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.

Application of Novel Short Tandem Repeat Typing for Wickerhamomyces anomalus Reveals Simultaneous Outbreaks within a Single Hospital.

Wickerhamomyces anomalus, previously known as Candida pelliculosa, occasionally causes candidemia in humans, primarily infecting neonates, and infants. The mortality rate of these invasive infections is high, and isolates with a reduced susceptibility to fluconazole have been reported. W. anomalus outbreaks are regularly reported in healthcare facilities, especially in neonatal intensive care units (NICUs). In order to rapidly genotype isolates with a high-resolution, we developed and applied a short tandem repeat (STR) typing scheme for W. anomalus. Six STR markers were selected and amplified in two multiplex PCRs, M3 and M6, respectively. In total, 90 W. anomalus isolates were typed, leading to the identification of 38 different genotypes. Four large clusters were found, unveiling simultaneous outbreak events spread across multiple units within the same hospital. STR typing results of 11 isolates were compared to whole-genome sequencing (WGS) single nucleotide polymorphism (SNP) calling, and the identified genotypic relationships were highly concordant. We performed antifungal susceptibility testing of these isolates, and a reduced susceptibility to fluconazole was found for two (2.3%) isolates. ERG11 genes of these two isolates were examined using WGS data, which revealed a novel I469L substitution in one isolate. By constructing a homology model for W. anomalus ERG11p, the substitution was found in close proximity to the fluconazole binding site. In summary, we showed multiple W. anomalus outbreak events by applying a novel STR genotyping scheme.

Structure-Property Relationships Governing Membrane-Penetrating Behaviour of Complex Coacervates.

Complex coacervates are phase-separated liquid droplets composed of oppositely charged multivalent molecules. The unique material properties of the complex coacervate interior favours the sequestration of biomolecules and facilitates reactions. Recently, it is shown that coacervates can be used for direct cytosolic delivery of sequestered biomolecules in living cells. Here, it is studied that the physical properties required for complex coacervates composed of oligo-arginine and RNA to cross phospholipid bilayers and enter liposomes penetration depends on two main parameters: the difference in ζ-potential between the complex coacervates and the liposomes, and the partitioning coefficient (Kp ) of lipids into the complex coacervates. Following these guidelines, a range of complex coacervates is found that is able to penetrate the membrane of living cells, thus paving the way for further development of coacervates as delivery vehicles of therapeutic agents.

Development and Application of a Short Tandem Repeat Multiplex Typing Assay for Candida tropicalis.

Candida tropicalis is a clinically important yeast that causes candidemia in humans with a high mortality rate. The yeast primarily infects immunocompromised patients, and causes outbreaks in health care facilities. Antifungal resistant isolates have been reported. We developed a short tandem repeat (STR) typing scheme for C. tropicalis to enable fast, cost-effective, and high-resolution genotyping. For the development of the typing scheme, 6 novel STR markers were selected, combined into 2 multiplex PCRs. In total, 117 C. tropicalis isolates were typed, resulting in the identification of 104 different genotypes. Subsequently, the outcome of STR typing of 10 isolates was compared to single nucleotide polymorphism (SNP) calling from whole-genome sequencing (WGS). Isolates with more than 111 SNPs were differentiated by the typing assay. Two isolates, which were identical according to SNP analysis, were separated by STR typing in 1 marker. To test specificity, the STR typing was applied to 15 related yeast species, and we found no amplification of these targets. For reproducibility testing, 2 isolates were independently typed five times, which showed identical results in each experiment. In summary, we developed a reliable and multiplex STR genotyping for C. tropicalis, which was found to correlate well to SNP calling by WGS. WGS analysis from and extensive collection of isolates is required to establish the precise resolution of this STR assay. IMPORTANCE Candida tropicalis frequently causes candidemia in immunocompromised patients. C. tropicalis infections have a high mortality rate, and the yeast is able to cause outbreaks in health care facilities. Further, antifungal resistant isolates are on the rise. Genotyping is necessary to investigate potential outbreaks. Here, we developed and applied a STR genotyping scheme in order to rapidly genotype isolates with a high-resolution. WGS SNP outcomes were highly comparable with STR typing results. Altogether, we developed a rapid, high-resolution, and specific STR genotyping scheme for C. tropicalis.

Growth, replication and division enable evolution of coacervate protocells.

