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Mammalian cells contain an elaborate network of organelles and molecular machines that orchestrate essential cellular processes. Visualization of this network at a molecular level is vital for understanding these cellular processes. Here we present a model system based on nerve growth factor (NGF)-differentiated PC12 cells (PC12+) and suitable for high resolution imaging of organelles and molecular machines in situ. We detail an optimized imaging pipeline that effectively combines correlative light and electron microscopy (CLEM), cryo-focused ion beam (cryo-FIB), cryo-electron tomography (cryo-ET), and sub-tomogram averaging to produce three-dimensional and molecular resolution snapshots of organelles and molecular machines in near-native cellular environments. Our studies demonstrate that cryo-ET imaging of PC12+ systems provides an accessible and highly efficient avenue for dissecting specific cellular processes in mammalian cells at high resolution.

During calcium-regulated exocytosis, the constitutive fusion machinery is 'clamped' in a partially assembled state until synchronously released by calcium. The protein machinery involved in this process is known, but the supra-molecular architecture and underlying mechanisms are unclear. Here, we use cryo-electron tomography analysis in nerve growth factor-differentiated neuro-endocrine (PC12) cells to delineate the organization of the release machinery under the docked vesicles. We find that exactly six exocytosis modules, each likely consisting of a single SNAREpin with its bound Synaptotagmins, Complexin, and Munc18 proteins, are symmetrically arranged at the vesicle-PM interface. Mutational analysis suggests that the symmetrical organization is templated by circular oligomers of Synaptotagmin. The observed arrangement, including its precise radial positioning, is in-line with the recently proposed 'buttressed ring hypothesis'.

Significance Neuronal cells maintain a small number of synaptic vesicles (SVs) that are “primed,” i.e., ready to release upon receiving the triggering signal to allow for tightly regulated and rapid release of neurotransmitters. The proteins involved in this process are known, but how they assemble and operate together is poorly understood. Here we report the visualization of protein organization under primed SVs in cultured neurons under native conditions. Using cryoelectron tomography analysis, we find that there is a symmetric arrangement of exactly six protein densities at the docking interface, suggesting the fusion machinery is well ordered and prearranged for fast and precise release of neurotransmitters., Controlled release of neurotransmitters stored in synaptic vesicles (SVs) is a fundamental process that is central to all information processing in the brain. This relies on tight coupling of the SV fusion to action potential-evoked presynaptic Ca2+ influx. This Ca2+-evoked release occurs from a readily releasable pool (RRP) of SVs docked to the plasma membrane (PM). The protein components involved in initial SV docking/tethering and the subsequent priming reactions which make the SV release ready are known. Yet, the supramolecular architecture and sequence of molecular events underlying SV release are unclear. Here, we use cryoelectron tomography analysis in cultured hippocampal neurons to delineate the arrangement of the exocytosis machinery under docked SVs. Under native conditions, we find that vesicles are initially “tethered” to the PM by a variable number of protein densities (∼10 to 20 nm long) with no discernible organization. In contrast, we observe exactly six protein masses, each likely consisting of a single SNAREpin with its bound Synaptotagmins and Complexin, arranged symmetrically connecting the “primed” vesicles to the PM. Our data indicate that the fusion machinery is likely organized into a highly cooperative framework during the priming process which enables rapid SV fusion and neurotransmitter release following Ca2+ influx.

Microalgae and cyanobacteria contribute roughly half of the global photosynthetic carbon assimilation. Faced with limited access to CO(2) in aquatic environments, which can vary daily or hourly, these microorganisms have evolved use of an efficient CO(2) concentrating mechanism (CCM) to accumulate high internal concentrations of inorganic carbon (C(i)) to maintain photosynthetic performance. For eukaryotic algae, a combination of molecular, genetic and physiological studies using the model organism Chlamydomonas reinhardtii, have revealed the function and molecular characteristics of many CCM components, including active C(i) uptake systems. Fundamental to eukaryotic C(i) uptake systems are C(i) transporters/channels located in membranes of various cell compartments, which together facilitate the movement of C(i) from the environment into the chloroplast, where primary CO(2) assimilation occurs. Two putative plasma membrane C(i) transporters, HLA3 and LCI1, are reportedly involved in active C(i) uptake. Based on previous studies, HLA3 clearly plays a meaningful role in HCO(3)(−) transport, but the function of LCI1 has not yet been thoroughly investigated so remains somewhat obscure. Here we report a crystal structure of the full length LCI1 membrane protein to reveal LCI1 structural characteristics, as well as in vivo physiological studies in an LCI1 loss-of-function mutant to reveal the C(i) species preference for LCI1. Together, these new studies demonstrate LCI1 plays an important role in active CO(2) uptake and that LCI1 likely functions as a plasma membrane CO(2) channel, possibly a gated channel.

