The HEAT repeat protein HPO-27 is a lysosome fission factor.

Lysosomes are degradation and signalling centres crucial for homeostasis, development and ageing1. To meet diverse cellular demands, lysosomes remodel their morphology and function through constant fusion and fission2,3. Little is known about the molecular basis of fission. Here we identify HPO-27, a conserved HEAT repeat protein, as a lysosome scission factor in Caenorhabditis elegans. Loss of HPO-27 impairs lysosome fission and leads to an excessive tubular network that ultimately collapses. HPO-27 and its human homologue MROH1 are recruited to lysosomes by RAB-7 and enriched at scission sites. Super-resolution imaging, negative-staining electron microscopy and in vitro reconstitution assays reveal that HPO-27 and MROH1 self-assemble to mediate the constriction and scission of lysosomal tubules in worms and mammalian cells, respectively, and assemble to sever supported membrane tubes in vitro. Loss of HPO-27 affects lysosomal morphology, integrity and degradation activity, which impairs animal development and longevity. Thus, HPO-27 and MROH1 act as self-assembling scission factors to maintain lysosomal homeostasis and function.

Structures of the TMC-1 complex illuminate mechanosensory transduction.

The initial step in the sensory transduction pathway underpinning hearing and balance in mammals involves the conversion of force into the gating of a mechanosensory transduction channel1. Despite the profound socioeconomic impacts of hearing disorders and the fundamental biological significance of understanding mechanosensory transduction, the composition, structure and mechanism of the mechanosensory transduction complex have remained poorly characterized. Here we report the single-particle cryo-electron microscopy structure of the native transmembrane channel-like protein 1 (TMC-1) mechanosensory transduction complex isolated from Caenorhabditis elegans. The two-fold symmetric complex is composed of two copies each of the pore-forming TMC-1 subunit, the calcium-binding protein CALM-1 and the transmembrane inner ear protein TMIE. CALM-1 makes extensive contacts with the cytoplasmic face of the TMC-1 subunits, whereas the single-pass TMIE subunits reside on the periphery of the complex, poised like the handles of an accordion. A subset of complexes additionally includes a single arrestin-like protein, arrestin domain protein (ARRD-6), bound to a CALM-1 subunit. Single-particle reconstructions and molecular dynamics simulations show how the mechanosensory transduction complex deforms the membrane bilayer and suggest crucial roles for lipid-protein interactions in the mechanism by which mechanical force is transduced to ion channel gating.

Cryo-electron microscopy structure of CLHM1 ion channel from Caenorhabditis elegans.

Calcium homeostasis modulators (CALHMs/CLHMs) comprise a family of pore-forming protein complexes assembling into voltage-gated, Ca2+ -sensitive, nonselective channels. These complexes contain an ion-conduction pore sufficiently wide to permit the passing of ATP molecules serving as neurotransmitters. While their function and structure information is accumulating, the precise mechanisms of these channel complexes remain to be full understood. Here, we present the structure of the Caenorhabditis elegans CLHM1 channel in its open state solved through single-particle cryo-electron microscopy at 3.7-Å resolution. The transmembrane region of the channel structure of the dominant class shows an assembly of 10-fold rotational symmetry in one layer, and its cytoplasmic region is involved in additional twofold symmetrical packing in a tail-to-tail manner. Furthermore, we identified a series of amino acid residues critical for the regulation of CeCLHM1 channel using functional assays, electrophysiological analyses as well as structural-based analysis. Our structure and function analyses provide new insights into the mechanisms of CALHM channels.

Cryo-EM structures of undocked innexin-6 hemichannels in phospholipids.

Gap junctions form intercellular conduits with a large pore size whose closed and open states regulate communication between adjacent cells. The structural basis of the mechanism by which gap junctions close, however, remains uncertain. Here, we show the cryo-electron microscopy structures of Caenorhabditis elegans innexin-6 (INX-6) gap junction proteins in an undocked hemichannel form. In the nanodisc-reconstituted structure of the wild-type INX-6 hemichannel, flat double-layer densities obstruct the channel pore. Comparison of the hemichannel structures of a wild-type INX-6 in detergent and nanodisc-reconstituted amino-terminal deletion mutant reveals that lipid-mediated amino-terminal rearrangement and pore obstruction occur upon nanodisc reconstitution. Together with molecular dynamics simulations and electrophysiology functional assays, our results provide insight into the closure of the INX-6 hemichannel in a lipid bilayer before docking of two hemichannels.

