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

2001 ◽  
Vol 153 (7) ◽  
pp. 1369-1380 ◽  
Author(s):  
John G. Marshall ◽  
James W. Booth ◽  
Vuk Stambolic ◽  
Tak Mak ◽  
Tamas Balla ◽  
...  
Keyword(s):  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Innate Immune ◽  
Pleckstrin Homology Domain ◽  
Type I ◽  
Phosphatidylinositol 3 Kinase ◽  
Two Factors ◽  
Green Fluorescent ◽  
Phagosomal Membrane ◽  
Phosphatidylinositol 3

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.

scholarly journals Tyrosine 1101 of Tie2 Is the Major Site of Association of p85 and Is Required for Activation of Phosphatidylinositol 3-Kinase and Akt

1998 ◽  
Vol 18 (7) ◽  
pp. 4131-4140 ◽  
Author(s):  
Christopher D. Kontos ◽  
Thomas P. Stauffer ◽  
Wen-Pin Yang ◽  
John D. York ◽  
Liwen Huang ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Vascular Development ◽  
Pi3 Kinase ◽  
Pleckstrin Homology Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Kinase ◽  
Embryonic Vascular Development ◽  
Phosphatidylinositol 3

ABSTRACT Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance. However, the signaling pathways responsible for the function of Tie2 remain unknown. In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity. Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast. Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase. Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2. These results suggest that activation of PI3-kinase and Akt may in part account for Tie2’s role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival.

scholarly journals Insights into the Dynamic Properties of Keratin Intermediate Filaments in Living Epithelial Cells

2001 ◽  
Vol 153 (3) ◽  
pp. 503-516 ◽  
Author(s):  
Kyeong Han Yoon ◽  
Miri Yoon ◽  
Robert D. Moir ◽  
Satya Khuon ◽  
Frederick W. Flitney ◽  
...  
Keyword(s):  
Intermediate Filaments ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Dynamic Properties ◽  
Cell Periphery ◽  
Type I ◽  
Wide Range ◽  
Different Types ◽  
Green Fluorescent ◽  
Keratin Intermediate Filaments

The properties of keratin intermediate filaments (IFs) have been studied after transfection with green fluorescent protein (GFP)-tagged K18 and/or K8 (type I/II IF proteins). GFP-K8 and -K18 become incorporated into tonofibrils, which are comprised of bundles of keratin IFs. These tonofibrils exhibit a remarkably wide range of motile and dynamic activities. Fluorescence recovery after photobleaching (FRAP) analyses show that they recover their fluorescence slowly with a recovery t1/2 of ∼100 min. The movements of bleach zones during recovery show that closely spaced tonofibrils (<1 μm apart) often move at different rates and in different directions. Individual tonofibrils frequently change their shapes, and in some cases these changes appear as propagated waveforms along their long axes. In addition, short fibrils, termed keratin squiggles, are seen at the cell periphery where they move mainly towards the cell center. The motile properties of keratin IFs are also compared with those of type III IFs (vimentin) in PtK2 cells. Intriguingly, the dynamic properties of keratin tonofibrils and squiggles are dramatically different from those of vimentin fibrils and squiggles within the same cytoplasmic regions. This suggests that there are different factors regulating the dynamic properties of different types of IFs within the same cytoplasmic regions.

scholarly journals Intracellular Movement of Green Fluorescent Protein–Tagged Phosphatidylinositol 3-Kinase in Response to Growth Factor Receptor Signaling

1999 ◽  
Vol 146 (4) ◽  
pp. 869-880 ◽  
Author(s):  
Helen Gillham ◽  
Matthew C.H.M. Golding ◽  
Rainer Pepperkok ◽  
William J. Gullick
Keyword(s):  
Green Fluorescent Protein ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Surface ◽  
Fluorescent Protein ◽  
Growth Factor Receptor ◽  
Phosphatidylinositol 3 Kinase ◽  
Epidermal Growth ◽  
Green Fluorescent ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase which has been implicated in mitogenesis, protein trafficking, inhibition of apoptosis, and integrin and actin functions. Here we show using a green fluorescent protein–tagged p85 subunit that phosphatidylinositol 3-kinase is distributed throughout the cytoplasm and is localized to focal adhesion complexes in resting NIH-3T3, A431, and MCF-7 cells. Ligand stimulation of an epidermal growth factor receptor/c-erbB-3 chimera expressed in these cells results in a redistribution of p85 to the cell membrane which is independent of the catalytic activity of the enzyme and the integrity of the actin cytoskeleton. The movement is, however, dependent on the phosphorylation status of the erbB-3 chimera. Using rhodamine-labeled epidermal growth factor we show that the phosphatidylinositol 3-kinase and the receptors colocalize in discrete patches on the cell surface. Low concentrations of ligand cause patching only at the periphery of the cells, whereas at high concentrations patches were seen over the whole cell surface. Using green fluorescent protein–tagged fragments of p85 we show that binding to the receptor requires the NH2-terminal part of the protein as well as its SH2 domains.

