Regulation of the Cell Biology of Antigen Cross-Presentation.

Antigen cross-presentation is an adaptation of the cellular process of loading MHC-I molecules with endogenous peptides during their biosynthesis within the endoplasmic reticulum. Cross-presented peptides derive from internalized proteins, microbial pathogens, and transformed or dying cells. The physical separation of internalized cargo from the endoplasmic reticulum, where the machinery for assembling peptide-MHC-I complexes resides, poses a challenge. To solve this problem, deliberate rewiring of organelle communication within cells is necessary to prepare for cross-presentation, and different endocytic receptors and vesicular traffic patterns customize the emergent cross-presentation compartment to the nature of the peptide source. Three distinct pathways of vesicular traffic converge to form the ideal cross-presentation compartment, each regulated differently to supply a unique component that enables cross-presentation of a diverse repertoire of peptides. Delivery of centerpiece MHC-I molecules is the critical step regulated by microbe-sensitive Toll-like receptors. Defining the subcellular sources of MHC-I and identifying sites of peptide loading during cross-presentation remain key challenges.

Antigen Presentation by Extracellular Vesicles from Professional Antigen-Presenting Cells.

The initiation and maintenance of adaptive immunity require multifaceted modes of communication between different types of immune cells, including direct intercellular contact, secreted soluble signaling molecules, and extracellular vesicles (EVs). EVs can be formed as microvesicles directly pinched off from the plasma membrane or as exosomes secreted by multivesicular endosomes. Membrane receptors guide EVs to specific target cells, allowing directional transfer of specific and complex signaling cues. EVs are released by most, if not all, immune cells. Depending on the type and status of their originating cell, EVs may facilitate the initiation, expansion, maintenance, or silencing of adaptive immune responses. This review focusses on EVs from professional antigen-presenting cells, their demonstrated and speculated roles, and their potential for cancer immunotherapy.

Extensive structural rearrangement of intraflagellar transport trains underpins bidirectional cargo transport.

Bidirectional transport in cilia is carried out by polymers of the IFTA and IFTB protein complexes, called anterograde and retrograde intraflagellar transport (IFT) trains. Anterograde trains deliver cargoes from the cell to the cilium tip, then convert into retrograde trains for cargo export. We set out to understand how the IFT complexes can perform these two directly opposing roles before and after conversion. We use cryoelectron tomography and in situ cross-linking mass spectrometry to determine the structure of retrograde IFT trains and compare it with the known structure of anterograde trains. The retrograde train is a 2-fold symmetric polymer organized around a central thread of IFTA complexes. We conclude that anterograde-to-retrograde remodeling involves global rearrangements of the IFTA/B complexes and requires complete disassembly of the anterograde train. Finally, we describe how conformational changes to cargo-binding sites facilitate unidirectional cargo transport in a bidirectional system.

Short-distance vesicle transport via phase separation.

In addition to long-distance molecular motor-mediated transport, cellular vesicles also need to be moved at short distances with defined directions to meet functional needs in subcellular compartments but with unknown mechanisms. Such short-distance vesicle transport does not involve molecular motors. Here, we demonstrate, using synaptic vesicle (SV) transport as a paradigm, that phase separation of synaptic proteins with vesicles can facilitate regulated, directional vesicle transport between different presynaptic bouton sub-compartments. Specifically, a large coiled-coil scaffold protein Piccolo, in response to Ca2+ and via its C2A domain-mediated Ca2+ sensing, can extract SVs from the synapsin-clustered reserve pool condensate and deposit the extracted SVs onto the surface of the active zone protein condensate. We further show that the Trk-fused gene, TFG, also participates in COPII vesicle trafficking from ER to the ER-Golgi intermediate compartment via phase separation. Thus, phase separation may play a general role in short-distance, directional vesicle transport in cells.

Transport mechanism and pharmacology of the human GlyT1.

The glycine transporter 1 (GlyT1) plays a crucial role in the regulation of both inhibitory and excitatory neurotransmission by removing glycine from the synaptic cleft. Given its close association with glutamate/glycine co-activated NMDA receptors (NMDARs), GlyT1 has emerged as a central target for the treatment of schizophrenia, which is often linked to hypofunctional NMDARs. Here, we report the cryo-EM structures of GlyT1 bound with substrate glycine and drugs ALX-5407, SSR504734, and PF-03463275. These structures, captured at three fundamental states of the transport cycle-outward-facing, occluded, and inward-facing-enable us to illustrate a comprehensive blueprint of the conformational change associated with glycine reuptake. Additionally, we identified three specific pockets accommodating drugs, providing clear insights into the structural basis of their inhibitory mechanism and selectivity. Collectively, these structures offer significant insights into the transport mechanism and recognition of substrate and anti-schizophrenia drugs, thus providing a platform to design small molecules to treat schizophrenia.