C-mannosylation supports folding and enhances stability of thrombospondin repeats.

Previous studies demonstrated importance of C-mannosylation for efficient protein secretion. To study its impact on protein folding and stability, we analyzed both C-mannosylated and non-C-mannosylated thrombospondin type 1 repeats (TSRs) of netrin receptor UNC-5. In absence of C-mannosylation, UNC-5 TSRs could only be obtained at low temperature and a significant proportion displayed incorrect intermolecular disulfide bridging, which was hardly observed when C-mannosylated. Glycosylated TSRs exhibited higher resistance to thermal and reductive denaturation processes, and the presence of C-mannoses promoted the oxidative folding of a reduced and denatured TSR in vitro. Molecular dynamics simulations supported the experimental studies and showed that C-mannoses can be involved in intramolecular hydrogen bonding and limit the flexibility of the TSR tryptophan-arginine ladder. We propose that in the endoplasmic reticulum folding process, C-mannoses orient the underlying tryptophan residues and facilitate the formation of the tryptophan-arginine ladder, thereby influencing the positioning of cysteines and disulfide bridging.

Electron microscopy snapshots of single particles from single cells.

Cryo-electron microscopy (cryo-EM) has become an indispensable tool for structural studies of biological macromolecules. Two additional predominant methods are available for studying the architectures of multiprotein complexes: 1) single-particle analysis of purified samples and 2) tomography of whole cells or cell sections. The former can produce high-resolution structures but is limited to highly purified samples, whereas the latter can capture proteins in their native state but has a low signal-to-noise ratio and yields lower-resolution structures. Here, we present a simple, adaptable method combining microfluidic single-cell extraction with single-particle analysis by EM to characterize protein complexes from individual Caenorhabditis elegans embryos. Using this approach, we uncover 3D structures of ribosomes directly from single embryo extracts. Moreover, we investigated structural dynamics during development by counting the number of ribosomes per polysome in early and late embryos. This approach has significant potential applications for counting protein complexes and studying protein architectures from single cells in developmental, evolutionary, and disease contexts.

Demonstrating Improved Multiple Transport-Mean-Free-Path Imaging Capabilities of Light Sheet Microscopy in the Quantification of Fluorescence Dynamics.

Optical microscopy constitutes, one of the most fundamental paradigms for the understanding of complex biological mechanisms in the whole-organism and live-tissue context. Novel imaging techniques such as light sheet fluorescence microscopy (LSFM) and optical projection tomography (OPT) combined with phase-retrieval algorithms (PRT) can produce highly resolved 3D images in multiple transport-mean-free-path scales. Our study aims to exemplify the microscopic capabilities of LSFM when imaging protein dynamics in Caenorhabditis elegans and the distribution of necrotic cells in cancer cell spheroids. To this end, we apply LSFM to quantify the spatio-temporal localization of the GFP-tagged aging and stress response factor DAF-16/FOXO in transgenic C. elegans. Our analysis reveals a linear nuclear localization of DAF-16::GFP across tissues in response to heat stress, using a system that outperforms confocal scanning fluorescent microscopy in imaging speed, 3D resolution and reduced photo-toxicity. Furthermore, we present how PRT can improve the depth-to-resolution-ratio when applied to image the far-red fluorescent dye DRAQ7 which stains dead cells in a T47D cancer cell spheroid recorded with a customized OPT/LSFM system. Our studies demonstrate that LSFM combined with our novel approaches enables higher resolution and more accurate 3D quantification than previously applied technologies, proving its advance as new gold standard for fluorescence microscopy.

Structure of a eukaryotic cyclic-nucleotide-gated channel.

Cyclic-nucleotide-gated channels are essential for vision and olfaction. They belong to the voltage-gated ion channel superfamily but their activities are controlled by intracellular cyclic nucleotides instead of transmembrane voltage. Here we report a 3.5-Å-resolution single-particle electron cryo-microscopy structure of a cyclic-nucleotide-gated channel from Caenorhabditis elegans in the cyclic guanosine monophosphate (cGMP)-bound open state. The channel has an unusual voltage-sensor-like domain, accounting for its deficient voltage dependence. A carboxy-terminal linker connecting S6 and the cyclic-nucleotide-binding domain interacts directly with both the voltage-sensor-like domain and the pore domain, forming a gating ring that couples conformational changes triggered by cyclic nucleotide binding to the gate. The selectivity filter is lined by the carboxylate side chains of a functionally important glutamate and three rings of backbone carbonyls. This structure provides a new framework for understanding mechanisms of ion permeation, gating and channelopathy of cyclic-nucleotide-gated channels and cyclic nucleotide modulation of related channels.