scholarly journals The Regularity of Sustained Firing Reveals Two Populations of Slowly Adapting Touch Receptors in Mouse Hairy Skin

2010 ◽  
Vol 103 (6) ◽  
pp. 3378-3388 ◽  
Author(s):  
Scott A. Wellnitz ◽  
Daine R. Lesniak ◽  
Gregory J. Gerling ◽  
Ellen A. Lumpkin
Keyword(s):  
Fluorescent Protein ◽  
Ex Vivo ◽  
Receptive Fields ◽  
Receptor Subtypes ◽  
Type I ◽  
Genetic Dissection ◽  
Merkel Cells ◽  
Simple Method ◽  
Green Fluorescent ◽  
Nerve Recordings

Touch is initiated by diverse somatosensory afferents that innervate the skin. The ability to manipulate and classify receptor subtypes is prerequisite for elucidating sensory mechanisms. Merkel cell–neurite complexes, which distinguish shapes and textures, are experimentally tractable mammalian touch receptors that mediate slowly adapting type I (SAI) responses. The assessment of SAI function in mutant mice has been hindered because previous studies did not distinguish SAI responses from slowly adapting type II (SAII) responses, which are thought to arise from different end organs, such as Ruffini endings. Thus we sought methods to discriminate these afferent types. We developed an epidermis-up ex vivo skin–nerve chamber to record action potentials from afferents while imaging Merkel cells in intact receptive fields. Using model-based cluster analysis, we found that two types of slowly adapting receptors were readily distinguished based on the regularity of touch-evoked firing patterns. We identified these clusters as SAI (coefficient of variation = 0.78 ± 0.09) and SAII responses (0.21 ± 0.09). The identity of SAI afferents was confirmed by recording from transgenic mice with green fluorescent protein–expressing Merkel cells. SAI receptive fields always contained fluorescent Merkel cells ( n = 10), whereas SAII receptive fields lacked these cells ( n = 5). Consistent with reports from other vertebrates, mouse SAI and SAII responses arise from afferents exhibiting similar conduction velocities, receptive field sizes, mechanical thresholds, and firing rates. These results demonstrate that mice, like other vertebrates, have two classes of slowly adapting light-touch receptors, identify a simple method to distinguish these populations, and extend the utility of skin–nerve recordings for genetic dissection of touch receptor mechanisms.

scholarly journals Vaccinia Virus F13L Protein with a Conserved Phospholipase Catalytic Motif Induces Colocalization of the B5R Envelope Glycoprotein in Post-Golgi Vesicles

2001 ◽  
Vol 75 (16) ◽  
pp. 7528-7542 ◽  
Author(s):  
Matloob Husain ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Recombinant Vaccinia Virus ◽  
Open Reading Frames ◽  
Type I ◽  
Golgi Vesicles ◽  
Infected Cells ◽  
Catalytic Motif ◽  
Green Fluorescent

ABSTRACT The wrapping of intracellular mature vaccinia virions by modifiedtrans-Golgi or endosomal cisternae to form intracellular enveloped virions is dependent on at least two viral proteins encoded by the B5R and F13L open reading frames. B5R is a type I integral membrane glycoprotein, whereas F13L is an unglycosylated, palmitylated protein with a motif that is conserved in a superfamily of phospholipid-metabolizing enzymes. Microscopic visualization of the F13L protein was achieved by fusing it to the enhanced green fluorescent protein (GFP). F13L-GFP was functional when expressed by a recombinant vaccinia virus in which it replaced the wild-type F13L gene or by transfection of uninfected cells with a plasmid vector followed by infection with an F13L deletion mutant. In uninfected or infected cells, F13L-GFP was associated with Golgi cisternae and post-Golgi vesicles containing the LAMP 2 late endosomal-lysosomal marker. Association of F13L-GFP with vesicles was dependent on an intact phospholipase catalytic motif and sites of palmitylation. The B5R protein was also associated with LAMP2-containing vesicles when F13L-GFP was coexpressed, but was largely restricted to Golgi cisternae in the absence of F13L-GFP or when the F13L moiety was mutated. We suggest that the F13L protein, like its human phospholipase D homolog, regulates vesicle formation and that this process is involved in intracellular enveloped virion membrane formation.

