Localized Biphasic Changes in Phosphatidylinositol-4,5-Bisphosphate at Sites of Phagocytosis

The PH domain of PLCδ can also bind to phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate in vitro, though with comparatively low affinity.

“In vitro, the PH domain of PLCδ can also bind to phosphatidylinositol-3,4-bisphosphate (3,4-PIP2) and phosphatidylinositol-3,4,5-trisphosphate (3,4,5-PIP3), though with comparatively low affinity (Kavran et al. 1998).”

• cites Kavran et al. 1998 sourcePartly supported

  The section on relative binding affinities says PLCδ-PH does not select against other phosphoinositides as strongly as some PH domains, which broadly supports binding beyond its preferred ligand. But the Discussion reports that, in vesicles containing 33% phosphatidylserine, PLCδ-PH bound PtdIns(3,4,5)P3 slightly more strongly than its preferred PtdIns(4,5)P2. The copy does not establish comparatively low affinity for both lipids named in the claim. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

PI3K appears essential for phagocytosis because inhibitors such as wortmannin impair particle engulfment.

“This kinase appears to be essential for phagocytosis since inhibitors such as wortmannin impair particle engulfment (Ninomiya et al. 1994; Araki et al. 1996; Cox et al. 1999).”

• cites Ninomiya et al. 1994 sourceSupported
• cites Araki et al. 1996 sourceSupported
• cites Cox et al. 1999 sourcePartly supported

  The Results section, “Wortmannin and LY294002 Inhibit Fcγ-mediated Phagocytosis,” reports concentration-dependent inhibition of particle engulfment by both PI3K inhibitors. But the paper finds weaker inhibition for smaller beads and cautions against generalizing to other phagocytic targets. It therefore supports the inhibitor finding, but not an unqualified requirement for PI3K in phagocytosis generally. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

The PH domain of PLCδ binds to 4,5-PIP2 in vitro and in vivo with high affinity and specificity.

“The PH domain of PLCδ was shown to bind to 4,5-PIP2 in vitro and in vivo with high affinity and specificity (Lemmon et al. 1995).”

• cites Lemmon et al. 1995 sourcePartly supported

  The Abstract reports that the isolated PLCδ1 PH domain binds PtdIns(4,5)P2 with high affinity and specificity. It does not report binding demonstrated in vivo, so the cited paper supports the binding claim but not that setting. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

PLCδ-PH-GFP associated predominantly with the plasmalemma of RAW cells, with little binding to organellar membranes.

“As reported for other cell types (Stauffer et al. 1998; Varnai and Balla 1998), PLCδ-PH-GFP associated predominantly with the plasmalemma of RAW cells, with little binding to organellar membranes (Fig. 1 A).”

• cites Stauffer et al. 1998 sourceNot checked — no full text of the source
• cites Varnai and Balla 1998 sourceSupported

PLCδ-PH-GFP is a specific probe for 4,5-PIP2 in macrophages.

“PLCδ-PH-GFP Is a Specific Probe for 4,5-PIP2 in Macrophages”

• Also stated in: § PLCδ-PH-GFP Is a Specific Probe for 4,5-PIP2 in Macrophages · plain

  “Therefore, we used a fusion of this domain with GFP (PLCδ-PH-GFP) to analyze phospholipid metabolism in RAW 264.7 cells.”

• cites Lemmon et al. 1995 sourcePartly supported

  The Abstract reports that the isolated PLCδ1 PH domain binds PtdIns(4,5)P2 with high affinity and binding specificity. It does not evaluate a GFP-tagged probe or establish that it specifically detects PtdIns(4,5)P2 in macrophages; it also reports binding to Ins(1,4,5)P3. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Actin assembly during phagocytosis is mediated by Rho family GTPases.

“Experiments using clostridial toxins or transfection of dominant-negative constructs have convincingly demonstrated that actin assembly during phagocytosis is mediated by Rho family GTPases (Cox et al. 1997; Hackam et al. 1997; Caron and Hall 1998; Massol et al. 1998).”

