Enhancing ubiquitin crystallization through surface-entropy reduction.

Ubiquitin has many attributes suitable for a crystallization chaperone, including high stability and ease of expression. However, ubiquitin contains a high surface density of lysine residues and the doctrine of surface-entropy reduction suggests that these lysines will resist participating in packing interactions and thereby impede crystallization. To assess the contributions of these residues to crystallization behavior, each of the seven lysines of ubiquitin was mutated to serine and the corresponding single-site mutant proteins were expressed and purified. The behavior of these seven mutants was then compared with that of the wild-type protein in a 384-condition crystallization screen. The likelihood of obtaining crystals varied by two orders of magnitude within this set of eight proteins. Some mutants crystallized much more readily than the wild type, while others crystallized less readily. X-ray crystal structures were determined for three readily crystallized variants: K11S, K33S and the K11S/K63S double mutant. These structures revealed that the mutant serine residues can directly promote crystallization by participating in favorable packing interactions; the mutations can also exert permissive effects, wherein crystallization appears to be driven by removal of the lysine rather than by addition of a serine. Presumably, such permissive effects reflect the elimination of steric and electrostatic barriers to crystallization.

A role for the polarity complex and PI3 kinase in branch formation within retinotectal arbors of zebrafish.

The developing zebrafish retinotectal arbors make many trial branches with synapses but most are retracted. With NMDA blockers, branches are withdrawn at a higher rate, and a synapse on a branch not only stabilizes that branch, but biases new branches to form nearby. Here, we tested whether new branch formation requires the polarity complex, which is essential for organizing the cytoskeleton in initial axon formation. The complex (PAR3, PAR6, and atypical protein kinase C [aPKC]) is downstream of phosphatidyl-inositol-3-kinase (PI3K), and its aPKC could be activated by retrograde arachidonic acid synaptic signaling. DiO-labeled arbors in zebrafish were imaged on day 3 (before treatment) and 1-2 days after treatment to suppress or inhibit PAR3, PAR6, or PI3K. Intraocular antisense (AS) oligos to PAR3 or PAR6 both severely limited branch addition, which was most evident in arbors with few branches before treatment. As a result of the inability to branch, arbor segments grew longer than in controls. Both PI3K inhibition (LY294002) and AS suppression of PI3Kα and PI3Kδ isoforms likewise limited branch addition but also decreased growth, as the sum of segment lengths was below normal after 2 days. Both the results support the idea that the polarity complex and PI3K participate in arbor branch formation. The PKC inhibitor Go6983 also severely restricted branch addition and growth, as did bisindolyl-maleimide and calphostin C reported previously, consistent with PKCζ, but not PKCµ, participation. These experiments suggest a mechanism whereby activity signaling could affect the branching of retinotectal arbors.

GAP43 phosphorylation is critical for growth and branching of retinotectal arbors in zebrafish.

Visual activity acts via NMDA Receptors to refine developing retinotectal maps by shaping retinal arbors. Retinal axons add and delete transient branches, and the dynamic rates increase when MK801 blocks NMDARs, as if this prevents release of a stabilizing signal. Ca(++) entry through NMDARs activates phospholipase A2 (cPLA2) to release arachidonic acid (AA), which taps into a presynaptic growth control mechanism. NCAM, L1, N-cadherin, and FGF all stimulate axon growth via AA activation of protein kinase C to phosphorylate GAP43 and polymerize/stabilize F-actin. Our previous results show that blocking cPLA2 mimics NMDAR blockers, whereas exogenous AA reverses the increased dynamics, and PKC inhibitors also arrest growth. To test whether this activity-driven F-actin control mechanism shapes retinotectal arbors in zebrafish, we used the alpha-1-tubulin promoter to express GAP43-GFP fusion proteins in retinal ganglion cells, and imaged arbors in time-lapse to test for effects of GAP43 levels and its phosphorylation. Overexpressing wildtype GAP43 gave faster growth and larger arbors (#branches, spatial extent, total length of branches) at three days and especially four days. Surprisingly, the N-terminal 20 amino acid segment alone caused the same increase in branching, but no increase in growth. Earlier studies implicate this region in activating G(o) resulting in collapse of growth cones, which is now known to precede branch initiation. In contrast, GAP43 with ser41 mutated to ala (S41A) to prevent phosphorylation did not increase either branching or growth but resulted in immature, elongated arbors even at four to five days. In support of this atrophic effect, only half of brain/spinal neurons expressing S41A successfully initiated axonal outgrowth (vs. nearly 100% for wtGAP43). These results suggest that the region around the ser41 phosphorylation site, which binds CaM and PIP2, promotes growth only when phosphorylated, and also activates the branching control region in the first 10-20 amino acids. Whereas phosphorylation introduces a bulky negative charge group, mutation of serine to arginine introduces a bulky positive charge. But this also produced the same growth and branching as phosphorylation, suggesting that the effect of phosphorylation is through hydrophilic bulk rather than negative charge, in agreement with other IQ motifs. The results implicate the cPLA2-AA-PKC-GAP43 pathway as part of an F-actin based mechanism that both stabilizes new synapses and initiates new branches near effective synapses.

