Restricted Accumulation of Phosphatidylinositol 3-Kinase Products in a Plasmalemmal Subdomain during Fcγ Receptor-Mediated Phagocytosis

Abstract

Phagocytosis is a highly localized and rapid event, requiring the generation of spatially and temporally restricted signals. Because phosphatidylinositol 3-kinase (PI3K) plays an important role in the innate immune response, we studied the generation and distribution of 3′ phosphoinositides (3′PIs) in macrophages during the course of phagocytosis. The presence of 3′PI was monitored noninvasively in cells transfected with chimeras of green fluorescent protein and the pleckstrin homology domain of either Akt, Btk, or Gab1. Although virtually undetectable in unstimulated cells, 3′PI rapidly accumulated at sites of phagocytosis. This accumulation was sharply restricted to the phagosomal cup, with little 3′PI detectable in the immediately adjacent areas of the plasmalemma. Measurements of fluorescence recovery after photobleaching were made to estimate the mobility of lipids in the cytosolic monolayer of the phagosomal membrane. Stimulation of phagocytic receptors induced a marked reduction of lipid mobility that likely contributes to the restricted distribution of 3′PI at the cup. 3′PI accumulation during phagocytosis was transient, terminating shortly after sealing of the phagosomal vacuole. Two factors contribute to the rapid disappearance of 3′PI: the dissociation of the type I PI3K from the phagosomal membrane and the persistent accumulation of phosphoinositide phosphatases.

A Large T Cell Invagination with CD2 Enrichment Resets Receptor Engagement in the Immunological Synapse

“…Elevations in the intracellular calcium concentration play a central role in exocytosis in nonsecretory cells (49), including the insertion of cell membrane mass by exocytosis before the formation of phagosomes (50). PI3K and Cdc42 activity were required for T cell invaginations, similar to phagocytosis (38,39) and macropinocytosis (40,44). Finally, the interaction of Pak with DLC1 was required to resolve T cell invaginations, consistent with a role of this interaction in the regulation of macropinocytosis (19).…”

Lateral Sequestration of Phosphatidylinositol 4,5-Bisphosphate by the Basic Effector Domain of Myristoylated Alanine-rich C Kinase Substrate Is Due to Nonspecific Electrostatic Interactions

“…Thus both local synthesis and sequestration could contribute to the accumulation of PIP 2 , as discussed elsewhere (8). If only local synthesis were important, the concentration of PIP 2 should decrease gradually from the membrane ruffle or nascent phagosome to the bulk plasma membrane because of diffusion (110). If local electrostatic sequestration by MARCKS is also important, sharp demarcation lines between regions of high PIP 2 concentration and the bulk of the plasma membrane could exist.…”

“…In fact, such demarcations (i.e. steep gradients) for polyphosphoinositides are indeed observed at the borders of membrane ruffles 3 and nascent phagosomes (109,110). The interpretation of these experiments, however, is not straightforward because the probe used to detect the lateral organization of PIP 2 in the plasma membrane, the PH domain of PLC-␦ 1 , is itself negatively charged when bound to PIP 2 , and a green fluorescent protein construct of this PH domain, like PIP 2 , is also electrostatically sequestered by the basic residues in the effector domain of MARCKS.…”

Neonatal FcR Expression in Bone Marrow-Derived Cells Functions to Protect Serum IgG from Catabolism

“…The only image manipulations performed were occasional cropping and linear brightness/contrast adjustments that were applied evenly across images from control and experimental samples. Image quantitation was performed using ImageJ software (25) as described previously (26). Briefly, for colocalization analysis, structures were outlined using ImageJ and the mean fluorescence intensity of at least five marker-positive structures (m) was corrected and normalized to background (b) using the formula (m Ϫ b)/b.…”