Author
Miriam Linsenmeier
Other affiliations: University of California, Los Angeles
Bio: Miriam Linsenmeier is an academic researcher from ETH Zurich. The author has contributed to research in topics: DEAD box & Analyte. The author has an hindex of 4, co-authored 11 publications receiving 91 citations. Previous affiliations of Miriam Linsenmeier include University of California, Los Angeles.
Topics: DEAD box, Analyte, Protein aggregation, RNA
Papers
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TL;DR: It is shown that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1, thereby aiding the assembly of large multivalent mRNP granules that are PBs.
Abstract: Processing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid-liquid phase separation and are implicated in the decay or storage of mRNAs. How PB assembly is regulated in cells remains unclear. Previously, we identified the ATPase activity of the DEAD-box protein Dhh1 as a key regulator of PB dynamics and demonstrated that Not1, an activator of the Dhh1 ATPase and member of the CCR4-NOT deadenylase complex inhibits PB assembly in vivo (Mugler et al., 2016). Here, we show that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1. Intriguingly, in vivo PB dynamics can be recapitulated in vitro, since Pat1 enhances the phase separation of Dhh1 and RNA into liquid droplets, whereas Not1 reverses Pat1-Dhh1-RNA condensation. Overall, our results uncover a function of Pat1 in promoting the multimerization of Dhh1 on mRNA, thereby aiding the assembly of large multivalent mRNP granules that are PBs.
48 citations
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TL;DR: The microfluidics approach represents an attractive platform to investigate the dynamics of compartmentalization in artificial cells in the absence and presence of network structures and shows that the characteristic time scale of phase separation decreases linearly with increasing the volume of the compartment.
Abstract: Cells can form membraneless organelles by liquid-liquid phase separation. As these organelles are highly dynamic, it is crucial to understand the kinetics of these phase transitions. Here, we use droplet-based microfluidics to mix reagents by chaotic advection and observe nucleation, growth, and coarsening in volumes comparable to cells (pL) and on timescales of seconds. We apply this platform to analyze the dynamics of synthetic organelles formed by the DEAD-box ATPase Dhh1 and RNA, which are associated with the formation of processing bodies in yeast. We show that the timescale of phase separation decreases linearly as the volume of the compartment increases. Moreover, the synthetic organelles coarsen into one single droplet via gravity-induced coalescence, which can be arrested by introducing a hydrogel matrix that mimics the cytoskeleton. This approach is an attractive platform to investigate the dynamics of compartmentalization in artificial cells.
46 citations
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TL;DR: A droplet microfluidic platform to increase the concentration of analytes in solution via reduction of the sample volume under well-defined conditions is developed and the droplet concentrator device, or DroMiCo, can quantify unlabeled proteins in nM concentrations and analyze multicomponent mixtures when coupled with a prefractionation step.
Abstract: We develop a droplet microfluidic platform to increase the concentration of analytes in solution via reduction of the sample volume under well-defined conditions. This approach improves the detection and quantification of analytes without requiring any a priori information on their structure nor physical chemical properties. Samples are compartmentalized and processed in water-in-oil droplets that are individually stored in cylindrical microwells located on top of a microfluidic channel. The individual droplets shrink over time due to water extraction in the surrounding oil, leading to an increase in the analyte concentration up to 100,000-fold within the droplet. We demonstrate the power of this approach for detection applications by quantifying a broad range of single analytes such as small molecules, proteins, nanoparticles, exosomes, and amyloid fibrils. With this setup, we can measure pM concentrations, corresponding to zeptomole (10-21 mol) amounts encapsulated in individual droplets. We further show that the droplet concentrator device, or DroMiCo, can quantify unlabeled proteins in nM concentrations and analyze multicomponent mixtures when coupled with a prefractionation step. We illustrate this concept by detecting femtomoles (10-15 mol) of soluble protein oligomers prefractionated by size exclusion chromatography. Finally, we apply the DroMiCo to the analysis of phase diagrams of macromolecules, including synthetic polymers and proteins. Specifically, we analyze the liquid-liquid phase separation of an in vitro model of cellular membraneless compartments, composed of a phase separating protein in the presence of defined concentrations of molecular modulators such as RNA and ATP.
33 citations
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TL;DR: The advantages of microfluidic technology for the analysis of several aspects of phase separation, including phase diagrams, dynamics of assembly and disassembly, rheological and surface properties, exchange of materials with the surrounding environment and the coupling between compartmentalization and biochemical reactions are described.
26 citations
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TL;DR: It is demonstrated that biomolecular condensates could sequester aggregation-prone proteins and prevent aberrant aggregation events, despite the local increase in their concentration, since the heterogenous composition of the condensate could prevent the formation of ordered fibrillar aggregates.
