Building programmable multicompartment artificial cells incorporating remotely activated protein channels using microfluidics and acoustic levitation

Intracellular compartments are functional units that support the metabolic processes within living cells, through spatiotemporal regulation of chemical reactions and biological processes. Consequently, as a step forward in the bottom-up creation of artificial cells, building analogous intracellular architectures is essential for the expansion of cell-mimicking functionality. Herein, we report the development of a droplet laboratory platform to engineer customised complex emulsion droplets as a multicompartment artificial cell chassis, using multiphase microfluidics and acoustic levitation. Such levitated constructs provide free-standing, dynamic, definable droplet networks for the encapsulation and organisation of chemical species. Equally, they can be remotely operated with pneumatic, heating, and magnetic elements for post-processing, including the incorporation of membrane proteins; alpha-hemolysin; and large-conductance mechanosensitive channel (MscL) and their activation. The assembly of droplet networks is three-dimensionally patterned with fluidic inputs configurations determining droplet contents and connectivity. Whilst acoustic manipulation can be harnessed to reconfigure the droplet network in situ. In addition, a mechanosensitive channel, MscL, can be repeatedly activated and deactivated in the levitated artificial cell by the application of acoustic and magnetic fields to modulate membrane tension on demand. This offers possibilities beyond one-time chemically mediated activation to provide repeated, non-contact control of membrane protein function. Collectively, this will expand our capability to program and operate increasingly sophisticated artificial cells as life-like materials.

Conservation of magnetite biomineralization genes in all domains of life and implications for magnetic sensing

Animals use geomagnetic fields for navigational cues, yet the sensory mechanism underlying magnetic perception remains poorly understood. One idea is that geomagnetic fields are physically transduced by magnetite crystals contained inside specialized receptor cells, but evidence for intracellular, biogenic magnetite in eukaryotes is scant. Certain bacteria produce magnetite crystals inside intracellular compartments, representing the most ancient form of biomineralization known and having evolved prior to emergence of the crown group of eukaryotes, raising the question of whether magnetite biomineralization in eukaryotes and prokaryotes might share a common evolutionary history. Here, we discover that salmonid olfactory epithelium contains magnetite crystals arranged in compact clusters and determine that genes differentially expressed in magnetic olfactory cells, contrasted to nonmagnetic olfactory cells, share ancestry with an ancient prokaryote magnetite biomineralization system, consistent with exaptation for use in eukaryotic magnetoreception. We also show that 11 prokaryote biomineralization genes are universally present among a diverse set of eukaryote taxa and that nine of those genes are present within the Asgard clade of archaea Lokiarchaeota that affiliates with eukaryotes in phylogenomic analysis. Consistent with deep homology, we present an evolutionary genetics hypothesis for magnetite formation among eukaryotes to motivate convergent approaches for examining magnetite-based magnetoreception, molecular origins of matrix-associated biomineralization processes, and eukaryogenesis.

Fast, live-cell imaging of 15 intracellular compartments by deep learning segmentation of super-resolution data

The number of colors that can be used in fluorescence microscopy to image the live-cell anatomy and organelles' interactions is far less than the number of intracellular organelles and compartments. Here, we report that deep convolutional neuronal networks can predict 15 subcellular structures from super-resolution spinning-disk microscopy images using only one dye, one laser excitation, and two detection channels. Comparing to the colocalization images, this method achieves pixel accuracies of over 91.7%, which not only bypasses the fundamental limitation of multi-color imaging but also accelerates the imaging speed by more than one order of magnitude.

A cryo-ET survey of microtubules and intracellular compartments in mammalian axons

The neuronal axon is packed with cytoskeletal filaments, membranes, and organelles, many of which move between the cell body and axon tip. Here, we used cryo-electron tomography to survey the internal components of mammalian sensory axons. We determined the polarity of the axonal microtubules (MTs) by combining subtomogram classification and visual inspection, finding MT plus and minus ends are structurally similar. Subtomogram averaging of globular densities in the MT lumen suggests they have a defined structure, which is surprising given they likely contain the disordered protein MAP6. We found the endoplasmic reticulum in axons is tethered to MTs through multiple short linkers. We surveyed membrane-bound cargos and describe unexpected internal features such as granules and broken membranes. In addition, we detected proteinaceous compartments, including numerous virus-like capsid particles. Our observations outline novel features of axonal cargos and MTs, providing a platform for identification of their constituents.

