, including Rab3-interacting molecule (RIM), RIM-binding proteins, ELKS, Munc13, Liprin, and CASK (calcium (Ca2+)/calmodulin (CaM) associated serine kinase) [68]. Their inter- and intramolecular interactions drive the formation of MLOs, which facilitates the release of neurotransmitter vesicles from the presynaptic neuron [69]. Meanwhile, in PSDs, phase-separation-mediated synaptic assembly relies on the scaffolding proteins PSD-95 and its participating partners GKAP, Shank3, and Homer [ 70‒ 73] . The valency with multi-component condensates is a major underlying determinative factor that controls the formation and function of PSD with localized material properties of PSD [48].

Given that synapses are required to be highly responsive to signals and stimulations, rising interest has been focused on regulating pre- and postsynaptic condensates. Considering the unique topological environment for the synaptic membranes, it is remarkable that curvature-binding proteins contribute significantly to this process in several ways. For example, the curvature of the postsynaptic membrane can influence the affinity of receptors for neurotransmitter molecules via multivalent curvature binding proteins [74]. The expression of IRSp53, as the I-BAR protein in the PSD protein complex, is known to affect the number and function of excitatory synapses [75]. A recent study provided direct in vitro evidence that IRSp53-specific multivalent interaction with its binding partners PSD-95 and Shank3 drives phase separation during PSD condensation formation [76]. Evidence also illustrates that IRSp53 deletion influences the stabilization of synaptic actin assembly, which affects the strength and duration of the signal transmitted across the synapse [77]. More future works are required in this exciting field to understand how the local curvature sensing protein orchestrates the localized PSD assembly and the progression of membrane deformation by coordinating the curvature-dependent molecular partition in a positive or feedback regulatory manner to guide the synaptic membrane curvature development.

Molecular Condensates at the Plant-Pathogen Interface for Defense Signaling

Although there is a cell wall structure in plant cells, the PM of plant cells still serves as the frontline platform, similar to the mammalian system, for biomolecular assembly and guiding signal transduction ( Figure 1). The PM’s geometry, fluidity, and topology are essential in regulating the functional formation of biomolecular condensation-based signaling hubs. In addition to protein-based condensations on the PM, the lipid bilayer offered a unique 2D-phase separating environment to compartmentalize or separate molecules by forming localized lipid-protein complexes, such as lipid rafts in mammals or nanodomains in plants. The spatially regulated molecular interactions on PM might tune the initial kinetic proofreading to initiate phase separation or create an energy state to maintain molecule association dynamics in a non-equilibrium free-energy minimum status.

Figure 1 .

Protein condensation on plant CW-PM-Cytoskeleton continuum upon stimuli

On resting state, nano- or micro-domain residing proteins like Remorin or GTP form of ROP6 lay within nano- or micro-domains. Upon stimuli like pathogen infection or abiotic stresses, specific proteins will be recruited into nano-or micro-domains through condensation.

We could better understand the PM-associated molecular condensation by integrating the macromolecular assembly principle with the previously well-known fluid mosaic model that emphasizes the importance of the dynamic property of phospholipids and transmembrane proteins floating around [78] and the PM-associated mechanical scaffolding structures, such as the cytoskeleton and surrounding polymer networks. For example, the detergent-based biochemical fractionation approach classifies PM into detergent-resistant (DRM) and detergent-sensitive (DRS) membrane fractions. It has been proposed that these DRMs work as lipid rafts enriched with saturated lipids, sterol, and sphingolipids to mediate cell signaling and live activities [ 79‒ 82] . Mechanically, DRM shows the compartmentation of a biophysically distinct biomolecule environment ensembled by a higher-order assembly of phospholipid acyl chains. DRM provides a confined package of the lipid chains and their associated proteins with limited motility and high surface tension [ 83‒ 85] . Recent high-resolution microscopy imaging techniques further demonstrated that plasma membrane consists of heterogeneously spatial-temporal coexisting micrometer-sized (microdomains) or nanometer-sized domains (nanodomains) [ 86‒ 88] . Lipids in those nanodomains are often enriched with saturated phospholipid, sterols, glycosylated lipids, and sphingolipids, which are tightly packed compared to their surroundings, making the nanodomain or microdomain regions with liquid-ordered phase [89]. With distinct biophysical properties, the phase separating nanodomains enable stimuli-activated retention and recruitment of diverse signaling regulators that are transiently engaged or with extended dwell time, allowing signaling activation, amplification, or suppression.

