Plasma membrane compartments, delimited by transmembrane proteins anchored to the membrane skeleton (anchored-protein picket model), would provide the membrane with fundamental mosaicism because they would affect the movement of practically all molecules incorporated in the cell membrane. Understanding such basic compartmentalized structures of the cell membrane is critical for further studies of a variety of membrane functions. Here, using both high temporal-resolution single particle tracking and single fluorescent molecule video imaging of an unsaturated phospholipid, DOPE, we found that plasma membrane compartments generally exist in various cell types, including CHO, HEPA-OVA, PtK2, FRSK, HEK293, HeLa, T24 (ECV304), and NRK cells. The compartment size varies from 30 to 230 nm, whereas the average hop rate of DOPE crossing the boundaries between two adjacent compartments ranges between 1 and 17 ms. The probability of passing a compartment barrier when DOPE is already at the boundary is also cell-type dependent, with an overall variation by a factor of approximately 7. These results strongly indicate the necessity for the paradigm shift of the concept on the plasma membrane: from the two-dimensional fluid continuum model to the compartmentalized membrane model in which its constituent molecules undergo hop diffusion over the compartments.

Membrane potential recordings, made from the circular smooth muscle layer of the gastric antrum taken from mutant mice which lacked the inositol trisphosphate (InsP3) type 1 receptor, were compared with those obtained from the stomach of control (wild‐type) mice. Immunostaining of gastric muscles indicated that the distribution and form of c‐kit positive cells were similar in wild‐type and mutant mice. Smooth muscles from wild‐type mice generated slow waves that in turn initiated spike potentials, while those from mutant mice were either quiescent or generated irregular bursts of spike potentials. In the presence of nifedipine, slow waves with reduced amplitude were generated in wild‐type mice, while all electrical activity was abolished in mutant mice. Acetylcholine depolarized and sodium nitroprusside hyperpolarized the membrane in muscles from both types of mice, being more effective in wild‐type mice. Noradrenaline produced similar hyperpolarizations in both types of mice. Transmural nerve stimulation evoked inhibitory junction potentials (IJPs) in both wild‐type and mutant mice. In wild‐type mice, the IJPs were reduced in amplitude by nitroarginine and converted to a cholinergic excitatory junction potential (EJP) by apamin. In mutant mice, the IJPs were unaffected by nitroarginine or atropine but were abolished by apamin. It is concluded that in antral smooth muscle, the expression of InsP3 type 1 receptors may be causally related to the generation of slow waves but not to the generation of action potentials. A lack of InsP3 receptors attenuates cholinergic excitatory and nitrergic inhibitory responses but does not alter the response to noradrenaline.

In eubacteria, PriA helicase detects the stalled DNA replication forks. This critical role of PriA is ascribed to its ability to bind to the 3 0 end of a nascent leading DNA strand in the stalled replication forks. The crystal structures in complexes with oligonucleotides and the combination of fluorescence correlation spectroscopy and mutagenesis reveal that the N-terminal domain of PriA possesses a binding pocket for the 3 0 -terminal nucleotide residue of DNA. The interaction with the deoxyribose 3 0 -OH is essential for the 3 0 -terminal recognition. In contrast, the direct interaction with 3 0 -end nucleobase is unexpected, considering the same affinity for oligonucleotides carrying the four bases at the 3 0 end. Thus, the N-terminal domain of PriA recognizes the 3 0 -end base in a base-non-selective manner, in addition to the deoxyribose and 5 0 -side phosphodiester group, of the 3 0 -terminal nucleotide to acquire both sufficient affinity and non-selectivity to find all of the stalled replication forks generated during DNA duplication. This unique feature is prerequisite for the proper positioning of the helicase domain of PriA on the unreplicated double-stranded DNA.

