生物物理学

Biomolecular condensates are macromolecular assemblies constituted of proteins that possess intrinsically disordered regions and RNA-binding ability together with nucleic acids. These compartments formed via liquid-liquid phase separation (LLPS) provide spatiotemporal control of crucial cellular processes such as RNA metabolism. The liquid-like state is dynamic and reversible, containing highly diffusible molecules, whereas gel, glass, and solid phases might not be reversible due to the strong intermolecular crosslinks. Neurodegeneration-associated proteins such as the prion protein (PrP) and Tau form liquid-like condensates that transition to gel- or solid-like structures upon genetic mutations and/or persistent cellular stress. Mounting evidence suggests that progression to a less dynamic state underlies the formation of neurotoxic aggregates. Understanding the dynamics of proteins and biomolecules in condensates by measuring their movement in different timescales is indispensable to characterize their material state and assess the kinetics of LLPS. Herein, we describe protein expression in E. coli and purification of full-length mouse recombinant PrP, our in vitro experimental system. Then, we describe a systematic method to analyze the dynamics of protein condensates by X-ray photon correlation spectroscopy (XPCS). We also present fluorescence recovery after photobleaching (FRAP)-optimized protocols to characterize condensates, including in cells. Next, we detail strategies for using fluorescence microscopy to give insights into the folding state of proteins in condensates. Phase-separated systems display non-equilibrium behavior with length scales ranging from nanometers to microns and timescales from microseconds to minutes. XPCS experiments provide unique insights into biomolecular dynamics and condensate fluidity. Using the combination of the three strategies detailed herein enables robust characterization of the biophysical properties and the nature of protein phase-separated states.

Dual-modal imaging, combining photoacoustic (PA) and ultrasound localization (UL) with microbubbles, holds substantial promise across biomedical fields such as oncology, neuroscience, nephrology, and immunology. The combination of PA and UL imaging faces challenges due to acquisition speed mismatches, limiting their combined efficacy. Here, we introduce a protocol that applies sparsity constraint optimization to accelerate dual-modal data acquisition, enabling in vivo super-resolution imaging of vascular and physiological structures at under two seconds per frame. The protocol provides detailed guidelines for constructing an interleaved PA/UL (PAUL) imaging system, covering material selection, system setup, and calibration, as well as methods for image acquisition, reconstruction, post-processing, and troubleshooting. This approach empowers the biomedical community to establish a rapid, dual-modal PAUL imaging platform, broadening biomedical applications and advancing imaging capabilities in clinical research.

The reduction in intracellular neuronal chloride concentration is a crucial event during neurodevelopment that shifts GABAergic signaling from depolarizing to hyperpolarizing. Alterations in chloride homeostasis are implicated in numerous neurodevelopmental disorders, including autism spectrum disorder (ASD). Recent advancements in biosensor technology allow the simultaneous determination of intracellular chloride concentration of multiple neurons. Here, we describe an optimized protocol for the use of the ratiometric chloride sensor SuperClomeleon (SClm) in organotypic hippocampal slices. We record chloride levels as fluorescence responses of the SClm sensor using two-photon microscopy. We discuss how the SClm sensor can be effectively delivered to specific cell types using virus-mediated transduction and describe the calibration procedure to determine the chloride concentration from SClm sensor responses.

Changes in neuronal conduction are common in disease states affecting peripheral nerves. These alterations can significantly impact nerve function and lead to sensorimotor disabilities. In vivo electromyography recording is a well-established electrophysiological method that has been used for decades to assess sensory and motor functions in the nervous system. Nerve studies are challenging to conduct in vivo in rodents, and the involvement of muscle activity makes it difficult to isolate and assess nerve function independently. This protocol provides a comprehensive guide for accurate ex vivo sciatic nerve dissection and handling from mice. It includes the creation of a three-compartment chamber and the establishment of electrophysiological protocols, which enable differential recordings and the analysis of compound action potentials from various nerve fibers. This setup allows researchers to study the specific effects of drugs and pathologies on nerves from a mechanistic perspective. The setup is a stand-alone apparatus that does not require the use of suction electrodes and the maintenance of negative pressure, which can affect the signal-to-noise ratio and recording stability.

Time-resolved cryo-EM (TRCEM) makes it possible to provide structural and kinetic information on a reaction of biomolecules before the equilibrium is reached. Several TRCEM methods have been developed in the past to obtain key insights into the mechanism of action of molecules and molecular machines on the time scale of tens to hundreds of milliseconds, which is unattainable by the normal blotting method. Here, we present our TRCEM setup utilizing a polydimethylsiloxane (PDMS)-based microfluidics chip assembly, comprising three components: a PDMS-based, internally SiO₂-coated micromixer, a glass-capillary microreactor, and a PDMS-based microsprayer for depositing the reaction product onto the EM grid. As we have demonstrated in recent experiments, this setup is capable of addressing problems of severe sample adsorption and ineffective mixing of fluids and leads to highly reproducible results in applications to the study of translation. As an example, we used our TRCEM sample preparation method to investigate the molecular mechanism of ribosome recycling mediated by High frequency of lysogenization X (HflX), which demonstrated the efficacy of the TRCEM device and its capability to yield biologically significant, reproducible information. This protocol has the promise to provide structural and kinetic information on pre-equilibrium intermediates in the 10–1,000 ms time range in applications to many other biological systems.