Cytoneme signaling provides essential contributions to mammalian tissue patterning.

During development, morphogens pattern tissues by instructing cell fate across long distances. Directly visualizing morphogen transport in situ has been inaccessible, so the molecular mechanisms ensuring successful morphogen delivery remain unclear. To tackle this longstanding problem, we developed a mouse model for compromised sonic hedgehog (SHH) morphogen delivery and discovered that endocytic recycling promotes SHH loading into signaling filopodia called cytonemes. We optimized methods to preserve in vivo cytonemes for advanced microscopy and show endogenous SHH localized to cytonemes in developing mouse neural tubes. Depletion of SHH from neural tube cytonemes alters neuronal cell fates and compromises neurodevelopment. Mutation of the filopodial motor myosin 10 (MYO10) reduces cytoneme length and density, which corrupts neuronal signaling activity of both SHH and WNT. Combined, these results demonstrate that cytoneme-based signal transport provides essential contributions to morphogen dispersion during mammalian tissue development and suggest MYO10 is a key regulator of cytoneme function.

A gut-secreted peptide suppresses arousability from sleep.

Suppressing sensory arousal is critical for sleep, with deeper sleep requiring stronger sensory suppression. The mechanisms that enable sleeping animals to largely ignore their surroundings are not well understood. We show that the responsiveness of sleeping flies and mice to mechanical vibrations is better suppressed when the diet is protein rich. In flies, we describe a signaling pathway through which information about ingested proteins is conveyed from the gut to the brain to help suppress arousability. Higher protein concentration in the gut leads to increased activity of enteroendocrine cells that release the peptide CCHa1. CCHa1 signals to a small group of dopamine neurons in the brain to modulate their activity; the dopaminergic activity regulates the behavioral responsiveness of animals to vibrations. The CCHa1 pathway and dietary proteins do not influence responsiveness to all sensory inputs, showing that during sleep, different information streams can be gated through independent mechanisms.

Engineering RNA export for measurement and manipulation of living cells.

A system for programmable export of RNA molecules from living cells would enable both non-destructive monitoring of cell dynamics and engineering of cells capable of delivering executable RNA programs to other cells. We developed genetically encoded cellular RNA exporters, inspired by viruses, that efficiently package and secrete cargo RNA molecules from mammalian cells within protective nanoparticles. Exporting and sequencing RNA barcodes enabled non-destructive monitoring of cell population dynamics with clonal resolution. Further, by incorporating fusogens into the nanoparticles, we demonstrated the delivery, expression, and functional activity of exported mRNA in recipient cells. We term these systems COURIER (controlled output and uptake of RNA for interrogation, expression, and regulation). COURIER enables measurement of cell dynamics and establishes a foundation for hybrid cell and gene therapies based on cell-to-cell delivery of RNA.

The gymnastics of intraflagellar transport complexes keeps trains running inside cilia.

The assembly and signaling properties of cilia rely on intraflagellar transport (IFT) trains moving proteins into, within, and out of cilia. A flurry of near-atomic models of the multiprotein complexes that make up IFT trains has revealed new conformational changes, which may underlie the switch between anterograde and retrograde intraflagellar transport.

Mechanism of IFT-A polymerization into trains for ciliary transport.

Intraflagellar transport (IFT) is the highly conserved process by which proteins are transported along ciliary microtubules by a train-like polymeric assembly of IFT-A and IFT-B complexes. IFT-A is sandwiched between IFT-B and the ciliary membrane, consistent with its putative role in transporting transmembrane and membrane-associated cargoes. Here, we have used single-particle analysis electron cryomicroscopy (cryo-EM) to determine structures of native IFT-A complexes. We show that subcomplex rearrangements enable IFT-A to polymerize laterally on anterograde IFT trains, revealing a cooperative assembly mechanism. Surprisingly, we discover that binding of IFT-A to IFT-B shields the preferred lipid-binding interface from the ciliary membrane but orients an interconnected network of ß-propeller domains with the capacity to accommodate diverse cargoes toward the ciliary membrane. This work provides a mechanistic basis for understanding IFT-train assembly and cargo interactions.

IFT-A structure reveals carriages for membrane protein transport into cilia.

