Intraflagellar transport complex structure and cargo interactions

Intraflagellar transport (IFT) is required for the assembly and maintenance of cilia, as well as the proper function of ciliary motility and signaling. IFT is powered by molecular motors that move along the axonemal microtubules, carrying large complexes of IFT proteins that travel together as so-called trains. IFT complexes likely function as adaptors that mediate interactions between anterograde/retrograde motors and ciliary cargoes, facilitating cargo transport between the base and tip of the cilium. Here, we provide an up-to-date review of IFT complex structure and architecture, and discuss how interactions with cargoes and motors may be achieved

Intraflagellar transport particle size scales inversely with flagellar length: revisiting the balance-point length control model

Chlamydomonas reinhardtii IFT particle trains, important for flagella maintenance and assembly, are observed to decrease in size as a function of cilia length

Cryo-focused ion beam sample preparation for imaging vitreous cells by cryo-electron tomography

Cryo-electron tomography (CET) is a well-established technique for imaging cellular and molecular structures at sub-nanometer resolution. As the method is limited to samples that are thinner than 500 nm, suitable sample preparation is required to attain CET data from larger cell volumes. Recently, cryo-focused ion beam (cryo-FIB) milling of plunge-frozen biological material has been shown to reproducibly yield large, homogeneously thin, distortion-free vitreous cross-sections for state-of-the-art CET. All eukaryotic and prokaryotic cells that can be plunge-frozen can be thinned with the cryo-FIB technique. Together with advances in low-dose microscopy, this has shifted the frontiers of in situ structural biology. In this protocol we describe the typical steps of the cryo-FIB technique, starting with fully grown cell cultures. Three recently investigated biological samples are given as examples

MemBrain: a deep learning-aided pipeline for detection of membrane proteins in cryo-electron tomograms

Cryo-electron tomography (cryo-ET) is an imaging technique that enables 3D visualization of the native cellular environment at sub-nanometer resolution, providing unpreceded insights into the molecular organization of cells. However, cryo-electron tomograms suffer from low signal-to-noise ratios and anisotropic resolution, which makes subsequent image analysis challenging. In particular, the efficient detection of membrane-embedded proteins is a problem still lacking satisfactory solutions. We present MemBrain - a new deep learning-aided pipeline that automatically detects membrane-bound protein complexes in cryo-electron tomograms. After subvolumes are sampled along a segmented membrane, each subvolume is assigned a score using a convolutional neural network (CNN), and protein positions are extracted by a clustering algorithm. Incorporating rotational subvolume normalization and using a tiny receptive field simplify the task of protein detection and thus facilitate the network training. MemBrain requires only a small quantity of training labels and achieves excellent performance with only a single annotated membrane (F1 score: 0.88). A detailed evaluation shows that our fully trained pipeline outperforms existing classical computer vision-based and CNN-based approaches by a large margin (F1 score: 0.92 vs. max. 0.63). Furthermore, in addition to protein center positions, MemBrain can determine protein orientations, which has not been implemented by any existing CNN-based method to date. We also show that a pre-trained MemBrain program generalizes to tomograms acquired using different cryo-ET methods and depicting different types of cells. MemBrain is a powerful and annotation-efficient tool for the detection of membrane protein complexes in cryo-ET data, with the potential to be used in a wide range of biological studies. It is generalizable to various kinds of tomograms, making it possible to use pretrained models for different tasks. Its efficiency in terms of required annotations also allows rapid training and fine-tuning of models. The corresponding code, pretrained models, and instructions for operating the MemBrain program can be found at: https://github.com/CellArchLab/MemBrain

The burden of pertussis in low- and middle-income countries since the inception of the Expanded Programme on Immunization (EPI) in 1974: a systematic review protocol

Abstract Background Vaccine against pertussis has been in use for several decades. Despite the widespread use of pertussis vaccine, evidence shows resurgence of pertussis in high-income countries. Pertussis surveillance data is largely missing from low- and middle-income countries (LMICs). Without data on trends of pertussis, it is difficult to review and amend pertussis control policies in any country. We propose conducting a systematic review to evaluate the burden and trends of pertussis in LMICs since 1974. Methods/design Common and medical subject heading (MeSH) terms for pertussis and LMICs will be used to search electronic databases for the relevant literature published between 1974 and December 2014. Only studies from LMICs that fulfils World Health Organisation (WHO) or CDC pertussis case definitions will be included. The studies must have a clear numerator and denominator in a well-defined population. Risk of bias will be evaluated by assessing all qualifying full-text articles for quality and eligibility using a modified quality score assessment tool. Standardised data extraction will be carried out after which descriptions of trends in the prevalence, incidence, as well as mortality rate and case fatality rate, will be done. Where sufficient data is available, the results will be stratified by age group, geography, location, vaccination and HIV status. Discussion The systematic review proposed by this protocol seeks to address the knowledge gap in the epidemiology of pertussis in LMICs for the first time. It is anticipated that the background epidemiological trends of pertussis in LMICs that our study will provide could be used in the planning for the control of pertussis. Systematic review registration PROSPERO CRD4201501515

The burden of laboratory-confirmed pertussis in low- and middle-income countries since the inception of the Expanded Programme on Immunisation (EPI) in 1974: a systematic review and meta-analysis

