Cryo-EM structure of cortical microtubules from human parasite Toxoplasma gondii identifies their microtubule inner proteins

In living cells, microtubules (MTs) play pleiotropic roles, which require very different mechanical properties. Unlike the dynamic MTs found in the cytoplasm of metazoan cells, the specialized cortical MTs from Toxoplasma gondii, a prevalent human pathogen, are extraordinarily stable and resistant to detergent and cold treatments. Using single-particle cryo-EM, we determine their ex vivo structure and identify three proteins (TrxL1, TrxL2 and SPM1) as bona fide microtubule inner proteins (MIPs). These three MIPs form a mesh on the luminal surface and simultaneously stabilize the tubulin lattice in both longitudinal and lateral directions. Consistent with previous observations, deletion of the identified MIPs compromises MT stability and integrity under challenges by chemical treatments. We also visualize a small molecule like density at the Taxol-binding site of β-tubulin. Our results provide the structural basis to understand the stability of cortical MTs and suggest an evolutionarily conserved mechanism of MT stabilization from the inside

Integrated model of the vertebrate augmin complex

Accurate segregation of chromosomes is required to maintain genome integrity during cell division. This feat is accomplished by the microtubule-based spindle. To build a spindle rapidly and with high fidelity, cells take advantage of branching microtubule nucleation, which rapidly amplifies microtubules during cell division. Branching microtubule nucleation relies on the hetero-octameric augmin complex, but lack of structure information about augmin has hindered understanding how it promotes branching. In this work, we combine cryo-electron microscopy, protein structural prediction, and visualization of fused bulky tags via negative stain electron microscopy to identify the location and orientation of each subunit within the augmin structure. Evolutionary analysis shows that augmin\u27s structure is highly conserved across eukaryotes, and that augmin contains a previously unidentified microtubule binding site. Thus, our findings provide insight into the mechanism of branching microtubule nucleation

Crystal structure and biochemical analyses reveal Beclin 1 as a novel membrane binding protein

The Beclin 1 gene is a haplo-insufficient tumor suppressor and plays an essential role in autophagy. However, the molecular mechanism by which Beclin 1 functions remains largely unknown. Here we report the crystal structure of the evolutionarily conserved domain (ECD) of Beclin 1 at 1.6 Å resolution. Beclin 1 ECD exhibits a previously unreported fold, with three structural repeats arranged symmetrically around a central axis. Beclin 1 ECD defines a novel class of membrane-binding domain, with a strong preference for lipid membrane enriched with cardiolipin. The tip of a surface loop in Beclin 1 ECD, comprising three aromatic amino acids, acts as a hydrophobic finger to associate with lipid membrane, consequently resulting in the deformation of membrane and liposomes. Mutation of these aromatic residues rendered Beclin 1 unable to stably associate with lipid membrane in vitro and unable to fully rescue autophagy in Beclin 1-knockdown cells in vivo. These observations form an important framework for deciphering the biological functions of Beclin 1

Design of composite membrane emergency drainage device for floating roof tank

Emergency drainage device is an important safety device for floating roof tank to timely drain the overloaded water in case of emergency. In practical application, a large amount of maintenance work is required for the commonlyused float type emergency drainage device with water seal structure. Especially for the float type emergency drainage device in dry weather conditions, frequent water filling is required, and there is potential safety hazard of water seal failure that is difficult to recover. According to the characteristics of PVA and PVDC materials, the solution of a new type of emergency drainage device in composite membrane was designed. Based on its flame retardant modification, PVA was compounded with PVDC, and thereby an emergency drainage device with composite membrane as its main body was designed to make up for the deficiency of float-type emergency drainage device with water seal structure. The field test results show that, since there is no water seal in the composite membrane emergency drainage device of the floating roof tank, the maintenance workload is reduced and the operation safety of the floating roof tank is improved

Structural mechanism of SARS-CoV-2 neutralization by two murine antibodies targeting the RBD

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has necessitated the rapid development of antibody-based therapies and vaccines as countermeasures. Here, we use cryoelectron microscopy (cryo-EM) to characterize two protective anti-SARS-CoV-2 murine monoclonal antibodies (mAbs) in complex with the spike protein, revealing similarities between epitopes targeted by human and murine B cells. The more neutralizing mAb, 2B04, binds the receptor-binding motif (RBM) of the receptor-binding domain (RBD) and competes with angiotensin-converting enzyme 2 (ACE2). By contrast, 2H04 binds adjacent to the RBM and does not compete for ACE2 binding. Naturally occurring sequence variants of SARS-CoV-2 and corresponding neutralization escape variants selected in vitro map to our structurally defined epitopes, suggesting that SARS-CoV-2 might evade therapeutic antibodies with a limited set of mutations, underscoring the importance of combination mAb therapeutics. Finally, we show that 2B04 neutralizes SARS-CoV-2 infection by preventing ACE2 engagement, whereas 2H04 reduces host cell attachment without directly disrupting ACE2-RBM interactions, providing distinct inhibitory mechanisms used by RBD-specific mAbs