Characterization of Metal Tolerance Proteins and Functional Analysis of GmMTP8.1 Involved in Manganese Tolerance in Soybean

Manganese is an essential micronutrient for plant growth but can be toxic to plants when it reaches excessive levels. Although metal tolerance proteins (MTPs), which belong to the cation diffusion facilitator (CDF) family, have been demonstrated to play critical roles in manganese (Mn) tolerance in plants, the characteristics and functions of GmMTP members in the response of soybean (Glycine max) to Mn toxicity have not been documented. In this study, growth inhibition was observed in soybean plants that were exposed to a toxic level of Mn in hydroponics, as reflected by the generation of brown spots, and decreased leaf chlorophyll concentration and plant fresh weight. Subsequent genome-wide analysis resulted in the identification of a total of 14 GmMTP genes in the soybean genome. Among these GmMTPs, 9 and 12 were found to be regulated by excess Mn in leaves and roots, respectively. Furthermore, the function of GmMTP8.1, a Mn-CDF homologue of ShMTP8 identified in the legume Stylosanthes hamata that is involved in Mn detoxification, was characterized. Subcellular localization analysis showed that GmMTP8.1 was localized to the endoplasmic reticulum (ER). Heterologous expression of GmMTP8.1 led to the restoration of growth of the Mn-hypersensitive yeast (Saccharomyces cerevisiae) mutant Δpmr1, which is made defective in Mn transport into the Golgi apparatus by P-type Ca/Mn-ATPase. Furthermore, GmMTP8.1 overexpression conferred tolerance to the toxic level of Mn in Arabidopsis (Arabidopsis thaliana). Under excess Mn conditions, concentrations of Mn in shoots but not roots were decreased in transgenic Arabidopsis, overexpressing GmMTP8.1 compared to the wild type. The overexpression of GmMTP8.1 also led to the upregulation of several transporter genes responsible for Mn efflux and sequestration in Arabidopsis, such as AtMTP8/11. Taken together, these results suggest that GmMTP8.1 is an ER-localized Mn transporter contributing to confer Mn tolerance by stimulating the export of Mn out of leaf cells and increasing the sequestration of Mn into intracellular compartments.

Cryo-EM of the yeast VO complex reveals distinct binding sites for macrolide V-ATPase inhibitors

Vacuolar-type adenosine triphosphatases (V-ATPases) are proton pumps found in almost all eukaryotic cells. These enzymes consist of a soluble catalytic V1 region that hydrolyzes ATP and a membrane-embedded VO region responsible for proton translocation. V-ATPase activity leads to acidification of endosomes, phagosomes, lysosomes, secretory vesicles, and the trans-Golgi network, with extracellular acidification occurring in some specialized cells. Small molecule inhibitors of V-ATPase have played a crucial role in elucidating numerous aspects of cell biology by blocking acidification of intracellular compartments, while therapeutic use of V-ATPase inhibitors has been proposed for treatment of cancer, osteoporosis, and some infections. Here, we determine structures of the isolated VO complex from Saccharomyces cerevisiae bound to two well-known macrolide inhibitors: bafilomycin A1 and archazolid A. The structures reveal different binding sites for the inhibitors on the surface of the proton-carrying c ring, with only a small amount of overlap between the two sites. Binding of both inhibitors is mediated primarily through van der Waals interactions in shallow pockets and suggests that the inhibitors block rotation of the ring. Together, these structures indicate the existence of a large chemical space available for V-ATPase inhibitors that block acidification by binding the c ring.

