Title

The role of calcium influx in deflagellation of Chlamydomonas reinhardtii: Isolation and characterization of a CIF (calcium influx factor)

Date of Completion

January 2000

Keywords

Biology, Molecular|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

The green alga Chlamydomonas reinhardtii deflagellates in response to certain environmental stimuli. Extracellular Ca2+ is required for this reaction, suggesting the involvement of a calcium influx factor (CIF) which triggers Ca2+ influx upon emptying of internal Ca2+ stores. I present evidence supporting the presence of a putative CIF in C. reinhardii. The CIF responds to depletion of intracellular Ca2+ stores by thapsigargin, an inhibitor of the cytoplasmic Ca2+-ATPase. Extracts from thapsigargin-treated C. reinhardii. cells showed three activities that are consistent with CIF-like behavior. They restored acid-induced deflagellation to the C. reinhardii. adfl (acid-deflagellation defective) mutant, which lacks this ability. They stimulated Ca2+-dependent Cl currents when microinjected into Xenopus laevis oocytes. Finally, the extracts triggered sustained increases in cytoplasmic Ca2+ levels when microinjected into Tradescantia virginiana L. stamen hair cells. We purified the putative CIF to a single peak on capillary electrophoresis. The purified CIF restored acid-induced deflagellation to the adfl cells and triggered increases in cytoplasmic Ca2+ levels in T. virginiana stamen hair cells. The polarity of the C. reinhardtii CIF is similar to CIFs reported by others from animal and fungal cells. ^ The CIF signal is also produced in response to other agents such as acid, G-protein activator Mastoparan, and calcium ionophore A23187, which are different agents for depleting internal calcium and therefore should cause CIF production, mobilizing calcium within the cells. Neomycin, a blocker of phospholipase C, has a negative effect on CIF production. We examined the chemical nature of CIF by comparing it with possible and suggested molecules in the literature that could be CIF(s) and by chemical treatment with alkaline phosphatase. We have compared the HPLC elution, UV spectra and CD spectra of those molecules to that of the native CIF from C. reinhardtii cells. Finally we subjected CIF from C. reinhardtii to Surface-Enhanced Laser Desorption/Ionization (SELDI) Protein Chip Mass Spectroscopy. We believe CIF in C. reinhardtii cells to be, at least in part, a peptide, of molecular weight close to 3000 daltons with absorbance in the far UV region and at about 295 mn. ^