The secretory-pathway Ca2+-ATPase SPCA1 is a thapsigargin-insensitive intracellular Ca2+ pump found mostly in the Golgi compartment. We have explored the contribution of this Ca2+ pump to cytosolic Ca2+ signaling in HeLa cells by using RNA-mediated interference to disrupt its expression. Removal of SPCA1 was confirmed by immunofluorescence with specific anti-SPCA1 antibodies. Measurements of the free Ca2+ concentration in the lumen of the Golgi apparatus by specifically targeting the Ca2+-sensitive luminescent protein aequorin to this organelle revealed that endogenous SPCA1 was responsible for Ca2+ uptake in a subfraction of the Golgi apparatus. HeLa cells lacking SPCA1 could still set up baseline Ca2+ spiking when stimulated with histamine, indicating that the SPCA1-containing Ca2+ store was not absolutely needed to set up these oscillations. However, baseline Ca2+ oscillations occurred less frequently than in control cells, pointing to a contribution of SPCA1 in the shaping of the cytosolic Ca2+ signal in HeLa cells.
The contribution of the SPCA1 Ca2+ pump to the Ca2+ accumulation in the Golgi apparatus of HeLa cells assessed via RNA-mediated interference
RIZZUTO, Rosario;
2003
Abstract
The secretory-pathway Ca2+-ATPase SPCA1 is a thapsigargin-insensitive intracellular Ca2+ pump found mostly in the Golgi compartment. We have explored the contribution of this Ca2+ pump to cytosolic Ca2+ signaling in HeLa cells by using RNA-mediated interference to disrupt its expression. Removal of SPCA1 was confirmed by immunofluorescence with specific anti-SPCA1 antibodies. Measurements of the free Ca2+ concentration in the lumen of the Golgi apparatus by specifically targeting the Ca2+-sensitive luminescent protein aequorin to this organelle revealed that endogenous SPCA1 was responsible for Ca2+ uptake in a subfraction of the Golgi apparatus. HeLa cells lacking SPCA1 could still set up baseline Ca2+ spiking when stimulated with histamine, indicating that the SPCA1-containing Ca2+ store was not absolutely needed to set up these oscillations. However, baseline Ca2+ oscillations occurred less frequently than in control cells, pointing to a contribution of SPCA1 in the shaping of the cytosolic Ca2+ signal in HeLa cells.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