DNA replication in archaea and eukaryotes is executed by family B DNA polymerases, which exhibit full activity when complexed with the DNA clamp, proliferating cell nuclear antigen (PCNA). This replication enzyme consists of the polymerase and exonuclease moieties responsible for DNA synthesis and editing (proofreading), respectively. Because of the editing activity, this enzyme ensures the high fidelity of DNA replication. However, it remains unclear how the PCNA-complexed enzyme temporally switches between the polymerizing and editing modes. Here, we present the threedimensional structure of the Pyrococcus furiosus DNA polymerase B-PCNA-DNA ternary complex, which is the core component of the replisome, determined by single particle electron microscopy of negatively stained samples. This structural view, representing the complex in the editing mode, revealed the whole domain configuration of the trimeric PCNA ring and the DNA polymerase, including protein-protein and protein-DNA contacts. Notably, besides the authentic DNA polymerase-PCNA interaction through a PCNAinteracting protein (PIP) box, a novel contact was found between DNA polymerase and the PCNA subunit adjacent to that with the PIP contact. This contact appears to be responsible for the configuration of the complex specific for the editing mode. The DNA was located almost at the center of PCNA and exhibited a substantial and particular tilt angle against the PCNA ring plane. The obtained molecular architecture of the complex, including the new contact found in this work, provides clearer insights into the switching mechanism between the two distinct modes, thus highlighting the functional significance of PCNA in the replication process.fidelity control | protein-DNA complex | replication fork | single particle analysis | structural bioinformatics

The 3D structure of the ternary complex, consisting of DNA ligase, the proliferating cell nuclear antigen (PCNA) clamp, and DNA, was investigated by single-particle analysis. This report presents the structural view, where the crescent-shaped DNA ligase with 3 distinct domains surrounds the central DNA duplex, encircled by the closed PCNA ring, thus forming a double-layer structure with dual contacts between the 2 proteins. The relative orientations of the DNA ligase domains, which remarkably differ from those of the known crystal structures, suggest that a large domain rearrangement occurs upon ternary complex formation. A second contact was found between the PCNA ring and the middle adenylation domain of the DNA ligase. Notably, the map revealed a substantial DNA tilt from the PCNA ring axis. This structure allows us to propose a switching mechanism for the replication factors operating on the PCNA ring.DNA replication ͉ electron microscopy ͉ single-particle analysis ͉ DNA sliding clamp ͉ protein-DNA complex D NA ligase plays essential roles in various DNA transactions, such as joining Okazaki fragments in DNA replication and nick-sealing at the final step of several DNA repair pathways, including nucleotide excision repair, base excision repair, and mismatch repair (1, 2). The enzyme catalyzes phosphodiester bond formation at the nicks generated within dsDNA, through the well-conserved 3-step reaction using either NAD ϩ or ATP as a cofactor (3). At the first step, the DNA ligase interacts with the nucleotide cofactor to form a covalent ligase-nucleoside monophosphate. At the second step, the bound AMP is transferred to the 5Ј terminus of the DNA, and finally, a phosphodiester bond is formed by a reaction between the 5Ј-DNAadenylate and the 3Ј-hydroxy group.To date, 3 crystal structures of ATP-dependent eukaryotictype DNA ligases have been reported. The structures of the 2 archaeal DNA ligases from Pyrococcus furiosus (PfuLig) and Sulfolobus solfataricus (SsoLig) were determined in the closed (4) and extended forms (5), respectively. The structure of the human DNA ligase 1 (hLigI) in complex with DNA (6) revealed that the enzyme entirely encircles the nicked DNA. All of these DNA ligases are in common composed of 3 domains, designated as the DNA binding domain (DBD), the adenylation domain (AdD), and the OB-fold domain (OBD), in the sequences from the N to C termini. Although the internal architectures of these domains are strikingly similar among the 3 DNA ligases, their relative domain orientations within each enzyme are quite different. Similar to many other replication factors, such as DNA polymerase and Flap endonuclease 1 (FEN1), DNA ligases exhibit the full activity by binding to proliferating cell nuclear antigen (PCNA). A small-angle X-ray scattering analysis revealed that the morphology of SsoLig in complex with PCNA coincides with the extended structure of SsoLig alone (5). However, the structure of the ternary Lig-PCNA-DNA complex remains unknown.PCNA interacts with various protein factors to contr...