Activity-driven sharpening of the retinotectal projection: the search for retrograde synaptic signaling pathways.

Patterned visual activity, acting via NMDA receptors, refines developing retinotectal maps by shaping individual retinal arbors. Because NMDA receptors are postsynaptic but the retinal arbors are presynaptic, there must be retrograde signals generated downstream of Ca(++) entry through NMDA receptors that direct the presynaptic retinal terminals to stabilize and grow or to withdraw. This review defines criteria for retrograde synaptic messengers, and then applies them to the leading candidates: nitric oxide (NO), brain-derived neurotrophic factor (BDNF), and arachidonic acid (AA). NO is not likely to be a general mechanism, as it operates only in selected projections of warm blooded vertebrates to speed up synaptic refinement, but is not essential. BDNF is a neurotrophin with strong growth promoting properties and complex interactions with activity both in its release and receptor signaling, but may modulate rather than mediate the retrograde signaling. AA promotes growth and stabilization of synaptic terminals by tapping into a pre-existing axonal growth-promoting pathway that is utilized by L1, NCAM, N-cadherin, and FGF and acts via PKC, GAP43, and F-actin stabilization, and it shares some overlap with BDNF pathways. The actions of both are consistent with recent demonstrations that activity-driven stabilization includes directed growth of new synaptic contacts. Certain nondiffusible factors (synapse-specific CAMs, ephrins, neurexin/neuroligin, and matrix molecules) may also play a role in activity-driven synapse stabilization. Interactions between these pathways are discussed.

Presynaptic protein kinase C controls maturation and branch dynamics of developing retinotectal arbors: possible role in activity-driven sharpening.

Visual activity refines developing retinotectal maps and shapes individual retinal arbors via an NMDA receptor-dependent mechanism. As retinal axons grow into tectum, they slow markedly and emit many transient side branches behind the tip, assuming a "bottlebrush" morphology. Some branches are stabilized and branch further, giving rise to a compact arbor. The dynamic rate of branch addition and deletion is increased twofold when MK801 is used to block NMDA receptors, as if this prevents release of a stabilizing signal such as arachidonic acid (AA) from the postsynaptic neuron. In optic tract, AA mediates NCAM and L1 stimulation of axon growth by activating presynaptic protein kinase C (PKC) to phosphorylate GAP-43 and stabilize F-actin, and, if present in tectum, this growth control pathway could be modulated by postsynaptic activation. To test for the effects on arbor morphology of blocking PKC or AA release, we examined DiO-labeled retinal axons of larval zebrafish with time-lapse videomicroscopy. Bath application of the selective PKC inhibitor bisindolylmaleimide from 2 or 3 days onward doubled the rate at which side branches were added and deleted, as seen with MK801, and also prevented maturation of the arbor so that it retained a "bottlebrush" morphology. In order to selectively block the PKC being transported to retinal terminals, we injected the irreversible inhibitor calphostin C into the eye from which the ganglion cells were labeled, and this produced both effects seen with bath application. In contrast, there were no effects of control injections, which included Ringers into the same eye and the same dose into the opposite eye (actually much closer to the tectum of interest), to rule out the possibility that the inhibitor leaked from the eye to act on tectal cells. For comparison, we examined arbors treated with the NMDA blocker MK801 at half-hour time-lapse intervals, and detected the twofold rise in rates of branch addition and deletion previously reported in Xenopus larvae, but not the structural effect seen with the PKC inhibitors. In addition, we could produce both effects seen with PKC inhibitors by using RHC80267 to block AA release from DAG lipase, indicating that AA is the main drive for PKC activation. Thus, the results show a distinct role of AA and presynaptic PKC in both maturation of arbor structure and in the dynamic control of branching. The effects on branch dynamics were present regardless of the level of maturity of arbor structure. The fact that they mimicked those of MK801 suggests that presynaptic PKC may be involved in the NMDA receptor-driven stabilization of developing retinal arbors.

