The mitochondrial intermembrane space protein mitofissin drives mitochondrial fission required for mitophagy.

Mitophagy plays an important role in mitochondrial homeostasis by selective degradation of mitochondria. During mitophagy, mitochondria should be fragmented to allow engulfment within autophagosomes, whose capacity is exceeded by the typical mitochondria mass. However, the known mitochondrial fission factors, dynamin-related proteins Dnm1 in yeasts and DNM1L/Drp1 in mammals, are dispensable for mitophagy. Here, we identify Atg44 as a mitochondrial fission factor that is essential for mitophagy in yeasts, and we therefore term Atg44 and its orthologous proteins mitofissin. In mitofissin-deficient cells, a part of the mitochondria is recognized by the mitophagy machinery as cargo but cannot be enwrapped by the autophagosome precursor, the phagophore, due to a lack of mitochondrial fission. Furthermore, we show that mitofissin directly binds to lipid membranes and brings about lipid membrane fragility to facilitate membrane fission. Taken together, we propose that mitofissin acts directly on lipid membranes to drive mitochondrial fission required for mitophagy.

Isolation of an archaeon at the prokaryote-eukaryote interface.

The origin of eukaryotes remains unclear1-4. Current data suggest that eukaryotes may have emerged from an archaeal lineage known as 'Asgard' archaea5,6. Despite the eukaryote-like genomic features that are found in these archaea, the evolutionary transition from archaea to eukaryotes remains unclear, owing to the lack of cultured representatives and corresponding physiological insights. Here we report the decade-long isolation of an Asgard archaeon related to Lokiarchaeota from deep marine sediment. The archaeon-'Candidatus Prometheoarchaeum syntrophicum' strain MK-D1-is an anaerobic, extremely slow-growing, small coccus (around 550 nm in diameter) that degrades amino acids through syntrophy. Although eukaryote-like intracellular complexes have been proposed for Asgard archaea6, the isolate has no visible organelle-like structure. Instead, Ca. P. syntrophicum is morphologically complex and has unique protrusions that are long and often branching. On the basis of the available data obtained from cultivation and genomics, and reasoned interpretations of the existing literature, we propose a hypothetical model for eukaryogenesis, termed the entangle-engulf-endogenize (also known as E3) model.

Subnanometer structure of medusavirus capsid during maturation using cryo-electron microscopy.

Medusavirus is a giant virus classified into an independent family of Mamonoviridae. Amoebae infected with medusavirus release immature particles in addition to virions. These particles were suggested to exhibit the maturation process of this virus, but the structure of these capsids during maturation remains unknown. Here, we apply a block-based reconstruction method in cryo-electron microscopy (cryo-EM) single particle analysis to these viral capsids, extending the resolution to 7-10 Å. The maps reveal a novel network composed of minor capsid proteins (mCPs) supporting major capsid proteins (MCPs). A predicted molecular model of the MCP fitted into the cryo-EM maps clarified the boundaries between the MCP and the underlining mCPs, as well as between the MCP and the outer spikes, and identified molecular interactions between the MCP and these components. Several structural changes of the mCPs under the fivefold vertices of the immature particles were observed, depending on the presence or absence of the underlying internal membrane. In addition, the lower part of the penton proteins on the fivefold vertices was also missing in mature virions. These dynamic conformational changes of mCPs indicate an important function in the maturation process of medusavirus.IMPORTANCEThe structural changes of giant virus capsids during maturation have not thus far been well clarified. Medusavirus is a unique giant virus in which infected amoebae release immature particles in addition to mature virus particles. In this study, we used cryo-electron microscopy to investigate immature and mature medusavirus particles and elucidate the structural changes of the viral capsid during the maturation process. In DNA-empty particles, the conformation of the minor capsid proteins changed dynamically depending on the presence or absence of the underlying internal membranes. In DNA-full particles, the lower part of the penton proteins was lost. This is the first report of structural changes of the viral capsid during the maturation process of giant viruses.

Structural insights into calcium-induced conformational changes in human gelsolin.