For example, plant biotic defense signaling molecule salicylic acid (SA) promotes the macromolecular assembly of REMORIN proteins, which are nanodomain assembly factors that drive PM compartmentalization and phase separation through nanoclustering REMORIN. REMORIN clustering results in the plasmodesmata (PD) closure and restriction of the virus spreading [18]. In addition, due to the REMORIN assembly and nanodomain-mediated PM compartmentalization, SA could repress auxin transporter PIN2 activity by condensing PIN2 on the PM, thereby contributing to defense-growth tradeoff phenomena [20]. Surprisingly, the nanodomains formed through the condensed assembly of REMORIN and nanodomain-associated signaling molecules at the PM interleaflet could also provide the phase-separating structures that perceive the bacterial outer membrane vesicles (OMVs). Nanodomain allows the insertion of OMVs at the nanodomain sites from the outer leaflet, thereafter activating plant immune defense responses [90]. As such, plant nanodomain structures contain clustered and condensed macromolecules and serve as surface hubs for regulating signal transductions. The local biochemical activities were modulated by separating residence molecules from surrounding biomolecules, providing restricted motility and reducing exchange with surroundings. During plant-pathogen communication, Xanthomonas campestris pv. campestris 8004 type III secretion system injects effector protein XopR into the plant cytoplasm. XopR protein then targets the PM using its amphipathic helix and positively charged residues. XopR proteins gradually form multivalent macromolecular condensates with progressive recruitment of PM-associated immune response regulators, evolving in assembly status and constituents in an infection-time-dependent manner. For example, XopR clusters type I FORMIN proteins trigger actin nucleation during early infection but inhibit FORMIN activity at the later infection stages through further condensation that inhibits FORMIN functions [25]. Different from cytoplasm and nucleoplasm, the PM surface is a fluidic 2D environment but is regulated by compartmentalization and PM-associated mechanical scaffolding structure, such as extracellular matrix-PM-actin cytoskeleton (AC) continuum by picket fence model in the mammalian system or cell wall (CW)-PM-AC continuum [91]. In plants, the CW creates tight anchors of the PM by connecting to the outer leaflet. At the same time, such anchoring effects are extended to the inner leaflet and stabilize the associated biomolecules, providing them with mechanical regulations using the CW-PM-AC continuum. The mechanical regulations include anchoring, stretching, and confinement forces provided by various changes in CW and AC networks that directly influence the molecule diffusion and interactions on the PM.

The functional connection between the nano-clustering of membrane proteins and cell signaling regulation on the PM by the dynamic partition of signaling regulators into nanodomain is an emerging research topic to understand the activation and amplification of signal transduction. During plant innate immune responses, REMORIN and actin nucleator type I FORMIN form complex condensation in an infection-time-dependent manner. During infection, multi-component condensates dynamically evolve into high-order assemblies from initial nanoclusters over time [42], which also depends on the cell wall composition and integrity of the actin cytoskeleton [22]. Molecular dynamics of several well-studied PM signaling molecules, including flagellum receptor FLS2, the auxin transporter PIN3, and the hormone receptor Brassinosteroid insensitive 1 (BRI1), is also tightly regulated by cell wall [92], which likely influences their nanoclustering and thereby immune activation, such as flg22-triggered FLS2 clustering [27]. In addition, plant growth hormone auxin triggers Rho of Plants 6 (ROP6) stabilization on the PM in a phosphatidylserine-dependent manner and, after that, nanoclustering [93]. ROP6 clustering can also be induced by osmatic stress that further elicits the activity of NADPH/respiratory burst oxidase protein D (RBOHD) for ROS generation [94], which might be mechanically regulated through changing surface tension and molecular packaging on the PM. Nanoclustering of surface biomolecules could mutually reinforce each other’s assembly, which likely creates the biomolecular mechanism underlying the sophisticated signaling crosstalks. For example, auxin-induced cell surface transmembrane receptor kinase 1 (TMK1) nanoclustering could stabilize flotillin1-associated nanodomain and promote flotillin1 nanoclustering, which further promote ROP6 nanoclustering and then trigger downstream cortical microtubule (CMT) reorganization. The ROP6-induced CMT stabilizes TMK1 and flotillin1 nanoclustering, forming a reinforced positive feedback loop to generate cell polarity [95]. A holistic understanding of how the mechanical regulation of nanodomain assembly and molecular partition by CW-PM-AC during diverse signaling would allow us to understand the complex cell signaling and crosstalk between environmental cues and mechanical cues for signaling transduction with precise regulations.