Intraflagellar transport (IFT) trains are massive molecular machines that traffic proteins between cilia and the cell body. Each IFT train is a dynamic polymer of two large complexes (IFT-A and -B) and motor proteins, posing a formidable challenge to mechanistic understanding. Here, we reconstituted the complete human IFT-A complex and obtained its structure using cryo-EM. Combined with AlphaFold prediction and genome-editing studies, our results illuminate how IFT-A polymerizes, interacts with IFT-B, and uses an array of ß-propeller and TPR domains to create "carriages" of the IFT train that engage TULP adaptor proteins. We show that IFT-A⋅TULP carriages are essential for cilia localization of diverse membrane proteins, as well as ICK-the key kinase regulating IFT train turnaround. These data establish a structural link between IFT-A's distinct functions, provide a blueprint for IFT-A in the train, and shed light on how IFT evolved from a proto-coatomer ancestor.

RBG Motif Bridge-Like Lipid Transport Proteins: Structure, Functions, and Open Questions.

The life of eukaryotic cells requires the transport of lipids between membranes, which are separated by the aqueous environment of the cytosol. Vesicle-mediated traffic along the secretory and endocytic pathways and lipid transfer proteins (LTPs) cooperate in this transport. Until recently, known LTPs were shown to carry one or a few lipids at a time and were thought to mediate transport by shuttle-like mechanisms. Over the last few years, a new family of LTPs has been discovered that is defined by a repeating ß-groove (RBG) rod-like structure with a hydrophobic channel running along their entire length. This structure and the localization of these proteins at membrane contact sites suggest a bridge-like mechanism of lipid transport. Mutations in some of these proteins result in neurodegenerative and developmental disorders. Here we review the known properties and well-established or putative physiological roles of these proteins, and we highlight the many questions that remain open about their functions.

Structural Mechanism of Transport of Mitochondrial Carriers.

Members of the mitochondrial carrier family [solute carrier family 25 (SLC25)] transport nucleotides, amino acids, carboxylic acids, fatty acids, inorganic ions, and vitamins across the mitochondrial inner membrane. They are important for many cellular processes, such as oxidative phosphorylation of lipids and sugars, amino acid metabolism, macromolecular synthesis, ion homeostasis, cellular regulation, and differentiation. Here, we describe the functional elements of the transport mechanism of mitochondrial carriers, consisting of one central substrate-binding site and two gates with salt-bridge networks on either side of the carrier. Binding of the substrate during import causes three gate elements to rotate inward, forming the cytoplasmic network and closing access to the substrate-binding site from the intermembrane space. Simultaneously, three core elements rock outward, disrupting the matrix network and opening the substrate-binding site to the matrix side of the membrane. During export, substrate binding triggers conformational changes involving the same elements but operating in reverse.

It's Better To Be Lucky Than Smart.

Bacterial cytoplasmic membrane vesicles provide a unique experimental system for studying active transport. Vesicles are prepared by lysis of osmotically sensitized cells (i.e., protoplasts or spheroplasts) and comprise osmotically intact, unit-membrane-bound sacs that are approximately 0.5-1.0 µm in diameter and devoid of internal structure. Their metabolic activities are restricted to those provided by the enzymes of the membrane itself, and each vesicle is functional. The energy source for accumulation of a particular substrate can be determined by studying which compounds or experimental conditions drive solute accumulation, and metabolic conversion of the transported substrate or the energy source is minimal. These properties of the vesicle system constitute a considerable advantage over intact cells, as the system provides clear definition of the reactions involved in the transport process. This discussion is not intended as a general review but is concerned with respiration-dependent active transport in membrane vesicles from Escherichia coli. Emphasis is placed on experimental observations demonstrating that respiratory energy is converted primarily into work in the form of a solute concentration gradient that is driven by a proton electrochemical gradient, as postulated by the chemiosmotic theory of Peter Mitchell.

Mitocytosis, a migrasome-mediated mitochondrial quality-control process.

Damaged mitochondria need to be cleared to maintain the quality of the mitochondrial pool. Here, we report mitocytosis, a migrasome-mediated mitochondrial quality-control process. We found that, upon exposure to mild mitochondrial stresses, damaged mitochondria are transported into migrasomes and subsequently disposed of from migrating cells. Mechanistically, mitocytosis requires positioning of damaged mitochondria at the cell periphery, which occurs because damaged mitochondria avoid binding to inward motor proteins. Functionally, mitocytosis plays an important role in maintaining mitochondrial quality. Enhanced mitocytosis protects cells from mitochondrial stressor-induced loss of mitochondrial membrane potential (MMP) and mitochondrial respiration; conversely, blocking mitocytosis causes loss of MMP and mitochondrial respiration under normal conditions. Physiologically, we demonstrate that mitocytosis is required for maintaining MMP and viability in neutrophils in vivo. We propose that mitocytosis is an important mitochondrial quality-control process in migrating cells, which couples mitochondrial homeostasis with cell migration.

Lipid Transport Across Bacterial Membranes.

