Membraneless Organelles Research Articles

Taylor Dispersion‐Induced Phase Separation for the Efficient Characterisation of Protein Condensate Formation

AbstractBiomolecular condensates have emerged as important structures in cellular function and disease, and are thought to form through liquid‐liquid phase separation (LLPS). Thorough and efficient in vitro experiments are therefore needed to elucidate the driving forces of protein LLPS and the possibility to modulate it with drugs. Here we present Taylor dispersion‐induced phase separation (TDIPS), a method to robustly measure condensation phenomena using a commercially available microfluidic platform. It uses only nanoliters of sample, does not require extrinsic fluorescent labels, and is straightforward to implement. We demonstrate TDIPS by screening the phase behaviour of two proteins that form biomolecular condensates in vivo, PGL‐3 and Ddx4. Uniquely accessible to this method, we find an unexpected re‐entrant behaviour at very low ionic strength, where LLPS is inhibited for both proteins. TDIPS can also probe the reversibility of assemblies, which was shown for both α‐synuclein and for lysozyme, relevant for health and biotechnology, respectively. Finally, we highlight how effective inhibition concentrations and partitioning of LLPS‐modifying compounds can be screened highly efficiently.

Membraneless organelles assembled by AuNPs-enzyme integration in non-photosynthetic bacteria: Achieving high specificity and selectivity for solar hydrogen production

The emergence of semiartificial photosynthesis holds great promise for improving light energy capture and conversion efficiency. However, achieving high product specificity and selectivity in semiartificial photosynthetic systems remains a challenge. In this study, we employed genetic engineering techniques to construct a membraneless organelle in E. coli, serving as the photoreaction carrier. The self-assembly of photosensitizers and hydrogenase within the carrier has successfully established a semiartificial photosynthetic model characterized by high product specificity and selectivity. In this model system, electrons were directly transferred to hydrogenase upon AuNPs light excitation in membraneless organelle, eliminating the need for additional electron mediators and bypassing the use of intracellular NADH/NAD+ as a reducing agent to avoid byproduct formation. The semiartificial photosynthetic system demonstrated an 11782-fold and 40-fold increase in hydrogen production compared to bare AuNPs and the empty vector E. coli hybrid system, respectively. This membraneless organelle holds the potential to robustly support the specific synthesis of high-energy-density, value-added compounds through solar energy conversion.

Biomolecular condensates of Chlorocatechol 1,2-Dioxygenase as prototypes of enzymatic microreactors for the degradation of polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are molecules with two or more fused aromatic rings that occur naturally in the environment due to incomplete combustion of organic substances. However, the increased demand for fossil fuels in recent years has increased anthropogenic activity, contributing to the environmental concentration of PAHs. The enzyme chlorocatechol 1,2-dioxygenase from Pseudomonas putida (Pp 1,2-CCD) is responsible for the breakdown of the aromatic ring of catechol, making it a potential player in bioremediation strategies. Pp 1,2-CCD can tolerate a broader range of substrates, including halogenated compounds, than other dioxygenases. Here, we report the construction of a chimera protein able to form biomolecular condensates with potential application in bioremediation. The chimera protein was built by conjugating Pp 1,2-CCD to low complex domains (LCDs) derived from the DEAD-box protein Dhh1. We showed that the chimera could undergo liquid-liquid phase separation (LLPS), forming a protein-rich liquid droplet under different conditions (variable protein and PEG8000 concentrations and pH values), in which the protein maintained its structure and main biophysical properties. The condensates were active against 4-chlorocatechol, showing that the chimera droplets preserved the enzymatic activity of the native protein. Therefore, it constitutes a prototype of a microreactor with potential use in bioremediation.

Spatially heterogeneous dynamics of droplets formed by arginine-rich dipeptides and poly-A RNA during liquid-liquid phase separation

Arginine-rich dipeptides (proline-arginine (PR)) are highly toxic products encoded by aberrant repeat expansion in the C9ORF72 gene, which is the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These dipeptides have the capability to undergo liquid-liquid phase separation (LLPS) with components inside the membrane-less organelles (MLO) that could have a detrimental effect on fundamental cellular processes, potentially leading to injury or death in motor neurons. In this study, Raman spectroscopy was used to investigate the spatial variation within these liquid droplets in vitro, revealing concentration gradients of RNA in droplets. The findings were compared with fluorescence recovery after photobleaching (FRAP), offering insights into the spatially heterogeneous dynamics within the droplets. Understanding the mechanism is essential for unraveling the pathogenesis of neuronal diseases and may provide future therapeutic strategies.