Gelsolin is known as one of the actin-binding proteins capable of severing and capping filamentous actin, and of undergoing structural changes in the presence of calcium ions to interact with actin filaments. In this study, single-particle 3D reconstruction using electron microscopy (EM) revealed that, in the presence of calcium, the structure of gelsolin undergoes structural changes before interacting with actin. These differences are subtle with similarities, as confirmed by the EM map. According to the results of the molecular dynamics simulations, these nuanced structural differences primarily manifest at the domain level when calcium is present. These results provide structural evidence that, in the presence of calcium, gelsolin enters a phase of conformational preparation to transition into the active state. This process enables gelsolin to bind to actin, whereupon gelsolin undergoes more drastic structural changes upon interaction with actin filaments, which allows it to participate in binding and severing to regulate the cytoskeleton. This is the first visualization of full-length gelsolin, and helps to clarify crucial aspects of the as of yet incompletely understood interaction between gelsolin and actin.

Structure and its transformation of elliptical nege-like virus Tanay virus.

Negeviruses that infect insects are recently identified virus species that are phylogenetically related to several plant viruses. They exhibit a unique virion structure, an elliptical core with a short projection. Negeviruses encode two structural proteins, a glycoprotein that forms a short projection, and an envelope protein that forms an elliptical core. The glycoprotein has been reported only in the negeviruses' genes, and not in phylogenetically related plant viruses' genes. In this report, we first describe the three-dimensional electron cryo-microscopy (cryo-EM) structure of Tanay virus (TANAV), one of the nege-like viruses. TANAV particle demonstrates a periodical envelope structure consisting of three layers surrounding the centred viral RNA. The elliptical core dynamically changes its shape under acidic and even low detergent conditions to form bullet-like or tubular shapes. The further cryo-EM studies on these transformed TANAV particles reveal their overall structural rearrangement. These findings suggest putative geometries of TANAV and its transformation in the life cycle, and the potential importance of the short projection for enabling cell entry to the insect hosts.

Particle Morphology of Medusavirus Inside and Outside the Cells Reveals a New Maturation Process of Giant Viruses.

Medusavirus, a giant virus, is phylogenetically closer to eukaryotes than the other giant viruses and has been recently classified as an independent species. However, details of its morphology and maturation process in host cells remain unclear. Here, we investigated the particle morphology of medusavirus inside and outside infected cells using conventional transmission electron microscopy (C-TEM) and cryo-electron microscopy (cryo-EM). The C-TEM of amoebae infected with the medusavirus showed four types of particles, i.e., pseudo-DNA-empty (p-Empty), DNA-empty (Empty), semi-DNA-full (s-Full), and DNA-full (Full). Time-dependent changes in the four types of particles and their intracellular localization suggested a new maturation process for the medusavirus. Viral capsids and viral DNAs are produced independently in the cytoplasm and nucleus, respectively, and only the empty particles located near the host nucleus can incorporate the viral DNA into the capsid. Therefore, all four types of particles were found outside the cells. The cryo-EM of these particles showed that the intact virus structure, covered with three different types of spikes, was preserved among all particle types, although with minor size-related differences. The internal membrane exhibited a structural array similar to that of the capsid, interacted closely with the capsid, and displayed open membrane structures in the Empty and p-Empty particles. The results suggest that these open structures in the internal membrane are used for an exchange of scaffold proteins and viral DNA during the maturation process. This new model of the maturation process of medusavirus provides insight into the structural and behavioral diversity of giant viruses. IMPORTANCE Giant viruses exhibit diverse morphologies and maturation processes. In this study, medusavirus showed four types of particle morphologies, both inside and outside the infected cells, when propagated in amoeba culture. Time-course analysis and intracellular localization of the medusavirus in the infected cells suggested a new maturation process via the four types of particles. Like the previously reported pandoravirus, the viral DNA of medusavirus is replicated in the host's nucleus. However, viral capsids are produced independently in the host cytoplasm, and only empty capsids near the nucleus can take up viral DNA. As a result, many immature particles were released from the host cell along with the mature particles. The capsid structure is well conserved among the four types of particles, except for the open membrane structures in the empty particles, suggesting that they are used to exchange scaffold proteins for viral DNAs. These findings indicate that medusavirus has a unique maturation process.

Atomic Structure of the Human Sapovirus Capsid Reveals a Unique Capsid Protein Conformation in Caliciviruses.