• cites Cox et al. 1997 sourceSupported
• cites Hackam et al. 1997 sourcePartly supported

  The Results report that inactivating Rho eliminated F-actin cups during Fcγ receptor–mediated phagocytosis, supporting Rho's role in actin assembly. The Discussion confines the conclusions to transformed cell lines; this paper alone does not establish the claim for phagocytosis generally. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites Caron and Hall 1998 sourceNot checked — no full text of the source

Recruitment and activation of Syk is critical for FcγR-mediated phagocytosis, as shown by the inability of Syk−/− macrophages to internalize IgG-opsonized particles.

“Recruitment and activation of this tyrosine kinase is critical for FcγR-mediated phagocytosis, as demonstrated by the inability of macrophages from Syk−/− mice to internalize IgG-opsonized particles (Crowley et al. 1997; Kiefer et al. 1998).”

• cites Crowley et al. 1997 sourceSupported
• cites Kiefer et al. 1998 sourcePartly supported

  The Results report that Syk is important for FcγR-mediated uptake of IgG-opsonized red blood cells, but they do not show an inability to internalize them: 15% of Syk-deficient macrophages completed phagocytosis after 10 minutes. The Discussion likewise describes a significant reduction, not a complete loss. Page numbers were not available in our copy. (LLM, conf=0.90)

  Experimental — accuracy not yet measured

The increase in 3,4,5-PIP3 predicted to occur during phagocytosis did not affect the distribution of PLCδ-PH-GFP.

“The increase in 3,4,5-PIP3 predicted to occur during phagocytosis, based on the reported activation of PI3K (Araki et al. 1996; Cox et al. 1999), did not affect the distribution of PLCδ-PH-GFP.”

• cites Araki et al. 1996 sourceSupported
• cites Cox et al. 1999 sourceSupported

Consumption of 4,5-PIP2 by PI3K must contribute to the dissociation of PLCδ-PH-GFP from phagosomes.

“Hence, consumption of 4,5-PIP2 by PI3K must contribute to the dissociation of PLCδ-PH-GFP.”

• cites Araki et al. 1996 sourcePartly supported

  The Introduction (around p. 1250) says PI3K phosphorylates phosphoinositides to produce phosphatidylinositol (3,4,5)-trisphosphate, and the paper links PI3K activity to completion of phagocytosis. It does not examine PLCδ-PH-GFP or show that PI3K’s consumption of 4,5-PIP2 causes that reporter to dissociate from phagosomes; that causal conclusion goes beyond this source. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites Cox et al. 1999 sourcePartly supported

  The Results (page unavailable) report rapid PIP3 accumulation during Fcγ receptor-mediated phagocytosis, consistent with increased PI3K activity. The Experimental Procedures describe generating PIP3 from 4,5-PIP2 for a laboratory standard. Neither passage establishes that PI3K consumes phagosomal 4,5-PIP2 or causes PLCδ-PH-GFP to dissociate. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

During pseudopod extension, PLCδ-PH-GFP accumulated in phagocytic cups compared with the rest of the plasmalemma.

“During pseudopod extension PLCδ-PH-GFP appeared to accumulate in phagocytic cups compared with the rest of the plasmalemma (Fig. 2 A, see also Fig. 4).”

In the phagosomal membrane, PLCδ-PH-GFP had peak intensities ranging from 1.3 to 2.3 times that of the plasma membrane.

“In the phagosomal membrane PLCδ-PH-GFP had peak intensities ranging from 1.3 to 2.3 times that of the plasma membrane (Fig. 4A and Fig. B).”

Akt-PH-GFP and Btk fusion proteins were almost entirely in the cytosol before and after treatment with ionomycin in RAW macrophages.

“As depicted for Akt-PH-GFP, these fusion proteins were almost entirely in the cytosol before and after treatment with ionomycin (Fig. 1C and Fig. D), implying that 3′-phosphorylated inositides are not abundant in unstimulated macrophages and that their contribution to the distribution of PLCδ-PH-GFP is likely negligible.”

Phagocytic cups labeled with PM-GFP had peak intensities ranging from 0.9 to 1.6 times that of the plasma membrane.

“In contrast, phagocytic cups labeled with PM-GFP had peak intensities ranging from 0.9 to 1.6 times that of the plasma membrane (Fig. 4C and Fig. D).”

Fully formed phagosomes were devoid of PLCδ-PH-GFP when using 8-μm IgG-opsonized latex beads.

