Nitric oxide, a free-radical gas produced endogenously by several mammalian cell types, has been implicated as a diffusible intercellular messenger subserving use-dependent modification of synaptic efficacy in the mature central nervous system. It has been suggested on theoretical grounds that nitric oxide might play an analogous role during the establishment of ordered connections by developing neurons. We report here that nitric oxide rapidly and reversibly inhibits growth of neurites of rat dorsal root ganglion neurons in vitro. In addition, we show that exposure to nitric oxide inhibits thioester-linked long-chain fatty acylation of neuronal proteins, possibly through a direct modification of substrate cysteine thiols. Our results demonstrate a potential role for nitric oxide in the regulation of process outgrowth and remodelling during neuronal development, which may be effected at least in part through modulation of dynamic protein fatty acylation in neuronal growth cones.

Thapsigargin, a sesquiterpene lactone with potent irritant and tumour-promoting activities, stimulates a rapid (within 15 s) transient increase in intracellular [Ca2+] in the NG115-401L neural cell line, as measured by the fluorescent indicator dye fura-2. This increase in cytoplasmic free [Ca2+] is concentration-dependent (ED50 around 20 nM) and occurs in the absence of extracellular Ca2+. Activation of NG115-401L cells by the inflammatory peptide bradykinin generates inositol phosphates, which parallel increases in intracellular [Ca2+]. However, the rise in cytoplasmic [Ca2+] stimulated by thapsigargin occurs in the absence of detectable production of inositol phosphates. Thapsigargin is unlike phorboid tumour promoters in that it has no action on two non-invasive indicators of phorbol stimulation of these cells, i.e. [3H]choline metabolite production and rise in intracellular pH. These data suggest that thapsigargin releases Ca2+ from an intracellular store by a novel mechanism, independent of the hydrolysis of phosphoinositides and concomitant activation of protein kinase C. Thus thapsigargin may provide a valuable tool for the analysis of intracellular signalling mechanisms.

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Large-scale structural genomics centres rely heavily on robotics to ensure that maximum throughput is achieved. However, the size and cost of these approaches is out of the reach of most academic structural biology efforts. A major challenge for such groups is to adapt current high-throughput schemes to a reasonable scale with the resources available. A flexible medium-throughput approach has been developed that is suitable for typical academic research groups. Following nested PCR, targets are routinely cloned into two Gateway expression vectors (pDEST15 for an N-terminal GST tag and pDEST17 for an N-terminal His tag). Expression of soluble recombinant protein in Escherichia coli is rapidly assessed in 96-well format. An eight-probe sonicator is utilized and a six-buffer lysis screen was incorporated to enhance solubility. Robotics is reserved for crystallization, since this is the key bottleneck for crystallography. Screening proteins with a 480-condition protocol using a Cartesian nanolitre-dispensing robot has increased crystallization success markedly, with an overall success rate (structures solved out of proteins screened) of 19%. The methods are robust and economical -- with the exception of the crystallization robot, investment in additional equipment has been minimal at 9000 US dollars. All protocols are designed for individuals so that graduate students and postdoctoral fellows gain expertise in every aspect of the structural pipeline, from cloning to crystallization.

ReferencesAggensteiner, M. and Reiser, G. (2003). Expression of the brain-specific membrane adapter protein p42 IP4 /centaurin a, a Ins(1,3,4,5)P 4 /PtdIns(3,4,5)P 3 binding protein, in developing rat brain. Brain Res. Dev. Brain Res. 142,[77][78][79][80][81][82][83][84][85][86][87]. Albertinazzi, C., Za, L., Paris, S. and de Curtis, I. (2003). ADP-ribosylation factor 6 and a functional PIX/p95-APP1 complex are required for Rac1B-mediated neurite outgrowth. Mol. Biol. Cell 14, 1295-1307. Allen, P., Ouimet, C. and Greengard, P. (1997 (1996). Identification and cloning of centaurin alpha: a novel phosphatidylinositol (3,4,5)-trisphosphate binding protein from rat brain.

Abstract. In neuronal growth cones, the advancing tips of elongating axons and dendrites, specific protein substrates appear to undergo cycles of posttranslational modification by covalent attachment and removal of long-chain fatty acids. We show here that ongoing fatty acylation can be inhibited selectively by longchain homologues of the antibiotic tunicamycin, a known inhibitor of N-linked glycosylation. Tunicamycin directly inhibits transfer of palmitate to protein in a cell-free system, indicating that tunicamycin inhibition of protein palmitoylation reflects an action of the drug separate from its previously established effects on glycosylation. Tunicamycin treatment of differentiated PC12 cells or dissociated rat sensory neurons, under conditions in which protein palmitoylation is inhibited, produces a prompt cessation of neurite elongation and induces a collapse of neuronal growth cones. These growth cone responses are rapidly reversed by washout of the antibiotic, even in the absence of protein synthesis, or by addition of serum. Two additional lines of evidence suggest that the effects of tunicamycin on growth cones arise from its ability to inhibit protein long-chain acylation, rather than its previously established effects on protein glycosylation and synthesis. The tunicamycin-induced impairment of growth cone function can be reversed by the addition of excess exogenous fatty acid, which reverses the inhibition of protein palmitoylation but has no effect on the inhibition of protein glycosylation. These results suggest an important role for dynamic protein acylation in growth cone-mediated extension of neuronal processes.

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In the mammalian cortex, the initial formation of synaptic connections is followed by a prolonged period during which synaptic circuits are functional, but retain an elevated capacity for activity‐dependent remodeling and functional plasticity. During this period, synaptic terminals appear fully mature, morphologically and physiologically. We show here, however, that synaptic terminals during this period are distinguished by their simultaneous accumulation of multiple growth‐associated proteins at levels characteristic of axonal growth cones, and proteins involved in synaptic transmitter release at levels characteristic of adult synapses. We show further that newly formed synapses undergo a switch in the dynamic S‐palmitoylation of proteins early in the critical period, which includes a large and specific decrease in the palmitoylation of GAP‐43 and other major substrates characteristic of growth cones. Previous studies have shown that a similar reduction in ongoing palmitoylation of growth cone proteins is sufficient to stop advancing axons in vitro, suggesting that a developmental switch in protein S‐palmitoylation serves to disengage the molecular machinery for axon extension in the absence of local triggers for remodeling during the critical period. Only much later does a decline in the availability of major growth cone components mark the molecular maturation of cortical synapses at the close of the critical period. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 423–437, 1999