Topological Cues in MLO Formation and Function

Curvature-guided Ras oligomerization and activation

Biological activities require complex cellular architectures that involve biomolecular interactions on the curving membrane sites. Curvature-sensing biomolecules can be oligomerized and even engage in larger scaffolds if the macromolecular assembly is triggered. For example, plasma membrane topology was recently found to regulate Ras nano-clustering and function on the cell surface. As the first discovered human oncogene, the role of Ras plays in tumorigenesis has been thoroughly studied in the past few decades [96]. The Ras superfamily includes three principal members: K-Ras, H-Ras, and Neuroblastoma Ras viral oncogene homolog (N-Ras). The Ras signaling pathway controls cell proliferation, differentiation, and survival. The mutation of Ras and the consequent amplification of Ras signaling can lead to cancerous phenotypes [97]. Thus, the Ras pathway is continuously regarded as a target for cancer therapy [98]. Ras signaling transduction is generated on the cell membrane, and Ras proteins are localized on the cell membrane [99]. Approximately 40% of Ras proteins form clusters on the membrane, which are ~9 nm in radius containing 6-7 Ras proteins each [100]. Ras condensates are believed to be the effector binding hubs of the rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. They are important for signal transduction [101].

Evidence suggests that Ras activity is likely coupled with physicochemical cues derived from membrane curvature. Using the single liposome curvature (SLiC) method, the N-Ras anchor was found to increase its membrane binding as the diameter of liposomes decreased [102]. In addition, lipid order and composition can regulate the curvature-induced N-Ras anchor recruitment [103]. Based on a nanobar platform with precisely controlled curvature sizes [104], the curvature-related Ras condensation was proved to depend on both the membrane lipid composition [60] and the activity of the Ras binding partner [105]. Curvature sensitivities of Ras condensates and their oncogenic mutants were tested on the nanobar platform, and the isoform specificity and the sensitivity of a Ras inhibitor were successfully characterized [61]. Further attempts to reveal the Ras condensation mechanism and decipher Ras regulations may be achieved via the nanofabrication platform that mimics the physiological-relevant radius of membrane curvatures.

Molecular condensation of curvature sensing proteins on the cell membrane

Many other membrane-bound proteins were also observed to enrich different membrane morphologies and to play a role in membrane deformation and functional compartmentalization. The amphipathic helix can insert into the lipid bilayer, and the Bin/amphiphysin/Rvs (BAR) domain can directly sense the curved lipid bilayer, both of which are well-studied curvature generating and sensing motifs. Based on the shapes of the dimeric BAR domain, they are classified into N-BAR, F-BAR, and I-BAR proteins [106]. These proteins tend to highly recognize topology-specific membrane structures which are generally divided into ‘positive curvature’ and ‘negative curvature’ [107]. First, membrane curvature can influence the membrane binding and the stability of BAR domain proteins. According to differential binding affinities towards the curvatures with different radii, highly curved membrane regions have been shown to significantly stabilize and enrich the BAR domains of both Endophilin and FBP17 [ 108, 109] . This curvature-mediated recruitment or insertion can increase local protein concentration and facilitate the potential nucleation of on-site phase separation if multivalent interactions are engaged. Second, membrane curvatures also alter the assembly pattern of the membrane-associated proteins. For instance, the F-BAR domain of human CIP4 (Cdc42 interacting protein 4) was observed to polymerize into helical coats on membrane tubules using cryo-electron microscopy. Lateral and tip-to-tip interactions of the dimeric F-BAR domain were visualized in the helical coats by 3D reconstitution, which indicates that the BAR domains form similarly oriented oligomers on curved membranes [110]. Therefore, the membrane curvature generated by BAR proteins and the sensing of curvatures by BAR proteins might not be strictly limited to a nanoscale that theoretically depends on the intrinsic BAR domain because a potential macromolecular oligomerization of BAR domain-containing proteins might enable an assembly of larger scaffolder (micro-scale) [111]. Compared to the minimum assembly states, larger scaffolded BAR proteins have different surface topological features or biochemical features of the ensembled oligomers that could influence the radius of the engaged bilayer in a feedback regulation manner. In addition, the protein crowding effect could also create steric constraints and trigger membrane bending [112]. How local crowding environments and mechanical forces could further influence the assembly of the curvature targeting and sensing domain-containing proteins needs to be further studied to understand the membrane topology-enabled lipid-protein condensation.