Sapovirus (SaV) is a member of the Caliciviridae family, which causes acute gastroenteritis in humans and animals. Human sapoviruses (HuSaVs) are genetically and antigenically diverse, but the lack of a viral replication system and structural information has hampered the development of vaccines and therapeutics. Here, we successfully produced a self-assembled virus-like particle (VLP) from the HuSaV GI.6 VP1 protein, and the first atomic structure was determined using single-particle cryo-electron microscopy (cryo-EM) at a 2.9-Å resolution. The atomic model of the VP1 protein revealed a unique capsid protein conformation in caliciviruses. All N-terminal arms in the A, B, and C subunits interacted with adjacent shell domains after extending through their subunits. The roof of the arched VP1 dimer was formed between the P2 subdomains by the interconnected ß strands and loops, and its buried surface was minimized compared to those of other caliciviruses. Four hypervariable regions that are potentially involved in the antigenic diversity of SaV formed extensive clusters on top of the P domain. Potential receptor binding regions implied by tissue culture mutants of porcine SaV were also located near these hypervariable clusters. Conserved sequence motifs of the VP1 protein, "PPG" and "GWS," may stabilize the inner capsid shell and the outer protruding domain, respectively. These findings will provide the structural basis for the medical treatment of HuSaV infections and facilitate the development of vaccines, antivirals, and diagnostic systems. IMPORTANCE SaV and norovirus, belonging to the Caliciviridae family, are common causes of acute gastroenteritis in humans and animals. SaV and norovirus infections are public health problems in all age groups, which occur explosively and sporadically worldwide. HuSaV is genetically and antigenically diverse and is currently classified into 4 genogroups consisting of 18 genotypes based on the sequence similarity of the VP1 proteins. Despite these detailed genetic analyses, the lack of structural information on viral capsids has become a problem for the development of vaccines or antiviral drugs. The 2.9-Å atomic model of the HuSaV GI.6 VLP presented here not only revealed the location of the amino acid residues involved in immune responses and potential receptor binding sites but also provided essential information for the design of stable constructs needed for the development of vaccines and antivirals.

Nasal delivery of single-domain antibody improves symptoms of SARS-CoV-2 infection in an animal model.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the disease COVID-19 can lead to serious symptoms, such as severe pneumonia, in the elderly and those with underlying medical conditions. While vaccines are now available, they do not work for everyone and therapeutic drugs are still needed, particularly for treating life-threatening conditions. Here, we showed nasal delivery of a new, unmodified camelid single-domain antibody (VHH), termed K-874A, effectively inhibited SARS-CoV-2 titers in infected lungs of Syrian hamsters without causing weight loss and cytokine induction. In vitro studies demonstrated that K-874A neutralized SARS-CoV-2 in both VeroE6/TMPRSS2 and human lung-derived alveolar organoid cells. Unlike other drug candidates, K-874A blocks viral membrane fusion rather than viral attachment. Cryo-electron microscopy revealed K-874A bound between the receptor binding domain and N-terminal domain of the virus S protein. Further, infected cells treated with K-874A produced fewer virus progeny that were less infective. We propose that direct administration of K-874A to the lung could be a new treatment for preventing the reinfection of amplified virus in COVID-19 patients.

Morphological and Taxonomic Properties of the Newly Isolated Cotonvirus japonicus, a New Lineage of the Subfamily Megavirinae.