Interleukin-1 (IL-1) Inhibits Growth of Cytomegalovirus in Human Marrow Stromal Cells: Inhibition Is Reversed Upon Removal of IL-1

Blood ◽  
1999 ◽  
Vol 94 (2) ◽  
pp. 572-578 ◽  
Author(s):  
Mineo Iwata ◽  
Jeff Vieira ◽  
Michael Byrne ◽  
Heidi Horton ◽  
Beverly Torok-Storb
Keyword(s):  
Stromal Cells ◽  
Neutralizing Antibodies ◽  
Fluorescent Protein ◽  
Interleukin 1 ◽  
Elongation Factor ◽  
Marrow Stromal Cells ◽  
Type I ◽  
Untreated Culture ◽  
Green Fluorescent ◽  
Induced Changes

Abstract A Toledo strain cytomegalovirus (CMV) containing the gene for green fluorescent protein (GFP) under the control of elongation factor-1 promoter was used to study infection of human marrow stromal cells. Two stromal cell lines were used: HS-5, which secretes copious amounts of known cytokines and interleukins; and HS-27a, which does not secrete these activities. CMV growth and spread was monitored by counting green plaques and quantitating GFP intensity. Initial studies indicated that, whereas HS-5 and 27a have similar susceptibilities to infection, as evidenced by the same number of GFP+ cells at day 2, HS-5 appears more resistant to growth and spread of CMV. Furthermore, conditioned media from HS-5 (HS-5 CM) inhibited CMV plaque formation in HS-27a, suggesting that factors secreted by HS-5 are responsible for limiting CMV growth. Neutralizing antibodies against interleukin-1 (IL-1) and IL-1β completely blocked the ability of HS-5 CM to limit viral growth, suggesting that IL-1, which is known to be present in HS-5 CM, is responsible for this effect. When exogenous IL-1β was added to CMV-infected HS-27a, both the number of plaques and the intensity of GFP was significantly reduced in IL-1–treated HS-27a compared with untreated HS-27a (the number of plaques by day 18 was 20 ± 3 v 151 ± 12/well, respectively; GFP intensity was 535 ± 165 v 6,516 ± 652/well, respectively, in 4 separate experiments). At day 21, when IL-1β–treated, CMV-infected cultures were passaged and then cultured in the absence of IL-1β, CMV growth progressed with the kinetics of the original untreated culture, indicating that the IL-1β effect is reversible. Because HS-27a expresses the type I IL-1 receptor, we speculate that the antiviral effects are mediated through IL-1–induced changes in cellular gene expression. DNA chip analysis of mRNA from IL-1β–treated and nontreated HS-27a cells has identified some candidate molecules.

Structural studies of the phosphatidylinositol 3-kinase (PI3K) SH3 domain in complex with a peptide ligand: role of the anchor residue in ligand binding

2010 ◽  
Vol 391 (1) ◽  
Author(s):  
Renu Batra-Safferling ◽  
Joachim Granzin ◽  
Susanne Mödder ◽  
Silke Hoffmann ◽  
Dieter Willbold
Keyword(s):  
Crystal Structure ◽  
Ligand Binding ◽  
Protein Interactions ◽  
Intracellular Signaling ◽  
Sh3 Domain ◽  
Peptide Ligand ◽  
Type I ◽  
Phosphatidylinositol 3 Kinase ◽  
Anchor Residue ◽  
Phosphatidylinositol 3

Abstract Src homology 3 (SH3) domains are mediators of protein-protein interactions. They comprise approximately 60 amino acid residues and are found in many intracellular signaling proteins. Here, we present the crystal structure of the SH3 domain from phosphatidylinositol 3-kinase (PI3K) in complex with the 12-residue proline-rich peptide PD1R (HSKRPLPPLPSL). The crystal structure of the PI3K SH3-PD1R complex at a resolution of 1.7 Å reveals type I ligand orientation of the bound peptide with an extended conformation where the central portion forms a left-handed type II polyproline (PPII) helix. The overall structure of the SH3 domain shows minimal changes on ligand binding. In addition, we also attempted crystallization with another peptide ligand (PD1) where the residue at anchor position P-3 is a tyrosine. The crystals obtained did not contain the PD1 ligand; instead, the ligand binding site is partially occupied by residues Arg18 and Trp55 from the symmetry-related PI3K SH3 molecule. Considering these crystal structures of PI3K SH3 together with published reports, we provide a comparative analysis of protein-ligand interactions that has helped us identify the individual residues which play an important role in defining target specificity.