“As in the case of opsonized RBCs, the larger beads were effectively internalized by RAW cells and, as before, fully formed phagosomes were devoid of PLCδ-PH-GFP (Fig. 2D and Fig. E, filled circle).”

The dissociation of actin paralleled the virtual loss of PLCδ-PH-GFP from the phagosomes.

“The dissociation of actin paralleled the virtual loss of PLCδ-PH-GFP from the phagosomes (Fig. 2 F), signaling depletion of 4,5-PIP2.”

Sites of F-actin accumulation coincided with regions where PLCδ-PH-GFP fluorescence was most intense.

“As shown in Fig. 2F and Fig. G (open arrows), sites of F-actin accumulation coincided with those regions where PLCδ-PH-GFP fluorescence was most intense.”

Activation of endogenous PLC by elevation of cytosolic Ca2+ using ionomycin caused extensive translocation of PLCδ-PH-GFP to the cytoplasm.

“Activation of endogenous PLC by elevation of cytosolic Ca2+ using ionomycin caused extensive translocation of the chimera to the cytoplasm (Fig. 1 B), suggesting that PLCδ-PH-GFP is predominantly anchored to the plasma membrane through 4,5-PIP2.”

The distribution of PM-GFP was unaltered upon addition of ionomycin.

“Importantly, the distribution of PM-GFP was unaltered upon addition of ionomycin (Fig. 1 F), indicating that activation of PLC did not induce wholesale redistribution of the plasmalemmal lipids.”

The accumulation of PLCδ-PH-GFP in phagocytic cups was significantly greater than that of PM-GFP (P < 0.005).

“Regardless of the mechanism of accumulation of PM-GFP, a different process must underlie at least part of the accumulation of PLCδ-PH-GFP, which was significantly greater (P < 0.005).”

The biphasic behavior of PLCδ-PH-GFP during phagocytosis closely resembled that of F-actin.

“The biphasic behavior of PLCδ-PH-GFP during phagocytosis closely resembled that of F-actin, which was revealed by staining with rhodamine-phalloidin.”

PM-GFP remained bound with phagosomes for at least 20 min after formation.

“In fact, PM-GFP remained bound with phagosomes for at least 20 min after formation (not illustrated).”

A biphasic change in fluorescence intensity was observed in the area of the membrane involved in phagocytosis.

“A biphasic change in fluorescence intensity was observed in the area of the membrane involved in phagocytosis.”

The average ratio of PLCδ-PH-GFP phagosomal to plasma membrane intensity was 1.61 ± 0.08 (mean ± SE; n = 64).

“The average ratio was 1.61 ± 0.08 (mean ± SE; n = 64, Fig. 4 E).”

The association of PM-GFP with the phagosomal membrane was persistent.

“However, in contrast to PLCδ-PH-GFP, the association of PM-GFP with the phagosomal membrane was persistent (Fig. 3 A, solid arrows).”

Once the phagocytic vacuole sealed, PLCδ-PH-GFP was no longer associated with the phagosomal membrane.

“In sharp contrast, once the phagocytic vacuole sealed, PLCδ-PH-GFP was no longer associated with the phagosomal membrane (Fig. 2 A, solid arrows).”

Upon particle internalization, F-actin was rapidly lost from sealed phagosomes.

“Upon particle internalization, F-actin was rapidly lost from the sealed phagosomes (indicated by filled circles in Fig. 2 G).”

In intermediate stages of phagosome formation, phagosomal cups had a base devoid of PLCδ-PH-GFP while the fluorescent chimera was clearly detectable in the pseudopods.

“In addition, the larger size of the beads facilitated the detection of intermediate stages of phagosome formation and it was often possible to find phagosomal cups where the base of the phagosome was devoid of PLCδ-PH-GFP, whereas the fluorescent chimera was clearly detectable in the pseudopods (Fig. 2D and Fig. E, open circle).”

U73122 inhibited phagocytosis in a dose-dependent fashion, with half-maximal effects at approximately 3 μM.

“We found that U73122 inhibited phagocytosis in a dose-dependent fashion, with half-maximal effects at ≈3 μM (Fig. 8 D).”

At the early stage, PLCδ-PH-CFP was enriched in the extending pseudopods.