Since 2003, various viruses from the subfamily Megavirinae in the family Mimiviridae have been isolated worldwide, including icosahedral mimiviruses and tailed tupanviruses. To date, the evolutionary relationship between tailed and nontailed mimiviruses has not been elucidated. Here, we present the genomic and morphological features of a newly isolated giant virus, Cotonvirus japonicus (cotonvirus), belonging to the family Mimiviridae. It contains a linear double-stranded DNA molecule of 1.47 Mb, the largest among the reported viruses in the subfamily Megavirinae, excluding tupanviruses. Among its 1,306 predicted open reading frames, 1,149 (88.0%) were homologous to those of the family Mimiviridae. Several nucleocytoplasmic large DNA virus (NCLDV) core genes, aminoacyl-tRNA synthetase genes, and the host specificity of cotonvirus were highly similar to those of Mimiviridae lineages A, B, and C; however, lineage A was slightly closer to cotonvirus than the others were. Moreover, based on its genome size, the presence of two copies of 18S rRNA-like sequences, and the period of its infection cycle, cotonvirus is the most similar to the tupanviruses among the icosahedral mimiviruses. Interestingly, the cotonvirus utilizes Golgi apparatus-like vesicles for virion factory (VF) formation. Overall, we showed that cotonvirus is a novel lineage of the subfamily Megavirinae. Our findings support the diversity of icosahedral mimiviruses and provide mechanistic insights into the replication, VF formation, and evolution of the subfamily Megavirinae. IMPORTANCE We have isolated a new virus of an independent lineage belonging to the family Mimiviridae, subfamily Megavirinae, from the fresh water of a canal in Japan, named Cotonvirus. In a proteomic tree, this new nucleocytoplasmic large DNA virus (NCLDV) is phylogenetically placed at the root of three lineages of the subfamily Megavirinae-lineages A (mimivirus), B (moumouvirus), and C (megavirus). Multiple genomic and phenotypic features of cotonvirus are more similar to those of tupanviruses than to those of the A, B, or C lineages, and other genomic features, while the host specificity of cotonvirus is more similar to those of the latter than of the former. These results suggest that cotonvirus is a unique virus that has chimeric features of existing viruses of Megavirinae and uses Golgi apparatus-like vesicles of the host cells for virion factory (VF) formation. Thus, cotonvirus can provide novel insights into the evolution of mimiviruses and the underlying mechanisms of VF formation.

Dynamic rotation of the protruding domain enhances the infectivity of norovirus.

Norovirus is the major cause of epidemic nonbacterial gastroenteritis worldwide. Lack of structural information on infection and replication mechanisms hampers the development of effective vaccines and remedies. Here, using cryo-electron microscopy, we show that the capsid structure of murine noroviruses changes in response to aqueous conditions. By twisting the flexible hinge connecting two domains, the protruding (P) domain reversibly rises off the shell (S) domain in solutions of higher pH, but rests on the S domain in solutions of lower pH. Metal ions help to stabilize the resting conformation in this process. Furthermore, in the resting conformation, the cellular receptor CD300lf is readily accessible, and thus infection efficiency is significantly enhanced. Two similar P domain conformations were also found simultaneously in the human norovirus GII.3 capsid, although the mechanism of the conformational change is not yet clear. These results provide new insights into the mechanisms of non-enveloped norovirus transmission that invades host cells, replicates, and sometimes escapes the hosts immune system, through dramatic environmental changes in the gastrointestinal tract.

Structural Fluctuations of the Human Proteasome α7 Homo-Tetradecamer Double Ring Imply the Proteasomal α-Ring Assembly Mechanism.

The 20S proteasome, which is composed of layered α and ß heptameric rings, is the core complex of the eukaryotic proteasome involved in proteolysis. The α7 subunit is a component of the α ring, and it self-assembles into a homo-tetradecamer consisting of two layers of α7 heptameric rings. However, the structure of the α7 double ring in solution has not been fully elucidated. We applied cryo-electron microscopy to delineate the structure of the α7 double ring in solution, revealing a structure different from the previously reported crystallographic model. The D7-symmetrical double ring was stacked with a 15° clockwise twist and a separation of 3 Å between the two rings. Two more conformations, dislocated and fully open, were also identified. Our observations suggest that the α7 double-ring structure fluctuates considerably in solution, allowing for the insertion of homologous α subunits, finally converting to the hetero-heptameric α rings in the 20S proteasome.

Structural determination of the large photosystem II-light-harvesting complex II supercomplex of Chlamydomonas reinhardtii using nonionic amphipol.

