Inhibition of tyrosine phosphorylation potentiates substrate-induced neurite growth

Protein tyrosine kinases (PTKs) have major roles in signal transduction and growth control. There are several lines of evidence implicating PTKs in the regulation of axon growth, and this has led to the suggestion that they are centrally involved in the transduction of neuronal growth signals. To test this idea, we assayed the effect of the compounds genistein and lavendustin, specific inhibitors of PTKs, on neurite growth. We find that genistein greatly reduces phosphotyrosine in neurons, as expected from its action on other cells. Surprisingly, administration of genistein or lavendustin potentiated substrate-induced neurite growth in at least several different neuronal types. Stimulation of neurite growth by genistein was abolished by vanadate, providing additional evidence that inhibition of PTKs is responsible for this effect. The potentiation of growth is rather general, in that it occurs on several different extracellular matrix substrates and on two different cell adhesion molecules. Both the initiation of neurite growth and the rate of neurite elongation appear to be potentiated. Our results do not provide evidence for models of substrate-induced signal transduction that involve PTKs asa positive and necessary step, but suggest that such kinases play aregulatory role in neurite elongation. © 1992 John Wiley & Sons, Inc.     

Inhibition of spindle elongation by taxol.

During anaphase B spindle elongation, interzonal microtubules lengthen to accomplish pole-pole separation, while at the same time remaining highly dynamic [Shelden and Wadsworth, J. Cell Sci. 97:273-281, 1990]. To further examine the role of microtubule polymerization and dynamics during spindle elongation, cells have been treated with taxol, which induces microtubule polymerization and stabilizes microtubules. Taxol was added to PtK1 cells 3 minutes after initial chromatid separation, so that the effect on anaphase B could be observed with minimal disruption to anaphase A movement. In 20 microM taxol, the rate and extent of pole-pole separation, measured from time-lapse video records, are reduced to 4% and 9.5% of controls, respectively. The organization of microtubules in taxol treated cells was examined using tubulin immunofluorescence and confocal fluorescence microscopy. Taxol induces a dramatic reorganization of interzonal microtubules resulting in a narrow gap, which is nearly completely lacking in MTs, across the center of the interzone. Furthermore, microtubules in taxol treated cells are resistant to nocodazole induced microtubule disassembly. Our results reveal that taxol rapidly inhibits anaphase B spindle elongation; inhibition is accompanied by a depletion of interdigitated interzonal microtubules and a reduction in microtubule dynamic behavior.     

Inhibition of tyrosine phosphorylation potentiates substrate-induced neurite growth.

Protein tyrosine kinases (PTKs) have major roles in signal transduction and growth control. There are several lines of evidence implicating PTKs in the regulation of axon growth, and this has led to the suggestion that they are centrally involved in the transduction of neuronal growth signals. To test this idea, we assayed the effect of the compounds genistein and lavendustin, specific inhibitors of PTKs, on neurite growth. We find that genistein greatly reduces phosphotyrosine in neurons, as expected from its action on other cells. Surprisingly, administration of genistein or lavendustin potentiated substrate-induced neurite growth in at least several different neuronal types. Stimulation of neurite growth by genistein was abolished by vanadate, providing additional evidence that inhibition of PTKs is responsible for this effect. The potentiation of growth is rather general, in that it occurs on several different extracellular matrix substrates and on two different cell adhesion molecules. Both the initiation of neurite growth and the rate of neurite elongation appear to be potentiated. Our results do not provide evidence for models of substrate-induced signal transduction that involve PTKs as a positive and necessary step, but suggest that such kinases play a regulatory role in neurite elongation.     

Inhibition of Trichomonas vaginalis replication by the microtubule stabilizer taxol

Taxol, a plant alkaloid stabilizer of microtubules, inhibits in vitro the replication of the human pathogenic flagellate Trichomonas vaginalis in a dose-dependent fashion. Micromolar concentrations of the drug induce massive assembly of microtubules, resistant to antimicrotubule agents, and block the mitosis of the protozoa at an early stage preceding the formation of the spindle fibers and the depolymerization of the axostylar microtubules. Some hypotheses involving the inactivation of the microtubule-organizing centers and the stabilization of axostylar microtubules are formulated to explain the mechanism of action of taxol.     

