Hirohide Takahashi

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

The Ca2+ sensor for rapid synaptic vesicle exocytosis, synaptotagmin 1 (syt), is largely composed of two Ca2+-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 Ca2+ and to drive evoked neurotransmitter release in cultured mouse neurons, uncovering a step in excitation-secretion coupling. Using atomic force microscopy, we found that the synergy between these C2 domains involved intra-molecular interactions between them. Thus, syt function is markedly affected by changes in the physical nature of the linker that connects its tandem C2 domains. Moreover, the linker mutations uncoupled syt-mediated regulation of evoked and spontaneous release, revealing that syt also acts as a fusion clamp before the Ca2+ trigger.The Ca2+ sensor for rapid synaptic vesicle exocytosis, synaptotagmin I (syt), is largely composed of two Ca2+-sensing C2-domains, C2A and C2B. We have 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 Ca2+ and to drive evoked neurotransmitter release in cultured mouse neurons, uncovering a step in excitation-secretion coupling. Atomic force microscopy experiments indicate that the synergy between these C2-domains involves intra-molecular interactions between them. Thus, syt function is profoundly affected by changes in the physical nature of the linker that connects its tandem C2-domains. Moreover, the linker mutations uncoupled syt-mediated regulation of evoked and spontaneous release, revealing that syt also acts as a fusion clamp prior to the Ca2+ trigger.

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. Spatio-temporal analyses using AFM, in combination with biochemical and cell biological approaches, will play important roles in the nano-biology of the nucleus, as most of nuclear structures and events occur in nanometer, piconewton and millisecond order. The new applications of AFM, such as recognition imaging, fast-scanning imaging, and a variety of modified cantilevers, are expected to be powerful techniques to reveal the nanostructure of the nucleus.

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. However, the molecular mechanism of phosphorylation‐induced ezrin activation has remained elusive. Here we use atomic force microscopy (AFM) to probe phosphorylation‐mediated activation of ezrin in single molecules. The phospho‐mimicking and non‐phosphorylatable mutant ezrin proteins were generated and purified to homogeneity. Comparative analyses of two ezrin mutants by AFM demonstrate the unfolding of the N‐ and C‐terminal domains upon the phospho‐activation. To measure the physical force underlying the inter‐domain contact during mechanical unfolding, we probed the defined region of ezrin using the N‐terminal ezrin coated onto the AFM tip. Comparative force measurements indicate that T567 phosphorylation‐induced unfolding of ezrin favors the inter‐molecular association. Taken together, these results provide molecular illustration of phosphorylation elicited functional activation of ERM proteins and indicate that stimulus‐induced protein conformational change can be used as a signaling mechanism orchestrating cellular dynamics.

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. We investigated the forces of interaction between synaptotagmin and lipid bilayers using single-molecule force spectroscopy. Glutathione-S-transferase-tagged proteins were attached to an atomic force microscope cantilever via a glutathione-derivatized polyethylene glycol linker. With wild-type C2AB, the force profile for a bilayer containing phosphatidylserine had both Ca²(+)-dependent and Ca²(+)-independent components. No force was detected when the bilayer lacked phosphatidylserine, even in the presence of Ca²(+). The binding characteristics of C2A and C2B indicated that the two C2 domains cooperate in binding synaptotagmin to the bilayer, and that the relatively weak Ca²(+)-independent force depends only on C2A. When the lysine residues K189-192 and K326, 327 were mutated to alanine, the strong Ca²(+)-dependent binding interaction was either absent or greatly reduced. We conclude that synaptotagmin binds to the bilayer via C2A even in absence of Ca²(+), and also that positively charged regions of both C2A and C2B are essential for the strong Ca²(+)-dependent binding of synaptotagmin to the bilayer.

Development of glutathione-coupled cantilever for the single-molecule force measurement by scanning force microscopy

The accuracy and the fidelity of a single‐molecule force measurement largely rely on how the molecule of interest is attached to the solid substrate surface (bead, cantilever, cover glass and etc.). A site‐specific attachment of a protein without affecting its structure and enzymatic function has been a major concern. Here, we established a glutathione‐coupled cantilever to which any glutathione S‐transferase (GST)‐fused proteins can be attached in a desired direction. The rupture force between glutathione and GST was ∼100 pN on average. By using this cantilever, we succeeded in measuring the interaction force between importin α and importin β.

