Astrocytes show unique anatomical, morphological, and metabolic features to take up substrates from the blood and metabolize them for local delivery to active synapses to sustain neuron function. In the present review, we specifically focus on key molecular aspects of energy and redox metabolism that facilitate this astrocyte-neuronal coupling in a controlled manner. Basal glycolysis is co-ordinated by the anaphase-promoting complex/cyclosome (APC/C)-Cdh1, a ubiquitin ligase that targets the proglycolytic enzyme 6-phosphofructokinase-2,6-bisphosphastate-3 (PFKFB3) for degradation. APC/C-Cdh1 activity is more robust in neurons than in astrocytes, which determine that PFKFB3 abundance and glycolytic rate are weaker in neurons. The low PFKFB3 activity in neurons facilitates glucose-6-phosphate oxidation via the pentose-phosphate pathway, which promotes antioxidant protection. Conversely, the high PFKFB3 activity in astrocytes allows the production and release of glycolytic lactate, which is taken up by neurons that use it as an oxidizable substrate. Importantly, the mitochondrial respiratory chain is tighter assembled in neurons than in astrocytes, thus the bioenergetic efficiency of mitochondria is higher in neurons. Because of this, the production of reactive oxygen species (mROS) by mitochondrial complex I is very low in neurons and very high in astrocytes. Such a naturally occurring high abundance of mROS in astrocytes physiologically determines a specific transcriptional fingerprint that contributes to sustaining cognitive performance. We conclude that the energy and redox metabolism of astrocytes must complementarily match that of neurons to regulate brain function and animal welfare.

Astrocytes are a type of non-neuronal, glial cells, anatomically placed in the intersection between the brain blood vessels and other neural cells-including neurons. Such a strategic situation confers these cells a unique opportunity to sense circulating molecules and adapt according to different organismal conditions. By acting as sentinel cells, astrocytes thus co-ordinate gene expression profiles, immune responses, signal transduction pathways, and metabolic programs that play essential roles in the formation of brain circuits to modulate neurotransmission and higher-order organismal functions.

Synaptic regulation of the primary inhibitory neurotransmitter γ-aminobutyric acid (GABA) is essential for brain function. Cerebral GABA homeostasis is tightly regulated through multiple mechanisms and is directly coupled to the metabolic collaboration between neurons and astrocytes. In this essay, we outline and discuss the fundamental roles of astrocytes in regulating synaptic GABA signaling. A major fraction of synaptic GABA is removed from the synapse by astrocytic uptake. Astrocytes utilize GABA as a metabolic substrate to support glutamine synthesis. The astrocyte-derived glutamine is subsequently transferred to neurons where it serves as the primary precursor of neuronal GABA synthesis. The flow of GABA and glutamine between neurons and astrocytes is collectively termed the GABA-glutamine cycle and is essential to sustain GABA synthesis and inhibitory signaling. In certain brain areas, astrocytes are even capable of synthesizing and releasing GABA to modulate inhibitory transmission. The majority of oxidative GABA metabolism in the brain takes place in astrocytes, which also leads to synthesis of the GABA-related metabolite γ-hydroxybutyric acid (GHB). The physiological roles of endogenous GHB remain unclear, but may be related to regulation of tonic inhibition and synaptic plasticity. Disrupted inhibitory signaling and dysfunctional astrocyte GABA handling are implicated in several diseases including epilepsy and Alzheimer's disease. Synaptic GABA homeostasis is under astrocytic control and astrocyte GABA uptake, metabolism, and recycling may therefore serve as relevant targets to ameliorate pathological inhibitory signaling.

