Kinase requirement for retinal growth cone motility.
Since cytoplasmic Ca2+ levels are reported to regulate neurite elongation, we tested whether calcium-activated kinases might be necessary for growth cone motility and neurite elongation in explant cultures of goldfish retina. Kinase inhibitors and activators were locally applied by micropipette to retinal growth cones and the responses were observed via phase-contrast videomicroscopy. In some cases, growth rates were also quantified over several hours after general application in the medium. The selective inhibitors of protein kinase C, calphostin C (0.1-1 microM) and chelerythrin (up to 50 microM), caused no obvious changes in growth cones or neurite elongation, and activators of PKC (phorbols, arachidonic acid, and diacylglycerol) also were generally without effects, although phorbols slowed the growth rate. Inhibitors of protein kinase A and tyrosine kinases also produced no obvious effects. The calmodulin antagonists, calmidazolium (0.1 microM), trifluoperazine (100 microM), and CGS9343B (50 microM), however, caused a reversible growth cone arrest with loss of filopodia and lamellipodia. The growth cone became a club-shaped swelling which sometimes moved a short distance back the shaft, leaving evacuated filaments at points of strong filopodial attachments. A similar reversible growth cone arrest occurred with the general kinase inhibitors: H7 at 200 but not at 100 microM, and staurosporine at 100 but not 10 nM, suggesting possible involvement of a calmodulin-dependent kinase (camK) rather than PKC. The selective inhibitor of camKII, KN-62 (tested up to 50 microM), produced no effects, but the specific myosin light-chain kinase (MLCK) inhibitors ML-7 (3-5 microM) and ML-9 (5-10 microM) reversibly reproduced the effect, suggesting that MLCK rather than camKII is necessary for growth cone motility. The MLCK inhibitors' effects both on growth cone morphology and on F-actin filaments (rhodamine-phalloidin staining) were similar to those caused by cytochalasin D (5 microM), and are discussed in light of findings that inhibiting MLCK disrupts actin filaments in astrocytes and fibroblasts.
Jian
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Department of Biological Sciences, State University of New York at Albany 12222.
Hidaka
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Schmidt
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D000199:Actins / Q000502:physiology; D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002147:Calmodulin / Q000037:antagonists & inhibitors; D015638:Cytochalasin D / Q000494:pharmacology; D004789:Enzyme Activation; D006054:Goldfish; D009416:Nerve Regeneration / Q000502:physiology*; D015739:Nocodazole / Q000494:pharmacology; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000502:physiology*
D000199:Actins; D002147:Calmodulin; D015638:Cytochalasin D; D010770:Phosphotransferases; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D015739:Nocodazole