Strains of the Burkholderia cepacia complex (Bcc) are Gram-negative opportunisitic bacteria that are capable of causing serious diseases, mainly in immunocompromised individuals. Bcc pathogens are intrinsically resistant to multiple antibiotics, including β-lactams, aminoglycosides, fluoroquinolones, and polymyxins. They are major pathogens in patients with cystic fibrosis (CF) and can cause severe necrotizing pneumonia, which is often fatal. Hopanoid biosynthesis is one of the major mechanisms involved in multiple antimicrobial resistance of Bcc pathogens. The hpnN gene of B. multivorans encodes an integral membrane protein of the HpnN family of transporters, which is responsible for shuttling hopanoids to the outer membrane. Here, we report crystal structures of B. multivorans HpnN, revealing a dimeric molecule with an overall butterfly shape. Each subunit of the transporter contains 12 transmembrane helices and two periplasmic loops that suggest a plausible pathway for substrate transport. Further analyses indicate that HpnN is capable of shuttling hopanoid virulence factors from the outer leaflet of the inner membrane to the periplasm. Taken together, our data suggest that the HpnN transporter is critical for multidrug resistance and cell wall remodeling in Burkholderia.

The mycobacterial cell wall is critical to the virulence of these pathogens. Recent work shows that the MmpL (mycobacterial membrane protein large) family of transporters contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the Mycobacterium tuberculosis MmpL proteins is controlled by a complex regulatory network, including the TetR family transcriptional regulators Rv3249c and Rv1816. Here we report the crystal structures of these two regulators, revealing dimeric, two-domain molecules with architecture consistent with the TetR family of regulators. Buried extensively within the C-terminal regulatory domains of Rv3249c and Rv1816, we found fortuitous bound ligands, which were identified as palmitic acid (a fatty acid) and isopropyl laurate (a fatty acid ester), respectively. Our results suggest that fatty acids may be the natural ligands of these regulatory proteins. Using fluorescence polarization and electrophoretic mobility shift assays, we demonstrate the recognition of promoter and intragenic regions of multiple mmpL genes by these proteins. Binding of palmitic acid renders these regulators incapable of interacting with their respective operator DNAs, which will result in derepression of the corresponding mmpL genes. Taken together, these experiments provide new perspectives on the regulation of the MmpL family of transporters.

The chloroplast division machinery is composed of numerous proteins that assemble as a large complex to divide double-membraned chloroplasts through binary fission. A key mediator of division-complex formation is ARC6, a chloroplast inner envelope protein and evolutionary descendant of the cyanobacterial cell division protein Ftn2. ARC6 connects stromal and cytosolic contractile rings across the two membranes through interaction with an outer envelope protein within the intermembrane space (IMS). The ARC6 IMS region bears a structurally uncharacterized domain of unknown function, DUF4101, that is highly conserved among ARC6 and Ftn2 proteins. Here we report the crystal structure of this domain from Arabidopsis thaliana ARC6. The domain forms an α/β barrel open towards the outer envelope membrane but closed towards the inner envelope membrane. These findings provide new clues into how ARC6 and its homologs contribute to chloroplast and cyanobacterial cell division.

Mycobacterium tuberculosis is a pathogenic bacterial species, which is neither Gram positive nor Gram negative. It has a unique cell wall, making it difficult to kill and conferring resistance to antibiotics that disrupt cell wall biosynthesis. Thus, the mycobacterial cell wall is critical to the virulence of these pathogens. Recent work shows that the mycobacterial membrane protein large (MmpL) family of transporters contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the Mycobacterium tuberculosis MmpL proteins is controlled by a complicated regulatory network system. Here we report crystallographic structures of two forms of the TetR‐family transcriptional regulator Rv0302, which participates in regulating the expression of MmpL proteins. The structures reveal a dimeric, two‐domain molecule with architecture consistent with the TetR family of regulators. Comparison of the two Rv0302 crystal structures suggests that the conformational changes leading to derepression may be due to a rigid body rotational motion within the dimer interface of the regulator. Using fluorescence polarization and electrophoretic mobility shift assays, we demonstrate the recognition of promoter and intragenic regions of multiple mmpL genes by this protein. In addition, our isothermal titration calorimetry and electrophoretic mobility shift experiments indicate that fatty acids may be the natural ligand of this regulator. Taken together, these experiments provide new perspectives on the regulation of the MmpL family of transporters.

