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2108 Janelia Publications

Showing 1-10 of 2108 results
05/16/22 | In situ cryo-electron tomography reveals the asymmetric architecture of mammalian sperm axonemes
Zhen Chen , Garrett A. Greenan , Momoko Shiozaki , Yanxin Liu , Will M. Skinner , Xiaowei Zhao , Shumei Zhao , Rui Yan , Caiying Guo , Zhiheng Yu , Polina V. Lishko , David A. Agard , Ronald D. Vale
bioRxiv. 2022 May 16:. doi: 10.1101/2022.05.15.492011

The flagella of mammalian sperm display non-planar, asymmetric beating, in contrast to the planar, symmetric beating of flagella from sea urchin sperm and unicellular organisms. The molecular basis of this difference is unclear. Here, we perform in situ cryo-electron tomography of mouse and human sperm axonemes, providing the highest resolution structural information to date. Our subtomogram averages reveal mammalian sperm- specific protein complexes within the outer microtubule doublets, the radial spokes and nexin-dynein regulatory complexes. The locations and structures of these complexes suggest potential roles in enhancing the mechanical strength of mammalian sperm axonemes and regulating dynein-based axonemal bending. Intriguingly, we find that each of the nine outer microtubule doublets is decorated with a distinct combination of sperm- specific complexes. We propose that this asymmetric distribution of proteins differentially regulates the sliding of each microtubule doublet and may underlie the asymmetric beating of mammalian sperm.

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05/15/22 | Combining multiple fluorescence imaging techniques in biology: when one microscope is not enough.
Hobson CM, Aaron JS
Molecular Biology of the Cell. 2022 May 15;33(6):tp1. doi: 10.1091/mbc.E21-10-0506

While fluorescence microscopy has proven to be an exceedingly useful tool in bioscience, it is difficult to offer simultaneous high resolution, fast speed, large volume, and good biocompatibility in a single imaging technique. Thus, when determining the image data required to quantitatively test a complex biological hypothesis, it often becomes evident that multiple imaging techniques are necessary. Recent years have seen an explosion in development of novel fluorescence microscopy techniques, each of which features a unique suite of capabilities. In this Technical Perspective, we highlight recent studies to illustrate the benefits, and often the necessity, of combining multiple fluorescence microscopy modalities. We provide guidance in choosing optimal technique combinations to effectively address a biological question. Ultimately, we aim to promote a more well-rounded approach in designing fluorescence microscopy experiments, leading to more robust quantitative insight.

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05/13/22 | Recovery mechanisms in the dragonfly righting reflex.
Wang ZJ, Melfi J, Leonardo A
Science. 2022 May 13;376(6594):754-758. doi: 10.1126/science.abg0946

Insects have evolved sophisticated reflexes to right themselves in mid-air. Their recovery mechanisms involve complex interactions among the physical senses, muscles, body, and wings, and they must obey the laws of flight. We sought to understand the key mechanisms involved in dragonfly righting reflexes and to develop physics-based models for understanding the control strategies of flight maneuvers. Using kinematic analyses, physical modeling, and three-dimensional flight simulations, we found that a dragonfly uses left-right wing pitch asymmetry to roll its body 180 degrees to recover from falling upside down in ~200 milliseconds. Experiments of dragonflies with blocked vision further revealed that this rolling maneuver is initiated by their ocelli and compound eyes. These results suggest a pathway from the dragonfly's visual system to the muscles regulating wing pitch that underly the recovery. The methods developed here offer quantitative tools for inferring insects' internal actions from their acrobatics, and are applicable to a broad class of natural and robotic flying systems.

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05/11/22 | Super-resolution microscopy reveals actomyosin dynamics in medioapical arrays.
Moore RP, Fogerson SM, Tulu US, Yu JW, Cox AH, Sican MA, Li D, Legant WR, Weigel AV, Crawford JM, Betzig E, Kiehart DP
Molecular Biology of the Cell. 2022 May 11:mbcE21110537. doi: 10.1091/mbc.E21-11-0537

Arrays of actin filaments (F-actin) near the apical surface of epithelial cells (medioapical arrays) contribute to apical constriction and morphogenesis throughout phylogeny. Here, super-resolution approaches (grazing incidence structured illumination, GI-SIM and lattice light sheet, LLSM) microscopy resolve individual, fluorescently labeled F-actin and bipolar myosin filaments that drive amnioserosa cell shape changes during dorsal closure in . In expanded cells, F-actin and myosin form loose, apically domed meshworks at the plasma membrane. The arrays condense as cells contract, drawing the domes into the plane of the junctional belts. As condensation continues, individual filaments are no longer uniformly apparent. As cells expand, arrays of actomyosin are again resolved - some F-actin turnover likely occurs, but a large fraction of existing filaments rearrange. In morphologically isotropic cells, actin filaments are randomly oriented and during contraction, are drawn together but remain essentially randomly oriented. In anisotropic cells, largely parallel actin filaments are drawn closer to one another. Our images offer unparalleled resolution of F-actin in embryonic tissue show that medioapical arrays are tightly apposed to the plasma membrane, are continuous with meshworks of lamellar F-actin and thereby constitute modified cell cortex. In concert with other tagged array components, super-resolution imaging of live specimens will offer new understanding of cortical architecture and function. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