Atomic structure of the innexin-6 gap junction channel determined by cryo-EM.

Innexins, a large protein family comprising invertebrate gap junction channels, play an essential role in nervous system development and electrical synapse formation. Here we report the cryo-electron microscopy structures of Caenorhabditis elegans innexin-6 (INX-6) gap junction channels at atomic resolution. We find that the arrangements of the transmembrane helices and extracellular loops of the INX-6 monomeric structure are highly similar to those of connexin-26 (Cx26), despite the lack of significant sequence similarity. The INX-6 gap junction channel comprises hexadecameric subunits but reveals the N-terminal pore funnel, consistent with Cx26. The helix-rich cytoplasmic loop and C-terminus are intercalated one-by-one through an octameric hemichannel, forming a dome-like entrance that interacts with N-terminal loops in the pore. These observations suggest that the INX-6 cytoplasmic domains are cooperatively associated with the N-terminal funnel conformation, and an essential linkage of the N-terminal with channel activity is presumably preserved across gap junction families.

A closed conformation of the Caenorhabditis elegans separase-securin complex.

The protease separase plays a key role in sister chromatid disjunction and centriole disengagement. To maintain genomic stability, separase activity is strictly regulated by binding of an inhibitory protein, securin. Despite its central role in cell division, the separase and securin complex is poorly understood at the structural level. This is partly owing to the difficulty of generating a sufficient quantity of homogeneous, stable protein. Here, we report the production of Caenorhabditis elegans separase-securin complex, and its characterization using biochemical methods and by negative staining electron microscopy. Single particle analysis generated a density map at a resolution of 21-24 Å that reveals a close, globular structure of complex connectivity harbouring two lobes. One lobe matches closely a homology model of the N-terminal HEAT repeat domain of separase, whereas the second lobe readily accommodates homology models of the separase C-terminal death and caspase-like domains. The globular structure of the C. elegans separase-securin complex contrasts with the more elongated structure previously described for the Homo sapiens complex, which could represent a different functional state of the complex, suggesting a mechanism for the regulation of separase activity through conformational change.

Classification using diffraction patterns for single-particle analysis.

An alternative method has been assessed; diffraction patterns derived from the single particle data set were used to perform the first round of classification in creating the initial averages for proteins data with symmetrical morphology. The test protein set was a collection of Caenorhabditis elegans small heat shock protein 17 obtained by Cryo EM, which has a tetrahedral (12-fold) symmetry. It is demonstrated that the initial classification on diffraction patterns is workable as well as the real-space classification that is based on the phase contrast. The test results show that the information from diffraction patterns has the enough details to make the initial model faithful. The potential advantage using the alternative method is twofold, the ability to handle the sets with poor signal/noise or/and that break the symmetry properties.

Solution structure of CEH-37 homeodomain of the nematode Caenorhabditis elegans.

The nematode Caenorhabditis elegans protein CEH-37 belongs to the paired OTD/OTX family of homeobox-containing homeodomain proteins. CEH-37 shares sequence similarity with homeodomain proteins, although it specifically binds to double-stranded C. elegans telomeric DNA, which is unusual to homeodomain proteins. Here, we report the solution structure of CEH-37 homeodomain and molecular interaction with double-stranded C. elegans telomeric DNA using nuclear magnetic resonance (NMR) spectroscopy. NMR structure shows that CEH-37 homeodomain is composed of a flexible N-terminal region and three α-helices with a helix-turn-helix (HTH) DNA binding motif. Data from size-exclusion chromatography and fluorescence spectroscopy reveal that CEH-37 homeodomain interacts strongly with double-stranded C. elegans telomeric DNA. NMR titration experiments identified residues responsible for specific binding to nematode double-stranded telomeric DNA. These results suggest that C. elegans homeodomain protein, CEH-37 could play an important role in telomere function via DNA binding.

