Intracellular Calcium and Calmodulin Involvement in Protoplast

45ca2+ uptake was compared between fusogenic and nonfusogenic Daucus carota L. protoplasts. Fusogenic protoplasts took 10 minutes to reach calcium equilibrium compared to 5 minutes in the nonfusogenic protoplasts. Intracellular stores of calcium were manipulated by isolating protoplasts in different calcium regimes. Lowering of intracellular calcium lowered fusion potential, while raising intracelluiar stores of calcium enhanced fusion potential. Regardless of the amount of calcium sequestered in a store, mobilization with A23187 increased fusion levels within 10 minutes. Calmodulin antagonists were potent inhibitors of protoplast fusion. This inhibition was obtained by treating cells with the calmodulin antagonists during protoplast isolation. A23187, however, only allowed a partial recovery from this inhibition, indicating that calcium flux alone was not sufficient for maximum fusion potential. On the basis of the evidence presented, we propose that calcium fluxes during protoplast isolation activate a calmodulin-mediated biochemical process that is necessary for the formation or maintenance of a fusion permissive state. The fusion of biological membranes is an integral component of many physiological phenomena. Intracellular fusion events are critical for membrane biogenesis and recycling (17). A much less common type of membrane fusion, however, is that involving plasma membranes from two different cells or protoplasts. In nature, plasma membrane fusion between two different cells is limited to a very few examples, including fertilization and myoblast fusion to form muscle fibers. We recently have added another example to this list. The growth conditions of a meristematic Daucus carota L. cell line were manipulated such that highly fusogenic protoplasts were obtained. These fusogenic protoplasts attain greater than 50% fusion levels with only a very mild calcium stimulus. Calcium is a requirement for fusion in the Daucus system; no other cations will stimulate fusion. Furthermore, the calcium chelator, EGTA, strongly inhibits protoplast fusion (5). The involvement of calcium in the fusion of bilayer membranes is an almost universal phenomenon (12, 20). An absolute requirement for calcium has been documented for membrane fusion during fertilization (24), secretory vesicle fusion (16), Sendai virus-induced erythrocyte fusion (13, 28), myoblast fusion (9), and Daucus fusogenic protoplasts (5). 'Suppported by the United States Department of Agriculture (83CRCR-1-1276). Paper No. 9812 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. 2Abbreviations: TFP, trifluoperazine; CPZ, chlorpromazine; W-13, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride; W12, N44-aminobutyl)-2-naphthalenesulfonamide hydrochloride. Separate roles for extraand intracellular calcium have been implied in various systems. Duzgunes et al. (12) have shown that acidic phospholipids of liposome bilayers bind external calcium resulting in an increase in both rate and maximum levels of fusion. Papahadjopoulos (20) hypothesizes that calcium alters the physical structure of the bilayer and that this alteration enhances fusion. Intracellular calcium has been implicated in Sendai virusinduced red blood cell fusion (13) and myoblast fusion (9), although the biochemical mechanism remains unclear. In myoblasts, preceding fusion, a transient increase in intracellular calcium is observed. If this increase is blocked, then fusion is inhibited (9). There is now substantial evidence that calcium regulation in a variety of eukaryotic cells is mediated by the calcium-binding protein calmodulin. Calmodulin has been implicated in mediating the calcium regulation of protein phosphorylation, microtubule assembly, cell motility, and calcium flux across cell membranes (for reviews see Cheung [8], Dieter [11]). Phenothiazinetype calmodulin antagonists bind to the calcium-activated form of calmodulin and inhibit its interaction with cellular target proteins (29). Consequently, these phenothiazine compounds have been used to study the participation of calcium-calmodulin in the regulation of calcium-dependent cellular events (18, 19). Recently, the phenothiazine drug TFP,2 was found to inhibit fusion in the myoblast system (2). In the present study, we have used "5Ca2l uptake methodologies to document differences in calcium regulation between fusogenic and nonfusogenic carrot protoplasts. Additional goals of this study were (a) to manipulate intracellular calcium and determine if fusion is affected, (b) to determine if calmodulin is involved in protoplast fusion, and (c) to determine whether calmodulin regulation of intracellular calcium fluxes is critical for the formation or maintenance of a fusion permissive state. MATERIALS AND METHODS Cell Cultures. Two different cell cultures were used for these studies. The first, denoted fusogenic, was originally derived from a wild carrot (Daucus carota L.) cell culture with the potential to form embryos. This cell line was maintained by biweekly subculture in wild carrot medium (5). To initiate the fusogenic cell line, 0.1 ml aliquots of a 61 -,um fraction were placed into 25 ml of fusion-inducing medium (5). Weekly serial transfers were made and cells were harvested between 4 to 5 d after transfer. Cells were always used after 2 to 3 weeks on fusion-inducing medium to ensure uniformity. The second cell line, denoted nonfusogenic, did not have the potential to form embryos. This cell line was serially transferred weekly and harvested 4 to 5 d after transfer. Protoplast Isolation. For fusion assays, protoplasts were isolated in 2% Driselase (Plenum Scientific Co., Hackensack, NJ), 0.4 molal sorbitol, I mm Mes at a final pH of 4.8 for 2 h on a