Since direct staining of growing hyphae with trifluoperazine (TFP) could result in the migration of protoplasm in hyphae of water mold Saprolegnia feraX, an improved staining method for fixation of growing hyphae was developed. When hyphae of Saprolegnia ferax were pre-fixed with paraformaldehyde, the TFP fluorescence showed a clear tip-high Ca(2+)-activated calmodulin (CaM) gradient. By indirect immunohistochemical staining, we proved that total CaM also mainly concentrated in the apex of hyphae.It is noticeble that when the hyphal tip growth ceased, the high intensity of TFP fluorescence usually disappeared in non-growing tips and became evident in the newly emerged growing tips, which implied that the concentration of CaM shifted in concord with the transfer of growing point in hyphae. 2×10(-5) mol/L CaM inhibitor TFP could reduce the tip growth rate and the percentage of growing hyphal tips to about 50%, and induce multibranching of hyphal tips. These findings strongly suggest that the high concentration of calmodulin in the hyphal tip may be involved in regulation of hyphal tip growth.
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