Membrane remodeling properties of the Parkinson's disease protein LRRK2.

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are responsible for late-onset autosomal dominant Parkinson's disease. LRRK2 has been implicated in a wide range of physiological processes including membrane repair in the endolysosomal system. Here, using cell-free systems, we report that purified LRRK2 directly binds acidic lipid bilayers with a preference for highly curved bilayers. While this binding is nucleotide independent, LRRK2 can also deform low-curvature liposomes into narrow tubules in a guanylnucleotide-dependent but Adenosine 5'-triphosphate-independent way. Moreover, assembly of LRRK2 into scaffolds at the surface of lipid tubules can constrict them. We suggest that an interplay between the membrane remodeling and signaling properties of LRRK2 may be key to its physiological function. LRRK2, via its kinase activity, may achieve its signaling role at sites where membrane remodeling occurs.

In situ architecture of the lipid transport protein VPS13C at ER-lysosome membrane contacts.

VPS13 is a eukaryotic lipid transport protein localized at membrane contact sites. Previous studies suggested that it may transfer lipids between adjacent bilayers by a bridge-like mechanism. Direct evidence for this hypothesis from a full-length structure and from electron microscopy (EM) studies in situ is still missing, however. Here, we have capitalized on AlphaFold predictions to complement the structural information already available about VPS13 and to generate a full-length model of human VPS13C, the Parkinson's disease-linked VPS13 paralog localized at contacts between the endoplasmic reticulum (ER) and endo/lysosomes. Such a model predicts an ∼30-nm rod with a hydrophobic groove that extends throughout its length. We further investigated whether such a structure can be observed in situ at ER-endo/lysosome contacts. To this aim, we combined genetic approaches with cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) to examine HeLa cells overexpressing this protein (either full length or with an internal truncation) along with VAP, its anchoring binding partner at the ER. Using these methods, we identified rod-like densities that span the space separating the two adjacent membranes and that match the predicted structures of either full-length VPS13C or its shorter truncated mutant, thus providing in situ evidence for a bridge model of VPS13 in lipid transport.

Cryo-ET reveals the macromolecular reorganization of S. pombe mitotic chromosomes in vivo.

Chromosomes condense during mitosis in most eukaryotes. This transformation involves rearrangements at the nucleosome level and has consequences for transcription. Here, we use cryo-electron tomography (cryo-ET) to determine the 3D arrangement of nuclear macromolecular complexes, including nucleosomes, in frozen-hydrated Schizosaccharomyces pombe cells. Using 3D classification analysis, we did not find evidence that nucleosomes resembling the crystal structure are abundant. This observation and those from other groups support the notion that a subset of fission yeast nucleosomes may be partially unwrapped in vivo. In both interphase and mitotic cells, there is also no evidence of monolithic structures the size of Hi-C domains. The chromatin is mingled with two features: pockets, which are positions free of macromolecular complexes; and "megacomplexes," which are multimegadalton globular complexes like preribosomes. Mitotic chromatin is more crowded than interphase chromatin in subtle ways. Nearest-neighbor distance analyses show that mitotic chromatin is more compacted at the oligonucleosome than the dinucleosome level. Like interphase, mitotic chromosomes contain megacomplexes and pockets. This uneven chromosome condensation helps explain a longstanding enigma of mitosis: a subset of genes is up-regulated.

128-pixel arrays of 4H-SiC UV APD with dual-frequency PECVD SiNx passivation.

In this paper, high-performance 1×128 linear arrays of 4H-SiC ultraviolet (UV) avalanche photodiode (APD) with dual-frequency plasma enhanced chemical vapor deposition (PECVD) passivation are demonstrated for the first time. The results show that SiNx dielectric deposited by dual-frequency PECVD can effectively reduce the leakage current at high bias voltages. Due to the improved 4H-SiC epi-layer material and SiNx passivation, the fabricated 22 mm-long 1×128 4H-SiC APD linear arrays exhibit an excellent performance with a high pixel yield of 100% and a small breakdown voltage variation of 0.2 V, which is the best result ever reported. At room temperature, the pixels have a gain of over 105 and a maximum quantum efficiency of 53.5% @ 285 nm. Besides the high uniformity of breakdown voltage for 128 pixels, the dark currents at 95% of breakdown voltage are all below 1 nA.

The photo-Nazarov reaction: scope and application.

