RESUMEN
Long-distance RNA transport enables local protein synthesis at metabolically-active sites distant from the nucleus. This process ensures an appropriate spatial organization of proteins, vital to polarized cells such as neurons. Here, we present a mechanism for RNA transport in which RNA granules "hitchhike" on moving lysosomes. In vitro biophysical modeling, live-cell microscopy, and unbiased proximity labeling proteomics reveal that annexin A11 (ANXA11), an RNA granule-associated phosphoinositide-binding protein, acts as a molecular tether between RNA granules and lysosomes. ANXA11 possesses an N-terminal low complexity domain, facilitating its phase separation into membraneless RNA granules, and a C-terminal membrane binding domain, enabling interactions with lysosomes. RNA granule transport requires ANXA11, and amyotrophic lateral sclerosis (ALS)-associated mutations in ANXA11 impair RNA granule transport by disrupting their interactions with lysosomes. Thus, ANXA11 mediates neuronal RNA transport by tethering RNA granules to actively-transported lysosomes, performing a critical cellular function that is disrupted in ALS.
Asunto(s)
Anexinas/metabolismo , Transporte Axonal/fisiología , Gránulos Citoplasmáticos/metabolismo , Lisosomas/metabolismo , ARN/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Animales Modificados Genéticamente , Anexinas/genética , Axones/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Mutación , Unión Proteica , Ratas/embriología , Ratas Sprague-Dawley , Transfección , Pez CebraRESUMEN
Parkinson's disease (PD) is an increasingly prevalent and currently incurable neurodegenerative disorder linked to the accumulation of α-synuclein (αS) protein aggregates in the nervous system. While αS binding to membranes in its monomeric state is correlated to its physiological role, αS oligomerization and subsequent aberrant interactions with lipid bilayers have emerged as key steps in PD-associated neurotoxicity. However, little is known of the mechanisms that govern the interactions of oligomeric αS (OαS) with lipid membranes and the factors that modulate such interactions. This is in large part due to experimental challenges underlying studies of OαS-membrane interactions due to their dynamic and transient nature. Here, we address this challenge by using a suite of microfluidics-based assays that enable in-solution quantification of OαS-membrane interactions. We find that OαS bind more strongly to highly curved, rather than flat, lipid membranes. By comparing the membrane-binding properties of OαS and monomeric αS (MαS), we further demonstrate that OαS bind to membranes with up to 150-fold higher affinity than their monomeric counterparts. Moreover, OαS compete with and displace bound MαS from the membrane surface, suggesting that disruption to the functional binding of MαS to membranes may provide an additional toxicity mechanism in PD. These findings present a binding mechanism of oligomers to model membranes, which can potentially be targeted to inhibit the progression of PD.
Asunto(s)
Membrana Dobles de Lípidos , alfa-Sinucleína , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Humanos , Unión Proteica , Multimerización de ProteínaRESUMEN
The physical characterization of proteins in terms of their sizes, interactions, and assembly states is key to understanding their biological function and dysfunction. However, this has remained a difficult task because proteins are often highly polydisperse and present as multicomponent mixtures. Here, we address this challenge by introducing single-molecule microfluidic diffusional sizing (smMDS). This approach measures the hydrodynamic radius of single proteins and protein assemblies in microchannels using single-molecule fluorescence detection. smMDS allows for ultrasensitive sizing of proteins down to femtomolar concentrations and enables affinity profiling of protein interactions at the single-molecule level. We show that smMDS is effective in resolving the assembly states of protein oligomers and in characterizing the size of protein species within complex mixtures, including fibrillar protein aggregates and nanoscale condensate clusters. Overall, smMDS is a highly sensitive method for the analysis of proteins in solution, with wide-ranging applications in drug discovery, diagnostics, and nanobiotechnology.
Asunto(s)
Proteínas , Imagen Individual de Molécula , Imagen Individual de Molécula/métodos , Proteínas/química , Proteínas/análisis , Soluciones , Difusión , Microfluídica/métodos , Hidrodinámica , Técnicas Analíticas Microfluídicas/métodosRESUMEN
The detection of proteins is of central importance to biomolecular analysis and diagnostics. Typical immunosensing assays rely on surface-capture of target molecules, but this constraint can limit specificity, sensitivity, and the ability to obtain information beyond simple concentration measurements. Here we present a surface-free, single-molecule microfluidic sensing platform for direct digital protein biomarker detection in solution, termed digital immunosensor assay (DigitISA). DigitISA is based on microchip electrophoretic separation combined with single-molecule detection and enables absolute number/concentration quantification of proteins in a single, solution-phase step. Applying DigitISA to a range of targets including amyloid aggregates, exosomes, and biomolecular condensates, we demonstrate that the assay provides information beyond stoichiometric interactions, and enables characterization of immunochemistry, binding affinity, and protein biomarker abundance. Taken together, our results suggest a experimental paradigm for the sensing of protein biomarkers, which enables analyses of targets that are challenging to address using conventional immunosensing approaches.
