In vivo analysis of 3-phosphoinositide dynamics duringDictyosteliumphagocytosis and chemotaxis

Abstract

Phagocytosis and chemotaxis are receptor-mediated processes that require extensive rearrangements of the actin cytoskeleton, and are controlled by lipid second messengers such as phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] and phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]. We used a panel of pleckstrin homology (PH) domains with distinct binding specificities for PtdIns(3,4,5)P3 and PtdIns(3,4)P2 to study the spatiotemporal dynamics of these phosphoinositides in vivo. During phagocytosis and macropinocytosis PtdIns(3,4,5)P3 levels transiently increased at sites of engulfment, followed by a rapid PtdIns(3,4)P2 production round the phagosome/macropinosome upon its internalisation, suggesting that PtdIns(3,4,5)P3 is degraded to PtdIns(3,4)P2. PTEN null mutants, which are defective in phagocytosis, showed normal rates of PtdIns(3,4,5)P3degradation, but unexpectedly an accelerated PtdIns(3,4)P2 degradation. During chemotaxis to cAMP only PtdIns(3,4,5)P3 was formed in the plasma membrane, and no PtdIns(3,4)P2 was detectable, showing that all PtdIns(3,4,5)P3 was degraded by PTEN to PtdIns(4,5)P2. Furthermore, we showed that different PtdIns(3,4,5)P3 binding PH domains gave distinct spatial and temporal readouts of the same underlying PtdIns(3,4,5)P3 signal, enabling distinct biological responses to one signal.

Self-organizing actin waves as planar phagocytic cup structures

“…18 This probe displayed no difference in labeling of the inner and external areas, or the waves separating them, indicating that PI (3,4,5)P3 is the phosphoinositide that distinguishes these two areas in wave-forming cells. When the waves propagate in one or the other direction, the inner and external areas undergo reciprocal changes in size with negligible changes in the total area of the substrate-attached Figure 4.…”

“…10,23 Figure 1A The domain of CRAC, a cytosolic regulator of adenylyl cyclase in Dictyostelium, 17 binds with high specificity to PIP3 and PI(3,4) P2. 16,18 Thus, PHcrac-GFP, a fusion of GFP to the PH domain of CRAC, labels the membrane of forming phagosomes and disappears shortly after the phagosome seals. [18][19][20] Using PHcrac-GFP together with mRFP-LimE∆, a probe for filamentous actin, 21 we examined cells in the process of generating actin waves on a planar surface.…”

“…16,18 Thus, PHcrac-GFP, a fusion of GFP to the PH domain of CRAC, labels the membrane of forming phagosomes and disappears shortly after the phagosome seals. [18][19][20] Using PHcrac-GFP together with mRFP-LimE∆, a probe for filamentous actin, 21 we examined cells in the process of generating actin waves on a planar surface. We found that these waves separate two different areas of the cell surface: an inner area that bound PHcrac-GFP, corresponding to the interior of a phagocytic cup, and an external area that was not labeled by PHcrac-GFP, corresponding to the plasma membrane external to a phagocytic cup.…”

Structure, Dynamics, Lipid Binding, and Physiological Relevance of the Putative GTPase-binding Domain of Dictyostelium Formin C

“…It has been shown that the concentration of various phosphoinositides changes during phagosome formation and the following maturation (35,64,65). In mammalian cells, the relative abundance of PIP45 is slightly higher at the nascent phagosome, whereas the level of PIP345 increases at the early phagosome, presumably followed by a rise in PIP35 at the later stages (35).…”

Novel protein Callipygian defines the back of migrating cells

“…Many of these asymmetries are conserved in migratory cells, such as Dictyostelium discoideum, mammalian leukocytes, fibroblasts, and axonal growth cones (2)(3)(4)(5)(6). In addition, these signaling molecules display characteristic localization patterns that are important for the regulation of other cellular behaviors, such as cytokinesis and phagocytosis, as well as in nonmigratory cells, such as epithelia (7)(8)(9)(10)(11)(12)(13).…”