Living and proliferating cells undergo repeated cycles of growth, replication and division, all orchestrated by complex molecular networks. How a minimal cell cycle emerged and helped primitive cells to evolve remains one of the biggest mysteries in modern science, and is an active area of research in chemistry. Protocells are cell-like compartments that recapitulate features of living cells and may be seen as the chemical ancestors of modern life. While compartmentalization is not strictly required for primitive, open-ended evolution of self-replicating systems, it gives such systems a clear identity by setting the boundaries and it can help them overcome three major obstacles of dilution, parasitism and compatibility. Compartmentalization is therefore widely considered to be a central hallmark of primitive life, and various types of protocells are actively investigated, with the ultimate goal of developing a protocell capable of autonomous proliferation by mimicking the well-known cell cycle of growth, replication and division. We and others have found that coacervates are promising protocell candidates in which chemical building blocks required for life are naturally concentrated, and chemical reactions can be selectively enhanced or suppressed. This feature article provides an overview of how growth, replication and division can be realized with coacervates as protocells and what the bottlenecks are. Considerations are given for designing chemical networks in coacervates that can lead to sustained growth, selective replication and controlled division, in a way that they are linked together like in the cell cycle. Ultimately, such a system may undergo evolution by natural selection of certain phenotypes, leading to adaptation and the gain of new functions, and we end with a brief discussion of the opportunities for coacervates to facilitate this.

ATP:Mg2+ shapes material properties of protein-RNA condensates and their partitioning of clients.

Many cellular condensates are heterotypic mixtures of proteins and RNA formed in complex environments. Magnesium ions (Mg2+) and ATP can impact RNA folding, and local intracellular levels of these factors can vary significantly. However, the effect of ATP:Mg2+ on the material properties of protein-RNA condensates is largely unknown. Here, we use an in vitro condensate model of nucleoli, made from nucleophosmin 1 (NPM1) proteins and ribosomal RNA (rRNA), to study the effect of ATP:Mg2+. While NPM1 dynamics remain unchanged at increasing Mg2+ concentrations, the internal RNA dynamics dramatically slowed until a critical point, where gel-like states appeared, suggesting the RNA component alone forms a viscoelastic network that undergoes maturation driven by weak multivalent interactions. ATP reverses this arrest and liquefies the gel-like structures. ATP:Mg2+ also influenced the NPM1-rRNA composition of condensates and enhanced the partitioning of two clients: an arginine-rich peptide and a small nuclear RNA. By contrast, larger ribosome partitioning shows dependence on ATP:Mg2+ and can become reversibly trapped around NPM1-rRNA condensates. Lastly, we show that dissipative enzymatic reactions that deplete ATP can be used to control the shape, composition, and function of condensates. Our results illustrate how intracellular environments may regulate the state and client partitioning of RNA-containing condensates.

Development of a Multiplex PCR Short Tandem Repeat Typing Scheme for Candida krusei.

Candida krusei is a human-pathogenic yeast that can cause candidemia with the lowest 90-day survival rate in comparison to other Candida species. Infections occur frequently in immunocompromised patients, and several C. krusei outbreaks in health care facilities have been described. Here, we developed a short tandem repeat (STR) typing scheme for C. krusei to allow the fast and cost-effective genotyping of an outbreak and compared the identified relatedness of 10 isolates to single nucleotide polymorphism (SNP) calling from whole-genome sequencing (WGS). From a selection of 14 novel STR markers, 6 were used to develop two multiplex PCRs. Additionally, three previously reported markers were selected for a third multiplex PCR. In total, 119 C. krusei isolates were typed using these nine markers, and 79 different genotypes were found. STR typing correlated well with WGS SNP typing, as isolates with the same STR genotype varied by 8 and 19 SNPs, while isolates that differed in all STR markers varied by at least tens of thousands of SNPs. The STR typing assay was found to be specific for C. krusei, stable in 100 subcloned generations, and comparable to SNP calling by WGS. In summary, this newly developed C. krusei STR typing scheme is a fast, reliable, easy-to-interpret, and cost-effective method compared to other typing methods. Moreover, the two newly developed multiplexes showed the same discriminatory power as all nine markers combined, indicating that multiplexes M3-1 and M9 are sufficient to type C. krusei.

Active coacervate droplets are protocells that grow and resist Ostwald ripening.

Active coacervate droplets are liquid condensates coupled to a chemical reaction that turns over their components, keeping the droplets out of equilibrium. This turnover can be used to drive active processes such as growth, and provide an insight into the chemical requirements underlying (proto)cellular behaviour. Moreover, controlled growth is a key requirement to achieve population fitness and survival. Here we present a minimal, nucleotide-based coacervate model for active droplets, and report three key findings that make these droplets into evolvable protocells. First, we show that coacervate droplets form and grow by the fuel-driven synthesis of new coacervate material. Second, we find that these droplets do not undergo Ostwald ripening, which we attribute to the attractive electrostatic interactions and translational entropy within complex coacervates, active or passive. Finally, we show that the droplet growth rate reflects experimental conditions such as substrate, enzyme and protein concentration, and that a different droplet composition (addition of RNA) leads to altered growth rates and droplet fitness. These findings together make active coacervate droplets a powerful platform to mimic cellular growth at a single-droplet level, and to study fitness at a population level.