Unphosphorylated MARCKS is involved in neurite initiation induced by insulin-like growth factor-I in SH-SY5Y cells

Myristoylated alanine-rich C kinase substrate (MARCKS) has been suggested to be involved in various aspects of neuronal cell differentiation, including neurite outgrowth. However, the precise mechanisms by which MARCKS phosphorylation is regulated, and how MARCKS contributes to neurite outgrowth, are poorly understood. Here, we found that treatment of SH-SY5Y cells with insulin-like growth factor-I (IGF-I) induced a rapid and transient decrease in the level of phosphorylated MARCKS (P-MARCKS) to below the basal level. The decrease in P-MARCKS induced by IGF-I was blocked by pretreatment of cells with phosphoinositide 3-kinase (PI3K) inhibitors, LY294002 and wortmannin. A decrease in P-MARCKS was also observed in cells treated with a Rho-dependent kinase (ROCK) inhibitor, Y27632. Furthermore, IGF-I induced transient inactivation of RhoA, an upstream effector of ROCK. We showed that MARCKS was translocated to the membrane and colocalized with F-actin at the lamellipodia and the tips of neurites in the cells stimulated with IGF-I. Finally, overexpression of wild-type MARCKS or an unphosphorylatable mutant of MARCKS enhanced the number of neurite-bearing cells relative to vector-transfected cells. Taken together, these findings suggest that unphosphorylated MARCKS is involved in neurite initiation, and highlight the important role played by MARCKS in organization of the actin cytoskeleton.     

Filopodia are required for cortical neurite initiation

Extension of neurites from a cell body is essential to form a functional nervous system; however, the mechanisms underlying neuritogenesis are poorly understood. Ena/VASP proteins regulate actin dynamics and modulate elaboration of cellular protrusions. We recently reported that cortical axon-tract formation is lost in Ena/VASP-null mice and Ena/VASP-null cortical neurons lack filopodia and fail to elaborate neurites. Here, we report that neuritogenesis in Ena/VASP-null neurons can be rescued by restoring filopodia formation through ectopic expression of the actin nucleating protein mDia2. Conversely, wild-type neurons in which filopodia formation is blocked fail to elaborate neurites. We also report that laminin, which promotes the formation of filopodia-like actin-rich protrusions, rescues neuritogenesis in Ena/VASP-deficient neurons. Therefore, filopodia formation is a key prerequisite for neuritogenesis in cortical neurons. Neurite initiation also requires microtubule extension into filopodia, suggesting that interactions between actin-filament bundles and dynamic microtubules within filopodia are crucial for neuritogenesis.     

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在改良的B     

Inhibition of mammalian translation initiation by volatile anesthetics

Volatile anesthetics are essential for modern medical practice, but sites and mechanisms of action for any of their numerous cellular effects remain largely unknown. Previous studies with yeast showed that volatile anesthetics induce nutrient-dependent inhibition of growth through mechanisms involving inhibition of mRNA translation. Studies herein show that the volatile anesthetic halothane inhibits protein synthesis in perfused rat liver at doses ranging from 2 to 6%. A marked disaggregation of polysomes occurs, indicating that inhibition of translation initiation plays a key role. Dose- and time-dependent alterations that decrease the function of a variety of translation initiation processes are observed. At 6% halothane, a rapid and persistent increase in phosphorylation of the alpha-subunit of eukaryotic translation initiation factor (eIF)2 occurs. This is accompanied by inhibition of activity of the guanine nucleotide exchange factor eIF2B that is responsible for GDP-GTP exchange on eIF2. At lower doses, neither eIF2alpha phosphorylation nor eIF2B activity is altered. After extended exposure to 6% halothane, alterations in two separate responses regulated by the target of rapamycin pathway occur: 1) redistribution of eIF4E from its translation-stimulatory association with eIF4G to its translation-inactive complex with eIF4E-binding protein-1; and 2) decreased phosphorylation of ribosomal protein S6 (rpS6) with a corresponding decrease in active forms of a kinase that phosphorylates rpS6 (p70(S6K1)). Changes in the association of eIF4E and eIF4G are observed only after extended exposure to low anesthetic doses. Thus dose- and time-dependent alterations in multiple processes permit liver cells to adapt translation to variable degrees and duration of stress imposed by anesthetic exposure.     

