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

In the paper

“As reported previously, F-actin accumulated transiently underneath the phagosomal cup, where it is thought to propel the extension of pseudopods (Greenberg et al. 1991).”

What the sources say

• Supported

  The Results (Fig. 3) describe F-actin around forming phagosomes at early time points that was barely visible by 30 minutes. The Discussion (around p. 765) describes F-actin accumulating at the bases of phagocytic cups and proposes that growth of the actin network pushes pseudopods outward. The citing sentence appropriately presents that mechanism as a hypothesis. (LLM, conf=0.92)

  cites Greenberg et al. 1991 source

In the paper

“To assess the relative abundance of these lipids, RAW macrophages were transfected with the GFP-tagged PH domains of either Akt (Fig. 1C and Fig. D) or Btk (not illustrated), which recognize specifically 3,4-PIP2 and 3,4,5-PIP3 (Kavran et al. 1998; Varnai et al. 1999).”

What the sources say

• Supported

  The Results report that the Akt/PKB PH domain recognizes PtdIns(3,4)P2 and PtdIns(3,4,5)P3. In “PH Domains Can Be Grouped According to Their Likely Physiological Ligand(s),” the paper places Btk’s PH domain among those that bind specifically to PtdIns(3,4,5)P3. The paper does not say Akt recognizes only PtdIns(3,4)P2; it recognizes both lipids. (LLM, conf=0.88)

  cites Kavran et al. 1998 source
• ?Not checked — no full text of the sourcecites Varnai et al. 1999 source

In the paper

“In addition to its role as a direct docking site for skeletal proteins, 4,5-PIP2 is also the primary substrate of phospholipase Cγ (PLCγ), which was shown to become activated upon engagement of FcγRs (Azzoni et al. 1992; Liao et al. 1992).”

What the sources say

• Partly supported

  The discussion following Fig. 4 (p. 1748) says FcγRIIIA stimulation induces PLCγ1 tyrosine phosphorylation and strongly suggests that PLCγ1 activation may account for the observed PIP2 hydrolysis. This supports the proposed link, but the paper does not establish PLCγ1 activation directly or identify PIP2 as its primary substrate; its experiments concern FcγRIIIA rather than FcγRs generally. (LLM, conf=0.86)

  cites Azzoni et al. 1992 sourceExperimental — accuracy not yet measured
• Partly supported

  The paper reports that cross-linking FcγRI or FcγRII in U937 cells leads to PLCγ1 phosphorylation and hydrolysis of phosphatidylinositol 4,5-bisphosphate (abstract, p. 3659). This supports receptor-linked PLCγ activity and identifies 4,5-PIP2 as a substrate, but the passage does not establish that it is PLCγ’s primary substrate. (LLM, conf=0.84)

  cites Liao et al. 1992 sourceExperimental — accuracy not yet measured

In the paper

“Clustering of FcγRs on the surface of opsonized particles is associated with the phosphorylation of tyrosine residues within a defined region known as the immunoreceptor tyrosine-based activation motif (Greenberg et al. 1993, Greenberg et al. 1994).”

What the sources say

• Partly supported

  Figure 5 (p. 532) reports that tyrosine phosphoproteins accumulate where ligand density is high on phagocytic particles, supporting an association between particle-bound ligand and tyrosine phosphorylation. The paper does not identify the phosphorylated residues as being within an immunoreceptor tyrosine-based activation motif; it describes the possible role of an Fc receptor-associated subunit in signaling as speculative. (LLM, conf=0.88)

  cites Greenberg et al. 1993 sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites Greenberg et al. 1994 source

In the paper

“These probes had been validated in other systems (Oancea et al. 1998; Stauffer et al. 1998; Varnai and Balla 1998) and their suitability was confirmed in RAW macrophages.”

What the sources say

• ?Not matched

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

  cites Oancea et al. 1998
• ?Not checked — no full text of the sourcecites Stauffer et al. 1998 source
• Supported

  The Results section, under “Plasma Membrane Localization of the PLCδ PH Domain Requires Interaction with PtdIns(4,5)P2,” reports that the PLCδ-PH–GFP probe localized to the plasma membrane in various cell types and that mutations disrupting its PtdIns(4,5)P2 binding prevented that localization. This supports the citation’s role in the multi-source statement that the probes had been validated in other systems; the RAW-macrophage confirmation is the citing authors’ own finding. (LLM, conf=0.91)

  cites Varnai and Balla 1998 source

In the paper

“PLCδ-PH-GFP can be used not only to monitor the distribution of 4,5-PIP2, but when expressed at sufficiently high levels, also to competitively inhibit other 4,5-PIP2–dependent processes.”

What the sources say

• Partly supported

  The Results section (‘PH Domains Can Inhibit the Agonist-induced Hydrolysis…’; page unavailable) reports that overexpressed PLCδ PH-domain–GFP greatly inhibited agonist-stimulated inositol phosphate formation. The Discussion proposes masking PtdIns(4,5)P2 or competing with PLC enzymes as possible mechanisms, but does not establish competitive inhibition or demonstrate inhibition of other PtdIns(4,5)P2-dependent processes beyond the PLC response tested. (LLM, conf=0.84)

  cites Varnai and Balla 1998 sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites Raucher et al. 2000 source

In the paper

“PLCγ is known to be recruited to the vicinity of activated receptors through interaction of its dual SH2 domains with phosphotyrosine residues on receptor–adaptor complexes (Rhee and Bae 1997).”