In photosynthetic organisms, photosystem II (PSII) is a large membrane protein complex, consisting of a pair of core complexes surrounded by an array of variable numbers of light-harvesting complex (LHC) II proteins. Previously reported structures of the PSII-LHCII supercomplex of the green alga Chlamydomonas reinhardtii exhibit significant structural heterogeneity, but recently improved purification methods employing ionic amphipol A8-35 have enhanced supercomplex stability, providing opportunities for determining a more intact structure. Herein, we present a 5.8 Å cryo-EM map of the C. reinhardtii PSII-LHCII supercomplex containing six LHCII trimers (C2S2M2L2). Utilizing a newly developed nonionic amphipol-based purification and stabilizing method, we purified the largest photosynthetic supercomplex to the highest percentage of the intact configuration reported to date. We found that the interprotein distances within the light-harvesting complex array in the green algal photosystem are larger than those previously observed in higher plants, indicating that the potential route of energy transfer in the PSII-LHCII supercomplex in green algae may be altered. Interestingly, we also observed an asymmetric PSII-LHCII supercomplex structure comprising C2S2M1L1 in the same sample. Moreover, we found a new density adjacent to the PSII core complex, attributable to a single-transmembrane helix. It was previously unreported in the cryo-EM maps of PSII-LHCII supercomplexes from land plants.

Ten antenna proteins are associated with the core in the supramolecular organization of the photosystem I supercomplex in Chlamydomonas reinhardtii.

Photosystem I (PSI) is a large pigment-protein complex mediating light-driven charge separation and generating a highly negative redox potential, which is eventually utilized to produce organic matter. In plants and algae, PSI possesses outer antennae, termed light-harvesting complex I (LHCI), which increase the energy flux to the reaction center. The number of outer antennae for PSI in the green alga Chlamydomonas reinhardtii is known to be larger than that of land plants. However, their exact number and location remain to be elucidated. Here, applying a newly established sample purification procedure, we isolated a highly pure PSI-LHCI supercomplex containing all nine LHCA gene products under state 1 conditions. Single-particle cryo-EM revealed the 3D structure of this supercomplex at 6.9 Å resolution, in which the densities near the PsaF and PsaJ subunits were assigned to two layers of LHCI belts containing eight LHCIs, whereas the densities between the PsaG and PsaH subunits on the opposite side of the LHCI belt were assigned to two extra LHCIs. Using single-particle cryo-EM, we also determined the 2D projection map of the lhca2 mutant, which confirmed the assignment of LHCA2 and LHCA9 to the densities between PsaG and PsaH. Spectroscopic measurements of the PSI-LHCI supercomplex suggested that the bound LHCA2 and LHCA9 proteins have the ability to increase the light-harvesting energy for PSI. We conclude that the PSI in C. reinhardtii has a larger and more distinct outer-antenna organization and higher light-harvesting capability than that in land plants.

Medusavirus, a Novel Large DNA Virus Discovered from Hot Spring Water.

Recent discoveries of new large DNA viruses reveal high diversity in their morphologies, genetic repertoires, and replication strategies. Here, we report the novel features of medusavirus, a large DNA virus newly isolated from hot spring water in Japan. Medusavirus, with a diameter of 260 nm, shows a T=277 icosahedral capsid with unique spherical-headed spikes on its surface. It has a 381-kb genome encoding 461 putative proteins, 86 of which have their closest homologs in Acanthamoeba, whereas 279 (61%) are orphan genes. The virus lacks the genes encoding DNA topoisomerase II and RNA polymerase, showing that DNA replication takes place in the host nucleus, whereas the progeny virions are assembled in the cytoplasm. Furthermore, the medusavirus genome harbored genes for all five types of histones (H1, H2A, H2B, H3, and H4) and one DNA polymerase, which are phylogenetically placed at the root of the eukaryotic clades. In contrast, the host amoeba encoded many medusavirus homologs, including the major capsid protein. These facts strongly suggested that amoebae are indeed the most promising natural hosts of medusavirus, and that lateral gene transfers have taken place repeatedly and bidirectionally between the virus and its host since the early stage of their coevolution. Medusavirus reflects the traces of direct evolutionary interactions between the virus and eukaryotic hosts, which may be caused by sharing the DNA replication compartment and by evolutionarily long lasting virus-host relationships. Based on its unique morphological characteristics and phylogenomic relationships with other known large DNA viruses, we propose that medusavirus represents a new family, MedusaviridaeIMPORTANCE We have isolated a new nucleocytoplasmic large DNA virus (NCLDV) from hot spring water in Japan, named medusavirus. This new NCLDV is phylogenetically placed at the root of the eukaryotic clades based on the phylogenies of several key genes, including that encoding DNA polymerase, and its genome surprisingly encodes the full set of histone homologs. Furthermore, its laboratory host, Acanthamoeba castellanii, encodes many medusavirus homologs in its genome, including the major capsid protein, suggesting that the amoeba is the genuine natural host from ancient times of this newly described virus and that lateral gene transfers have repeatedly occurred between the virus and amoeba. These results suggest that medusavirus is a unique NCLDV preserving ancient footprints of evolutionary interactions with its hosts, thus providing clues to elucidate the evolution of NCLDVs, eukaryotes, and virus-host interaction. Based on the dissimilarities with other known NCLDVs, we propose that medusavirus represents a new viral family, Medusaviridae.