Ionic Effect on the Microenvironment of Biomolecular Condensates.

Biomolecules such as proteins and RNA could organize to form condensates with distinct microenvironments through liquid-liquid phase separation (LLPS). Recent works have demonstrated that the microenvironment of biomolecular condensates plays a crucial role in mediating biological activities, such as the partition of biomolecules, and the subphase organization of the multiphasic condensates. Ions could influence the phase transition point of LLPS, following the Hofmeister series. However, the ion-specific effect on the microenvironment of biomolecular condensates remains unknown. In this study, we utilized fluorescence lifetime imaging microscopy (FLIM), fluorescence recovery after photobleaching (FRAP), and microrheology techniques to investigate the ion effect on the microenvironment of condensates. We found that ions significantly affect the microenvironment of biomolecular condensates: salting-in ions increase micropolarity and reduce the microviscosity of the condensate, while salting-out ions induce opposing effects. Furthermore, we manipulate the miscibility and multilayering behavior of condensates through ion-specific effects. In summary, our work provides the first quantitative survey of the microenvironment of protein condensates in the presence of ions from the Hofmeister series, demonstrating how ions impact micropolarity, microviscosity, and viscoelasticity of condensates. Our results bear implications on how membrane-less organelles would exhibit varying microenvironments in the presence of continuously changing cellular conditions.

Phase-separated droplets swim to their dissolution

Biological macromolecules can condense into liquid domains. In cells, these condensates form membraneless organelles that can organize chemical reactions. However, little is known about the physical consequences of chemical activity in and around condensates. Working with model bovine serum albumin (BSA) condensates, we show that droplets swim along chemical gradients. Active BSA droplets loaded with urease swim toward each other. Passive BSA droplets show diverse responses to externally applied gradients of the enzyme’s substrate and products. In all these cases, droplets swim toward solvent conditions that favor their dissolution. We call this behavior “dialytaxis”, and expect it to be generic, as conditions which favor dissolution typically reduce interfacial tension, whose gradients are well-known to drive droplet motion through the Marangoni effect. These results could potentially suggest alternative physical mechanisms for active transport in living cells, and may enable the design of fluid micro-robots.

Enhanced Catalytic Activity of a de novo Enzyme in a Coacervate Phase

Biomolecular condensates are membraneless organelles that orchestrate various metabolic pathways in living cells. Understanding how these crowded structures regulate enzyme reactions remains yet challenging due to their dynamic and intricate nature. Coacervate microdroplets formed by associative liquid‐liquid phase separation of oppositely charged polyions have emerged as relevant condensate models to study enzyme catalysis. Enzyme reactions within these droplets show altered kinetics, influenced by factors such as enzyme and substrate partitioning, crowding, and interactions with coacervate components; it is often challenging to disentangle the contributions of each. Here, we investigate the peroxidase activity of a de novo enzyme within polysaccharide‐based coacervates. By comparing the reaction kinetics in buffer, in a suspension of coacervates and in the bulk coacervate phase collected after centrifugation of the droplets, we show that the coacervate phase significantly increases the enzyme catalytic efficiency. We demonstrate that the main origin of this enhanced activity lies in macromolecular crowding coupled to changes in the conformational dynamics of the enzyme within the coacervate environment. Altogether, these findings underline the crucial role of the coacervate matrix in enzyme catalysis, beyond simple partitioning effects. The observed boost in enzyme activity within the coacervate phase provides insights for designing biocatalytically active synthetic organelles.

Altering biomolecular condensates as a potential mechanism that mediates cannabidiol effect on glioblastoma.

Glioblastoma (GBM) is an extremely aggressive primary brain tumor with poor prognosis, short survival time post-diagnosis and high recurrence. Currently, no cure for GBM exists. The identification of an effective therapeutic modality for GBM remains a high priority amongst medical professionals and researches. In recent studies, inhalant cannabidiol (CBD) has demonstrated promise in effectively inhibiting GBM tumor growth. However, exactly how CBD treatment affects the physiology of these tumor cells remains unclear. Stress granules (SG) (a sub-class of biomolecular condensates (BMC)) are dynamic, membrane-less intracellular microstructures which contain proteins and nucleic acids. The formation and signaling of SGs and BMCs plays a significant role in regulating malignancies. This study investigates whether inhaled CBD may play an intervening role towards SGs in GBM tumor cells. Integrated bioinformatics approaches were preformed to gain further insights. This includes use of Immunohistochemistry and flow cytometry to measure SGs, as well as expression and phosphorylation of eukaryotic initiation factor-2α (eIF2α). The findings of this study reveal that CBD receptors (and co-regulated genes) have the potential to play an important biological role in the formation of BMCs within GBM. In this experiment, CBD treatment significantly increased the volume of TIAR-1. This increase directly correlated with elevation in both eIF2α expression and p-eIF2α in CBD treated tissues in comparison to the placebo group (p < 0.05). These results suggest that inhalant CBD significantly up-regulated SGs in GBM, and thus support a theory of targeting BMCs as a potential therapeutic substrate for treating GBM.