Micro-computed tomography (micro-CT) is a powerful, non-destructive imaging technique that creates high-resolution 3D images of the internal structures of small animal models such as mice and rats. Familiarizing oneself with micro-CT imaging and data analysis can be overwhelming without easy-to-follow, clear instructions. Training on new instruments is often a task exclusive to a select subset of researchers, leaving the majority of potential trainees without a technical grasp of how to navigate the instructions. This protocol on the use of micro-CT aims to bridge that gap by providing a clear, step-by-step guide to acquire and analyze micro-CT images from mice for quantitative data. By exclusively detailing the necessary procedural steps from start to finish and overcoming complex user interfaces during imaging operations and analysis, this protocol will equip new micro-CT users with the ability to measure mouse body composition (bone, body fat, and lean muscle mass) and identify and quantify lung fibrosis. This approach applies to researchers with a basic understanding of medical imaging, animal care, and software analysis.

Voltage clamp fluorometry (VCF) is a powerful technique in which the voltage of a cell’s membrane is clamped to control voltage-sensitive membrane proteins while simultaneously measuring fluorescent signals from a protein of interest. By combining fluorescence measurements with electrophysiology, VCF provides real-time measurement of a protein’s motions, which gives insight into its function. This protocol describes the use of VCF to study a membrane protein, the voltage-sensing phosphatase (VSP). VSP is a 3 and 5 phosphatidylinositol phosphate (PIP) phosphatase coupled to a voltage sensing domain (VSD). The VSD of VSP is homologous to the VSD of ion channels, with four transmembrane helices (S1–S4). The S4 contains the gating charge arginine residues that sense the membrane’s electric field. Membrane depolarization moves the S4 into a state that activates the cytosolic phosphatase domain. To monitor the movement of S4, the environmentally sensitive fluorophore tetramethylrhodamine-6-maleimide (TMRM) is attached extracellularly to the S3-S4 loop. Using VCF, the resulting fluorescence signals from the S4 movement measure the kinetics of activation and repolarization, as well as the voltage dependence of the VSD. This protocol details the steps to express VSP in Xenopus laevis oocytes and then acquire and analyze the resulting VCF data. VCF is advantageous as it provides voltage control of VSP in a native membrane while quantitatively assessing the functional properties of the VSD.

Gap junctions are transmembrane protein channels that enable the exchange of small molecules such as ions, second messengers, and metabolites between adjacent cells. Gap junctions are found in various mammalian organs, including skin, endothelium, liver, pancreas, muscle, and central nervous system (CNS). In the CNS, they mediate coupling between neural cells including glial cells, and the resulting panglial networks are vital for brain homeostasis. Tracers of sufficiently small molecular mass can diffuse across gap junctions and are used to visualize the extent of cell-to-cell coupling in situ by delivering them to a single cell through sharp electrodes or patch-clamp micropipettes. Here, we describe a protocol for pre-labeling and identification of astrocytes in acute mouse forebrain slices using Sulforhodamine 101 (SR101). Fluorescent cells can then be targeted for whole-cell patch-clamp, which allows for further confirmation of astroglial identity by assessing their electrophysiological properties, as well as for passive dialysis with a tracer such as biocytin. Slices can then be subjected to chemical fixation and immunostaining to detect dye-coupled networks. This protocol provides a method for the identification of astrocytes in live tissue through SR101 labeling. Alternatively, transgenic reporter mice can also be used to identify astrocytes. While we illustrate the use of this protocol for the study of glial networks in the mouse brain, the general principles are applicable to other species, tissues, and cell types.

The physiological role of a-synuclein (a-syn), an intrinsically disordered presynaptic neuronal protein, is believed to impact the release of neurotransmitters through interactions with the SNARE complex. However, under certain cellular conditions that are not well understood, a-syn will self-assemble into β-sheet-rich fibrils that accumulate and form insoluble neuronal inclusions. Studies of patient-derived brain tissues have concluded that these inclusions are associated with Parkinson’s disease, the second most common neurodegenerative disorder, and other synuclein-related diseases called synucleinopathies. In addition, repetitions of specific mutations to the SNCA gene, the gene that encodes a-syn, result in an increased disposition for synucleinopathies. The latest advances in cryo-EM structure determination and real-space helical reconstruction methods have resulted in over 60 in vitro structures of a-syn fibrils solved to date, with a handful of these reaching a resolution below 2.5 Å. Here, we provide a protocol for a-syn protein expression, purification, and fibrilization. We detail how sample quality is assessed by negative stain transmission electron microscopy (NS-TEM) analysis and followed by sample vitrification using the Vitrobot Mark IV vitrification robot. We provide a detailed step-by-step protocol for high-resolution cryo-EM structure determination of a-syn fibrils using RELION and a series of specialized helical reconstruction tools that can be run within RELION. Finally, we detail how ChimeraX, Coot, and Phenix are used to build and refine a molecular model into the high-resolution cryo-EM map. This workflow resulted in a 2.04 Å structure of a-syn fibrils with excellent resolution of residues 36–97 and an additional island of density for residues 15–22 that had not been previously reported. This workflow should serve as a starting point for individuals new to the neurodegeneration and structural biology fields. Together, this procedure lays the foundation for advanced structural studies of a-syn and other amyloid fibrils.

Interstitial fluid (ISF) is a promising diagnostic sample due to its extensive biomolecular content while being safer and less invasive to collect than blood. However, existing ISF sampling methods are time-consuming, require specialized equipment, and yield small amounts of fluid (<5 μL). We have recently reported a simple and minimally invasive technique for rapidly sampling larger quantities of dermal ISF using a microneedle (MN) array to generate micropores in the skin from which ISF is extracted using a vacuum-assisted skin patch. Here, we present step-by-step protocols for fabricating the MN array and skin patch, as well as for using them to sample ISF from human skin. Using this technique, an average of 20.8 μL of dermal ISF can be collected within 25 min, which is a ∼6-fold improvement over existing ISF sampling methods. Furthermore, the technique is well-tolerated and does not require the use of expensive or specialized equipment. The ability to collect ample volumes of ISF in a quick and minimally invasive manner will facilitate the analysis of ISF for biomarker discovery and its use for diagnostic testing.