Coordinated conformational changes in the V1 complex during V-ATPase reversible dissociation

Vacuolar-type ATPases (V-ATPases) are rotary enzymes that acidify intracellular compartments in eukaryotic cells. These multi-subunit complexes consist of a cytoplasmic V1 region that hydrolyzes ATP and a membrane-embedded VO region that transports protons. V-ATPase activity is regulated by reversible dissociation of the two regions, with the isolated V1 and VO complexes becoming autoinhibited upon disassembly and subunit C subsequently detaching from V1. In yeast, assembly of the V1 and VO regions is mediated by the RAVE complex through an unknown mechanism. We used cryoEM of yeast V-ATPase to determine structures of the intact enzyme, the dissociated but complete V1 complex, and the V1 complex lacking subunit C. Upon separation, V1 undergoes a dramatic conformational rearrangement, with its rotational state becoming incompatible for reassembly with VO. Loss of subunit C allows V1 to match the rotational state of VO, suggesting how RAVE could reassemble V1 and VO by recruiting subunit C.

Dynamic and cell-specific transport networks for intracellular copper ions

ABSTRACT Copper (Cu) homeostasis is essential for the development and function of many organisms. In humans, Cu misbalance causes serious pathologies and has been observed in a growing number of diseases. This Review focuses on mammalian Cu(I) transporters and highlights recent studies on regulation of intracellular Cu fluxes. Cu is used by essential metabolic enzymes for their activity. These enzymes are located in various intracellular compartments and outside cells. When cells differentiate, or their metabolic state is otherwise altered, the need for Cu in different cell compartments change, and Cu has to be redistributed to accommodate these changes. The Cu transporters SLC31A1 (CTR1), SLC31A2 (CTR2), ATP7A and ATP7B regulate Cu content in cellular compartments and maintain Cu homeostasis. Increasing numbers of regulatory proteins have been shown to contribute to multifaceted regulation of these Cu transporters. It is becoming abundantly clear that the Cu transport networks are dynamic and cell specific. The comparison of the Cu transport machinery in the liver and intestine illustrates the distinct composition and dissimilar regulatory response of their Cu transporters to changing Cu levels.

The Distribution and Role of the CFTR Protein in the Intracellular Compartments

Cystic fibrosis is a hereditary disease that mainly affects secretory organs in humans. It is caused by mutations in the gene encoding CFTR with the most common phenylalanine deletion at position 508. CFTR is an anion channel mainly conducting Cl− across the apical membranes of many different epithelial cells, the impairment of which causes dysregulation of epithelial fluid secretion and thickening of the mucus. This, in turn, leads to the dysfunction of organs such as the lungs, pancreas, kidney and liver. The CFTR protein is mainly localized in the plasma membrane; however, there is a growing body of evidence that it is also present in the intracellular organelles such as the endosomes, lysosomes, phagosomes and mitochondria. Dysfunction of the CFTR protein affects not only the ion transport across the epithelial tissues, but also has an impact on the proper functioning of the intracellular compartments. The review aims to provide a summary of the present state of knowledge regarding CFTR localization and function in intracellular compartments, the physiological role of this localization and the consequences of protein dysfunction at cellular, epithelial and organ levels. An in-depth understanding of intracellular processes involved in CFTR impairment may reveal novel opportunities in pharmacological agents of cystic fibrosis.

Spatiotemporal proteomic profiling of the pro-inflammatory response to lipopolysaccharide in the THP-1 human leukaemia cell line

Protein localisation and translocation between intracellular compartments underlie almost all physiological processes. The hyperLOPIT proteomics platform combines mass spectrometry with state-of-the-art machine learning to map the subcellular location of thousands of proteins simultaneously. We combine global proteome analysis with hyperLOPIT in a fully Bayesian framework to elucidate spatiotemporal proteomic changes during a lipopolysaccharide (LPS)-induced inflammatory response. We report a highly dynamic proteome in terms of both protein abundance and subcellular localisation, with alterations in the interferon response, endo-lysosomal system, plasma membrane reorganisation and cell migration. Proteins not previously associated with an LPS response were found to relocalise upon stimulation, the functional consequences of which are still unclear. By quantifying proteome-wide uncertainty through Bayesian modelling, a necessary role for protein relocalisation and the importance of taking a holistic overview of the LPS-driven immune response has been revealed. The data are showcased as an interactive application freely available for the scientific community.