Integral membrane proteins are involved in a plethora of biological processes including cellular signalling, molecular transport, and catalysis. Many of these functions are mediated by non-covalent interactions with other proteins, substrates, metabolites, and surrounding lipids. Uncovering such interactions and deciphering their effect on protein activity is essential for understanding the regulatory mechanisms underlying integral membrane protein function. However, the detection of such dynamic complexes has proven to be challenging using traditional approaches in structural biology. Native mass spectrometry has emerged as a powerful technique for the structural characterisation of membrane proteins and their complexes, enabling the detection and identification of protein-binding partners. In this review, we discuss recent native mass spectrometry-based studies that have characterised non-covalent interactions of membrane proteins in the presence of detergents or membrane mimetics. We additionally highlight recent progress towards the study of membrane proteins within native membranes and provide our perspective on how these could be combined with recent developments in instrumentation to investigate increasingly complex biomolecular systems.

Proteins and RNAs are fundamental parts of biological systems, and their interactions affect many essential cellular processes. Therefore, it is crucial to understand at a molecular and at a systems level how proteins and RNAs form complexes and mutually affect their functions. In the present mini-review, we will first provide an overview of different mass spectrometry (MS)-based methods to study the RNA-binding proteome (RBPome), most of which are based on photochemical cross-linking. As we will show, some of these methods are also able to provide higher-resolution information about binding sites, which are important for the structural characterisation of protein-RNA interactions. In addition, classical structural biology techniques such as nuclear magnetic resonance (NMR) spectroscopy and biophysical methods such as electron paramagnetic resonance (EPR) spectroscopy and fluorescence-based methods contribute to a detailed understanding of the interactions between these two classes of biomolecules. We will discuss the relevance of such interactions in the context of the formation of membrane-less organelles (MLOs) by liquid-liquid phase separation (LLPS) processes and their emerging importance as targets for drug discovery.

Mass spectrometry (MS) is now established as an analytical tool to interrogate the structure and dynamics of proteins and their assemblies. An array of MS-based technologies has been developed, with each providing unique information pertaining to protein structure, and forming the heart of integrative structural biology studies. This special issue includes a collection of review articles that discuss both established and emerging structural MS methodologies, along with examples of how these technologies are being deployed to interrogate protein structure and function. Combined, this collection highlights the immense potential of the structural MS toolkit in the study of molecular mechanisms underpinning cellular homeostasis and disease.

Inline low-energy electron holography (LEEH) in conjunction with sample preparation by electrospray ion beam deposition (ES-IBD) has recently emerged as a promising method for the sub-nanometre-scale single-molecule imaging of biomolecules. The single-molecule nature of the LEEH measurement allows for the mapping of the molecules' conformational space and thus for the imaging of structurally variable biomolecules, thereby providing valuable complementary information to well-established biomolecular structure determination methods. Here, after briefly tracing the development of inline LEEH in bioimaging, we present the state-of-the-art of native ES-IBD + LEEH as a method of single-protein imaging, discuss its applications, specifically regarding the imaging of structurally flexible protein systems and the amplitude and phase information encoded in a low-energy electron hologram, and provide an outlook regarding the considerable possibilities for the future advancement of the approach.

Integral membrane proteins (IMPs) perform a range of diverse functions and their dysfunction underlies numerous pathological conditions. Consequently, IMPs constitute most drug targets, and the elucidation of their mechanism of action has become an intense field of research. Historically, IMP studies have relied on their extraction from membranes using detergents, which have the potential to perturbate their structure and dynamics. To circumnavigate this issue, an array of membrane mimetics has been developed that aim to reconstitute IMPs into native-like lipid environments that more accurately represent the biological membrane. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has emerged as a versatile tool for probing protein dynamics in solution. The continued development of HDX-MS methodology has allowed practitioners to investigate IMPs using increasingly native-like membrane mimetics, and even pushing the study of IMPs into the in vivo cellular environment. Consequently, HDX-MS has come of age and is playing an ever-increasingly important role in the IMP structural biologist toolkit. In the present mini-review, we discuss the evolution of membrane mimetics in the HDX-MS context, focusing on seminal publications and recent innovations that have led to this point. We also discuss state-of-the-art methodological and instrumental advancements that are likely to play a significant role in the generation of high-quality HDX-MS data of IMPs in the future.