The reaction conditions and scope of the photo-Nazarov reaction of aryl vinyl ketones were investigated. In contrast to the conventional acid-catalyzed methods, this photolytic electrocyclization proceeds in the neutral or basic conditions. Irradiating substrates bearing various aromatic rings, acid-sensitive groups, cyclohexenyl, cycloheptenyl, and unsaturated pyran with UV-light (254 nm) smoothly yielded hexahydrofluorenones and related structures. This photo-Nazarov reaction could also be applicable to the substrates carrying ß-alkyl groups on the enone, which gave corresponding polycyclic rings containing quaternary centers. These photo-electrocyclized products may prove useful for synthesizing a variety of natural products and their derivatives. Further application of this mild photo-Nazarov reaction in the synthesis of taiwaniaquinol B was achieved.

Total synthesis of gracilamine.

The total synthesis of gracilamine, a pentacyclic Amaryllidaceae alkaloid, was achieved from simple building blocks. The synthesis features a mild photo-Nazarov reaction, intramolecular 1,4-addition, and an intramolecular Mannich reaction. This approach not only confirms the C6 stereochemistry of natural gracilamine, and also provides a novel solution to prepare its derivatives and structurally related natural products.

Recent technical advances in cellular cryo-electron tomography.

Understanding the in situ structure, organization, and interactions of macromolecules is essential for elucidating their functions and mechanisms of action. Cellular cryo-electron tomography (cryo-ET) is a cutting-edge technique that reveals in situ molecular-resolution architectures of macromolecules in their lifelike states. It also provides insights into the three-dimensional distribution of macromolecules and their spatial relationships with various subcellular structures. Thus, cellular cryo-ET bridges the gap between structural biology and cell biology. With rapid advancements, this technique achieved substantial improvements in throughput, automation, and resolution. This review presents the fundamental principles and methodologies of cellular cryo-ET, highlighting recent developments in sample preparation, data collection, and image processing. We also discuss emerging trends and potential future directions. As cellular cryo-ET continues to develop, it is set to play an increasingly vital role in structural cell biology.

Lysosome damage triggers acute formation of ER to lysosomes membrane tethers mediated by the bridge-like lipid transport protein VPS13C.

Based on genetic studies, lysosome dysfunction is thought to play a pathogenetic role in Parkinson's disease (PD). Here we show that VPS13C, a bridge-like lipid transport protein and a PD gene, is a sensor of lysosome stress/damage. Upon lysosome membrane perturbation, VPS13C rapidly relocates from the cytosol to the surface of lysosomes where it tethers their membranes to the ER. This recruitment depends on Rab7 and requires release of a brake, most likely an intramolecular interaction within VPS13C, which hinders access of its VAB domain to lysosome-bound Rab7. While another PD protein, LRRK2, is also recruited to stressed/damaged lysosomes, its recruitment occurs at much later stages and by different mechanisms. Given the putative role of VPS13 proteins in bulk lipid transport, these findings suggest lipid delivery to lysosomes by VPS13C is part of an early response to lysosome damage.

Biomimetic synthesis of equisetin and (+)-fusarisetin A.

(+)-Fusarisetin A belongs to a group of acyl tetramic acid natural products that show potential anticancer activity. Equisetin, a biogenetically related acyl tetramic acid, contains the basic skeleton of (+)-fusarisetin A. We proposed that equisetin and (+)-fusarisetin A share a biosynthetic pathway that starts with naturally occurring (S)-serine and an unsaturated fatty acid. In support of this hypothesis, we have demonstrated that a cyclization sequence involving an intramolecular Diels-Alder reaction followed by a Dieckmann cyclization of polyenoylamino acid yielded equisetin. The aerobic oxidation of equisetin, promoted by either Mn(III)/O2 or a reactive oxygen species (ROS) produced by visible-light chemistry, gave peroxyfusarisetin, which could be easily reduced to (+)-fusarisetin A. We report herein detailed information on the biogenetic synthesis of equisetin and (+)-fusarisetin A.

Heterogeneous non-canonical nucleosomes predominate in yeast cells in situ.

Nuclear processes depend on the organization of chromatin, whose basic units are cylinder-shaped complexes called nucleosomes. A subset of mammalian nucleosomes in situ (inside cells) resembles the canonical structure determined in vitro 25 years ago. Nucleosome structure in situ is otherwise poorly understood. Using cryo-electron tomography (cryo-ET) and 3D classification analysis of budding yeast cells, here we find that canonical nucleosomes account for less than 10% of total nucleosomes expected in situ. In a strain in which H2A-GFP is the sole source of histone H2A, class averages that resemble canonical nucleosomes both with and without GFP densities are found ex vivo (in nuclear lysates), but not in situ. These data suggest that the budding yeast intranuclear environment favors multiple non-canonical nucleosome conformations. Using the structural observations here and the results of previous genomics and biochemical studies, we propose a model in which the average budding yeast nucleosome's DNA is partially detached in situ.