“At this stage, PLCδ-PH-CFP was enriched in the extending pseudopods (Fig. 6 D1, inset C).”

The association of C1δ-YFP with the phagosome declined gradually thereafter, becoming undetectable after several minutes.

“The association of C1δ-YFP with the phagosome declined gradually thereafter (Fig. 6 D7.5), becoming undetectable after several minutes.”

Paired experiments in which GFP alone was transfected showed no effect on phagocytosis.

“As before, paired experiments in which GFP alone was transfected showed no effect on phagocytosis.”

Actin remodeling under the cup was obliterated in cells treated with U73122, with virtually no F-actin accumulation despite normal adherence of RBCs.

“virtually no accumulation was noted in cells treated with the PLC blocker, despite normal adherence of RBCs to their surface (Fig. 9E and Fig. F).”

Treatment of RAW cells with TPA or DiC8 induced displacement of C1δ-GFP and C1δ-YFP to the plasmalemma.

“Likewise, treatment of the cells with TPA (Fig. 6 C) or DiC8 (not shown) induced displacement of C1δ-GFP and C1δ-YFP to the plasmalemma.”

Soluble unmodified GFP had no significant effect on phagocytosis.

“In contrast, the soluble, unmodified form of GFP had no significant effect on phagocytosis (Fig. 7 A).”

Expression of PM-5′-phosphatase-GFP reduced the phagocytic index of RAW macrophages by 42 ± 2% (n = 12, P < 0.0001).

“We found that expression of PM-5′-phosphatase-GFP reduced the phagocytic index of RAW macrophages by 42 ± 2% (n = 12, P < 0.0001; Fig. 7 A).”

PIPKIα dissociated from the phagosomal membrane shortly after completion of phagocytosis.

“The kinase dissociated from the phagosomal membrane shortly after completion of phagocytosis (Fig. 4F and Fig. G, solid arrow).”

In unstimulated RAW macrophages, PLCγ2 was mostly cytosolic with slight accumulation in ruffling membranes.

“In unstimulated RAW macrophages, PLCγ2 was mostly cytosolic with slight accumulation in ruffling membranes (not illustrated).”

Expression of PLCδ-PH-GFP in RAW macrophages reduced the phagocytic index moderately (30 ± 2%; n = 12; P < 0.0001).

“In cells expressing PLCδ-PH-GFP the phagocytic index was reduced moderately (30 ± 2%; n = 12), yet significantly (P < 0.0001; Fig. 7 A).”

Macrophages transfected with PLCγ-SH2-GFP or PLCz had a markedly impaired ability to internalize opsonized RBCs, with phagocytic index dropping by more than 50% (n = 12; P < 0.0001).

“As illustrated in Fig. 8 F, macrophages transfected with PLCγ-SH2-GFP or PLCz had a markedly impaired ability to internalize opsonized RBCs. In both instances the phagocytic index dropped by >50% (n = 12; P < 0.0001).”

ET-18-OCH3 reduced phagocytosis in a dose-dependent fashion, with half-maximal inhibition at approximately 25 μM.

“Importantly, ET-18-OCH3, a structurally and mechanistically unrelated antagonist of PLC, also reduced phagocytosis in a dose-dependent fashion, with half-maximal inhibition at ≈25 μM (Fig. 8 E).”

PLCδ-PH-CFP fluorescence diminished progressively in the inner bilayer of the pseudopods.

“In comparison, PLCδ-PH-CFP fluorescence diminished progressively in the inner bilayer of the pseudopods, i.e., the one more closely apposed to the particle and later becomes the phagosomal membrane (Fig. 6 D3.5, inset C).”

Translocation of the C1δ chimeras to the plasma membrane persisted at 15°C.

“Translocation of the C1δ chimeras to the plasma membrane persisted at 15°C (Fig. 6 C).”

Shortly after addition of opsonized particles, PLCδ-PH-GFP accumulated on the membrane of the developing phagosome.

“Shortly after addition of the opsonized particles, PLCδ-PH-GFP was found to accumulate on the membrane of the developing phagosome.”

Upon activation of endogenous PLC by calcium-ionomycin, a sizable fraction of C1δ-GFP translocated to the plasma membrane.