Chaperone regulation of biomolecular condensates

Biomolecular condensation allows for the dynamic organization of molecules in time and space. Condensate formation is regulated through many mechanisms including the action of molecular chaperones. While molecular chaperones have long been viewed through the lens of their roles in protein folding, misfolding, and quality control, their ability to manipulate protein-protein interactions is increasingly recognized to play a major role in the precise control of condensate biology. In this review we highlight recent studies investigating the roles of canonical and non-canonical chaperones in regulating condensate formation, material state, and dispersal. We discuss the broadening of longstanding conceptions of chaperone functions to include condensate regulation, and the discovery of previously unappreciated chaperone activities in well-known proteins. We close by considering the biological activities being uncovered during the ongoing upheaval at the boundary between chaperone biology and biomolecular condensation.

Spatial heterogeneity in the photobiology of phototrophs—questions and methods

Photosynthesis which harvests energy from the Sun, whether it occurs in prokaryotes or eukaryotes, is a key biological process that ultimately nourishes the biosphere. The molecular efficiencies of the photo-physical and physiological processes are intricately tied not only to the photo-physics/enzymatic kinetics of the proteins involved, but also to their spatial co-localization in membrane microdomains or in cell compartments (e.g., in membrane-less organelles). Similar heterogeneity in function can be found also between cells in isogenic cell cultures (phenotypic heterogeneity) or in filaments of phototrophic cells (e.g., heterocysts/vegetative cells in nitrogen fixing cyanobacteria). This review paper delves into the connection between the spatial (co)-localization of biomolecules (lipids, RNA, DNA, proteins, membranes compartments) and their functionality in situ. We highlight recent methodological advances in the field (e.g., super-resolution microscopy, Raman micro-spectroscopy, nanoSIMS, microsensors) and showcase applications of these methods in understanding heterogeneity on single-cell and on population-scale level. This paper thus aims to highlight the avenues that will help to unravel the molecular, cellular and ecological mechanisms in photobiology by combining up-to-date microscopy techniques with more traditional functional approaches.

YAP condensates are highly organized hubs

YAP/TEAD signaling is essential for organismal development, cell proliferation, and cancer progression. As a transcriptional coactivator, how YAP activates its downstream target genes is incompletely understood. YAP forms biomolecular condensates in response to hyperosmotic stress, concentrating transcription-related factors to activate downstream target genes. However, whether YAP forms condensates under other signals, how YAP condensates organize and function, and how YAP condensates activate transcription in general are unknown. Here, we report that endogenous YAP forms sub-micron scale condensates in response to Hippo pathway regulation and actin cytoskeletal tension. YAP condensates are stabilized by the transcription factor TEAD1, and recruit BRD4, a coactivator that is enriched at active enhancers. Using single-particle tracking, we found that YAP condensates slowed YAP diffusion within condensate boundaries, a possible mechanism for promoting YAP target search. These results reveal that YAP condensate formation is a highly regulated process that is critical for YAP/TEAD target gene expression.

