Tsapi EM et al. (2020) Pan Afr Med J "Prevalence of onchocerciasis after seven years of continuous community-directed treatment with ivermectin in the Ntui health district, Centre region, Cameroon."

Todjom FG, Gamago GA, Teukeng FFD, Tsapi EM, Pone JW

[Pan Afr Med J, 2020]

Introduction: onchocerciasis is one of the major infectious diseases caused by Onchocerca volvulus. This parasite is responsible for chronic cutaneous and ocular diseases affecting more than 37 million people of whom 99% are in Africa. The study was conducted in the health district of Ntui from June to September 2016 to determine the prevalence of O. volvulus infection after seven years of massive administration of ivermectin. Methods: two cutaneous snips were made at the iliac crests level in volunteers. These tissues were incubated in physiological saline water and were examined for parasitological investigations in the laboratory. Results: a total of 310 participants were randomly selected, of whom 170 (54.8%) were women and 140 (45.1%) were men aged 6 to 83 years, thus giving a sex ratio of 1.2 in favour of women. After parasitological analysis, 26 participants had microfilaraemia, of whom 15 (10.7%) were men and 11 (6.4%) were women. The most infected age group was 16 to 26 years (12.5%). The highest infection rates were found among farmers (11%) and participants living in the village of Essougly (26.6%). No significant differences in prevalence values between the different groups were noted, whatever the parameter considered. Conclusion: the prevalence of onchocerciasis in the health district of Ntui has declined from a hyperendemic to a hypoendemic state after seven years of massive administration of ivermectin. However, careful monitoring of onchocerciasis should be continued to prevent the area from returning to its original hyperendemicity.

Shu X et al. (2011) PLoS Biol "A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms."

Ramko EB, Shu X, Jin Y, Qi Y, Lev-Ram V, Davidson MW, Deerinck TJ, Tsien RY, Ellisman MH

[PLoS Biol, 2011]

Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce "miniSOG" (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.

Santella A et al. (2022) Elife "Cross-modality synthesis of EM time series and live fluorescence imaging."

Kolotuev I, Santella A, Kizilyaprak C, Bao Z

[Elife, 2022]

Analyses across imaging modalities allow the integration of complementary spatiotemporal information about brain development, structure and function. However, systematic atlasing across modalities is limited by challenges to effective image alignment. We combine highly spatially resolved electron microscopy (EM) and highly temporally resolved time-lapse fluorescence microscopy (FM) to examine the emergence of a complex nervous system in C. elegans embryogenesis. We generate an EM time series at four classic developmental stages and create a landmark-based co-optimization algorithm for cross-modality image alignment, which handles developmental heterochrony among datasets to achieve accurate single-cell level alignment. Synthesis based on the EM series and time-lapse FM series carrying different cell-specific markers reveals critical dynamic behaviors across scales of identifiable individual cells in the emergence of the primary neuropil, the nerve ring, as well as a major sensory organ, the amphid. Our study paves the way for systematic cross-modality data synthesis in C. elegans and demonstrates a powerful approach that may be applied broadly.

Markert SM et al. (2016) Neurophotonics "Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome."

Mulcahy B, Britz S, Markert SM, Lang M, Witvliet D, Proppert S, Sauer M, Stigloher C, Bessereau JL, Zhen M

[Neurophotonics, 2016]

Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses.

Rahman MM et al. (2021) Bio Protoc "A Workflow for High-pressure Freezing and Freeze Substitution of the Caenorhabditis elegans Embryo for Ultrastructural Analysis by Conventional and Volume Electron Microscopy."

Chang IY, Narayan K, Rahman MM, Cohen-Fix O

[Bio Protoc, 2021]

The free-living nematode <i>Caenorhabditis elegans</i> is a popular model system for studying developmental biology. Here we describe a detailed protocol to high-pressure freeze the <i>C. elegans</i> embryo (either <i>ex vivo</i> after dissection, or within the intact worm) followed by quick freeze substitution. Processed samples are suitable for ultrastructural analysis by conventional electron microscopy (EM) or newer volume EM (vEM) approaches such as Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). The ultrastructure of cellular features such as the nuclear envelope, chromosomes, endoplasmic reticulum and mitochondria are well preserved after these experimental procedures and yield accurate 3D models for visualization and analysis ( Chang <i>et al.</i>, 2020 ). This protocol was used in the 3D reconstruction of membranes and chromosomes after pronuclear meeting in the <i>C. elegans</i> zygote ( Rahman <i>et al.</i>, 2020 ).

Demura K et al. (2020) Sci Adv "Cryo-EM structures of calcium homeostasis modulator channels in diverse oligomeric assemblies."