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05/10/22 | Rescue of behavioral and electrophysiological phenotypes in a Pitt-Hopkins syndrome mouse model by genetic restoration of expression.
Kim H, Gao EB, Draper A, Berens NC, Vihma H, Zhang X, Higashi-Howard A, Ritola KD, Simon JM, Kennedy AJ, Philpot BD
eLife. 2022 May 10;11:. doi: 10.7554/eLife.72290

Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder caused by monoallelic mutation or deletion in the () gene. Individuals with PTHS typically present in the first year of life with developmental delay and exhibit intellectual disability, lack of speech, and motor incoordination. There are no effective treatments available for PTHS, but the root cause of the disorder, haploinsufficiency, suggests that it could be treated by normalizing gene expression. Here, we performed proof-of-concept viral gene therapy experiments using a conditional mouse model of PTHS and found that postnatally reinstating expression in neurons improved anxiety-like behavior, activity levels, innate behaviors, and memory. Postnatal reinstatement also partially corrected EEG abnormalities, which we characterized here for the first time, and the expression of key TCF4-regulated genes. Our results support a genetic normalization approach as a treatment strategy for PTHS, and possibly other TCF4-linked disorders.

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05/09/22 | Gene structure-based homology search identifies highly divergent putative effector gene family.
Stern DL, Han C
Genome Biology and Evolution. 2022 May 09:. doi: 10.1093/gbe/evac069

Homology of highly divergent genes often cannot be determined from sequence similarity alone. For example, we recently identified in the aphid Hormaphis cornu a family of rapidly evolving bicycle genes, which encode novel proteins implicated as plant gall effectors, and sequence similarity search methods yielded few putative bicycle homologs in other species. Coding sequence-independent features of genes, such as intron-exon boundaries, often evolve more slowly than coding sequences, however, and can provide complementary evidence for homology. We found that a linear logistic regression classifier using only structural features of bicycle genes identified many putative bicycle homologs in other species. Independent evidence from sequence features and intron locations supported homology assignments. To test the potential roles of bicycle genes in other aphids, we sequenced the genome of a second gall-forming aphid, Tetraneura nigriabdominalis, and found that many bicycle genes are strongly expressed in the salivary glands of the gall forming foundress. In addition, bicycle genes are strongly overexpressed in the salivary glands of a non-gall forming aphid, Acyrthosiphon pisum, and in the non-gall forming generations of Hormaphis cornu. These observations suggest that Bicycle proteins may be used by multiple aphid species to manipulate plants in diverse ways. Incorporation of gene structural features into sequence search algorithms may aid identification of deeply divergent homologs, especially of rapidly evolving genes involved in host-parasite interactions.

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05/05/22 | Actin nano-architecture of phagocytic podosomes
J. Cody Herron , Shiqiong Hu , Takashi Watanabe , Ana T. Nogueira , Bei Liu , Megan Kern , Jesse Aaron , Aaron Taylor , Michael Pablo , Teng-Leong Chew , Timothy C. Elston , Klaus M. Hahn
bioRxiv. 2022 May 05:. doi: 10.1101/2022.05.04.490675

Podosomes are actin-enriched adhesion structures important for multiple cellular processes, including migration, bone remodeling, and phagocytosis. Here, we characterized the structure and organization of phagocytic podosomes using interferometric photoactivated localization microscopy (iPALM), a super-resolution microscopy technique capable of 15-20 nm resolution, together with structured illumination microscopy (SIM) and localization-based superresolution microscopy. Phagocytic podosomes were observed during frustrated phagocytosis, a model in which cells attempt to engulf micro-patterned IgG antibodies. For circular patterns, this resulted in regular arrays of podosomes with well-defined geometry. Using persistent homology, we developed a pipeline for semi-automatic identification and measurement of podosome features. These studies revealed an "hourglass" shape of the podosome actin core, a protruding "knob" at the bottom of the core, and two actin networks extending from the core. Additionally, the distributions of paxillin, talin, myosin II, α-actinin, cortactin, and microtubules relative to actin were characterized.