What the sources say

• Partly supported

  In “Activation of PLC-γ by Protein Tyrosine Kinases” (page unavailable), the paper says PLC-γ binds phosphorylated receptors or receptor components through its SH2 domains. “PLC Isoforms and Structural Organization” (page unavailable) confirms that PLC-γ has two SH2 domains. The copy does not establish binding to receptor–adaptor complexes or that both SH2 domains participate in a given interaction. (LLM, conf=0.82)

  cites Rhee and Bae 1997 sourceExperimental — accuracy not yet measured

In the paper

“While wortmannin-treated cells fail to internalize particles, they nevertheless display marked actin reassembly under the adherent RBCs (Araki et al. 1996; Cox et al. 1999; and our own observations, not shown).”

What the sources say

• Supported

  The Results (p. 1254) report that wortmannin inhibited internalization of erythrocytes, while treated macrophages extended actin-rich pseudopodia partway around them. The paper also reports more F-actin-rich phagocytic cups in treated cells than in controls, supporting the claim about actin beneath adherent RBCs. (LLM, conf=0.93)

  cites Araki et al. 1996 source
• Partly supported

  In “Actin Assembly in Response to Fc” (page unavailable), the paper reports that wortmannin did not visibly reduce F-actin accumulation beneath attached particles, including IgG-coated erythrocytes. It also reports inhibited phagocytosis, but not a complete failure to internalize particles: inhibition depended on particle size and was less effective for smaller beads. (LLM, conf=0.86)

  cites Cox et al. 1999 sourceExperimental — accuracy not yet measured

In the paper

“both PKCα and PKCδ copurify with isolated phagosomal membranes (Allen and Aderem 1995; Brumell et al. 1999) and some inhibitors of PKC activity have been reported to interfere with phagocytosis (Zheleznyak and Brown 1992; Karimi et al. 1999).”

What the sources say

• Partly supported

  The Discussion reports that PKCα associates with forming phagosomes and remains on maturing phagosomes. It does not report PKCδ or show that both isoforms copurify with isolated phagosomal membranes. The separate claim about PKC inhibitors is cited to other papers in the citing sentence, so it is not assessed against Allen and Aderem. (LLM, conf=0.85)

  cites Allen and Aderem 1995 sourceExperimental — accuracy not yet measured
• Partly supported

  The passage on phagosomal membranes reports that PKCδ accumulates on purified phagosomal membranes. It says conventional PKC isoforms have been found on phagosomes, but does not identify PKCα or establish its copurification. The inhibitor clause is attributed to other citations, not Brumell et al. (1999). (LLM, conf=0.87)

  cites Brumell et al. 1999 sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites Zheleznyak and Brown 1992 source

In the paper

“The average ratio in this case was 1.25 ± 0.08 (n = 35, Fig. 4 E), which is significantly different from unity (t test, P ≤ 0.005).”

In the paper

“PM-GFP was preferentially targeted to the plasma membrane, although internal structures were also decorated (Fig. 1 E).”

In the paper

“In this system, fluorescence was seen to surround the phagosome throughout the sealing process and to remain therein long after (Fig. 3D and Fig. E, open and filled circles, respectively).”

In the paper

“In this case, the density of phagosomal and plasmalemmal PM-GFP was virtually identical, whereas PLCδ-PH-GFP was essentially undetectable in the phagosome (Fig. 4 E).”

In the paper

“Soon after addition of the opsonized particles, pseudopods labeled with PLCδ-PH-GFP extended from the macrophage surface and made contact with the RBCs (Fig. 2 A, open arrows).”

In the paper

“Importantly, C1δ-YFP labeling was usually strongest immediately after phagosome closure, at which time PLCδ-PH-CFP was completely absent from the phagosome (Fig. 6 D6).”

In the paper

“The initial accumulation of PLCδ-PH-GFP was transient and was followed immediately by its abrupt dissociation from formed phagosomes (Fig. 2, see also Fig. 6 D).”

In the paper

“In contrast, cells treated with U73122 were unable to extend pseudopods or form pedestals, even though RBCs appeared tightly attached to the surface of the macrophages (Fig. 9 B).”

In the paper

“In brief, PLCδ-PH-GFP localized almost exclusively to the plasmalemma of untreated cells, but redistributed to the cytosol when PLC was activated (Fig. 1A and Fig. B).”

In the paper

“In contrast, U73343 had no significant effect at up to 6 μM and had only a minor antagonistic effect at 12 μM (Fig. 8 D).”

In the paper

“The accumulation of the 4,5-PIP2 probe exceeded that of PM-GFP, implying that it is not simply due to folding of the membrane during pseudopod extension (Fig. 2 Fig. 3 Fig. 4).”

In the paper

“Treatment with 6 μM U73122 for 15 min precluded accumulation of C1δ-GFP at the sites of RBC attachment (Fig. 8A and Fig. B).”