Structures of radial spokes and associated complexes important for ciliary motility

© 2020, The Author(s), under exclusive licence to Springer Nature America, Inc. In motile cilia, a mechanoregulatory network is responsible for converting the action of thousands of dynein motors bound to doublet microtubules into a single propulsive waveform. Here, we use two complementary cryo-EM strategies to determine structures of the major mechanoregulators that bind ciliary doublet microtubules in Chlamydomonas reinhardtii. We determine structures of isolated radial spoke RS1 and the microtubule-bound RS1, RS2 and the nexin−dynein regulatory complex (N-DRC). From these structures, we identify and build atomic models for 30 proteins, including 23 radial-spoke subunits. We reveal how mechanoregulatory complexes dock to doublet microtubules with regular 96-nm periodicity and communicate with one another. Additionally, we observe a direct and dynamically coupled association between RS2 and the dynein motor inner dynein arm subform c (IDAc), providing a molecular basis for the control of motor activity by mechanical signals. These structures advance our understanding of the role of mechanoregulation in defining the ciliary waveform

Determination of a novel parvovirus pathogen associated with massive mortality in adult tilapia.

Tilapia is one of the most important economic and fastest-growing species in aquaculture worldwide. In 2015, an epidemic associated with severe mortality occurred in adult tilapia in Hubei, China. The causative pathogen was identified as Tilapia parvovirus (TiPV) by virus isolation, electron microscopy, experimental challenge, In situ hybridization (ISH), indirect immunofluorescence (IFA), and viral gene sequencing. Electron microscopy revealed large numbers of parvovirus particles in the organs of diseased fish, including kidney, spleen, liver, heart, brain, gill, intestine, etc. The virions were spherical in shape, non-enveloped and approximately 30nm in diameter. The TiPV was isolated and propagated in tilapia brain cells (TiB) and induced a typical cytopathic effect (CPE) after 3 days post-infection (dpi). This virus was used to experimentally infect adult tilapia and clinical disease symptoms similar to those observed naturally were replicated. Additionally, the results of ISH and IFA showed positive signals in kidney and spleen tissues from TiPV-infected fish. To identify TiPV-specific sequences, the near complete genome of TiPV was obtained and determined to be 4269 bp in size. Phylogenetic analysis of the NS1 sequence revealed that TiPV is a novel parvovirus, forms a separate branch in proposed genus Chapparvovirus of Parvoviridae. Results presented here confirm that TiPV is a novel parvovirus pathogen that can cause massive mortality in adult tilapia. This provides a basis for the further studies to define the epidemiology, pathology, diagnosis, prevention and treatment of this emerging viral disease

Paracrine Action of Mesenchymal Stem Cells Revealed by Single Cell Gene Profiling in Infarcted Murine Hearts

<div><p>Background</p><p>Mesenchymal stem cells (MSCs) have been recently demonstrated as a promising stem cell type to rescue damaged myocardium after acute infarction. One of the most important mechanisms underlying their therapeutic effects is the secretion of paracrine factors. However, the expression profile of paracrine factors of MSCs in infarcted hearts, especially at single cell level, is poorly defined.</p><p>Methods and Results</p><p>We aimed to depict the transcriptional profile of paracrine factors secreted by MSCs <i>in vivo</i>, with particular interest in the comparison between normal and infarcted hearts. Bone marrow mesenchymal stem cells were isolated and injected into mice hearts immediately after infarction surgery. Bioluminescence imaging (BLI) indicated a proportion of cells still alive even up to 10 days post surgery. Paralleled with survived cells, cardiac function was significantly improved after MSC injection compared to that in PBS-injected mice, indicated by MRI and histology. Despite increased number of vessels in MSC-injected hearts, endothelial cells and cardiomyocytes transdifferentiation were not observed in infarcted hearts 5 days after infarction. Furthermore, laser capture microdissection (LCM) followed by high through-put real time PCR was employed in our study, uncovering that the injected MSCs, compared to local cardiomyocytes, displayed elevated levels of secreted factors. To further investigate the regulation of those factors, we performed single cell analysis to dissect the gene expression profile of MSCs at single cell level in infarcted and normal hearts, respectively. Consistent with the <i>in vivo</i> observation, a similar regulation pattern of those factors was detected in cultured MSCs under hypoxia.</p><p>Conclusions</p><p>Our study, for the first time, elucidated gene expression profiles, as well as regulation of paracrine factors, of MSCs at single cell level <i>in vivo</i>, indicating that paracrine factors from MSCs account for the improvement of cardiac function after infarction.</p></div

Immunostaining for CD31 and Actinin 2 in infarcted hearts with transplanted MSCs.

<p>(A) Representative images of immunostaining against CD31 (red fluorescence) in infarcted hearts. Magnification: 100× in upper and middle panels, 400× in lower panel which is the magnification of the box in upper panel. (B) Statistical analysis of vessel density. The value was an average of 8 slides per each mouse, total 8 mice per each group. (C) Representative images of immunostaining against Actinin 2 (red fluorescence) in infarcted hearts.</p