Abstract Background An effective vaccine against Bordetella pertussis was introduced into the Expanded Programme on Immunisation (EPI) by WHO in 1974, leading to a substantial global reduction in pertussis morbidity and mortality. In low- and middle-income countries (LMICs), however, the epidemiology of pertussis remains largely unknown. This impacts negatively on pertussis control strategies in these countries. This study aimed to systematically and comprehensively review published literature on the burden of laboratory-confirmed pertussis in LMICs over the 45 years of EPI. Methods Electronic databases were searched for relevant literature (1974 to December 2018) using common and MeSH terms for pertussis. Studies using PCR, culture or paired serology to confirm Bordetella pertussis and parapertussis in symptomatic individuals were included if they had clearly defined numerators and denominators to determine prevalence and mortality rates. Results Eighty-two studies (49,167 participants) made the inclusion criteria. All six WHO regions were represented with most of the studies published after 2010 and involving mainly upper middle-income countries (n = 63; 77%). PCR was the main diagnostic test after the year 2000. The overall median point prevalence of PCR-confirmed Bordetella pertussis was 11% (interquartile range (IQR), 5–27%), while culture-confirmed was 3% (IQR 1–9%) and paired serology a median of 17% (IQR 3–23%) over the period. On average, culture underestimated prevalence by 85% (RR = 0.15, 95% CI, 0.10–0.22) compared to PCR in the same studies. Risk of pertussis increased with HIV exposure [RR, 1.4 (95% CI, 1.0–2.0)] and infection [RR, 2.4 (95% CI, 1.1–5.1)]. HIV infection and exposure were also related to higher pertussis incidences, higher rates of hospitalisation and pertussis-related deaths. Pertussis mortality and case fatality rates were 0.8% (95% CI, 0.4–1.4%) and 6.5% (95% CI, 4.0–9.5%), respectively. Most deaths occurred in infants less than 6 months of age. Conclusions Despite the widespread use of pertussis vaccines, the prevalence of pertussis remains high in LMIC over the last three decades. There is a need to increase access to PCR-based diagnostic confirmation in order to improve surveillance. Disease control measures in LMICs must take into account the persistent significant infant mortality and increased disease burden associated with HIV infection and exposure

VIPP2 interacts with VIPP1 and HSP22E/F at chloroplast membranes and modulates a retrograde signal for HSP22E/F gene expression

VIPP proteins aid thylakoid biogenesis and membrane maintenance in cyanobacteria, algae, and plants. Some members of the Chlorophyceae contain two VIPP paralogs termed VIPP1 and VIPP2, which originate from an early gene duplication event during the evolution of green algae. VIPP2 is barely expressed under nonstress conditions but accumulates in cells exposed to high light intensities or H2O2, during recovery from heat stress, and in mutants with defective integration (alb3.1) or translocation (secA) of thylakoid membrane proteins. Recombinant VIPP2 forms rod-like structures in vitro and shows a strong affinity for phosphatidylinositol phosphate. Under stress conditions, >70% of VIPP2 is present in membrane fractions and localizes to chloroplast membranes. A vipp2 knock-out mutant displays no growth phenotypes and no defects in the biogenesis or repair of photosystem II. However, after exposure to high light intensities, the vipp2 mutant accumulates less HSP22E/F and more LHCSR3 protein and transcript. This suggests that VIPP2 modulates a retrograde signal for the expression of nuclear genes HSP22E/F and LHCSR3. Immunoprecipitation of VIPP2 from solubilized cells and membrane-enriched fractions revealed major interactions with VIPP1 and minor interactions with HSP22E/F. Our data support a distinct role of VIPP2 in sensing and coping with chloroplast membrane stress

Dissecting the molecular organization of the translocon-associated protein complex

In eukaryotic cells, one-third of all proteins must be transported across or inserted into the endoplasmic reticulum (ER) membrane by the ER protein translocon. The translocon-associated protein (TRAP) complex is an integral component of the translocon, assisting the Sec61 protein-conducting channel by regulating signal sequence and transmembrane helix insertion in a substrate-dependent manner. Here we use cryo-electron tomography (CET) to study the structure of the native translocon in evolutionarily divergent organisms and disease-linked TRAP mutant fibroblasts from human patients. The structural differences detected by subtomogram analysis form a basis for dissecting the molecular organization of the TRAP complex. We assign positions to the four TRAP subunits within the complex, providing insights into their individual functions. The revealed molecular architecture of a central translocon component advances our understanding of membrane protein biogenesis and sheds light on the role of TRAP in human congenital disorders of glycosylation

Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity

Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-remodeling functions. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket on one end of the ring. Inside the ring's lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Using cryo-correlative light and electron microscopy (cryo-CLEM), we observe oligomeric VIPP1 coats encapsulating membrane tubules within the Chlamydomonas chloroplast. Our work provides a structural foundation for understanding how VIPP1 directs thylakoid biogenesis and maintenance

Field-free deterministic ultra fast creation of skyrmions by spin orbit torques

Magnetic skyrmions are currently the most promising option to realize current-driven magnetic shift registers. A variety of concepts to create skyrmions were proposed and demonstrated. However, none of the reported experiments show controlled creation of single skyrmions using integrated designs. Here, we demonstrate that skyrmions can be generated deterministically on subnanosecond timescales in magnetic racetracks at artificial or natural defects using spin orbit torque (SOT) pulses. The mechanism is largely similar to SOT-induced switching of uniformly magnetized elements, but due to the effect of the Dzyaloshinskii-Moriya interaction (DMI), external fields are not required. Our observations provide a simple and reliable means for skyrmion writing that can be readily integrated into racetrack devices