Resistance-nodulation-cell division efflux pumps are integral membrane proteins that catalyze the export of substrates across cell membranes. Within the hydrophobe-amphiphile efflux subfamily, these resistance-nodulation-cell division proteins largely form trimeric efflux pumps. The drug efflux process has been proposed to entail a synchronized motion between subunits of the trimer to advance the transport cycle, leading to the extrusion of drug molecules. Here we use X-ray crystallography and single-molecule fluorescence resonance energy transfer imaging to elucidate the structures and functional dynamics of the Campylobacter jejuni CmeB multidrug efflux pump. We find that the CmeB trimer displays a very unique conformation. A direct observation of transport dynamics in individual CmeB trimers embedded in membrane vesicles indicates that each CmeB subunit undergoes conformational transitions uncoordinated and independent of each other. On the basis of our findings and analyses, we propose a model for transport mechanism where CmeB protomers function independently within the trimer., Multidrug efflux pumps significantly contribute for bacteria resistance to antibiotics. Here the authors present the structure of Campylobacter jejuni CmeB pump combined with functional FRET assays to propose a transport mechanism where each CmeB protomers is functionally independent from the trimer.

As one of the world's most prevalent enteric pathogens, Campylobacter jejuni is a major causative agent of human enterocolitis and is responsible for more than 400 million cases of diarrhea each year. The impact of this pathogen on children is of particular significance. Campylobacter has developed resistance to many antimicrobial agents via multidrug efflux machinery. The CmeABC tripartite multidrug efflux pump, belonging to the resistance-nodulation-cell division (RND) superfamily, plays a major role in drug resistant phenotypes of C. jejuni. This efflux complex spans the entire cell envelop of C. jejuni and mediates resistance to various antibiotics and toxic compounds. We here report the crystal structure of C. jejuni CmeC, the outer membrane component of the CmeABC tripartite multidrug efflux system. The structure reveals a possible mechanism for substrate export.

The Rv1217c-Rv1218c multidrug efflux system, which belongs to the ATP-binding cassette superfamily, recognizes and actively extrudes a variety of structurally unrelated toxic chemicals and mediates the intrinsic resistance to these antimicrobials in Mycobacterium tuberculosis. The expression of Rv1217c-Rv1218c is controlled by the TetR-like transcriptional regulator Rv1219c, which is encoded by a gene immediately upstream of rv1218c. To elucidate the structural basis of Rv1219c regulation, we have determined the crystal structure of Rv1219c, which reveals a dimeric two-domain molecule with an entirely helical architecture similar to members of the TetR family of transcriptional regulators. The N-terminal domains of the Rv1219c dimer are separated by a large center-to-center distance of 64 A. The C-terminal domain of each protomer possesses a large cavity. Docking of small compounds to Rv1219c suggests that this large cavity forms a multidrug binding pocket, which can accommodate a variety of structurally unrelated antimicrobial agents. The internal wall of the multidrug binding site is surrounded by seven aromatic residues, indicating that drug binding may be governed by aromatic stacking interactions. In addition, fluorescence polarization reveals that Rv1219c binds drugs in the micromolar range.

The potential of the folic acid biosynthesis pathway as a target for the development of antibiotics has been clinically validated. However, many pathogens have developed resistance to these antibiotics, prompting a re-evaluation of potential drug targets within the pathway. The ydaH gene of Alcanivorax borkumensis encodes an integral membrane protein of the AbgT family of transporters for which no structural information was available. Here we report the crystal structure of A. borkumensis YdaH, revealing a dimeric molecule with an architecture distinct from other families of transporters. YdaH is a bowl-shaped dimer with a solvent-filled basin extending from the cytoplasm to halfway across the membrane bilayer. Each subunit of the transporter contains nine transmembrane helices and two hairpins that suggest a plausible pathway for substrate transport. Further analyses also suggest that YdaH could act as an antibiotic efflux pump and mediate bacterial resistance to sulfonamide antimetabolite drugs.