Inhibition of DNA replication initiation by silver nanoclusters

Silver nanoclusters (AgNCs) have outstanding physicochemical characteristics, including the ability to interact with proteins and DNA. Given the growing number of diagnostic and therapeutic applications of AgNCs, we evaluated the impact of AgNCs on DNA replication and DNA damage response in cell-free extracts prepared from unfertilized Xenopus laevis eggs. We find that, among a number of silver nanomaterials, AgNCs uniquely inhibited genomic DNA replication and abrogated the DNA replication checkpoint in cell-free extracts. AgNCs did not affect nuclear membrane or nucleosome assembly. AgNCs-supplemented extracts showed a strong defect in the loading of the mini chromosome maintenance (MCM) protein complex, the helicase that unwinds DNA ahead of replication forks. FLAG-AgNCs immunoprecipitation and mass spectrometry analysis of AgNCs associated proteins demonstrated direct interaction between MCM and AgNCs. Our studies indicate that AgNCs directly prevent the loading of MCM, blocking pre-replication complex (pre-RC) assembly and subsequent DNA replication initiation. Collectively, our findings broaden the scope of silver nanomaterials experimental applications, establishing AgNCs as a novel tool to study chromosomal DNA replication.     

Inhibition of initiation of DNA synthesis by aminoglycoside antibiotics

In a $\text{dna}\text{C}{}^{\mathrm{ts}}$ mutant of $\text{E. coli}$, the reinitiation of DNA synthesis, which occurred by the shift of the culture from a restrictive temperature to a permissive temperature, was markedly prevented by habakacin, dibekacin, kanamycin, and gentamicin. On the contrary, chloramphenicol did not inhibit the reinitiation synthesis for 30 min. In a parallel experiment, leucine uptake into protein was profoundly blocked by chloramphenicol, but only slightly by habekacin. Habekacin did not significantly affect DNA elongation of the cells at a restrictive temperature. We propose that inhibition of initiation of replication by aminoglycoside antibiotics is related to their lethality.     

Regulation of the neurite growth

Recent achievement of molecular biology and neurophysiology resulting in remarkable progress in the understanding of processes of neurodifferentiation, playing the relevant role both in an ontogenesis and working of adult brain. New representations have been formed about a role of cytoskeletal proteins in mechanisms of neurites growth. In the review we discussed recent data regarding mechanisms of neuronal differentiation regulation. The special interest is given to a problem of influence of electrical fields on neuronal growth and differentiation. The mechanisms of signal transduction from a cell surface to nuclear are in detail analyzed. The classification of the physical and chemical factors of growth neurites regulation is given.     

Control of growth cone motility and neurite outgrowth by SPIN90

SPIN90 is an F-actin binding protein thought to play important roles in regulating cytoskeletal dynamics. It is known that SPIN90 is expressed during the early stages of neuronal development, but details of its localization and function in growth cones have not been fully investigated. Our immunocytochemical data show that SPIN90 is enriched throughout growth cones and neuronal shafts in young hippocampal neurons. We also found that its localization correlates with and depends upon the presence of F-actin. Detailed observation of primary cultures of hippocampal neurons revealed that SPIN90 knockout reduces both growth cone areas and in the numbers of filopodia, as compared to wild-type neurons. In addition, total neurite length, the combined lengths of the longest (axonal) and shorter (dendritic) neurites, was smaller in SPIN90 knockout neurons than wild-type neurons. Finally, Cdc42 activity was down-regulated in SPIN90 knockout neurons. Taken together, our findings suggest that SPIN90 plays critical roles in controlling growth cone dynamics and neurite outgrowth.     

Inhibition of replicon initiation by 12-O-tetradecanoylphorbol-13-acetate

To investigate the inhibition of DNA replication by tumor promoters, we incubated HeLa cells with 12-O-tetradecanoylphorbol-13-acetate (TPA; 10^?8 to 10^?5 g/ml) and quantified DNA synthesis on alkaline sucrose gradients. TPA was found to selectively inhibit replicon initiation without affecting DNA chain elongation in replicons that had already initiated. No inhibition of DNA synthesis was seen when cells were exposed to the nonpromoting derivative of TPA, 4-α-phorbol 12,13-didecanoate. Superoxide dismutase did not prevent the TPA-induced inhibition of initiation.     