“Upon activation of endogenous PLC by calcium-ionomycin, a sizable fraction of C1δ-GFP translocated to the plasma membrane (Fig. 6 B).”

Overnight treatment with 5 mM neomycin reduced the phagocytic index by 30 ± 8% (n = 15, P < 0.001).

“Overnight treatment with 5 and 10 mM neomycin reduced the phagocytic index by 30 ± 8 and 45 ± 5%, respectively (n = 15, P < 0.001; Fig. 7 A).”

Overnight treatment with 10 mM neomycin reduced the phagocytic index by 45 ± 5% (n = 15, P < 0.001).

“Overnight treatment with 5 and 10 mM neomycin reduced the phagocytic index by 30 ± 8 and 45 ± 5%, respectively (n = 15, P < 0.001; Fig. 7 A).”

PIPKIα concentrates in phagocytic cups.

“PIPKIα Concentrates in Phagocytic Cups”

• Also stated in: § PIPKIα Concentrates in Phagocytic Cups · boosted

  “Strikingly, upon addition of opsonized and fluorescently labeled particles, PIPKIα migrated to the nascent phagosome (Fig. 4F and Fig. G, open arrow).”

Interpolation of cell-associated fluorescence against calibration curves of recombinant EGFP yields intracellular concentrations of approximately 25 μM in cells expressing the probe.

“interpolation of the cell-associated fluorescence in such calibration curves yielded intracellular concentrations of ≈25 μM.”

PLCγ2 accumulated intensely at sites of phagocytosis.

“Significantly, we found that PLCγ2 accumulated intensely at sites of phagocytosis (Fig. 5A and Fig. B).”

In otherwise untreated RAW cells, C1δ-GFP was exclusively associated with endomembranes, concentrating in a juxtanuclear complex.

“In otherwise untreated cells, C1δ-GFP was exclusively associated with endomembranes, concentrating in a juxtanuclear complex (Fig. 6 A).”

Similar inhibition of pseudopod and actin cup formation was obtained by treating cells with 80 μM ET-18-OCH3 in the presence of 10% serum.

“Similar results were obtained by treating cells with 80 μM ET-18-OCH3 in the presence of 10% serum (not depicted).”

The abundance of 3′-phosphorylated inositides in resting cells was negligible compared with 4,5-PIP2.

“the abundance of the latter in resting cells was found to be negligible compared with 4,5-PIP2 (Fig. 1 C).”

The dissociation of PLCδ-PH-CFP from the inner bilayer was accompanied by recruitment of C1δ-YFP.

“The dissociation of PLCδ-PH-CFP from the inner bilayer was accompanied by recruitment of C1δ-YFP (Fig. 6 D3.5, inset Y).”

C1δ-YFP was only weakly associated with the early phagocytic cup.

“C1δ-YFP was only weakly associated with the early phagocytic cup (Fig. 6 D1, inset Y).”

Inactivation of PLC with pharmacological agents and dominant-inhibitory forms of PLCγ effectively impaired phagocytosis.

“We found that inactivation of PLC with pharmacological agents and using dominant-inhibitory forms of PLCγ effectively impaired phagocytosis.”

We detected an exquisitely localized biphasic change in the concentration of 4,5-PIP2 in the region of the plasmalemma that evolves to become the phagosome.

“In summary, we detected an exquisitely localized biphasic change in the concentration of 4,5-PIP2 in the region of the plasmalemma that evolves to become the phagosome.”

Our results reveal the induction of localized and transient changes in 4,5-PIP2 metabolism in the nascent phagosome.

“In conclusion, our results reveal the induction of localized and transient changes in 4,5-PIP2 metabolism in the nascent phagosome.”

This biphasic change is accompanied by an equally localized, yet somewhat delayed and longer lasting accumulation of DAG.

“This is accompanied by an equally localized, yet somewhat delayed and longer lasting accumulation of DAG.”

Activation of PLC did not induce wholesale redistribution of the plasmalemmal lipids.

“Importantly, the distribution of PM-GFP was unaltered upon addition of ionomycin (Fig. 1 F), indicating that activation of PLC did not induce wholesale redistribution of the plasmalemmal lipids.”

A different process must underlie at least part of the accumulation of PLCδ-PH-GFP.