Recent work demonstrates that the MmpL (mycobacterial membrane protein large) transporters are dedicated to the export of mycobacterial lipids for cell wall biosynthesis. An MmpL transporter frequently works with an accessory protein, belonging to the MmpS (mycobacterial membrane protein small) family, to transport these key virulence factors. One such efflux system in Mycobacterium tuberculosis is the MmpS5-MmpL5 transporter. The expression of MmpS5-MmpL5 is controlled by the MarR-like transcriptional regulator Rv0678, whose open reading frame is located downstream of the mmpS5-mmpL5 operon. To elucidate the structural basis of Rv0678 regulation, we have determined the crystal structure of this regulator, to 1.64 Å resolution, revealing a dimeric two-domain molecule with an architecture similar to members of the MarR family of transcriptional regulators. Rv0678 is distinct from other MarR regulators in that its DNA-binding and dimerization domains are clustered together. These two domains seemingly cooperate to bind an inducing ligand that we identified as 2-stearoylglycerol, which is a fatty acid glycerol ester. The structure also suggests that the conformational change leading to substrate-mediated derepression is primarily caused by a rigid body rotational motion of the entire DNA-binding domain of the regulator toward the dimerization domain. This movement results in a conformational state that is incompatible with DNA binding. We demonstrate using electrophoretic mobility shift assays that Rv0678 binds to the mmpS5-mmpL5, mmpS4-mmpL4, and the mmpS2-mmpL2 promoters. Binding by Rv0678 was reversed upon the addition of the ligand. These findings provide new insight into the mechanisms of gene regulation in the MarR family of regulators.

Active efflux of antimicrobial agents is one of the most important strategies used by bacteria to defend against antimicrobial factors present in their environment. Mediating many cases of antibiotic resistance are transmembrane efflux pumps, composed of one or more proteins. The Neisseria gonorrhoeae MtrCDE tripartite multidrug efflux pump, belonging to the hydrophobic and amphiphilic efflux resistance-nodulation-cell division (HAE-RND) family, spans both the inner and outer membranes of N. gonorrhoeae and confers resistance to a variety of antibiotics and toxic compounds. We here describe the crystal structure of N. gonorrhoeae MtrE, the outer membrane component of the MtrCDE tripartite multidrug efflux system. This trimeric MtrE channel forms a vertical tunnel extending down contiguously from the outer membrane surface to the periplasmic end, indicating that our structure of MtrE depicts an open conformational state of this channel.

Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually-transmitted disease gonorrhea. The control of this disease has been compromised by the increasing proportion of infections due to antibiotic-resistant strains, which are growing at an alarming rate. The MtrCDE tripartite multidrug efflux pump, belonging to the hydrophobic and amphiphilic efflux resistance-nodulation-cell division (HAE-RND) family, spans both the inner and outer membranes of N. gonorrhoeae and confers resistance to a variety of antibiotics and toxic compounds. We here report the crystal structure of the inner membrane MtrD multidrug efflux pump, which reveals a novel structural feature that is not found in other RND efflux pumps.

The Rv1217c–Rv1218c multidrug efflux system, which belongs to the ATP-binding cassette superfamily, recognizes and actively extrudes a variety of structurally unrelated toxic chemicals and mediates the intrinsic resistance to these antimicrobials in Mycobacterium tuberculosis. The expression of Rv1217c–Rv1218c is controlled by the TetR-like transcriptional regulator Rv1219c, which is encoded by a gene immediately upstream of rv1218c. To elucidate the structural basis of Rv1219c regulation, we have determined the crystal structure of Rv1219c, which reveals a dimeric two-domain molecule with an entirely helical architecture similar to members of the TetR family of transcriptional regulators. The N-terminal domains of the Rv1219c dimer are separated by a large center-to-center distance of 64 Å. The C-terminal domain of each protomer possesses a large cavity. Docking of small compounds to Rv1219c suggests that this large cavity forms a multidrug binding pocket, which can accommodate a variety of structurally unrelated antimicrobial agents. The internal wall of the multidrug binding site is surrounded by seven aromatic residues, indicating that drug binding may be governed by aromatic stacking interactions. In addition, fluorescence polarization reveals that Rv1219c binds drugs in the micromolar range.

SummaryNeisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually transmitted disease gonorrhea. The control of this disease has been compromised by the increasing proportion of infections due to antibiotic-resistant strains, which are growing at an alarming rate. N. gonorrhoeae MtrF is an integral membrane protein that belongs to the AbgT family of transporters for which no structural information is available. Here, we describe the crystal structure of MtrF, revealing a dimeric molecule with architecture distinct from all other families of transporters. MtrF is a bowl-shaped dimer with a solvent-filled basin extending from the cytoplasm to halfway across the membrane bilayer. Each subunit of the transporter contains nine transmembrane helices and two hairpins, posing a plausible pathway for substrate transport. A combination of the crystal structure and biochemical functional assays suggests that MtrF is an antibiotic efflux pump mediating bacterial resistance to sulfonamide antimetabolite drugs.