The interplay between calcineurin inhibitors (CNIs) and WNK bodies

KS-WNK1 is a kidney-specific scaffolding isoform of the With-No-Lysine (WNK) kinase signaling pathway. It is the predominant WNK1 isoform in the kidney, lacks a functional kinase domain, and is highly expressed in the distal convoluted tubule (DCT) where it drives the formation of biomolecular condensates called WNK bodies. During hypokalemia, WNK bodies bring together KS-WNK1, L-WNK1, WNK4, and SPAK, facilitating activation of the kinase cascade. This leads to increased downstream phosphorylation of the SLC12 cotransporter NCC. Mechanisms governing the reversibility and size of WNK body condensate scaffolds are poorly understood. We propose that phosphatases regulate KS-WNK1 and WNK body formation--a likely candidate being the phosphatase calcineurin (PP3). Prior work suggests that calcineurin dephosphorylates and inhibits the WNK-SPAK-NCC pathway, whereas calcineurin inhibitors (CNIs) such as tacrolimus increase phosphorylation and activation of the pathway. Here, we tested the hypothesis that CNIs also increase the abundance of KS-WNK1, causing increased WNK body size and abundance, and activation of the WNK-SPAK-NCC pathway. To study the effect of endogenous WNK kinases on KS-WNK1 function, WT HEK293 cells or HEK293 L-WNK1 KO cells were transiently transfected with KS-WNK1-HA-mRuby, treated with tacrolimus and studied by immunofluorescence confocal microscopy and immunoblotting of supernatant and detergent-insoluble pellet fractions. In both WT and KO cells, tacrolimus significantly increased WNK body abundance and size. Consistent with its localization within WNK bodies, KS-WNK1 protein was enriched in the pellet fraction by 10-fold in both WT and KO cells. KS-WNK1 protein abundance was increased by tacrolimus in a dose- (0-400ng/ml) and time-dependent manner (0-72h), an effect that was independent of endogenous L-WNK1 expression. Consistent with previous studies, SPAK abundance and phosphorylation was increased with tacrolimus treatment in a dose and time-dependent manner. Interestingly, SPAK localization was shifted into the pellet fraction in WT cells expressing KS-WNK1, whereas it was localized in the supernatant in L-WNK1 KO cells. The tacrolimus-induced increase in KS-WNK1 and pSPAK correlated with increased SLC12 cotransporter phosphorylation in WT cells. These preliminary results suggest that tacrolimus promotes WNK body formation and activity via KS-WNK1, and that SPAK localization in HEK cell-reconstituted WNK bodies requires full length WNK1. There is growing evidence that biomolecular condensates concentrate biomolecules to optimize signaling pathways and enhance kinase specificity through spatiotemporal control. Here we propose a novel concept that WNK bodies are biomolecular condensates that are regulated by calcineurin inhibitors. NIH K08DK118211, U54DK137329, ASN Gottschalk Award, P30DK081943. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Essential roles of the nucleolus during early embryonic development: a regulatory hub for chromatin organization.

The nucleolus is the most prominent liquid droplet-like membrane-less organelle in mammalian cells. Unlike the nucleolus in terminally differentiated somatic cells, those in totipotent cells, such as murine zygotes or two-cell embryos, have a unique nucleolar structure known as nucleolus precursor bodies (NPBs). Previously, it was widely accepted that NPBs in zygotes are simply passive repositories of materials that will be gradually used to construct a fully functional nucleolus after zygotic genome activation (ZGA). However, recent research studies have challenged this simplistic view and demonstrated that functions of the NPBs go beyond ribosome biogenesis. In this review, we provide a snapshot of the functions of NPBs in zygotes and early two-cell embryos in mice. We propose that these membrane-less organelles function as a regulatory hub for chromatin organization. On the one hand, NPBs provide the structural platform for centric and pericentric chromatin remodelling. On the other hand, the dynamic changes in nucleolar structure control the release of the pioneer factors (i.e. double homeobox (Dux)). It appears that during transition from totipotency to pluripotency, decline of totipotency and initiation of fully functional nucleolus formation are not independent events but are interconnected. Consequently, it is reasonable to hypothesize that dissecting more unknown functions of NPBs may shed more light on the enigmas of early embryonic development and may ultimately provide novel approaches to improve reprogramming efficiency.

The joint action of yeast eisosomes and membraneless organelles in response to ethanol stress

Elevated ethanol concentrations in yeast affect the plasma membrane. The plasma membrane in yeast has many lipid-protein complexes, such as Pma1 (MCP), Can1 (MCC), and the eisosome complex. We investigated the response of eisosomes, MCPs, and membraneless structures to ethanol stress. We found a correlation between ethanol stress and proton flux with quick acidification of the medium. Moreover, ethanol stress influences the symporter expression in stressed cells. We also suggest that acute stress from ethanol leads to increases in eisosome size and SG number: we hypothesized that eisosomes may protect APC symporters and accumulate an mRNA decay protein in ethanol-stressed cells. Our findings suggest that the joint action of these factors may provide a protective effect on cells under ethanol stress.

Lighting ATR/Chk1 by mesoscale TopBP1 condensates

Biomolecular condensation has gained considerable attention as a fundamental mechanism in cell signaling and various biological processes. A recent study by Egger et al. provides valuable insights into the constituents of topoisomerase IIβ binding protein 1 (TopBP1) condensates and sheds light on the mechanism of Chk1 activation by ataxia telangiectasia-mutated and Rad3-related (ATR) at the interface of these condensates.