Shimada H, Kusakizako T, Demura K, Yamashita K, Hiraizumi M, Nomura K, Nishizawa T, Taruno A, Nureki O, Shihoya W

[Sci Adv, 2020]

Calcium homeostasis modulator (CALHM) family proteins are Ca²⁺-regulated adenosine triphosphate (ATP)-release channels involved in neural functions including neurotransmission in gustation. Here, we present the cryo-electron microscopy (EM) structures of killifish CALHM1, human CALHM2, and <i>Caenorhabditis elegans</i> CLHM-1 at resolutions of 2.66, 3.4, and 3.6 A, respectively. The CALHM1 octamer structure reveals that the N-terminal helix forms the constriction site at the channel pore in the open state and modulates the ATP conductance. The CALHM2 undecamer and CLHM-1 nonamer structures show the different oligomeric stoichiometries among CALHM homologs. We further report the cryo-EM structures of the chimeric construct, revealing that the intersubunit interactions at the transmembrane domain (TMD) and the TMD-intracellular domain linker define the oligomeric stoichiometry. These findings advance our understanding of the ATP conduction and oligomerization mechanisms of CALHM channels.

Partlow EA et al. (2019) Elife "A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex."

Leschziner AE, Chappie JS, Baker RW, Hollopeter G, Partlow EA, Beacham GM

[Elife, 2019]

Endocytosis of transmembrane proteins is orchestrated by the AP2 clathrin adaptor complex. AP2 dwells in a closed, inactive state in the cytosol, but adopts an open, active conformation on the plasma membrane. Membrane-activated complexes are also phosphorylated, but the significance of this mark is debated. We recently proposed that NECAP negatively regulates AP2 by binding open and phosphorylated complexes (Beacham <i>et al</i>., 2018). Here, we report high-resolution cryo-EM structures of NECAP bound to phosphorylated AP2. The site of AP2 phosphorylation is directly coordinated by residues of the NECAP PHear domain that are predicted from genetic screens in <i>C. elegans</i>. Using membrane mimetics to generate conformationally open AP2, we find that a second domain of NECAP binds these complexes and cryo-EM reveals both domains of NECAP engaging closed, inactive AP2. Assays <i>in vitro</i> and <i>in vivo</i> confirm these domains cooperate to inactivate AP2. We propose that phosphorylation marks adaptors for inactivation.

Shen QT et al. (2014) J Cell Biol "Structural analysis and modeling reveals new mechanisms governing ESCRT-III spiral filament assembly."

Mitchell JC, Schuh AL, Ahlquist P, Hanna M, Audhya A, Cui Q, Otegui MS, Wang L, Shen QT, Zheng Y, Quinney K

[J Cell Biol, 2014]

The scission of biological membranes is facilitated by a variety of protein complexes that bind and manipulate lipid bilayers. ESCRT-III (endosomal sorting complex required for transport III) filaments mediate membrane scission during the ostensibly disparate processes of multivesicular endosome biogenesis, cytokinesis, and retroviral budding. However, mechanisms by which ESCRT-III subunits assemble into a polymer remain unknown. Using cryogenic electron microscopy (cryo-EM), we found that the full-length ESCRT-III subunit Vps32/CHMP4B spontaneously forms single-stranded spiral filaments. The resolution afforded by two-dimensional cryo-EM combined with molecular dynamics simulations revealed that individual Vps32/CHMP4B monomers within a filament are flexible and able to accommodate a range of bending angles. In contrast, the interface between monomers is stable and refractory to changes in conformation. We additionally found that the carboxyl terminus of Vps32/CHMP4B plays a key role in restricting the lateral association of filaments. Our findings highlight new mechanisms by which ESCRT-III filaments assemble to generate a unique polymer capable of membrane remodeling in multiple cellular contexts.

Yi X et al. (2018) J Biol Chem "Electron microscopy snapshots of single particles from single cells."

Dickinson DJ, Chang Y, Taylor DW, Verbeke EJ, Yi X

[J Biol Chem, 2018]

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 multi-protein 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 <i>Caenorhabditis elegans</i> embryos. Using this approach, we uncover three-dimensional 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.

Schierenberg E et al. (1986) Exp Cell Res "Computer-aided three-dimensional reconstruction of nematode embryos from EM serial sections."

Carlson C, Sidio W, Cole TS, Schierenberg E

[Exp Cell Res, 1986]

Embryos of the nematode Caenorhabditis elegans were serially sectioned and photographed in the electron microscope (EM). The micrographs were used to produce three-dimensional (3D) reconstructions. Size and position of each nucleus were entered into a computer, displayed as spheres, and were color-coded to indicate lineage membership. Location in space and position in the cell cycle are generally adequate criteria to identify cells. The reconstructions allow visualization of lineage-related topographic patterns and ultrastructural analysis of