Abstract: Biomolecular condensates are emerging as an efficient strategy developed by cells to control biochemical reactions in space and time by locally modifying composition and environment. Yet, local increase in protein concentration within these compartments could promote aberrant aggregation events, including the nucleation and growth of amyloid fibrils. Understanding protein stability within the crowded and heterogeneous environment of biological condensates is therefore crucial, not only when the aggregation-prone protein is the scaffold element of the condensates but also when proteins are recruited as client molecules within the compartments. Here, we investigate the partitioning and aggregation kinetics of the amyloidogenic peptide Abeta42 (Aβ-42), the peptide strongly associated with Alzheimer's disease, recruited into condensates based on low complexity domains (LCDs) derived from the DEAD-box proteins Laf1, Dbp1 and Ddx4, which are associated with biological membraneless organelles. We show that interactions between Aβ-42 and the scaffold proteins promote sequestration and local increase of the peptide concentration within the condensates. Yet, heterotypic interactions within the condensates inhibit the formation of amyloid fibrils. These results demonstrate that biomolecular condensates could sequester aggregation-prone proteins and prevent aberrant aggregation events, despite the local increase in their concentration. Biomolecular condensates could therefore work not only as hot-spots of protein aggregation but also as protective reservoirs, since the heterogenous composition of the condensates could prevent the formation of ordered fibrillar aggregates.
25 citations
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01 Jul 1986
TL;DR: Structures in Other Domains The methodology of structural analysis discussed in this article has been applied beyond the narrow realm of natural language syntax that we have discussed in this paper, and it has been found that variation in the types of sentences that are used, whether during the course of children's acquisition of their native languages or in the centuries-long periods of linguistic change, are best characterized not as super cial and haphazard alterations, but rather in terms of parametric modi cations to the fundamental underlying grammatical rules and constraints.
Abstract: Structures in Other Domains The methodology of structural analysis discussed in this article has been applied beyond the narrow realm of natural language syntax that we have discussed in this article. Within the study of language, similar methods of analysis have been pervasively applied to the study of sounds (phonology), words (morphology), and meanings (semantics), yielding a range of of abstract structural representations whose properties bear considerable explanatory burden. There are a wealth of cases in each of these domains analogous to those discussed here, though space prevents us from going in these (see Akmajian, Demers, Farmer and Harnish 1995 for a traditional overview, and Jackendo 1994 for one more focused on connections with cognitive science). Additionally, these representations have shed substantial light on the processes of language acquisition and language change. It has been found that variation in the types of sentences that are used, whether during the course of children's acquisition of their native languages or in the centuries-long periods of linguistic change, are best characterized not as super cial and haphazard alterations, but rather in terms of parametric modi cations to the fundamental underlying grammatical rules and constraints. Moving outside the domain of language, one application of these same methods has been in the study of music cognition. Just as the representations of linguistic theory arise out of an attempt to model speakers' intuitions about well-formedness and possible meanings of the sentences of their
761 citations
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01 Nov 2020TL;DR: This Review describes how synthetic peptides afford tunable scaffolds for biomineralization, drug delivery and tissue growth and discusses recent conceptual and experimental advances in self-assembling artificial peptidic materials.
Abstract: Natural biomolecular systems have evolved to form a rich variety of supramolecular materials and machinery fundamental to cellular function. The assembly of these structures commonly involves interactions between specific molecular building blocks, a strategy that can also be replicated in an artificial setting to prepare functional materials. The self-assembly of synthetic biomimetic peptides thus allows the exploration of chemical and sequence space beyond that used routinely by biology. In this Review, we discuss recent conceptual and experimental advances in self-assembling artificial peptidic materials. In particular, we explore how naturally occurring structures and phenomena have inspired the development of functional biomimetic materials that we can harness for potential interactions with biological systems. As our fundamental understanding of peptide self-assembly evolves, increasingly sophisticated materials and applications emerge and lead to the development of a new set of building blocks and assembly principles relevant to materials science, molecular biology, nanotechnology and precision medicine. The self-assembly of biomimetic peptides can mimic complex natural systems involving whole proteins. This Review describes how synthetic peptides afford tunable scaffolds for biomineralization, drug delivery and tissue growth.
175 citations
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TL;DR: An important role is established for eIF4A, and potentially other DEAD-box proteins, as ATP-dependent RNA chaperones that limit the condensation of RNA, analogous to the function of proteins like HSP70 in combatting protein aggregates.
154 citations
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105 citations
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TL;DR: An emerging all‐aqueous microfluidic technology derived from micrometer‐scaled manipulation of LLPS is presented and enables the state‐of‐art design of advanced biomaterials with exquisite structural proficiency and diversified biological functions.
Abstract: Living cells have evolved over billions of years to develop structural and functional complexity with numerous intracellular compartments that are formed due to liquid-liquid phase separation (LLPS). Discovery of the amazing and vital roles of cells in life has sparked tremendous efforts to investigate and replicate the intracellular LLPS. Among them, all-aqueous emulsions are a minimalistic liquid model that recapitulates the structural and functional features of membraneless organelles and protocells. Here, an emerging all-aqueous microfluidic technology derived from micrometer-scaled manipulation of LLPS is presented; the technology enables the state-of-art design of advanced biomaterials with exquisite structural proficiency and diversified biological functions. Moreover, a variety of emerging biomedical applications, including encapsulation and delivery of bioactive gradients, fabrication of artificial membraneless organelles, as well as printing and assembly of predesigned cell patterns and living tissues, are inspired by their cellular counterparts. Finally, the challenges and perspectives for further advancing the cell-inspired all-aqueous microfluidics toward a more powerful and versatile platform are discussed, particularly regarding new opportunities in multidisciplinary fundamental research and biomedical applications.
98 citations