scholarly journals Inhibitory receptor FcγRIIb mediates the effects of IgG on a phagosome acidification and a sequential dephosphorylation system comprising SHIPs and Inpp4a

2017 ◽  
Vol 23 (4) ◽  
pp. 401-409 ◽  
Author(s):  
Tomohiro Segawa ◽  
Kaoru Hazeki ◽  
Kiyomi Nigorikawa ◽  
Atsuko Nukuda ◽  
Tomoki Tanizawa ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescence Probe ◽  
Pleckstrin Homology Domain ◽  
Raw 264.7 Macrophages ◽  
Lipid Phosphatases ◽  
Src Homology 2 ◽  
Lipid Phosphatase ◽  
Src Homology ◽  
Src Homology 2 Domain ◽  
Green Fluorescent

The relative abundance of phosphoinositide (PI) species on the phagosome membrane fluctuates over the course of phagocytosis. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 rapidly increase in the forming of the phagocytic cup, following which they disappear after sealing of the cup. In the present study, we monitored the clearance of these PI species using the enhanced green fluorescent protein-fused pleckstrin homology domain of Akt, a fluorescence probe that binds both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in Raw 264.7 macrophages. The clearance of PIs was much faster when the phagocytosed particles were coated with IgG. The effect of IgG was not observed in the macrophages deficient in FcγRIIb, an inhibitory IgG receptor. To identify the lipid phosphatases responsible for the FcγRIIb-accelerated PI clearance, we prepared a panel of lipid phosphatase-deficient cells. The lack of a PI 5-phosphatase Src homology 2 domain-containing inositol-5-phosphatase (SHIP)1 or SHIP2 impaired the FcγRIIb-accelerated clearance of PIs. The lack of a PI 4-phosphatase Inpp4a also impaired the accelerated PIs clearance. In the FcγRIIb- and Inpp4a-deficient cells, acidification of the formed phagosome was slowed. These results suggested that FcγRIIb drives the sequential dephosphorylation system comprising SHIPs and Inpp4a, and accelerates phagosome acidification.

scholarly journals Statin-induced Ras Activation Integrates the Phosphatidylinositol 3-Kinase Signal to Akt and MAPK for Bone Morphogenetic Protein-2 Expression in Osteoblast Differentiation

2006 ◽  
Vol 282 (7) ◽  
pp. 4983-4993 ◽  
Author(s):  
Nandini Ghosh-Choudhury ◽  
Chandi Charan Mandal ◽  
Goutam Ghosh Choudhury
Keyword(s):  
Bone Morphogenetic Protein ◽  
Osteoblast Differentiation ◽  
Dominant Negative ◽  
Bone Morphogenetic Protein 2 ◽  
Regulatory Subunit ◽  
Type I ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Morphogenetic Protein ◽  
Phosphatidylinositol 3

Lovastatin promotes osteoblast differentiation by increasing bone morphogenetic protein-2 (BMP-2) expression. We demonstrate that lovastatin stimulates tyrosine phosphorylation of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K), leading to an increase in its kinase activity in osteoblast cells. Inhibition of PI3K ameliorated expression of the osteogenic markers alkaline phosphatase, type I collagen, osteopontin, and BMP-2. Expression of dominant-negative PI3K and PTEN, an inhibitor of PI3K signaling, significantly attenuated lovastatin-induced transcription of BMP-2. Akt kinase was also activated in a PI3K-dependent manner. However, our data suggest involvement of an additional signaling pathway. Lovastatin-induced Erk1/2 activity contributed to BMP-2 transcription. Inhibition of PI3K abrogated Erk1/2 activity in response to lovastatin, indicating the presence of a signal relay between them. We provide, as a mechanism of this cross-talk, the first evidence that lovastatin stimulates rapid activation of Ras, which associates with and activates PI3K in the plasma membrane, which in turn regulates Akt and Erk1/2 to induce BMP-2 expression for osteoblast differentiation.

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