“Regardless of the mechanism of accumulation of PM-GFP, a different process must underlie at least part of the accumulation of PLCδ-PH-GFP, which was significantly greater (P < 0.005).”

Chimeric constructs of GFP with defined lipid-binding domains of proteins can monitor the distribution of phospholipids and their metabolites in transfected cells.

“Recent and elegant reports have introduced the use of chimeric constructs of GFP with defined lipid-binding domains of proteins to monitor the distribution of phospholipids and their metabolites in transfected cells (Oancea and Meyer 1998; Oancea et al. 1998; Stauffer et al. 1998; Varnai et al. 1999; Czech 2000).”

• cites Oancea and Meyer 1998 sourceNot checked — no full text of the source
• cites Oancea et al. 1998Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Stauffer et al. 1998 sourceNot checked — no full text of the source

The loss of PLCδ-PH-GFP is due to local changes in the availability of 4,5-PIP2.

“Rather, the loss of PLCδ-PH-GFP is occurring due to local changes in the availability of 4,5-PIP2.”

Rapid membrane turnover is not a likely explanation for the loss of PLCδ-PH-GFP from nascent phagosomes.

“These data indicate that rapid membrane turnover is not a likely explanation for the loss of PLCδ-PH-GFP from nascent phagosomes.”

PLCδ-PH-GFP is predominantly anchored to the plasma membrane through 4,5-PIP2.

“Activation of endogenous PLC by elevation of cytosolic Ca2+ using ionomycin caused extensive translocation of the chimera to the cytoplasm (Fig. 1 B), suggesting that PLCδ-PH-GFP is predominantly anchored to the plasma membrane through 4,5-PIP2.”

The onset of 4,5-PIP2 conversion preceded phagosomal sealing.

“This suggests that the onset of 4,5-PIP2 conversion preceded phagosomal sealing.”

The observations are consistent with a role of 4,5-PIP2 in controlling actin remodeling during phagocytosis.

“These observations are consistent with a role of 4,5-PIP2 in controlling actin remodeling during phagocytosis.”

The C1δ domain reaches the plasmalemma by diffusion across the cytosol, after net dissociation from endomembrane binding sites.

“This is consistent with the notion that the C1δ domain reaches the plasmalemma by diffusion across the cytosol, after net dissociation from endomembrane binding sites.”

• AssumptionRests on an assumption: At 15°C, vesicular traffic essentially ceases in mammalian cells.

The localized activation of 4,5-PIP2 metabolism in the forming phagosome suggests a functional role for this phosphoinositide in phagocytosis.

“The localized activation of 4,5-PIP2 metabolism in the forming phagosome suggests a functional role for this phosphoinositide in phagocytosis.”

Degradation of 4,5-PIP2 contributes to the subsequent cytoskeletal remodeling.

“The temporal sequence of phosphoinositide metabolism suggests that accumulation of 4,5-PIP2 is involved in the initial recruitment of actin to the phagocytic cup, while its degradation contributes to the subsequent cytoskeletal remodeling.”

Translocation of the C1δ fusion proteins during PLC activation occurs in response to the release of endogenous DAG and not to the concomitant increases in IP3 and calcium concentration.

“This implies that translocation of the C1δ fusion proteins during PLC activation occurs in response to the release of endogenous DAG and not to the concomitant increases in IP3 and calcium concentration.”

Unhindered availability of 4,5-PIP2 is required for optimal phagocytosis.

“Jointly, these observations suggest that unhindered availability of 4,5-PIP2 is required for optimal phagocytosis.”

C1δ-GFP is appropriate for detection of DAG.

“C1δ-GFP seems similarly appropriate for the detection of DAG.”

Bulk exchange of membrane during maturation is unlikely to account for the disappearance of PLCδ-PH-GFP from the sealed phagosome.

“This quantitation confirms that bulk exchange of membrane during maturation is unlikely to account for the disappearance of PLCδ-PH-GFP from the sealed phagosome.”

Accumulation of 4,5-PIP2 is involved in the initial recruitment of actin to the phagocytic cup.

“The temporal sequence of phosphoinositide metabolism suggests that accumulation of 4,5-PIP2 is involved in the initial recruitment of actin to the phagocytic cup, while its degradation contributes to the subsequent cytoskeletal remodeling.”

