KINASE REQUIREMENT FOR RETINAL GROWTH CONE MOTILITY

Since cytoplasmic Ca2+ levels are reported to regulate neurite elongat ion, we tested whether calcium-activated kinases might be necessary fo r growth cone motility and neurite elongation in explant cultures of g oldfish retina. Kinase inhibitors and activators were locally applied by micropipette to retinal growth cones and the responses were observe d via phase-contrast videomicroscopy. In some cases, growth rates were also quantified over several hours after general application in the m edium. The selective inhibitors of protein kinase C, calphostin C (0.1 -1 mu M) and chelerythrin (up to 50 mu M), caused no obvious changes i n growth cones or neurite elongation, and activators of PKC (phorbols, arachidonic acid, and diacylglycerol) also were generally without eff ects, although phorbols slowed the growth rate. Inhibitors of protein kinase A and tyrosine kinases also produced no obvious effects. The ca lmodulin antagonists, calmidazolium (0.1 mu M), trifluoperazine (100 m u M), and CGS9343B (50 mu M), however, caused a reversible growth cone arrest with loss of filopodia and lamellipodia. The growth cone becam e a club-shaped swelling which sometimes moved a short distance back t he shaft, leaving evacuated filaments at points of strong filopodial a ttachments. A similar reversible growth cone arrest occurred with the general kinase inhibitors: H7 at 200 but not at 100 mu M, and staurosp orine at 100 but not 10 nM, suggesting possible involvement of a calmo dulin-dependent kinase (camK) rather than PKC. The selective inhibitor of camKII, KN-62 (tested up to 50 mu M), produced no effects, but the specific myosin light-chain kinase (MLCK) inhibitors ML-7 (3-5 mu M) and ML-9 (5-10 mu M) reversibly reproduced the effect, suggesting that MLCK rather than camKII is necessary for growth cone motility. The ML CK inhibitors' effects both on growth cone morphology and on F-actin f ilaments (rhodaminephalloidin staining) were similar to those caused b y cytochalasin D (5 mu M), and are discussed in light of findings that inhibiting MLCK disrupts actin filaments in astrocytes and fibroblast s. (C) 1994 John Wiley and Sons, Inc.