Asymmetric synthesis and biosynthetic implications of (+)-fusarisetin†A.

Starting from equisetin, the asymmetric synthesis of (+)-fusarisetin A has been accomplished in a one-pot transformation including a biomimetic oxidation and an intramolecular Diels-Alder/Roskamp reaction. Peroxyfusarisetin is proposed as a plausible biosynthetic intermediate based on studies of the oxidation of equisetin.

Cryo-ET detects bundled triple helices but not ladders in meiotic budding yeast.

In meiosis, cells undergo two sequential rounds of cell division, termed meiosis I and meiosis II. Textbook models of the meiosis I substage called pachytene show that nuclei have conspicuous 100-nm-wide, ladder-like synaptonemal complexes and ordered chromatin loops. It remains unknown if these cells have any other large, meiosis-related intranuclear structures. Here we present cryo-ET analysis of frozen-hydrated budding yeast cells before, during, and after pachytene. We found no cryo-ET densities that resemble dense ladder-like structures or ordered chromatin loops. Instead, we found large numbers of 12-nm-wide triple-helices that pack into ordered bundles. These structures, herein called meiotic triple helices (MTHs), are present in meiotic cells, but not in interphase cells. MTHs are enriched in the nucleus but not enriched in the cytoplasm. Bundles of MTHs form at the same timeframe as synaptonemal complexes (SCs) in wild-type cells and in mutant cells that are unable to form SCs. These results suggest that in yeast, SCs coexist with previously unreported large, ordered assemblies.

A collection of yeast cellular electron cryotomography data.

BACKGROUND: Cells are powered by a large set of macromolecular complexes, which work together in a crowded environment. The in situ mechanisms of these complexes are unclear because their 3D distribution, organization, and interactions are largely unknown. Electron cryotomography (cryo-ET) can address these knowledge gaps because it produces cryotomograms-3D images that reveal biological structure at ∼4-nm resolution. Cryo-ET uses no fixation, dehydration, staining, or plastic embedment, so cellular features are visualized in a life-like, frozen-hydrated state. To study chromatin and mitotic machinery in situ, we subjected yeast cells to genetic and chemical perturbations, cryosectioned them, and then imaged the cells by cryo-ET. FINDINGS: Here we share >1,000 cryo-ET raw datasets of cryosectioned budding yeast Saccharomyces cerevisiaecollected as part of previously published studies. These data will be valuable to cell biologists who are interested in the nanoscale organization of yeasts and of eukaryotic cells in general. All the unpublished tilt series and a subset of corresponding cryotomograms have been deposited in the EMPIAR resource for the community to use freely. To improve tilt series discoverability, we have uploaded metadata and preliminary notes to publicly accessible Google Sheets, EMPIAR, and GigaDB. CONCLUSIONS: Cellular cryo-ET data can be mined to obtain new cell-biological, structural, and 3D statistical insights in situ. These data contain structures not visible in traditional electron-microscopy data. Template matching and subtomogram averaging of known macromolecular complexes can reveal their 3D distributions and low-resolution structures. Furthermore, these data can serve as testbeds for high-throughput image-analysis pipelines, as training sets for feature-recognition software, for feasibility analysis when planning new structural-cell-biology projects, and as practice data for students.

Growth of graphene with large single-crystal domains by Ni foam-assisted structure and its high-gain field-effect transistors.

High-quality graphene materials and high-performance graphene transistors have attracted much attention in recent years. To obtain high-performance graphene transistors, large single-crystal graphene is needed. The synthesis of large-domain-sized single-crystal graphene requires low nucleation density; this can lead to a lower growth rate. In this study, a Ni-foam assisted structure was developed to control the nucleation density and growth rate of graphene by tuning the flow dynamics. Lower nucleation density and high growth rate (∼50 µm min-1) were achieved with a 4 mm-gap Ni foam. With the graphene transistor fabrication process, a pre-deposited Au film as the protective layer was used during the graphene transfer. Graphene transistors showed good current saturation with drain differential conductance as low as 0.04 S mm-1 in the strong saturation region. For the devices with gate length of 2 µm, the intrinsic cut-off frequency f T and maximum oscillation frequency f max were 8.4 and 16.3 GHz, respectively, with f max/f T = 1.9 and power gain of up to 6.4 dB at 1 GHz. The electron velocity saturation induced by the surface optical phonons of SiO2 substrates was analyzed. Electron velocity saturation and ultra-thin Al2O3 gate dielectrics were thought to be the reasons for the good current saturation and high power gain of the graphene transistors.