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

Background In the R6/2 mouse model of Huntingtons 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. At least some of these features are hairpin loops, as judged by cross-sectional analysis and sensitivity to cleavage by mung bean nuclease. Single-molecule force measurements showed that the convoluted DNA was very resistant to untangling. In vitro replication by PCR was markedly reduced, and TseI restriction enzyme digestion was also hindered by the abnormal DNA structures. However, significantly, the DNA gained sensitivity to cleavage by the Type III restriction-modification enzyme, EcoP15I. Conclusions/Significance “Super-long” CAG repeats are found in a number of neurological diseases and may also appear through CAG repeat instability. We suggest that unusual DNA structures associated with super-long CAG repeats decrease transcriptional efficiency in vitro. We also raise the possibility that if these structures occur in vivo, they may play a role in the aetiology of CAG repeat diseases such as HD.

Spatial Control of Proton Pump H,K-ATPase Docking at the Apical Membrane by Phosphorylation-coupled Ezrin-Syntaxin 3 Interaction

Background: Polarized acid secretion in gastric parietal cells requires ezrin and its phosphorylation at Ser-66. Results: Phosphorylation of Ser-66 induces ezrin conformational change, which enables ezrin to interact with syntaxin 3. Conclusion: Conformational change of ezrin provides a spatial cue for apical trafficking of H,K-ATPase. Significance: Ezrin conformation orchestrates the polarized vesicle trafficking in epithelial cells. 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. However, little is known regarding the molecular mechanism(s) by which ezrin operates in gastric acid secretion. Here we show that phosphorylation of Ser-66 induces a conformational change of ezrin that enables its association with syntaxin 3 (Stx3) and provides a spatial cue for H,K-ATPase trafficking. This conformation-dependent association is specific for Stx3, and the binding interface is mapped to the N-terminal region. Biochemical analyses show that inhibition of ezrin phosphorylation at Ser-66 prevents ezrin-Stx3 association and insertion of H,K-ATPase into the apical plasma membrane of parietal cells. Using atomic force microscopic analyses, our study revealed that phosphorylation of Ser-66 induces unfolding of ezrin molecule to allow Stx3 binding to its N terminus. Given the essential role of Stx3 in polarized secretion, our study presents the first evidence in which phosphorylation-induced conformational rearrangement of the ezrin molecule provides a spatial cue for polarized membrane trafficking in epithelial cells.

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. Protocols to prepare nucleic acids, nucleic acid-protein complexes, reconstituted chromatin, the cell nucleus, and the nucleolus are included, as well as protocols describing how to prepare the AFM substrate and the AFM tip. Finally, we describe how to perform conventional imaging, high-speed imaging, recognition imaging, force spectroscopy, and nanoindentation experiments.

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 Ca2+-triggered vesicle exocytosis. CAPS binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and SNARE proteins and is proposed to promote SNARE protein complex assembly for vesicle docking and priming. We characterized purified soluble CAPS as mainly monomer in equilibrium with small amounts of dimer. However, the active form of CAPS bound to PC12 cell membranes or to liposomes containing PI(4,5)P2 and Q-SNARE proteins was mainly dimer. CAPS dimer formation required its C2 domain based on mutation or deletion studies. Moreover, C2 domain mutations or deletions resulted in a loss of CAPS function in regulated vesicle exocytosis, indicating that dimerization is essential for CAPS function. Comparison of the CAPS C2 domain to a structurally defined Munc13-1 C2A domain dimer revealed conserved residues involved in CAPS dimerization. We conclude that CAPS functions as a C2 domain-mediated dimer in regulated vesicle exocytosis. The unique tandem C2-PH domain of CAPS may serve as a PI(4,5)P2-triggered switch for dimerization. CAPS dimerization may be coupled to oligomeric SNARE complex assembly for vesicle docking and priming.

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. In this study, we determine the capability of each ESCRT complex that harbors a ubiquitin-binding domain to bind a reconstituted integral membrane cargo (VAMP2), which has been covalently linked to mono-ubiquitin. We demonstrate that ESCRT-0, but not ESCRT-I or ESCRT-II, is able to associate stably with the mono-ubiquitinated cargo within a lipid bilayer. Moreover, we show that the ubiquitin-binding domains in both Hrs and STAM must be intact to enable cargo binding. These results indicate that the two subunits of ESCRT-0 function together to bind and sequester cargoes for downstream sorting into intralumenal vesicles.