Asunto(s)
Técnicas Biosensibles , Técnicas Biosensibles/métodos , Inmunoensayo , Biomarcadores/análisis , Amiloide , Microfluídica/métodosRESUMEN
Phase transitions of cellular proteins and lipids play a key role in governing the organisation and coordination of intracellular biology. The frequent juxtaposition of proteinaceous biomolecular condensates to cellular membranes raises the intriguing prospect that phase transitions in proteins and lipids could be co-regulated. Here we investigate this possibility in the ribonucleoprotein (RNP) granule-ANXA11-lysosome ensemble, where ANXA11 tethers RNP granule condensates to lysosomal membranes to enable their co-trafficking. We show that changes to the protein phase state within this system, driven by the low complexity ANXA11 N-terminus, induce a coupled phase state change in the lipids of the underlying membrane. We identify the ANXA11 interacting proteins ALG2 and CALC as potent regulators of ANXA11-based phase coupling and demonstrate their influence on the nanomechanical properties of the ANXA11-lysosome ensemble and its capacity to engage RNP granules. The phenomenon of protein-lipid phase coupling we observe within this system offers an important template to understand the numerous other examples across the cell whereby biomolecular condensates closely juxtapose cell membranes.
RESUMEN
Multisomes are multicompartmental structures formed by a lipid-stabilized network of aqueous droplets, which are contained by an outer oil phase. These biomimetic structures are emerging as a versatile platform for soft matter and synthetic biology applications. While several methods for producing multisomes have been described, including microfluidic techniques, approaches for generating biocompatible, monodisperse multisomes in a reproducible manner remain challenging to implement due to low throughput and complex device fabrication. Here, we report on a robust method for the dynamically controlled generation of multisomes with controllable sizes and high monodispersity from lipid-based double emulsions. The described microfluidic approach entails the use of three different phases forming a water/oil/water (W/O/W) double emulsion stabilized by lipid layers. We employ a gradient of glycerol concentration between the inner core and outer phase to drive the directed osmosis, allowing the swelling of lamellar lipid layers resulting in the formation of small aqueous daughter droplets at the interface of the inner aqueous core. By adding increasing concentrations of glycerol to the outer aqueous phase and subsequently varying the osmotic gradient, we show that key structural parameters, including the size of the internal droplets, can be specifically controlled. Finally, we show that this approach can be used to generate multisomes encapsulating small-molecule cargo, with potential applications in synthetic biology, drug delivery, and as carriers for active materials in the food and cosmetics industries.
Asunto(s)
Lípidos/química , Emulsiones/síntesis química , Emulsiones/química , Membrana Dobles de Lípidos/química , Aceites/química , Tamaño de la Partícula , Propiedades de Superficie , Agua/químicaRESUMEN
Liquid-liquid phase separation of proteins underpins the formation of membraneless compartments in living cells. Elucidating the molecular driving forces underlying protein phase transitions is therefore a key objective for understanding biological function and malfunction. Here we show that cellular proteins, which form condensates at low salt concentrations, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, can reenter a phase-separated regime at high salt concentrations. By bringing together experiments and simulations, we demonstrate that this reentrant phase transition in the high-salt regime is driven by hydrophobic and non-ionic interactions, and is mechanistically distinct from the low-salt regime, where condensates are additionally stabilized by electrostatic forces. Our work thus sheds light on the cooperation of hydrophobic and non-ionic interactions as general driving forces in the condensation process, with important implications for aberrant function, druggability, and material properties of biomolecular condensates.
Asunto(s)
Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Transición de Fase , Proteínas/química , Electricidad Estática , Animales , Anexinas/química , Proteínas de Ciclo Celular/química , Proteínas de Unión al ADN/química , Humanos , Proteína FUS de Unión a ARN/química , Factores de Transcripción SOXB1/química , Células Sf9 , Spodoptera , Factores de Transcripción/químicaRESUMEN
This paper demonstrates a microfluidic system that automates i) formation of a lipid bilayer at the interface between a pair of nanoliter-sized aqueous droplets in oil, ii) exchange of one droplet of the pair to form a new bilayer, and iii) current measurements on single proteins. A new microfluidic architecture is introduced - a set of traps designed to localize the droplets with respect to each other and with respect to the recording electrodes. The system allows for automated execution of experimental protocols by active control of the flow on chip with the use of simple external valves. Formation of stable artificial lipid bilayers, incorporation of α-hemolysin into the bilayers and electrical measurements of ionic transport through the protein pore are demonstrated.
Asunto(s)
Técnicas Electroquímicas/métodos , Proteínas Hemolisinas/análisis , Membrana Dobles de Lípidos/química , Técnicas Analíticas Microfluídicas/instrumentación , Alcanos/química , Automatización , Técnicas Electroquímicas/instrumentación , Electrodos , Proteínas Hemolisinas/metabolismo , Membrana Dobles de Lípidos/metabolismo , Aceites/química , Fosfatidilcolinas/química , Staphylococcus aureus/metabolismoRESUMEN
We demonstrate an integrated system for rapid and automated generation of multiple, chemically distinct populations of ~10(3)-10(4) sub-nanoliter droplets. Generation of these 'libraries of droplets' proceeds in the following automated steps: i) generation of a sequence of micro-liter droplets of individually predetermined composition, ii) injection of these 'parental' droplets onto a chip, iii) transition from a mm- to a µm-scale of the channels and splitting each of the parental drops with a flow-focusing module into thousands of tightly monodisperse daughter drops and iv) separation of such formed homogeneous populations with plugs of a third immiscible fluid. This method is compatible both with aspiration of microliter portions of liquid from a 96-well plate with a robotic station and with automated microfluidic systems that generate (~µL) droplets of preprogrammed compositions. The system that we present bridges the techniques that provide elasticity of protocols executed on microliter droplets with the techniques for high-throughput screening of small (~pL, ~nL) droplet libraries. The method that we describe can be useful in exploiting the synergy between the ability to rapidly screen distinct chemical environments and to perform high-throughput studies of single cells or molecules and in digital droplet PCR systems.