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04/29/22 | Cryo-EM structure of the EBV ribonucleotide reductase BORF2 and mechanism of APOBEC3B inhibition.
Shaban NM, Yan R, Shi K, Moraes SN, Cheng AZ, Carpenter MA, McLellan JS, Yu Z, Harris RS
Science Advances. 2022 Apr 29;8(17):eabm2827. doi: 10.1126/sciadv.abm2827

Viruses use a plethora of mechanisms to evade immune responses. A recent example is neutralization of the nuclear DNA cytosine deaminase APOBEC3B by the Epstein-Barr virus (EBV) ribonucleotide reductase subunit BORF2. Cryo-EM studies of APOBEC3B-BORF2 complexes reveal a large >1000-Å binding surface composed of multiple structural elements from each protein, which effectively blocks the APOBEC3B active site from accessing single-stranded DNA substrates. Evolutionary optimization is suggested by unique insertions in BORF2 absent from other ribonucleotide reductases and preferential binding to APOBEC3B relative to the highly related APOBEC3A and APOBEC3G enzymes. A molecular understanding of this pathogen-host interaction has potential to inform the development of drugs that block the interaction and liberate the natural antiviral activity of APOBEC3B. In addition, given a role for APOBEC3B in cancer mutagenesis, it may also be possible for information from the interaction to be used to develop DNA deaminase inhibitors.

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04/29/22 | The PV2 cluster of parvalbumin neurons in the murine periaqueductal gray: connections and gene expression.
Leemann S, Babalian A, Girard F, Davis F, Celio MR
Brain Structure and Function. 2022 Apr 29:. doi: 10.1007/s00429-022-02491-0

The PV2 (Celio 1990), a cluster of parvalbumin-positive neurons located in the ventromedial region of the distal periaqueductal gray (PAG) has not been previously described as its own entity, leading us to study its extent, connections, and gene expression. It is an oval, bilateral, elongated cluster composed of approximately 475 parvalbumin-expressing neurons in a single mouse hemisphere. In its anterior portion it impinges upon the paratrochlear nucleus (Par4) and in its distal portion it is harbored in the posterodorsal raphe nucleus (PDR). It is known to receive inputs from the orbitofrontal cortex and from the parvafox nucleus in the ventrolateral hypothalamus. Using anterograde tracing methods in parvalbumin-Cre mice, the main projections of the PV2 cluster innervate the supraoculomotor periaqueductal gray (Su3) of the PAG, the parvafox nucleus of the lateral hypothalamus, the gemini nuclei of the posterior hypothalamus, the septal regions, and the diagonal band in the forebrain, as well as various nuclei within the reticular formation in the midbrain and brainstem. Within the brainstem, projections were discrete, but involved areas implicated in autonomic control. The PV2 cluster expressed various peptides and receptors, including the receptor for Adcyap1, a peptide secreted by one of its main afferences, namely, the parvafox nucleus. The expression of GAD1 and GAD2 in the region of the PV2, the presence of Vgat-1 in a subpopulation of PV2-neurons as well as the coexistence of GAD67 immunoreactivity with parvalbumin in terminal endings indicates the inhibitory nature of a subpopulation of PV2-neurons. The PV2 cluster may be part of a feedback controlling the activity of the hypothalamic parvafox and the Su3 nuclei in the periaqueductal gray.

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04/27/22 | Hormone-controlled changes in the differentiation state of post-mitotic neurons.
Lai Y, Miyares RL, Liu L, Chu S, Lee T, Yu H
Current Biology. 2022 Apr 27:. doi: 10.1016/j.cub.2022.04.027

While we think of neurons as having a fixed identity, many show spectacular plasticity. Metamorphosis drives massive changes in the fly brain; neurons that persist into adulthood often change in response to the steroid hormone ecdysone. Besides driving remodeling, ecdysone signaling can also alter the differentiation status of neurons. The three sequentially born subtypes of mushroom body (MB) Kenyon cells (γ, followed by α'/β', and finally α/β) serve as a model of temporal fating. γ neurons are also used as a model of remodeling during metamorphosis. As γ neurons are the only functional Kenyon cells in the larval brain, they serve the function of all three adult subtypes. Correspondingly, larval γ neurons have a similar morphology to α'/β' and α/β neurons-their axons project dorsally and medially. During metamorphosis, γ neurons remodel to form a single medial projection. Both temporal fate changes and defects in remodeling therefore alter γ-neuron morphology in similar ways. Mamo, a broad-complex, tramtrack, and bric-à-brac/poxvirus and zinc finger (BTB/POZ) transcription factor critical for temporal specification of α'/β' neurons, was recently described as essential for γ remodeling. In a previous study, we noticed a change in the number of adult Kenyon cells expressing γ-specific markers when mamo was manipulated. These data implied a role for Mamo in γ-neuron fate specification, yet mamo is not expressed in γ neurons until pupariation, well past γ specification. This indicates that mamo has a later role in ensuring that γ neurons express the correct Kenyon cell subtype-specific genes in the adult brain.

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