Multidomain scaffolding proteins organize the molecular machinery of neurotransmitter vesicle dynamics during synaptogenesis and synaptic activity. We find that domains of five active zone proteins converge on an interaction node that centers on the N-terminal region of Munc13-1 and includes the zinc-finger domain of Rim1, the C-terminal region of Bassoon, a segment of CAST1/ELKS2, and the third coiled-coil domain (CC3) of either Aczonin/Piccolo or Bassoon. This multidomain complex may constitute a center for the physical and functional integration of the protein machinery at the active zone. An additional connection between Aczonin and Bassoon is mediated by the second coiled-coil domain of Aczonin. Recombinant Aczonin-CC3, expressed in cultured neurons as a green fluorescent protein fusion protein, is targeted to synapses and suppresses vesicle turnover, suggesting involvements in synaptic assembly as well as activity. Our findings show that Aczonin, Bassoon, CAST1, Munc13, and Rim are closely and multiply interconnected, they indicate that Aczonin-CC3 can actively participate in neurotransmitter vesicle dynamics, and they highlight the N-terminal region of Munc13-1 as a hub of protein interactions by adding three new binding partners to its mechanistic potential in the control of synaptic vesicle priming.
The protein machinery of neurotransmitter exocytosis requires efficient orchestration in space and time, for speed and precision of neurotransmission and also for synaptic ontogeny and plasticity. However, its spatial organization in situ is virtually unknown. Aczonin/Piccolo is a putative organizer protein of mammalian active zones. We determined by immunogold electron microscopy (EM) (i) the spatial arrangement (i.e., topology) of 11 segments of the Aczonin polypeptide in situ, and correlated it to (ii) the positioning of Aczonin-interacting domains of Bassoon, CAST/ELKS, Munc13, and RIM and (iii) the ultrastructurally defined presynaptic macromolecular aggregates known as dense projections and synaptic ribbons. At conventional synapses, Aczonin assumes a compact molecular topology within a layer 35 to 80 nm parallel to the plasma membrane (PM), with a "trunk" sitting on the dense projection top and a C-terminal "arm" extending down toward the PM and sideward to the dense projection periphery. At ribbon synapses, Aczonin occupies the whole ribbon area. Bassoon colocalizes with Aczonin at conventional synapses but not at ribbon synapses. At both conventional and ribbon synapses, CAST, Munc13, and RIM are segregated from Aczonin, closer to the PM, and Aczonin is positioned such that it may control the access of neurotransmitter vesicles to the fusion site.scaffolding protein | protein structure N eurotransmitter release is confined to a specialized area of the presynaptic plasma membrane (PM) known as the active zone. Calcium-dependent exocytosis at the synapse is an elaborate, multistep process that is fast and precise, yet highly restrained and subject to modulation (i.e., presynaptic plasticity) (1). A solubilization-resistant lattice of proteins associated with the inner face of the presynaptic PM, the cytomatrix at the active zone (CAZ), is apparent on electron microscopy (EM) imaging and seems to integrate and organize many proteins of the presynaptic machinery (2).When mammalian conventional synapses are stained with heavy metal ions, EM reveals regularly spaced, cone-shaped structures of a size of approximately 50 nm attached to the presynaptic PM, which are known as dense projections (DPs) or, collectively, the "presynaptic particle web" or "presynaptic grid." It has been possible to isolate DP-like particles by subcellular fractionation, and even to reconstitute similar aggregates after previous solubilization, and many proteins of the presynaptic molecular machinery have been detected in them (3). These findings suggest that the presynaptic machinery may be organized into modular units of homogeneous size and shape, and possibly also of defined stoichiometry and spatial organization of its constituent proteins. An organized positioning of individual proteins and protein domains could allow these supramolecular assemblies to function as "molecular machines" and contribute to the high speed, precision, and fine-tuning that is characteristic for synaptic exocytosis. Morphological organization of active z...
Early processes underlying plant gravity sensing were investigated in rhizoids of Chara globularis under microgravity conditions provided by parabolic flights of the A300-Zero-G aircraft and of sounding rockets. By applying centrifugal forces during the microgravity phases of sounding rocket flights, lateral accelerations of 0.14g, but not of 0.05g, resulted in a displacement of statoliths. Settling of statoliths onto the subapical plasma membrane initiated the gravitropic response. Since actin controls the positioning of statoliths and restricts sedimentation of statoliths in these cells, it can be calculated that lateral actomyosin forces in a range of 2 3 10 214 N act on statoliths to keep them in place. These forces represent the threshold value that has to be exceeded by any lateral acceleration stimulus for statolith sedimentation and gravisensing to occur. When rhizoids were gravistimulated during parabolic plane flights, the curvature angles of the flight samples, whose sedimented statoliths became weightless for 22 s during the 31 microgravity phases, were not different from those of in-flight 1g controls. However, in ground control experiments, curvature responses were drastically reduced when the contact of statoliths with the plasma membrane was intermittently interrupted by inverting gravistimulated cells for less than 10 s. Increasing the weight of sedimented statoliths by lateral centrifugation did not enhance the gravitropic response. These results provide evidence that graviperception in characean rhizoids requires contact of statoliths with membrane-bound receptor molecules rather than pressure or tension exerted by the weight of statoliths.
Gravitropically tip-growing rhizoids and protonemata of characean algae are well-established unicellular plant model systems for research on gravitropism. In recent years, considerable progress has been made in the understanding of the cellular and molecular mechanisms underlying gravity sensing and gravity-oriented growth. While in higher-plant statocytes the role of cytoskeletal elements, especially the actin cytoskeleton, in the mechanisms of gravity sensing is still enigmatic, there is clear evidence that in the characean cells actin is intimately involved in polarized growth, gravity sensing, and the gravitropic response mechanisms. The multiple functions of actin are orchestrated by a variety of actin-binding proteins which control actin polymerisation, regulate the dynamic remodelling of the actin filament architecture, and mediate the transport of vesicles and organelles. Actin and a steep gradient of cytoplasmic free calcium are crucial components of a feedback mechanism that controls polarized growth. Experiments performed in microgravity provided evidence that actomyosin is a key player for gravity sensing: it coordinates the position of statoliths and, upon a change in the cell's orientation, directs sedimenting statoliths to specific areas of the plasma membrane, where contact with membrane-bound gravisensor molecules elicits short gravitropic pathways. In rhizoids, gravitropic signalling leads to a local reduction of cytoplasmic free calcium and results in differential growth of the opposite subapical cell flanks. The negative gravitropic response of protonemata involves actin-dependent relocation of the calcium gradient and displacement of the centre of maximal growth towards the upper flank. On the basis of the results obtained from the gravitropic model cells, a similar fine-tuning function of the actomyosin system is discussed for the early steps of gravity sensing in higher-plant statocytes.
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