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De novo formation of the double-membrane compartment autophagosome is seeded by small vesicles carrying membrane protein autophagy-related 9 (ATG9), the function of which remains unknown. Here we find that ATG9A scrambles phospholipids of membranes in vitro. Cryo-EM structures of human ATG9A reveal a trimer with a solvated central pore, which is connected laterally to the cytosol through the cavity within each protomer. Similarities to ABC exporters suggest that ATG9A could be a transporter that uses the central pore to function. Moreover, molecular dynamics simulation suggests that the central pore opens laterally to accommodate lipid headgroups, thereby enabling lipids to flip. Mutations in the pore reduce scrambling activity and yield markedly smaller autophagosomes, indicating that lipid scrambling by ATG9A is essential for membrane expansion. We propose ATG9A acts as a membrane-embedded funnel to facilitate lipid flipping and to redistribute lipids added to the outer leaflet of ATG9 vesicles, thereby enabling growth into autophagosomes.Cryo-EM analyses together with liposome and cellular assays reveal that human ATG9A forms a trimer that mediates phospholipid flipping and promotes autophagosome membrane expansion.

Bioorthogonal chemistry capable of operating in live animals is needed to investigate biological processes such as cell death and immunity. Recent studies have identified a gasdermin family of pore-forming proteins that executes inflammasome-dependent and -independent pyroptosis 1 – 5 . Pyroptosis is proinflammatory, but its effect on antitumour immunity is unknown. Here we establish a bioorthogonal chemical system, in which a cancer-imaging probe phenylalanine trifluoroborate (Phe-BF 3 ) that can enter cells desilylates and ‘cleaves’ a designed linker that contains a silyl ether. This system enabled the controlled release of a drug from an antibody–drug conjugate in mice. When combined with nanoparticle-mediated delivery, desilylation catalysed by Phe-BF 3 could release a client protein—including an active gasdermin—from a nanoparticle conjugate, selectively into tumour cells in mice. We applied this bioorthogonal system to gasdermin, which revealed that pyroptosis of less than 15% of tumour cells was sufficient to clear the entire 4T1 mammary tumour graft. The tumour regression was absent in immune-deficient mice or upon T cell depletion, and was correlated with augmented antitumour immune responses. The injection of a reduced, ineffective dose of nanoparticle-conjugated gasdermin along with Phe-BF 3 sensitized 4T1 tumours to anti-PD1 therapy. Our bioorthogonal system based on Phe-BF 3 desilylation is therefore a powerful tool for chemical biology; our application of this system suggests that pyroptosis-induced inflammation triggers robust antitumour immunity and can synergize with checkpoint blockade. In mouse models of cancer, a biorthogonal chemical system based on desilylation catalysed by phenylalanine trifluoroborate enables the controlled release of gasdermin to induce pyroptosis selectively in tumour cells

The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9’s ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.Cryo-EM and liposome assays reveal that Atg9 functions as a lipid scramblase, transporting phospholipids between inner and outer liposome leaflets. Analyses of mutants in yeast support a role for this activity in autophagy.

Intensiometric genetically encoded biosensors, based on allosteric modulation of the fluorescence of a single fluorescent protein, are powerful tools for enabling imaging of neural activities and other cellular biochemical events. The archetypical example of such biosensors is the GCaMP series of Ca 2+ biosensors, which have been steadily improved over the past two decades and are now indispensable tools for neuroscience. However, no other biosensors have reached levels of performance, or had revolutionary impacts within specific disciplines, comparable to that of the Ca 2+ biosensors. Of the many reasons why this has been the case, a critical one has been a general black-box view of biosensor structure and mechanism. With this Perspective, we aim to summarize what is known about biosensor structure and mechanisms and, based on this foundation, provide guidelines to accelerate the development of a broader range of biosensors with performance comparable to that of the GCaMP series. Using extensive knowledge about the structures and mechanisms of genetically encoded fluorescent biosensors, this Perspective provides guidelines to aid and accelerate the development of an increasingly broad range of high-performance imaging tools.

Many genetically encoded biosensors use Förster resonance energy transfer (FRET) to dynamically report biomolecular activities. While pairs of cyan and yellow fluorescent proteins (FPs) are most commonly used as FRET partner fluorophores, respectively, green and red FPs offer distinct advantages for FRET, such as greater spectral separation, less phototoxicity, and lower autofluorescence. We previously developed the green-red FRET pair Clover and mRuby2, which improves responsiveness in intramolecular FRET reporters with different designs. Here we report the engineering of brighter and more photostable variants, mClover3 and mRuby3. mClover3 improves photostability by 60% and mRuby3 by 200% over the previous generation of fluorophores. Notably, mRuby3 is also 35% brighter than mRuby2, making it both the brightest and most photostable monomeric red FP yet characterized. Furthermore, we developed a standardized methodology for assessing FP performance in mammalian cells as stand-alone markers and as FRET partners. We found that mClover3 or mRuby3 expression in mammalian cells provides the highest fluorescence signals of all jellyfish GFP or coral RFP derivatives, respectively. Finally, using mClover3 and mRuby3, we engineered an improved version of the CaMKIIα reporter Camuiα with a larger response amplitude.

Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP.

Genetically encoded biosensors based on fluorescent proteins (FPs) are a reliable tool for studying the various biological processes in living systems. The circular permutation of single FPs led to the development of an extensive class of biosensors that allow the monitoring of many intracellular events. In circularly permuted FPs (cpFPs), the original N- and C-termini are fused using a peptide linker, while new termini are formed near the chromophore. Such a structure imparts greater mobility to the FP than that of the native variant, allowing greater lability of the spectral characteristics. One of the common principles of creating genetically encoded biosensors is based on the integration of a cpFP into a flexible region of a sensory domain or between two interacting domains, which are selected according to certain characteristics. Conformational rearrangements of the sensory domain associated with ligand interaction or changes in the cellular parameter are transferred to the cpFP, changing the chromophore environment. In this review, we highlight the basic principles of such sensors, the history of their creation, and a complete classification of the available biosensors.

Comprehensive genotype–phenotype mapping of the green fluorescent protein shows that the local fitness peak is narrow, shaped by a high prevalence of epistatic interactions, providing for the loss of fluorescence when the joint effect of mutations exceeds a threshold. Genotype to phenotype mapping of a model protein Fyodor Kondrashov and colleagues report comprehensive genotype–phenotype mapping across an entire protein, based on analysis of the fitness landscape of green fluorescent protein (GFP) using a molecular barcoding and sequencing approach. They find that the fitness landscape is characterized by locally narrow regions, combined with a high prevalence of epistatic interactions, providing for the loss of fluorescence when the joint effect of mutations exceeds a threshold. Fitness landscapes 1 , 2 depict how genotypes manifest at the phenotypic level and form the basis of our understanding of many areas of biology 2 , 3 , 4 , 5 , 6 , 7 , yet their properties remain elusive. Previous studies have analysed specific genes, often using their function as a proxy for fitness 2 , 4 , experimentally assessing the effect on function of single mutations and their combinations in a specific sequence 2 , 5 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 or in different sequences 2 , 3 , 5 , 16 , 17 , 18 . However, systematic high-throughput studies of the local fitness landscape of an entire protein have not yet been reported. Here we visualize an extensive region of the local fitness landscape of the green fluorescent protein from Aequorea victoria (avGFP) by measuring the native function (fluorescence) of tens of thousands of derivative genotypes of avGFP. We show that the fitness landscape of avGFP is narrow, with 3/4 of the derivatives with a single mutation showing reduced fluorescence and half of the derivatives with four mutations being completely non-fluorescent. The narrowness is enhanced by epistasis, which was detected in up to 30% of genotypes with multiple mutations and mostly occurred through the cumulative effect of slightly deleterious mutations causing a threshold-like decrease in protein stability and a concomitant loss of fluorescence. A model of orthologous sequence divergence spanning hundreds of millions of years predicted the extent of epistasis in our data, indicating congruence between the fitness landscape properties at the local and global scales. The characterization of the local fitness landscape of avGFP has important implications for several fields including molecular evolution, population genetics and protein design.

The low photostability of fluorescent proteins is a limiting factor in many applications of fluorescence microscopy. Here we present StayGold, a green fluorescent protein (GFP) derived from the jellyfish Cytaeis uchidae . StayGold is over one order of magnitude more photostable than any currently available fluorescent protein and has a cellular brightness similar to mNeonGreen. We used StayGold to image the dynamics of the endoplasmic reticulum (ER) with high spatiotemporal resolution over several minutes using structured illumination microscopy (SIM) and observed substantially less photobleaching than with a GFP variant optimized for stability in the ER. Using StayGold fusions and SIM, we also imaged the dynamics of mitochondrial fusion and fission and mapped the viral spike proteins in fixed cells infected with severe acute respiratory syndrome coronavirus 2. As StayGold is a dimer, we created a tandem dimer version that allowed us to observe the dynamics of microtubules and the excitatory post-synaptic density in neurons. StayGold will substantially reduce the limitations imposed by photobleaching, especially in live cell or volumetric imaging. StayGold is over one order of magnitude more photostable than current fluorescent proteins

Proteins smaller than about 50 kDa are currently too small to be imaged at high resolution by cryo-electron microscopy (cryo-EM), leaving most protein molecules in the cell beyond the reach of this powerful structural technique. Here we use a designed protein scaffold to bind and symmetrically display 12 copies of a small 26 kDa protein, green fluorescent protein (GFP). We show that the bound cargo protein is held rigidly enough to visualize it at a resolution of 3.8 Å by cryo-EM, where specific structural features of the protein are visible. The designed scaffold is modular and can be modified through modest changes in its amino acid sequence to bind and display diverse proteins for imaging, thus providing a general method to break through the lower size limitation in cryo-EM. Proteins smaller than about 50 kDa are currently too small to be imaged at high resolution by cryo‐electron microscopy (cryo‐EM). Here authors design a protein scaffold that binds 12 copies of a small 26 kDa protein (GFP), which allowed visualizing GFP at a resolution of 3.8Å by cryo‐EM.