Hydrophobic Monomers Recognize Microenvironments in Hydrogel Microspheres during Free-Radical-Seeded Emulsion Polymerization.

The three-dimensional structure of nanocomposite microgels was precisely determined by cryo-electron micrography. Several nanocomposite microgels that differ with respect to their nanocomposite structure, which were obtained from seeded emulsion polymerization in the presence of microgels, were used as model nanocomposite materials for cryo-electron micrography. The obtained three-dimensional segmentation images of these nanocomposite microgels provide important insights into the interactions between the hydrophobic monomers and the microgels, that is, hydrophobic styrene monomers recognize molecular-scale differences in polarity within the microgels during the emulsion polymerization. This result led to the formation of unprecedented multi-layered nanocomposite microgels, which promise substantial potential in colloidal applications.

Evolutionary transformation of mouthparts from particle-feeding to piercing carnivory in Viper copepods: Review and 3D analyses of a key innovation using advanced imaging techniques.

BACKGROUND: Novel feeding adaptations often facilitate adaptive radiation and diversification. But the evolutionary origins of such feeding adaptations can be puzzling if they require concordant change in multiple component parts. Pelagic, heterorhabdid copepods (Calanoida) exhibit diverse feeding behaviors that range from simple particle feeding to a highly specialized form of carnivory involving piercing mouthparts that likely inject venom. We review the evolutionary history of heterorhabdid copepods and add new high-resolution, 3D anatomical analyses of the muscular system, glands and gland openings associated with this remarkable evolutionary transformation. RESULTS: We examined four heterorhabdid copepods with different feeding modes: one primitive particle-feeder (Disseta palumbii), one derived and specialized carnivore (Heterorhabdus subspinifrons), and two intermediate taxa (Mesorhabdus gracilis and Heterostylites longicornis). We used two advanced, high-resolution microscopic techniques - serial block-face scanning electron microscopy and two-photon excitation microscopy - to visualize mouthpart form and internal anatomy at unprecedented nanometer resolution. Interactive 3D graphical visualizations allowed putative homologues of muscles and gland cells to be identified with confidence and traced across the evolutionary transformation from particle feeding to piercing carnivory. Notable changes included: a) addition of new gland cells, b) enlargement of some (venom producing?) glands, c) repositioning of gland openings associated with hollow piercing fangs on the mandibles, d) repurposing of some mandibular-muscle function to include gland-squeezing, and e) addition of new muscles that may aid venom injection exclusively in the most specialized piercing species. In addition, live video recording of all four species revealed mandibular blade movements coupled to cyclic contraction of some muscles connected to the esophagus. These behavioral and 3D morphological observations revealed a novel injection system in H. subspinifrons associated with piercing (envenomating?) carnivory. CONCLUSIONS: Collectively, these results suggest that subtle changes in mandibular tooth form, and muscle and gland form and location, facilitated the evolution of a novel, piercing mode of feeding that accelerated diversification of the genus Heterorhabdus. They also highlight the value of interactive 3D animations for understanding evolutionary transformations of complex, multicomponent morphological systems.

Mutational and Combinatorial Control of Self-Assembling and Disassembling of Human Proteasome α Subunits.