Regulation of neurite growth by tumour necrosis superfamily member RANKL

RANKL (receptor-activator of NF-κB ligand, TNFSF11) is a member of the TNF superfamily that regulates bone remodelling and the development of the thymus, lymph nodes and mammary glands. While RANKL and its membrane bound receptor RANK (TNFRSF11A) are expressed in the adult central nervous system and have been implicated in thermoregulation, the potential function of RANK signalling in the developing nervous system remains unexplored. Here, we show that RANK is expressed by sympathetic and sensory neurons of the developing mouse peripheral nervous system and that activating RANK signalling in these neurons during perinatal development by either treating cultured neurons with soluble RANKL or overexpressing RANK in the neurons inhibited neurotrophin-promoted neurite growth without affecting neurotrophin-promoted neuronal survival. RANKL is expressed in tissues innervated by these neurons, and studies in compartment cultures demonstrated that RANKL is capable of acting directly on neurites to inhibit growth locally. Enhancing RANK signalling in cultured neurons resulted in NF-κB activation and phosphorylation of the p65 NF-κB subunit on serine 536. Transfecting neurons with a series of mutated signalling proteins showed that NF-κB activation and p65 phosphorylation occurred by an IKKβ-dependent mechanism and that blockade of this signalling pathway prevented neurite growth inhibition by RANKL. These findings reveal that RANKL is a novel negative regulator of neurite growth from developing PNS neurons and that it exerts its effects by IKKβ-dependent activation of NF-κB.     

Inhibition of neurite extension by overexpression of individual domains of LIM kinase 1

Lin-11, Isl-1 and Mec-3 (LIM) kinases are serine/threonine kinases that phosphorylate cofilin, an actin depolymerizing protein. LIM kinases have a highly modular structure composed of two N-terminal LIM domains (LIM 1/2), a PSD-95, Dlg and ZO-1 (PDZ) domain and a C-terminal protein kinase domain. Here, we overexpressed individual domains of mouse LIM kinase 1 (LIMK1) in PC12 cells and investigated their effects on neurite outgrowth. Although none of the LIMK1 domains had an effect on spontaneous neurite outgrowth, the N-terminal LIM 1/2 domains strongly inhibited differentiation of PC12 cells after stimulation with both nerve growth factor (NGF) and the Rho-kinase inhibitor Y-27632. In contrast, the overexpressed PDZ domain reduced neurite outgrowth only when differentiation had been induced by Y-27632, but not by NGF. Our data suggest that the different non-catalytic N-terminal domains of LIMK1 contribute to the regulation of neurite extension by using distinct signal transduction pathways.     

Control of neurite outgrowth and growth cone motility by phosphatidylinositol-3-kinase

Phosphatidylinositol-3-kinase (PI-3K) has been reported to affect neurite outgrowth both in vivo and in vitro. Here we investigated the signaling pathways by which PI-3K affects neurite outgrowth and growth cone motility in identified snail neurons in vitro. Inhibition of PI-3K with wortmannin (2 microM) or LY 294002 (25 microM) resulted in a significant elongation of filopodia and in a slow-down of neurite outgrowth. Experiments using cytochalasin and blebbistatin, drugs that interfere with actin polymerization and myosin II activity, respectively, demonstrated that filopodial elongation resulting from PI-3K inhibition was dependent on actin polymerization. Inhibition of strategic kinases located downstream of PI-3K, such as Akt, ROCK, and MEK, also caused significant filopodial elongation and a slow-down in neurite outgrowth. Another growth cone parameter, filopodial number, was not affected by inhibition of PI-3K, Akt, ROCK, or MEK. A detailed study of growth cone behavior showed that the filopodial elongation induced by inhibiting PI-3K, Akt, ROCK, and MEK was achieved by increasing two motility parameters: the rate with which filopodia extend (extension rate) and the time that filopodia spend elongating. Whereas the inhibition of ROCK or Akt (both activated by the lipid kinase activity of PI-3K) and MEK (activated by the protein kinase activity of PI-3K) had additive effects, simultaneous inhibition of Akt and ROCK showed no additive effect. We further demonstrate that the effects on filopodial dynamics investigated were calcium-independent. Taken together, our results suggest that inhibition of PI-3K signaling results in filopodial elongation and a slow-down of neurite advance, reminiscent of growth cone searching behavior.