In the paper

“It redistributed to the plasma membrane when endogenous PLC was activated with ionomycin, leading to production of DAG synthesis (Fig. 6A and Fig. B), as well as upon addition of phorbol esters (Fig. 6 C) or exogenous DAG (not shown).”

In the paper

“our own unpublished observations also failed to demonstrate an absolute requirement for PKC.”

In the paper

“We found that phagocytosis was markedly inhibited when 4,5-PIP2 was sequestered by addition of neomycin or by overexpression of PLCδ-PH domain. Similarly, enzymatic degradation of 4,5-PIP2 also resulted in diminution of the phagocytic index (Fig. 7).”

In the paper

“During this process phagocytes internalize opsonized particles into a vacuole by an actin-dependent mechanism (Kwiatkowska and Sobota 1999).”

What the sources say

• ?Not checked — no full text of the sourcecites Kwiatkowska and Sobota 1999 source

In the paper

“This phospholipid is thought to govern the distribution and function of a variety of cytoskeletal and signaling proteins that bear pleckstrin homology (PH) domains or lysine/arginine-rich hydrophobic motifs, which interact directly and specifically with the headgroup of 4,5-PIP2 (Bottomley et al. 1998; Hinchliffe et al. 1998; Martin 1998; Czech 2000).”

What the sources say

• ?Not checked — no full text of the sourcecites Bottomley et al. 1998 source
• ?Not checked — no full text of the sourcecites Hinchliffe et al. 1998 source
• ?Not checked — no full text of the sourcecites Martin 1998 source

In the paper

“After internalization, the phagosomal vacuole is converted into an effective microbicidal organelle by progressive fusion with endosomes and lysosomes (Beron et al. 1995).”

What the sources say

• ?Not checked — no full text of the sourcecites Beron et al. 1995 source

In the paper

“These properties place constraints on the effectiveness of conventional biochemical analysis, since in a population of cells, receptor clustering and particle internalization occur asynchronously and only in a subdomain of the plasmalemma.”

In the paper

“These include the Fcγ receptors (FcγRs), that bind specifically to the IgG found on antibody-coated particles (Daeron 1997).”

What the sources say

• ?Not checked — no full text of the sourcecites Daeron 1997 source

In the paper

“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.”

In the paper

“These observations suggest that DAG is locally synthesized by PLC-mediated hydrolysis of 4,5-PIP2 during phagosome formation and is subsequently converted in the sealed phagosome to other metabolites.”

In the paper

“We conclude that inhibition of PLC arrests phagocytosis at an early stage, and is required for actin remodeling during pseudopod formation.”

In the paper

“This behavior contrasted strikingly with that of PM-GFP, which remained associated with phagosomes for the duration of our analysis (Fig. 3), ruling out rapid membrane turnover as the process responsible for the loss of PLCδ-PH-GFP.”

In the paper

“The concomitant appearance of DAG argues in favor of a central role of PLC in the depletion of 4,5-PIP2 from phagosomes (Fig. 6 D).”

In the paper

“Instead, we attribute the concentration of PLCδ-PH-GFP in the phagocytic cups to increased local synthesis of 4,5-PIP2. In support of this conclusion, we noticed local recruitment of PIPKIα to phagocytic cups (Fig. 4).”

In the paper

“A similar mechanism is likely involved during phagocytosis since, when transfected into RAW cells, the dual SH2 domains of PLCγ1 fused to GFP (PLCγ-SH2-GFP) were often seen to concentrate around phagocytic cups (Fig. 5C and Fig. D).”

In the paper

“The inability of cells to form pseudopods and actin cups upon incubation with U73122 and ET-18-OCH3 is not the result of a detrimental effect of the drug on cellular viability. These effects were fully reversible. Moreover, in the presence of the drug, pinocytosis of FITC-dextran appeared to be unimpaired (data not shown).”

In the paper

“The latter results, at least in part, from activation of PLC, although a contribution of phospholipase D and phosphatidate phosphohydrolase is also possible (Kusner et al. 1999).”

What the sources say

• ?Not checked — no full text of the sourcecites Kusner et al. 1999 source

In the paper

“Accordingly, addition of exogenous DAG or its analogues augments phagocytosis (Karimi et al. 1999), an effect that has been attributed to PKC isoforms.”

What the sources say

• ?Not checked — no full text of the sourcecites Karimi et al. 1999 source

In the paper

“This phospholipid is a regulator of actin capping, nucleation of de novo F-actin, and anchoring, cross-linking, and severing proteins (Hinchliffe et al. 1998), and actin remodeling is essential to the phagocytic process (Kwiatkowska and Sobota 1999).”

What the sources say

• ?Not checked — no full text of the sourcecites Hinchliffe et al. 1998 source
• ?Not checked — no full text of the sourcecites Kwiatkowska and Sobota 1999 source

In the paper

“Thus we tentatively postulate that local accumulation of 4,5-PIP2 might promote the recruitment of cytoskeletal proteins, stimulating F-actin anchorage and remodeling and contributing to the formation of the phagocytic cup (Fig. 10).”