Eukaryotic proteasomes harbor heteroheptameric α-rings, each composed of seven different but homologous subunits α1-α7, which are correctly assembled via interactions with assembly chaperones. The human proteasome α7 subunit is reportedly spontaneously assembled into a homotetradecameric double ring, which can be disassembled into single rings via interaction with monomeric α6. We comprehensively characterized the oligomeric state of human proteasome α subunits and demonstrated that only the α7 subunit exhibits this unique, self-assembling property and that not only α6 but also α4 can disrupt the α7 double ring. We also demonstrated that mutationally monomerized α7 subunits can interact with the intrinsically monomeric α4 and α6 subunits, thereby forming heterotetradecameric complexes with a double-ring structure. The results of this study provide additional insights into the mechanisms underlying the assembly and disassembly of proteasomal subunits, thereby offering clues for the design and creation of circularly assembled hetero-oligomers based on homo-oligomeric structural frameworks.

Single-Nanoparticle Tracking with Angstrom Localization Precision and Microsecond Time Resolution.

Gold nanoparticles (AuNPs) have been used as a contrast agent for optical imaging of various single biomolecules. Because AuNPs have high scattering efficiency without photobleaching, biomolecular dynamics have been observed with nanometer localization precision and sub-millisecond time resolution. To understand the working principle of biomolecular motors in greater detail, further improvement of the localization precision and time resolution is necessary. Here, we investigated the lower limit of localization precision achievable with AuNPs and the fundamental law, which determines the localization precision. We first used objective-lens-type total internal reflection dark-field microscopy to obtain a scattering signal from an isolated AuNP. The localization precision was inversely proportional to the square root of the photon number, which is consistent with theoretical estimation. The lower limit of precision for a 40 nm AuNP was ∼0.3 nm with 1 ms time resolution and was restricted by detector saturation. To achieve higher localization precision, we designed and constructed an annular illumination total internal reflection dark-field microscopy system with an axicon lens, which can illuminate the AuNPs at high laser intensity without damaging the objective lens. In addition, we used high image magnification to avoid detector saturation. Consequently, we achieved 1.3 Å localization precision for 40 nm AuNPs and 1.9 Å localization precision for 30 nm AuNPs at 1 ms time resolution. Furthermore, even at 33 µs time resolution, localization precisions at 5.4 Å for 40 nm AuNPs and at 1.7 nm for 30 nm AuNPs were achieved. We then observed motion of head of kinesin-1 labeled with AuNP at microsecond time resolution. Transition cycles of bound/unbound states and tethered diffusion of unbound head during stepping motion on microtubule were clearly captured with higher time resolution or smaller AuNP than those used in previous studies, indicating applicability to single-molecule imaging of biomolecular motors.

Seeded Emulsion Polymerization of Styrene in the Presence of Water-Swollen Hydrogel Microspheres.

In a previous study, we have ascertained that the charge distribution in hydrogel microspheres (microgels) plays a crucial role in controlling the nanocomposite structure of the polystyrene obtained from the seeded emulsion polymerization (SEP) of styrene in the presence of microgels. However, all these polymerizations were conducted at high temperature, where most of these microgels were dehydrated and deswollen. In the present study, we initially verified that the nanocomposite microgels can be synthesized even when the seed microgels are swollen and hydrated during the SEP of styrene. These highly swollen microgels were used as the nucleation sites for the polystyrene, and subsequently the propagation of the hydrophobic polystyrenes proceeded within water-swollen microgels.

Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis.

Serratia marcescens chitinase A is a linear molecular motor that hydrolyses crystalline chitin in a processive manner. Here, we quantitatively determined the rate constants of elementary reaction steps, including binding (kon), translational movement (ktr), and dissociation (koff) with single-molecule fluorescence imaging. The kon for a single chitin microfibril was 2.1 × 109 M-1 µm-1 s-1. The koff showed two components, k (3.2 s-1, 78%) and k (0.38 s-1, 22%), corresponding to bindings to different crystal surfaces. From the kon, k, k and ratio of fast and slow dissociations, dissociation constants for low and high affinity sites were estimated as 2.0 × 10-9 M µm and 8.1 × 10-10 M µm, respectively. The ktr was 52.5 nm s-1, and processivity was estimated as 60.4. The apparent inconsistency between high turnover (52.5 s-1) calculated from ktr and biochemically determined low kcat (2.6 s-1) is explained by a low ratio (4.8%) of productive enzymes on the chitin surface (52.5 s-1 × 0.048 = 2.5 s-1). Our results highlight the importance of single-molecule analysis in understanding the mechanism of enzymes acting on a solid-liquid interface.