Abstract. We have examined the cytoskeletal architecture and its relationship with synaptic vesicles in synapses by quick-freeze deep-etch electron microscopy (QF.DE). The main cytoskeletal elements in the presynaptic terminals (neuromuscular junction, electric organ, and cerebellar cortex) were actin filaments and microtubules. The actin filaments formed a network and frequently were associated closely with the presynaptic plasma membranes and active zones. Short, linking strands ~30 nm long were found between actin and synaptic vesicles, between microtubules and synaptic vesicles. Fine strands (30-60 nm) were also found between synaptic vesicles. Frequently spherical structures existed in the middle of the strands between synaptic vesicles. Another kind of strand (~100 nm long, thinner than the actin filaments) between synaptic vesicles and plasma membranes was also observed. We have examined the molecular structure of synapsin ~ and its relationship with actin filaments, microtubules, and synaptic vesicles in vitro using the low angle rotary shadowing technique and QF.DE. The synapsin 1, *47 nm long, was composed of a head (~14 nm diam) and a tail (~33 nm long), having a tadpole-like appearance. The high resolution provided by QF.DE revealed that a single synapsin 1 cross-linked actin filaments and linked actin filaments with synaptic vesicles, forming ~30-nm short strands. The head was on the actin and the tail was attached to the synaptic vesicle or actin filament. Microtubules were also cross-linked by a single syr~apsin 1, which also connected a microtubule to synaptic vesicles, forming •30 nm strands. The spherical head was on the microtubules and the tail was attached to the synaptic vesicles or to microtubules. Synaptic vesicles incubated with synapsin 1 were linked with each other via fine short fibrils and frequently we identified spherical structures from which two or three fibrils radiated and cross-linked synaptic vesicles.We have ex,~mined the localization of synapsin 1 using ultracryomicrotomy and colloidal gold-immunocytochemistr:~ ~ anti-synapsin 1 IgG. Synapsin 1 was exclusively localized in the regions occupied by synaptic vesicles. Statistical analyses indicated that synapsin 1 is located mostly at least *30 nm away from the presynaptic membrane. These data derived via, three different approaches suggest that synapsin 1 could be a main element of short linkages between actin filaments and synaptic vesicles, and between microtubules and synaptic vesicles; and between synaptic vesicles in the nerve terminals. The longer strands (*100 nm): associated with presynaptic membrane could consist of other proteins, most probably fodrin, judging from its structure. Because phosphorylation of synapsin 1 by Ca++/calmodulin-dependent kinase detaches synapsin 1 from vesicles it could release synaptic vesicles from actin filaments, microtubules and other synaptic vesicles, and thus increase the mobility of synaptic vesicles to the premy.naptie' membrane after depolarization dependent influx of Ca. ~+ into ...

Caldesmon was originally purified from gizzard smooth muscle as a major calmodulin-binding protein which also interacts with actin filaments. It has an alternative binding ability to either calmodulin or actin filaments depending upon the concentration of Ca2+ ("flip-flop binding"). Two forms of caldesmon (Mr's in the range of 120-150 kDa and 70-80 kDa) have been demonstrated in a wide variety of smooth muscles and nonmuscle cells. Immunohistochemical studies suggest that caldesmon is colocalized with actin filaments in vivo. Considering its abundance, the Ca2+-dependent flip-flop binding ability to either calmodulin or actin filaments, and its intracellular localization, caldesmon is expected to be involved in contractile events. Recent results from our laboratory have led to the conclusion that caldesmon regulates the smooth muscle and nonmuscle actin-myosin interaction and the smooth muscle actin-high Mr actin-binding protein (ABP or filamin) interactin in a flip-flop manner. It might function in cell motility by regulating the contractile system.

We investigated the expression of two molecular weight forms of caldesmon in a wide range of tissues and cells.

Several hybrid DFT methods were applied to full geometry optimizations of the CaMn4O4X(H2O)4 (XOH1− (1) or O2− (2)) cluster in the oxygen evolving complex (OEC) of photosystem II (PSII) to elucidate Mn‐Mn, Mn‐Ca, and Mn‐O distances on a theoretical ground. The computed Mn‐Mn distances were compared with previous (London and Berlin) X‐ray diffraction (XRD), and Berkeley and Berlin EXAFS results, together with the recent high‐resolution XRD structure by Umena and coworkers. Present computational results by the hybrid DFT methods have elucidated several differences among these accumulated results. These DFT results led us to reassign the Mn‐Mn and Mn‐Ca distances by the EXAFS experiments, which became consistent with the results obtained by the high‐resolution XRD structure. A characteristic feature revealed via the optimized Mn‐O distances was that the degree of symmetry breaking of the Mn1‐O(57)‐Mn4 bond is not so remarkable under the UBHandHLYP approximation but it can be large by other hybrid DFT methods. The computational results for 2 indicated reduction of the Mn3‐Mn4 distance with the deprotonation of the bridging oxo group. The hybrid DFT results for 1 are not inconsistent with an experimental proposal based on the new XRD structure, namely a protonated μ3‐oxygen at the internal O(57) site of the cluster in the S1 state. On the other hand, the reduction of Mn ions (not degradation of whole cluster structure) by the X‐ray irradiation still remains an important issue for refinements of the XRD structure. The computational results are discussed in relation to those of the electron spin echo envelope modulation (ESEEM) and possible pathways for water splitting reaction. Implications of the present DFT structures are discussed in relation to the previous DFT and related computational results, together with recent XRD results for cubane‐like model clusters for OEC of PSII. © 2012 Wiley Periodicals, Inc.

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