The presence of 4,5-PIP2 and active PLCγ at the phagosome is essential for effective particle ingestion.

“The presence of 4,5-PIP2 and active PLCγ at the phagosome was shown to be essential for effective particle ingestion.”

• AssumptionRests on an assumption: Neomycin sulfate can compete with endogenous ligands for binding to 4,5-PIP2.

The reduction in phagocytic index by PM-5′-phosphatase-GFP may not be entirely attributable to phosphatase activity, because transfection with PM-GFP alone produced a modest, reproducible inhibition of phagocytosis.

“This effect may not be entirely attributable to the phosphatase activity of the construct, since transfection with PM-GFP alone produced a modest, yet reproducible inhibition of phagocytosis (Fig. 7 A).”

Effective scavenging of 4,5-PIP2 by PLCδ-PH-GFP would only be expected to occur if the concentration of PLCδ-PH-GFP can approximate or exceed that of its phosphoinositide ligand.

“Effective scavenging of 4,5-PIP2 by PLCδ-PH-GFP would only be expected to occur if the concentration of the latter can approximate or exceed that of its phosphoinositide ligand.”

The transient association of PIPKIα with the nascent phagosome is consistent with the notion that 4,5-PIP2 is locally accumulated during FcγR-mediated phagocytosis.

“The transient association of PIPKIα with the nascent phagosome is consistent with the notion that 4,5-PIP2 is locally accumulated during FcγR-mediated phagocytosis.”

The yeast 4,5-PIP2-specific 5′-phosphatase INP54p reduces the content of 4,5-PIP2 in COS cells.

“This yeast phosphatase has been shown to reduce the content of 4,5-PIP2 in COS cells (Raucher et al. 2000).”

• cites Raucher et al. 2000 sourceNot checked — no full text of the source

PLCδ-PH-GFP appears to be exquisitely selective for 4,5-PIP2 in RAW cells.

“Therefore, PLCδ-PH-GFP appears to be exquisitely selective for 4,5-PIP2 in RAW cells.”

U73122 and ET-18-OCH3 are structurally different PLC-specific antagonists.

“we used U73122 and ET-18-OCH3, two structurally different PLC-specific antagonists (Chen et al. 1996b).”

• cites Chen et al. 1996bNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

A concentration of approximately 25 μM PLCδ-PH-GFP should suffice to exert a significant scavenging effect on 4,5-PIP2, which is estimated to exist in the micromolar range.

“This concentration should suffice to exert a significant scavenging effect on 4,5-PIP2, which can be estimated to exist in the micromolar range, based on existing determinations of phospholipid content in several cell types.”

• AssumptionRests on an assumption: 4,5-PIP2 exists in the micromolar range in cells.

PLCγ is an important regulator of FcγR-mediated phagocytosis.

“Therefore, these findings suggest that PLCγ is an important regulator of FcγR-mediated phagocytosis.”

The rapid recruitment of PIPKIα to the nascent phagosome suggests that this enzyme contributes to the early phase of 4,5-PIP2 accumulation.

“The rapid recruitment of PIPKIα to the nascent phagosome suggests that this enzyme contributes to the early phase of 4,5-PIP2 accumulation.”

Our findings are consistent with the notion that 4,5-PIP2 can act as a second messenger in signal transduction.

“Together, our findings are consistent with the growing notion that 4,5-PIP2 can act as a second messenger in signal transduction.”

DAG is more likely than IP3 to play a critical role in transducing the phagocytic response.

“While the involvement of IP3 in phagocytosis is open to question, DAG is more likely to play a critical role in transducing the response.”

Net changes in the focal concentration of the phosphoinositide contribute to remodeling of F-actin in the phagosomal cup.

“These observations suggest that net changes in the focal concentration of the phosphoinositide contribute to remodeling of F-actin in the phagosomal cup (Fig. 10).”

One or more products of 4,5-PIP2 metabolism, including DAG and 3,4,5-PIP3, are likely to play essential roles in pseudopod extension and phagosome closure.

“In addition, one or more products of its metabolism, including DAG and 3,4,5-PIP3, are likely to play essential roles in pseudopod extension and phagosome closure.”