Fireflies at one hundred plus: a new look at flash control.

The mysterious process by which fireflies can control their flashing has inspired over a century of careful observation but has remained elusive. Many studies have implicated oxygen as the controlling element in the photochemical reaction, and the discovery of nitric oxide synthetase (NOS) in the lantern has suggested that nitric oxide (NO) may control oxygen access to the light-emitting photocytes, thereby triggering the flash. However, there are several drawbacks to oxygen as a controlling agent, and in view of the prominence of peroxisomes in lantern morphology and biochemistry, we suggest that it is hydrogen peroxide that triggers the flash, and we present a model by which this may take place.

Myosin light chain phosphorylation and growth cone motility.

According to the treadmill hypothesis, the rate of growth cone advance depends upon the difference between the rates of protrusion (powered by actin polymerization at the leading edge) and retrograde F-actin flow, powered by activated myosin. Myosin II, a strong candidate for powering the retrograde flow, is activated by myosin light chain (MLC) phosphorylation. Earlier results showing that pharmacological inhibition of myosin light chain kinase (MLCK) causes growth cone collapse with loss of F-actin-based structures are seemingly inconsistent with the treadmill hypothesis, which predicts faster growth cone advance. These experiments re-examine this issue using an inhibitory pseudosubstrate peptide taken from the MLCK sequence and coupled to the fatty acid stearate to allow it to cross the membrane. At 5-25 microM, the peptide completely collapsed growth cones from goldfish retina with a progressive loss of lamellipodia and then filopodia, as seen with pharmacological inhibitors, but fully reversible. Lower concentrations (2.5 microM) both simplified the growth cone (fewer filopodia) and caused faster advance, doubling growth rates for many axons (51-102 microm/h; p <.025). Rhodamine-phalloidin staining showed reduced F-actin content in the faster growing growth cones, and marked reductions in collapsed ones. At higher concentrations, there was a transient advance of individual filopodia before collapse (also seen with the general myosin inhibitor, butanedione monoxime, which did not accelerate growth). The rho/rho kinase pathway modulates MLC dephosphorylation by myosin-bound protein phosphatase 1 (MPP1), and manipulations of MPP1 also altered motility. Lysophosphatidic acid (10 microM), which causes inhibition of MPP1 to accumulate activated myosin II, caused a contracted collapse (vs. that due to loss of F-actin) but was ineffective after treatment with low doses of peptide, demonstrating that the peptide acts via MLC phosphorylation. Inhibiting rho kinase with Y27632 (100 microM) to disinhibit the phosphatase increased the growth rate like the MLCK peptide, as expected. These results suggest that: varying the level of MLCK activity inversely affects the rate of growth cone advance, consistent with the treadmill hypothesis and myosin II powering of retrograde F-actin flow; MLCK activity in growth cones, as in fibroblasts, contributes strongly to controlling the amount of F-actin; and the phosphatase is already highly active in these cultures, because rho kinase inhibition produces much smaller effects on growth than does MLCK inhibition.