High-speed atomic force microscopic observation of ATP-dependent rotation of the AAA+ chaperone p97.

p97 (also called VCP and CDC-48) is an AAA+ chaperone, which consists of a substrate/cofactor-binding N domain and two ATPase domains (D1 and D2), and forms a homo-hexameric ring. p97 plays crucial roles in a variety of cellular processes such as the ubiquitin-proteasome pathway, the endoplasmic reticulum-associated protein degradation, autophagy, and modulation of protein aggregates. Mutations in human p97 homolog VCP are linked to neurodegenerative diseases. The key mechanism of p97 in these various functions has been proposed to be the disassembly of protein complexes. To understand the molecular mechanism of p97, we studied the conformational changes of hexameric CDC-48.1, a Caenorhabditis elegans p97 homolog, using high-speed atomic force microscopy. In the presence of ATP, the N-D1 ring repeatedly rotates ~23 ± 8° clockwise and resets relative to the D2 ring. Mutational analysis reveals that this rotation is induced by ATP binding to the D2 domain.

Conserved eukaryotic fusogens can fuse viral envelopes to cells.

Caenorhabditis elegans proteins AFF-1 and EFF-1 [C. elegans fusion family (CeFF) proteins] are essential for developmental cell-to-cell fusion and can merge insect cells. To study the structure and function of AFF-1, we constructed vesicular stomatitis virus (VSV) displaying AFF-1 on the viral envelope, substituting the native fusogen VSV glycoprotein. Electron microscopy and tomography revealed that AFF-1 formed distinct supercomplexes resembling pentameric and hexameric "flowers" on pseudoviruses. Viruses carrying AFF-1 infected mammalian cells only when CeFFs were on the target cell surface. Furthermore, we identified fusion family (FF) proteins within and beyond nematodes, and divergent members from the human parasitic nematode Trichinella spiralis and the chordate Branchiostoma floridae could also fuse mammalian cells. Thus, FF proteins are part of an ancient family of cellular fusogens that can promote fusion when expressed on a viral particle.

Imaging of mitotic spindle dynamics in Caenorhabditis elegans embryos.

Development of the nematode Caenorhabditis elegans is highly reproducible, and the cell division patterns are virtually invariant. Transparency of the eggshell and cells enables the observation of intracellular events with a high temporal and spatial resolution. These unique features, along with the sophisticated genetic techniques, make this organism one of the most attractive model systems for dissecting regulatory mechanisms of dynamic cellular behaviors, such as mitosis, at an organismal level. In this chapter, we describe immunofluorescence and live imaging methods for analyzing mitotic spindle regulation. In particular, we present the use of double- or triple-labeled fluorescent strains for high-resolution two-dimensional and three-dimensional live imaging to analyze dynamic behaviors of mitotic spindles.

AMPH-1/Amphiphysin/Bin1 functions with RME-1/Ehd1 in endocytic recycling.

RME-1/EHD1 (receptor mediated endocytosis/Eps15 homology-domain containing 1) family proteins are key residents of the recycling endosome, which are required for endosome-to-plasma membrane transport in Caenorhabditis elegans and mammals. Recent studies suggest similarities between the RME-1/EHD proteins and the Dynamin GTPase superfamily of mechanochemical pinchases, which promote membrane fission. Here we show that endogenous C. elegans AMPH-1, the only C. elegans member of the Amphiphysin/BIN1 family of BAR (Bin1-Amphiphysin-Rvs161p/167p)-domain-containing proteins, colocalizes with RME-1 on recycling endosomes in vivo, that amph-1-deletion mutants are defective in recycling endosome morphology and function, and that binding of AMPH-1 Asn-Pro-Phe(Asp/Glu) sequences to the RME-1 EH-domain promotes the recycling of transmembrane cargo. We also show a requirement for human BIN1 (also known as Amphiphysin 2) in EHD1-regulated endocytic recycling. In vitro, we find that purified recombinant AMPH-1-RME-1 complexes produce short, coated membrane tubules that are qualitatively distinct from those produced by either protein alone. Our results indicate that AMPH-1 and RME-1 cooperatively regulate endocytic recycling, probably through functions required for the production of cargo carriers that exit the recycling endosome for the cell surface.

Caenorhabditis elegans innexins regulate active zone differentiation.

In a genetic screen for active zone defective mutants in Caenorhabditis elegans, we isolated a loss-of-function allele of unc-7, a gene encoding an innexin/pannexin family gap junction protein. Innexin UNC-7 regulates the size and distribution of active zones at C. elegans neuromuscular junctions. Loss-of-function mutations in another innexin, UNC-9, cause similar active zone defects as unc-7 mutants. In addition to presumptive gap junction localizations, both UNC-7 and UNC-9 are also localized perisynaptically throughout development and required in presynaptic neurons to regulate active zone differentiation. Our mosaic analyses, electron microscopy, as well as expression studies suggest a novel and likely nonjunctional role of specific innexins in active zone differentiation in addition to gap junction formations.