Biophysical Characterization Technologies of Biomolecule Condensates

Nanofabrication for studying mechanical regulation by topological cues

A series of technologies have been applied to study in vivo biomolecular condensates ( Table 1). For studying the topological cues, the ability to create precise nano-scale patterns and curvatures has allowed scientists to control the mechanical environment of cells in a highly controlled manner. This has led to new insights into how cells sense and respond to changes in their mechanical environment, which is critical for understanding cellular processes such as endocytosis, signaling protein clustering, cell migration, proliferation, and differentiation.

Table 1 In vivo characterization technologies of biomolecule condensates

Methods	Biophysical properties	Advantages	References
Single molecule tracking (SMT)	Dynamics and mobility	SMP is used to study the dynamic formation and dissociation of molecule condensates and the interactions between different molecules within the condensate.	[ 22, 25, 113]
Intermolecular interactions
Super-resolution microscopy	Size and shape	Technologies like STED microscopy, PALM, 3D SIM, and STORM allow the imaging of biological samples with resolutions beyond the diffraction limit of light, which is approximately 200 nanometers for visible light.	[ 114‒ 116]
Dynamics and mobility
Internal structures
Fluorescence recovery after photo-bleaching (FRAP)	Mobility and diffusivity within condensates	FRAP allows for the quantification of the mobility of molecule condensates, which can be used to calculate their diffusion coefficient binding constants, and FRAP is a relatively simple technique that can be easily performed in most research laboratories.	[ 20, 25, 117]
The molecule exchange rate between condensates and the environment

Up to date, a few groups have started to investigate how the geometrical cues of membranes influence molecular condensations by using nanofabrication or nanomaterials. For example, nanopores with a 200-nm diameter can recruit the TCR condensations at the tips of microvilli protrusions, thereby facilitating T-cell signaling [118]. Another nanobridge was designed for T-cell activation. ZAP70-associated condensations were enriched along nanoridges [119]. A nanobead platform is applied to induce inward curvature on the cell membrane. WASP condensations are preferentially enriched at the necks of these membrane invaginations, followed by recruitment of the Arp2/3 complex, stimulating actin polymerization and cytoskeleton assembly [120]. The nanobar or nanopillars generated using the nanofabrication method [121] is an emerging technology that makes it possible to create nanostructures with precise control of size, curvature, and shape close to the cellular membrane structures. Future interdisciplinary studies using nanofabrication or comparable nanomaterials allow a better understanding of the underlying mechanisms by which the mechanical features of the biointerface between cell and environment, cancer and immune cells, or pathogen and host cells, regulate molecular condensation for generating desired signal transduction.

In vitro reconstitution on lipid bilayer

Technologies including AFM, SFA, and liquid TEM have been widely used in studying in vitro biomolecular condensates ( Table 2). Furthermore, in vitro reconstitution of macromolecular assembly on lipid bilayers recapitulates direct molecular interactions and biochemical reactions to study how the physiochemical environments influence biomolecular phase separation and function on the two-dimensional membrane surface. The artificial lipid-bilayer-based reconstitution has greatly enhanced our understanding of the molecular mechanism of membrane-bound condensation and the subsequent development of bottom-up biology-enabled synthetic engineering to control and optimize signal transduction.

