Fig. 1. (A) Internal generation and maintenance of the Ca²⁺ gradient in growing hyphae of N. crassa. This model is based upon extensive screening of inhibitors of Ca²⁺ signaling and PLC effects on hyphal growth, morphology, cytoplasmic and vesicular Ca²⁺ gradients, as well as direct evidence of IP₃-activated Ca²⁺ channels using the BLM technique. Treatment with the Ca²⁺ ATPase inhibitor cyclopiazonic acid inhibited growth (hyphal widening was observed) and increased cytoplasmic [Ca²⁺] behind the apex, consistent with Ca²⁺ sequestration into endoplasmic reticulum behind the growing apex (Silverman-Gavrila and Lew, 2001). Microinjection of IP₃ receptor agonists (IP₃ and Adenophostin A) behind the tip inhibited growth transiently, caused subapical branching, and affected the tip-high cytoplasmic Ca²⁺ gradient; these effects were not observed after microinjection of the biologically inactive L-IP₃ (Silverman-Gavrila and Lew, 2001). IP₃-induced subapical branching was similar to subapical branching induced by ionophoretic injection of Ca²⁺ (Silverman-Gavrila and Lew, 2000), suggesting that IP₃-activated Ca²⁺ release occurs in growing hyphae. An inhibitor of the IP₃ receptor, 2-APB inhibited hyphal elongation and dissipated the tip high cytoplasmic [Ca²⁺] gradient. Thus, tip-localized IP₃ production due to a stretch-activated phospholipase C could activate vesicular Ca²⁺ channels at the growing apex to generate the tip-high [Ca²⁺] gradient. The Ca²⁺ would induce fusion of wall vesicles at the apex, before being sequestered behind the tip via the Ca²⁺ ATPase. (B) The BLM technique used to identify and measure IP₃-activated Ca² channel activity in membrane vesicles isolated. from N. crassa. Hyphae are first homogenized to release the endomembranes. Following a series of centrifugations, subcellular fractions of membranes are isolated. Vesicles are added to the cis-chamber and fused to the lipid bilayer formed across an aperture in a septum that separates two chambers: cis and trans. The electronics are configured as a high gain current to voltage converter capable of measuring picoAmpere currents through ion channels. With only one permeant ion, Ca²⁺, only Ca²⁺ channels will be observed. Agonist addition to the cis compartment would activate channels oriented with their ligand binding site facing the cis compartment.