Maintenance of the intestinal tube in Caenorhabditis elegans: the role of the intermediate filament protein IFC-2.

The Caenorhabditis elegans intestinal lumen is surrounded by a dense cytoplasmic network that is laterally attached to the junctional complex and is referred to as the endotube. It localizes to the terminal web region which anchors the microvillar actin filament bundles and is particularly rich in intermediate filaments. To examine their role in intestinal morphogenesis and function, C. elegans reporter strains were generated expressing intestine-specific CFP-tagged intermediate filament polypeptide IFB-2. When these animals were treated with dsRNA against intestinal intermediate filament polypeptide IFC-2, the endotube developed multiple bubble-shaped invaginations that protruded into the enterocytic cytoplasm. The irregularly widened lumen remained surrounded by a continuous IFB-2::CFP-labeled layer. Comparable but somewhat mitigated phenotypic changes were also noted in wild-type N2 worms treated with ifc-2 (RNAi). Junctional complexes were ultrastructurally and functionally normal and the apical domain of intestinal cells was also not altered. These observations demonstrate that IFC-2 is important for structural maintenance of the intestinal tube but is not needed for establishment of the endotube and epithelial cell polarity.

Single-molecule force spectroscopy reveals a stepwise unfolding of Caenorhabditis elegans giant protein kinase domains.

Myofibril assembly and disassembly are complex processes that regulate overall muscle mass. Titin kinase has been implicated as an initiating catalyst in signaling pathways that ultimately result in myofibril growth. In titin, the kinase domain is in an ideal position to sense mechanical strain that occurs during muscle activity. The enzyme is negatively regulated by intramolecular interactions occurring between the kinase catalytic core and autoinhibitory/regulatory region. Molecular dynamics simulations suggest that human titin kinase acts as a force sensor. However, the precise mechanism(s) resulting in the conformational changes that relieve the kinase of this autoinhibition are unknown. Here we measured the mechanical properties of the kinase domain and flanking Ig/Fn domains of the Caenorhabditis elegans titin-like proteins twitchin and TTN-1 using single-molecule atomic force microscopy. Our results show that these kinase domains have significant mechanical resistance, unfolding at forces similar to those for Ig/Fn beta-sandwich domains (30-150 pN). Further, our atomic force microscopy data is consistent with molecular dynamic simulations, which show that these kinases unfold in a stepwise fashion, first an unwinding of the autoinhibitory region, followed by a two-step unfolding of the catalytic core. These data support the hypothesis that titin kinase may function as an effective force sensor.

Nanoscale organization of the MEC-4 DEG/ENaC sensory mechanotransduction channel in Caenorhabditis elegans touch receptor neurons.

Hearing, touch and proprioception are thought to involve direct activation of mechano-electrical transduction (MeT) channels. In Caenorhabditis elegans touch receptor neurons (TRNs), such channels contain two pore-forming subunits (MEC-4 and MEC-10) and two auxiliary subunits (MEC-2 and MEC-6). MEC-4 and MEC-10 belong to a large superfamily of ion channel proteins (DEG/ENaCs) that form nonvoltage-gated, amiloride-sensitive Na+ channels. In TRNs, unique 15-protofilament microtubules and an electron-dense extracellular matrix have been proposed to serve as gating tethers critical for MeT channel activation. We combined high-pressure freezing and serial-section immunoelectron microscopy to determine the position of MeT channels relative to putative gating tethers. MeT channels were visualized using antibodies against MEC-4 and MEC-2. This nanometer-resolution view of a sensory MeT channel establishes structural constraints on the mechanics of channel gating. We show here that MEC-2 and MEC-5 collagen, a putative extracellular tether, occupy overlapping but distinct domains in TRN neurites. Although channels decorate all sides of TRN neurites; they are not associated with the distal endpoints of 15-protofilament microtubules hypothesized to be gating tethers. These specialized microtubules, which are unique to TRNs, assemble into a cross-linked bundle connected by a network of kinked filaments to the neurite membrane. We speculate that the microtubule bundle converts external point loads into membrane stretch which, in turn, facilitates MeT channel activation.