Cryo-EM structures of an LRRC8 chimera with native functional properties reveal heptameric assembly.

Volume-regulated anion channels (VRACs) mediate volume regulatory Cl and organic solute efflux from vertebrate cells. VRACs are heteromeric assemblies of LRRC8A-E proteins with unknown stoichiometries. Homomeric LRRC8A and LRRC8D channels have a small pore, hexameric structure.

Structural basis for activation and gating of IP receptors.

A pivotal component of the calcium (Ca) signaling toolbox in cells is the inositol 1,4,5-triphosphate (IP) receptor (IPR), which mediates Ca release from the endoplasmic reticulum (ER), controlling cytoplasmic and organellar Ca concentrations. IPRs are co-activated by IP and Ca, inhibited by Ca at high concentrations, and potentiated by ATP. However, the underlying molecular mechanisms are unclear.

High-Speed Force Spectroscopy for Single Protein Unfolding.

Single-molecule force spectroscopy (SMFS) measurements allow for quantification of the molecular forces required to unfold individual protein domains. Atomic force microscopy (AFM) is one of the long-established techniques for force spectroscopy (FS). Although FS at conventional AFM pulling rates provides valuable information on protein unfolding, in order to get a more complete picture of the mechanism, explore new regimes, and combine and compare experiments with simulations, we need higher pulling rates and μs-time resolution, now accessible via high-speed force spectroscopy (HS-FS).

α-Helix Unwinding as Force Buffer in Spectrins.

Spectrins are cytoskeletal proteins located at the inner face of the plasma membrane, making connections between membrane anchors and the actin cortex, and between actin filaments. Spectrins share a common structure forming a bundle of 3 α-helices and play a major role during cell deformation. Here, we used high-speed force spectroscopy and steered molecular dynamics simulations to understand the mechanical stability of spectrin, revealing a molecular force buffering function.

Lysenin Toxin Membrane Insertion Is pH-Dependent but Independent of Neighboring Lysenins.

Pore-forming toxins form a family of proteins that act as virulence factors of pathogenic bacteria, but similar proteins are found in all kingdoms of life, including the vertebrate immune system. They are secreted as soluble monomers that oligomerize on target membranes in the so-called prepore state; after activation, they insert into the membrane and adopt the pore state. Lysenin is a pore-forming toxin from the earthworm Eisenida foetida, of which both the soluble and membrane-inserted structures are solved.

A new self-completing questionnaire for motor and non-motorsymptoms in Parkinson's disease (MASAC-PD31).

Parkinson's disease (PD) patients may suffer from various motor symptoms as well as non-motor symptoms. In order to identify and grade these symptoms within the limited time in outpatient clinic, a new self-completing questionnaire (MASAC-PD31) was developed. The questionnaire consists of two parts.

The Vesicle Priming Factor CAPS Functions as a Homodimer via C2 Domain Interactions to Promote Regulated Vesicle Exocytosis.

Neurotransmitters and peptide hormones are secreted by regulated vesicle exocytosis. CAPS (also known as CADPS) is a 145-kDa cytosolic and peripheral membrane protein required for vesicle docking and priming steps that precede Ca-triggered vesicle exocytosis. CAPS binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P) and SNARE proteins and is proposed to promote SNARE protein complex assembly for vesicle docking and priming.

Glasslike Membrane Protein Diffusion in a Crowded Membrane.

Many functions of the plasma membrane depend critically on its structure and dynamics. Observation of anomalous diffusion in vivo and in vitro using fluorescence microscopy and single particle tracking has advanced our concept of the membrane from a homogeneous fluid bilayer with freely diffusing proteins to a highly organized crowded and clustered mosaic of lipids and proteins. Unfortunately, anomalous diffusion could not be related to local molecular details given the lack of direct and unlabeled molecular observation capabilities.

Hrs and STAM function synergistically to bind ubiquitin-modified cargoes in vitro.

The turnover of integral membrane proteins requires a specialized transport pathway mediated by components of the endosomal sorting complex required for transport (ESCRT) machinery. In most cases, entry into this pathway requires that cargoes undergo ubiquitin-modification, thereby facilitating their sequestration on endosomal membranes by specific, ubiquitin-binding ESCRT subunits. However, requirements underlying initial cargo recognition of mono-ubiquitinated cargos remain poorly defined.

Analyses of nuclear proteins and nucleic acid structures using atomic force microscopy.

Since the inception of atomic force microscopy (AFM) in 1986, the value of this technology for exploring the structure and biophysical properties of a variety of biological samples has been increasingly recognized. AFM provides the opportunity to both image samples at nanometer resolution and also measure the forces on the surface of the sample. Here, we describe a variety of methods for studying nuclear samples including single nucleic acid molecules, higher-order chromatin structures, the nucleolus, and the nucleus.

Spatial control of proton pump H,K-ATPase docking at the apical membrane by phosphorylation-coupled ezrin-syntaxin 3 interaction.

The digestive function of the stomach depends on acidification of the gastric lumen. Acid secretion into the lumen is triggered by activation of a cAMP-dependent protein kinase (PKA) cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. A coupling protein is ezrin whose phosphorylation at Ser-66 by PKA is required for parietal cell activation.

Activation-induced structural change in the GluN1/GluN3A excitatory glycine receptor.

