The role of different scinderin domains in the control of actin filament dynamics and regulated exocytosis.

Abstract

In chromaffin cells, cortical F-actin disassembly in response to stimulation allows the movement of secretory vesicles towards exocytotic sites. Scinderin (Sc), a Ca2+-dependent F-actin severing protein, controls F-actin dynamics. The Sc gene has previously been cloned in our laboratory. Sc has six domains with three actin-binding sites in domains 1, 2 and 5, two PIP2 in domains 1 and 2 and two Ca2+-binding sites. In order to obtain additional data on the role of Sc domains in secretion, we performed experiments with a human growth hormone reporter gene (hGH) system for regulated exocytosis, and different GFP-fusion Sc constructs: Sc1--6, Sc1--2 (the first two domains of Sc), Sc5 (5th domain of Sc), ScL5 (5th domain lacking the third actin binding site of Sc), and ScABS3. Cortical F-actin distribution evaluated by rhodamine-phalloidine staining, in resting and stimulated cells (56 mM K+), showed that cells overexpressing Sc1--6 or Sc1--2 had an increased F-actin disassembly upon stimulation, whereas cells overexpressing Sc5 or ScABS3 had a decreased F-actin disassembly. No significant difference in F-actin assembly/disassembly was obtained for cells overexpressing ScL5. Increased hGH release in response to stimulus was found for cells overexpressing Sc1--6 or Sc1--2 when compared to cells transfected with vector alone. Cells overexpressing Sc5 or ScABS3 showed decreased hGH release in response to stimulus. These results suggest that during secretion the N-terminal half of the protein involved in F-actin severing and the C-terminal domains of Sc are responsible for actin polymerization. Thus, it appears that Sc functions as a molecular switch in the control of actin dynamics during secretion.