Transfection of bone marrow-derived neutrophils
BM-PMNs were transfected by nucleofection using the Amaxa TM P3 Primary Cell 4DNucleofector
TM X kit L and a 4D-Nucleofector™ System (Lonza AG). Briefly, 2.5x106 cells
were resuspended in 100 ?l of nucleofection solution P3, transferred to a nucleofection
cuvette, and after addition of 2-5 ?g of the indicated expression vector, the cells were
immediately pulsed using the human monocytes setup in the 4D-Nucleofector™ System. After
addition of 300 ?l of RPMI medium (no serum), the cells were transferred to an 8-well Lab-Tek
chambered #1.0 borosilicate coverglass system or to 1.5 ml polypropylene tubes containing
RPMI medium. The cells were recovered and used in fluorescence or functional assays 2
hours after transfection. Transfection efficiency was ?20% and transfected cells not preactivated
by the nucleofection procedure and were perfectly functional (Fig. 7A).
Cytoskeleton remodeling is important for the regulation of vesicular transport associated with exocytosis but a direct association between granular secretory proteins and actin remodeling molecules has not been shown and this mechanism remains obscure. Using a proteomic approach, we identified the RhoA-GAP GMIP as a factor that associates with the Rab27aeffector JFC1, and modulates vesicular transport and exocytosis. GMIP downregulation induced RhoA activation and actin polymerization. Importantly, GMIP-downregulated cells showed impaired vesicular transport and exocytosis while inhibition of the RhoA-signaling pathway induced actin depolymerization and facilitated exocytosis. We show that RhoA activity polarizes around JFC1-containing secretory granules suggesting that it may control directionality of granule movement. Using quantitative live-cell microscopy, we show that JFC1-containing secretory organelles move in areas near the plasma membrane deprived of polymerized actin and that dynamic vesicles maintain an actin-free environment in their surroundings. Supporting a role for JFC1 in RhoA inactivation and actin remodeling during exocytosis, JFC1-knockout neutrophils showed increased RhoA activity and azurophilic granules were unable to traverse cortical actin in cells lacking JFC1. We propose that during exocytosis, actin-depolymerization commences near the secretory organelle not the plasma membrane and that secretory granules use a JFC1- and GMIP-dependent molecular mechanism to traverse cortical actin.