The in situ structures of mono-, di-, and trinucleosomes in human heterochromatin.

The in situ three-dimensional organization of chromatin at the nucleosome and oligonucleosome levels is unknown. Here we use cryo-electron tomography to determine the in situ structures of HeLa nucleosomes, which have canonical core structures and asymmetric, flexible linker DNA. Subtomogram remapping suggests that sequential nucleosomes in heterochromatin follow irregular paths at the oligonucleosome level. This basic principle of higher-order repressive chromatin folding is compatible with the conformational variability of the two linker DNAs at the single-nucleosome level.

Natural chromatin is heterogeneous and self-associates in vitro.

The 30-nm fiber is commonly formed by oligonucleosome arrays in vitro but rarely found inside cells. To determine how chromatin higher-order structure is controlled, we used electron cryotomography (cryo-ET) to study the undigested natural chromatin released from two single-celled organisms in which 30-nm fibers have not been observed in vivo: picoplankton and yeast. In the presence of divalent cations, most of the chromatin from both organisms is condensed into a large mass in vitro. Rare irregular 30-nm fibers, some of which include face-to-face nucleosome interactions, do form at the periphery of this mass. In the absence of divalent cations, picoplankton chromatin decondenses into open zigzags. By contrast, yeast chromatin mostly remains condensed, with very few open motifs. Yeast chromatin packing is largely unchanged in the absence of linker histone and mildly decondensed when histones are more acetylated. Natural chromatin is therefore generally nonpermissive of regular motifs, even at the level of oligonucleosomes.

Development of a one-step strip test for the diagnosis of chicken infectious bursal disease.

A rapid diagnostic strip for chicken infectious bursal disease (IBD) was developed based on membrane chromatography using high-affinity monoclonal antibodies directed to chicken infectious bursal disease virus (IBDV). The diagnostic strip has high specificity for detection of chicken IBDV antigen and recognizes a variety of the virus isolates, including virulent and attenuated strains, with no cross-reactivity to other viruses, such as Newcastle disease virus, Marek's disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, and egg-drop-syndrome virus. The results showed that its specificity was highly consistent with the agar-gel precipitation test (AGP). The diagnostic strip detected as low as 800 median egg lethal dose (ELD50) viruses in the IBDV BC6/85-infected sample, which was comparable with AC-ELISA (400 ELD50) and 32 times more sensitive than the AGP test (2.56 x 10(4) ELD50). In experimental infection, IBDV was detected in the bursa as early as 36 hr postinfection with the diagnostic strip before the clinical signs and gross lesions appeared. It takes only 1-2 min to do a strip test to detect chicken IBDV antigen after the specimen is grounded in a whirl pack with finger massage.

A photo-induced C-O bond formation methodology to construct tetrahydroxanthones.

A metal-free, photo-induced C-O bond formation methodology was developed to construct tetrahydroxanthones. This mild and efficient methodology was based on intramolecular oxygen trapping of the reactive species produced by photolytic activation of a C-Cl bond. We believe this method could be used in the synthesis of related xanthone-type natural products.

Development of immunoassays for the detection of sulfamethazine in swine urine.

The use of sulfonamides, such as sulfamethazine (SM2), in pig production is recognized as a public health risk as it inevitably results in sulfamethazine residues in pork. This study is aimed at establishing rapid, simple, reliable methods, with both sensitivity and specificity, for detecting sulfamethazine residues. For this purpose, monoclonal antibodies against sulfamethazine were prepared and characterized. No cross-reaction of the monoclonal antibodies was identified with other sulfonamides or analytes. Based on the competitive immunoassay principle, an indirect competitive ELISA kit (SM2 kit) and a rapid detection strip for detecting sulfamethazine residues were developed using monoclonal antibodies and the colloidal gold technique. The indirect competitive ELISA kit and the strip assay could be performed within 2 h and 5-10 min, respectively. The results showed that the detection limits were 1 ng ml(-1) for the indirect competitive ELISA kit and 8 ng ml(-1) with the unaided eye and 1 ng ml(-1) with the strip reader for the rapid strip assay. Comparing the HPLC method with the SM2-kit or the strip in pig urine spiked with standards of SM2, the difference was <4.6% for SM2-kit and 4.3% for the strip. The two methods are suitable for the rapid screening of sulfamethazine residues in swine urine. Experimental data revealed that the two methods, especially the strip, proved to be sensitive, specific, rapid and easy to use for the quantitative, semi-quantitative or qualitative detection of SM2 residues in swine urine.