Unlike GluN2-containing N-methyl-d-aspartate (NMDA) receptors, which require both glycine and glutamate for activation, receptors composed of GluN1 and GluN3 subunits are activated by glycine alone. Here, we used atomic force microscopy (AFM) imaging to examine the response to activation of the GluN1/GluN3A excitatory glycine receptor. GluN1 and GluN3A subunits were shown to interact intimately within transfected tsA 201 cells.

EIF4G1 gene mutations are not a common cause of Parkinson's disease in the Japanese population.

Pathogenic mutations in the EIF4G1 gene were recently reported as a cause of autosomal dominant parkinsonism. To assess the frequency of EIF4G1 mutations in the Japanese population we sequenced the entire gene coding region (31 exons) in 95 patients with an apparent autosomal dominant inherited form of Parkinson's disease. We detected three novel point mutations located in a poly-glutamic acid repeat within exon 10.

Linker mutations reveal the complexity of synaptotagmin 1 action during synaptic transmission.

The Ca(2+) sensor for rapid synaptic vesicle exocytosis, synaptotagmin 1 (syt), is largely composed of two Ca(2+)-sensing C2 domains, C2A and C2B. We investigated the apparent synergy between the tandem C2 domains by altering the length and rigidity of the linker that connects them. The behavior of the linker mutants revealed a correlation between the ability of the C2 domains to penetrate membranes in response to Ca(2+) and to drive evoked neurotransmitter release in cultured mouse neurons, uncovering a step in excitation-secretion coupling.

[The development and validation of a new comprehensive self-completing questionnaire for symptoms in Parkinson's disease (MASAC-PD 31)].

Background: Patients with Parkinson's disease (PD) suffer from various symptoms. In order to identify untreated symptoms within the limited time of a clinical interview, we developed a new self-completing questionnaire (MASAC-PD 31). The questionnaire consists of two parts (5 domains, 31 items); Part I intended at rating the motor symptoms and activities of daily living (ADL) during both "on" and "off" periods, and Part II aimed at screening and assessing mainly the non-motor symptoms, such as sleep-related difficulties, autonomic symptoms, cognition, mood and others.

Unusual structures are present in DNA fragments containing super-long Huntingtin CAG repeats.

Background: In the R6/2 mouse model of Huntington's disease (HD), expansion of the CAG trinucleotide repeat length beyond about 300 repeats induces a novel phenotype associated with a reduction in transcription of the transgene.

Methodology/principal Findings: We analysed the structure of polymerase chain reaction (PCR)-generated DNA containing up to 585 CAG repeats using atomic force microscopy (AFM). As the number of CAG repeats increased, an increasing proportion of the DNA molecules exhibited unusual structural features, including convolutions and multiple protrusions.

Interaction of synaptotagmin with lipid bilayers, analyzed by single-molecule force spectroscopy.

Synaptotagmin I is the major Ca²(+) sensor for membrane fusion during neurotransmitter release. The cytoplasmic domain of synaptotagmin consists of two C2 domains, C2A and C2B. On binding Ca²(+), the tips of the two C2 domains rapidly and synchronously penetrate lipid bilayers.

Single-molecule anatomy by atomic force microscopy and recognition imaging.

Atomic force microscopy (AFM) has been a useful technique to visualize cellular and molecular structures at single-molecule resolution. The combination of imaging and force modes has also allowed the characterization of physical properties of biological macromolecules in relation to their structures. Furthermore, recognition imaging, which is obtained under the TREC(TM) (Topography and RECognition) mode of AFM, can map a specific protein of interest within an AFM image.

Mutation analysis of the PINK1 gene in 391 patients with Parkinson disease.

Objectives: To determine the frequency, distribution, and clinical features of Parkinson disease (PD) with PINK1 mutations.

Design: Retrospective clinical and genetic review.

Setting: University hospital.

Nuclear architecture and chromatin dynamics revealed by atomic force microscopy in combination with biochemistry and cell biology.

The recent technical development of atomic force microscopy (AFM) has made nano-biology of the nucleus an attractive and promising field. In this paper, we will review our current understanding of nuclear architecture and dynamics from the structural point of view. Especially, special emphases will be given to: (1) How to approach the nuclear architectures by means of new techniques using AFM, (2) the importance of the physical property of DNA in the construction of the higher-order structures, (3) the significance and implication of the linker and core histones and the nuclear matrix/scaffold proteins for the chromatin dynamics, (4) the nuclear proteins that contribute to the formation of the inner nuclear architecture.

Single-molecule detection of phosphorylation-induced plasticity changes during ezrin activation.

Ezrin-radixin-moesin protein family provides a regulated link between the cortical actin cytoskeleton and the plasma membrane. Phosphorylation of ezrin has been functionally linked to membrane dynamics and plasticity. Our recent study demonstrated that phosphorylation of the conserved T567 residue of ezrin alters the physiology of gastric parietal cells.

[A case of multiple cranial neuropathy after Campylobacter jejuni infection].

We report a patient who developed overlapping symptoms of ophthalmoplegia and oropharyngeal palsy after Campylobacter jejuni infection. A 15-year-old man had diarrhea and fever, and developed dysarthria, diplopia and ptosis two weeks later. He did not show ataxia, weakness or abnormal tendon reflexes in the extremities during the clinical course.