Table 2 In vitro characterization technologies of biomolecule condensates

Methods	Biophysical properties	Advantages	References
Atomic force microscopy (AFM)	Surface structure	Produce 3D images of the condensates. Study the condensates under different temperatures. Quantitative measurements of surface roughness.	[ 122, 123]
Stiffness and viscosity
Surface forces apparatus (SFA)	Surface forces	SFA is a highly sensitive technique to detect small forces with piconewton precision and the changes in the biophysical properties of the condensate’s surface. Study the interactions between the condensates and solid substrates or other condensates, which can provide insight into the forces driving the formation and stability of the condensates.	[ 25, 124, 125]
Adhesion strength
Cohesion strength
Liquid TEM	Movement	Liquid TEM allows for imaging at the nanometer scale and can provide information about the chemical composition and structural organization of the condensates at the molecular level.	[126]
Viscosity

GUV and SLB for membrane-associated protein assembly

GUV (giant unilamellar vesicles) and SLB (supported lipid bilayers) are two widely used in vitro systems to study membrane-associated protein assembly. The biomolecular assembly of membrane-associated proteins is able to be studied in a controlled and reproducible manner to understand multivalent protein condensation and catalytic activities in diverse cellular environments.

In vitro reconstitution on GUV involves the creation of artificial vesicles with a single lipid bilayer using various techniques, including electroporation, extrusion, and sonication [ 127‒ 129] . These vesicles are then used as a model system because of their comparable size to cells and their aqueous interior. By adding different proteins, direct visualization of membrane deformations on GUV was reported in many recent studies. For example, the I-BAR domain of IRSp53 can deform the phosphatidylinositol 4,5-biphosphate-containing GUV membranes into inwards tubulations under the concentration of several micromolar [ 130‒ 132] . Different from GUV, SLB in vitro reconstitution involves the creation of a supported lipid bilayer on a solid substrate, such as a glass slide, which provides a 2D view of protein assembly. This is done by anchoring a thin layer of membrane-associated proteins onto the lipid layer under more controlled conditions. As the proteins are immobilized on the substrate and restricted by lateral movement, they can be more easily visualized and manipulated [ 24, 55] .

Concluding Remarks

In conclusion, 2D-phase separation on biomembrane and mechanical regulation of phase separation are rapidly evolving research fields that have gained significant attention in recent years. The ability to control the mechanical environment of biological systems using nanofabrication techniques coupled with single molecular analysis assays has allowed scientists to gain new insights into how cellular biomolecular condensation processes sense and respond to changes in their mechanical environment. The study of MLO in lipid bilayers, in particular, has provided a new understanding of how the mechanical properties of the bilayer or the bilayer-connected scaffolding structure affect the behavior of proteins and how these proteins form distinct regions or phases within the membrane. The research of MLOs in the bilayer has expanded the understanding of how cells compartmentalize their biomolecule to generate signaling hubs and how mechanical forces regulate this process. This is critical for understanding numerous cellular processes and signal transductions for cell development and defense, and has the potential to lead to new strategies for combating cancer and pathogenic infection.

Furthermore, the ability to manipulate and characterize the mechanical environment of cells using nanofabrication techniques and to quantitatively analyze biomolecules in single molecular approaches has opened up new possibilities for developing diagnostic tools, biosensors, and synthetic engineering to control signaling and cell behaviors.

In summary, the field of mechanical regulation of phase separation is still in its infancy, and many open questions need to be addressed. However, the advances made so far have provided a new level of understanding of how mechanical forces regulate cellular behavior and have the potential to lead to new strategies for cancer therapy and tissue engineering.

COMPETING INTERESTS

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding Statement

This work was supported by the grants from the Human Frontier Science Program Foundation (No. RGY0088/2021 to W.Z.), the Start-up Grant from Nanyang Technological University to W.Z., Ministry of Education, Singapore (Nos. RG112/20, RG95/21, RG93/22, MOE-MOET32020-0001 to W.Z.), National Research Foundation, Singapore (No. NRF2019-NRF-ISF003-3292 to W.Z.), Singapore MOE Tier 2 (Nos. MOE-T2EP30121-0015 and MOE-T2EP30122-0021 to Y.M.), MOE Tier 3 (No. MOE2019-T3-1-012 to Y.M.), National Research Foundation Singapore under its Open Fund-Individual Research Grant (No. MOH-000955 to Y.M.), and National Research Foundation-Quantum Sensors for Health and Life Sciences (No. NRF2021-QEP2-03-P10 to Y.M.).