Abstract 3073: Gene Regulation By RNP Granule Formation In Atherosclerosis

Background: Atherosclerosis, the leading cause of cardiovascular disease related death, is a chronic inflammatory condition incited by the accumulation of low-density lipoproteins (LDL) and recruitment of macrophages in the arterial intima. Macrophages become lipid laden foam cells that persist in the arterial vasculature, inciting chronic inflammation and plaque formation. A better understanding of the mechanisms underlying this maladaptive inflammatory response may reveal novel therapeutic strategies for intervention. Hypothesis: We hypothesize that the transformation into a foam cell state, a process that requires transcriptional rewiring, is facilitated by stress granules (SG). SGs are cytoplasmic membrane-less organelles that sequester translation initiation factors, mRNAs, and noncoding RNAs due to stress. We previously reported that SG formation is increased in plaque macrophages of atherosclerotic mice and can be induced in vitro during cellular cholesterol loading. However, the consequences of SG formation and the transcripts sequestered by these organelles is unknown. Methods and Results: To investigate whether SGs influence macrophage phenotype, we used siRNA to knockdown G3bp1 and treated with Dil-oxLDL. We found that G3bp1 knockdown reduced macrophage lipid loading and induced transcriptional changes associated with canonical M2 anti-inflammatory macrophage polarization. Furthermore, RNA-seq of macrophages treated with si G3bp1 and oxLDL showed downregulation of pathways involved in phagosome formation and S100 family signaling. RNA immunoprecipitation of G3BP1 from macrophages treated with an ER stressor revealed preferential binding of G3BP1 to transcripts implicated in cholesterol metabolism. Altogether, these data support a role for SGs in maintaining a pro-inflammatory state and regulating cholesterol pathways in plaque macrophages. Conclusion: Our data support a role for SGs in repressing the translation of RNA transcripts in lipid-laden foam cells that accumulate in atherosclerotic plaque. Identification of the specific proteins and RNAs sequestered in SGs will provide insight into how they regulate gene expression and cellular phenotypic switching that prevents the resolution of inflammation.

NRPB1 is a WNK-like pseudokinase that modulates the activity of the WNK4-SPAK/OSR1 pathway with implications in the physiology of the distal convoluted tubule

The pseudokinase NRBP1 and the TSC22D protein family have been implicated in the regulation of cell proliferation and protein turnover. Preliminary observations from one of our laboratories (DA) revealed that, during osmotic stress, NRBP1 and TSC22D proteins interact with WNK1 in vitro. Mouse renal transcriptomic databases show that TSC22D1.1 and TSC22D2 expression is enriched in the distal convoluted tubule (DCT) of the nephron, where WNK kinases play an essential role in the regulation of Na+ reabsorption. WNK4 is the mayor catalytically active WNK in this nephron segment. It is known that WNK’s undergo liquid-liquid phase separation and form biomolecular condensates now called “WNK-bodies” that are observed in the DCT under certain conditions. In this work, our objective was to study the expression and subcellular localization of NRBP1 and TSC22D proteins in the kidney and to investigate their effect on the WNK4-SPAK pathway. We hypothesized that NRBP1 and TSC22D proteins may modulate the activity of the WNK-SPAK pathway. By immunofluorescent staining, we confirmed that TSC22D1.1 and TSC22D2 expression is enriched in the DCT. At baseline, they were localized to the apical membrane and, under conditions in which DCT WNK bodies are observed (mice in low K+ diet or mice with Familial Hyperkalemic Hypertension (FHHt)), they colocalized with WNKs and SPAK in the condensates. NRBP1 was also observed in condensates under these conditions. Increased TSC22D1.1 and TSC22D2 abundance was observed in the FHHt mouse model by immunoblot. In HEK293 transfected cells, the co-expression of NRBP1 and TSC22D1.1, TSC22D2, or TSC22D4 with WNK4 and SPAK increased pSPAK levels, as well as WNK4 T-loop phosphorylation. We then investigated the interactions between NRBP1, TSC22D2, and WNK4. The SPAK-WNK interaction is well characterized and involves RFXV motifs present in WNK kinases and a Conserved C-Terminal (CCT) domains present in SPAK. Long TSC22D proteins possess a conserved RFXV motif, NRBP1 possesses a conserved CCT domain, and WNK kinases possess two CCT domains in addition to the RFXV motifs. Thus, we tested the interaction of WNK4 with TSC22D2 and NRBP1 and observed that mutations on the CCT domain of WNK4 dramatically decreased WNK4’s activity and TSC22D2 binding. Interaction with NRBP1 was not affected. However, the overexpression of TSC22D2 and NRBP1 restored WNK4-CCT mutant activity. In conclusion, TSC22D1.1 and TSC22D2 proteins are enriched in the DCT and, under certain conditions that are known to activate the WNK pathway, colocalize with WNK4, SPAK, and NRPB1 in WNK bodies, demonstrating in vivo interaction of these proteins and suggesting a role in DCT physiology. Increased WNK activation is observed in the presence of NRBP1 and TSC22D1.1/TSC22D2. CONACyT, Mexico. IMIN, Mexico. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Distinguishing individual photobodies using Oligopaints reveals thermo-sensitive and -insensitive phytochrome B condensation at distinct subnuclear locations