The movement of lipids within and between membranes in bacteria is essential for building and maintaining the bacterial cell envelope. Moving lipids to their final destination is often energetically unfavorable and does not readily occur spontaneously. Bacteria have evolved several protein-mediated transport systems that bind specific lipid substrates and catalyze the transport of lipids across membranes and from one membrane to another. Specific protein flippases act in translocating lipids across the plasma membrane, overcoming the obstacle of moving relatively large and chemically diverse lipids between leaflets of the bilayer. Active transporters found in double-membraned bacteria have evolved sophisticated mechanisms to traffic lipids between the two membranes, including assembling to form large, multiprotein complexes that resemble bridges, shuttles, and tunnels, shielding lipids from the hydrophilic environment of the periplasm during transport. In this review, we explore our current understanding of the mechanisms thought to drive bacterial lipid transport.

Structural Biology of Cilia and Intraflagellar Transport.

Cilia are ubiquitous microtubule-based eukaryotic organelles that project from the cell to generate motility or function in cellular signaling. Motile cilia or flagella contain axonemal dynein motors and other complexes to achieve beating. Primary cilia are immotile and act as signaling hubs, with receptors shuttling between the cytoplasm and ciliary compartment. In both cilia types, an intraflagellar transport (IFT) system powered by unique kinesin and dynein motors functions to deliver the molecules required to build cilia and maintain their functions. Cryo-electron tomography has helped to reveal the organization of protein complex arrangement along the axoneme and the structure of anterograde IFT trains as well as the structure of primary cilia. Only recently, single-particle analysis (SPA) cryo-electron microscopy has provided molecular details of the protein organization of ciliary components, helping us to understand how they bind to microtubule doublets and how mechanical force propagated by dynein conformational changes is converted into ciliary beating. Here we highlight recent structural advances that are leading to greater knowledge of ciliary function.

Structural and Mechanistic Principles of ABC Transporters.

ATP-binding cassette (ABC) transporters constitute one of the largest and most ancient protein superfamilies found in all living organisms. They function as molecular machines by coupling ATP binding, hydrolysis, and phosphate release to translocation of diverse substrates across membranes. The substrates range from vitamins, steroids, lipids, and ions to peptides, proteins, polysaccharides, and xenobiotics. ABC transporters undergo substantial conformational changes during substrate translocation. A comprehensive understanding of their inner workings thus requires linking these structural rearrangements to the different functional state transitions. Recent advances in single-particle cryogenic electron microscopy have not only delivered crucial information on the architecture of several medically relevant ABC transporters and their supramolecular assemblies, including the ATP-sensitive potassium channel and the peptide-loading complex, but also made it possible to explore the entire conformational space of these nanomachines under turnover conditions and thereby gain detailed mechanistic insights into their mode of action.

Biosynthesis and Export of Bacterial Glycolipids.

Complex carbohydrates are essential for many biological processes, from protein quality control to cell recognition, energy storage, and cell wall formation. Many of these processes are performed in topologically extracellular compartments or on the cell surface; hence, diverse secretion systems evolved to transport the hydrophilic molecules to their sites of action. Polyprenyl lipids serve as ubiquitous anchors and facilitators of these transport processes. Here, we summarize and compare bacterial biosynthesis pathways relying on the recognition and transport of lipid-linked complex carbohydrates. In particular, we compare transporters implicated in O antigen and capsular polysaccharide biosyntheses with those facilitating teichoic acid and N-linked glycan transport. Further, we discuss recent insights into the generation, recognition, and recycling of polyprenyl lipids.

Context-specific regulation of extracellular vesicle biogenesis and cargo selection.

To coordinate, adapt and respond to biological signals, cells convey specific messages to other cells. An important aspect of cell-cell communication involves secretion of molecules into the extracellular space. How these molecules are selected for secretion has been a fundamental question in the membrane trafficking field for decades. Recently, extracellular vesicles (EVs) have been recognized as key players in intercellular communication, carrying not only membrane proteins and lipids but also RNAs, cytosolic proteins and other signalling molecules to recipient cells. To communicate the right message, it is essential to sort cargoes into EVs in a regulated and context-specific manner. In recent years, a wealth of lipidomic, proteomic and RNA sequencing studies have revealed that EV cargo composition differs depending upon the donor cell type, metabolic cues and disease states. Analyses of distinct cargo 'fingerprints' have uncovered mechanistic linkages between the activation of specific molecular pathways and cargo sorting. In addition, cell biology studies are beginning to reveal novel biogenesis mechanisms regulated by cellular context. Here, we review context-specific mechanisms of EV biogenesis and cargo sorting, focusing on how cell signalling and cell state influence which cellular components are ultimately targeted to EVs.