Photobodies (PBs) are membraneless subnuclear organelles that self-assemble via concentration-dependent liquid-liquid phase separation (LLPS) of the plant photoreceptor and thermosensor phytochrome B (PHYB). The current PHYB LLPS model posits that PHYB phase separates randomly in the nucleoplasm regardless of the cellular or nuclear context. Here, we established a robust Oligopaints method in Arabidopsis to determine the positioning of individual PBs. We show surprisingly that even in PHYB overexpression lines – where PHYB condensation would be more likely to occur randomly – PBs positioned at twelve distinct subnuclear locations distinguishable by chromocenter and nucleolus landmarks, suggesting that PHYB condensation occurs nonrandomly at preferred seeding sites. Intriguingly, warm temperatures reduce PB number by inducing the disappearance of specific thermo-sensitive PBs, demonstrating that individual PBs possess different thermosensitivities. These results reveal a nonrandom PB nucleation model, which provides the framework for the biogenesis of spatially distinct individual PBs with diverse environmental sensitivities within a single plant nucleus.

Dynamic structure of E. coli cytoplasm: supramolecular complexes and cell aging impact spatial distribution and mobility of proteins

Protein diffusion is a critical factor governing the functioning and organization of a cell’s cytoplasm. In this study, we investigate the influence of (poly)ribosome distribution, cell aging, protein aggregation, and biomolecular condensate formation on protein mobility within the E. coli cytoplasm. We employ nanoscale single-molecule displacement mapping (SMdM) to determine the spatial distribution of the proteins and to meticulously track their diffusion. We show that the distribution of polysomes does not impact the lateral diffusion coefficients of proteins. However, the degradation of mRNA induced by rifampicin treatment leads to an increase in protein mobility within the cytoplasm. Additionally, we establish a significant correlation between cell aging, the asymmetric localization of protein aggregates and reduced diffusion coefficients at the cell poles. Notably, we observe variations in the hindrance of diffusion at the poles and the central nucleoid region for small and large proteins, and we reveal differences between the old and new pole of the cell. Collectively, our research highlights cellular processes and mechanisms responsible for spatially organizing the bacterial cytoplasm into domains with different structural features and apparent viscosity.

Probing the surface charge of condensates using microelectrophoresis

Biomolecular condensates play an important role in cellular organization. Coacervates are commonly used models that mimic the physicochemical properties of biomolecular condensates. The surface of condensates plays a key role in governing molecular exchange between condensates, accumulation of species at the interface, and the stability of condensates against coalescence. However, most important surface properties, including the surface charge and zeta potential, remain poorly characterized and understood. The zeta potential of coacervates is often measured using laser doppler electrophoresis, which assumes a size-independent electrophoretic mobility. Here, we show that this assumption is incorrect for liquid-like condensates and present an alternative method to study the electrophoretic mobility of coacervates and in vitro condensate models by microelectrophoresis and single-particle tracking. Coacervates have a size-dependent electrophoretic mobility, originating from their fluid nature, from which a well-defined zeta potential is calculated. Interestingly, microelectrophoresis measurements reveal that polylysine chains are enriched at the surface of polylysine/polyaspartic acid complex coacervates, which causes the negatively charged protein ɑ-synuclein to adsorb and accumulate at the interface. Addition of ATP inverts the surface charge, displaces ɑ-synuclein from the surface and may help to suppress its interface-catalyzed aggregation. Together, these findings show how condensate surface charge can be measured and altered, making this microelectrophoresis platform combined with automated single-particle tracking a promising characterization technique for both biomolecular condensates and coacervate protocells.