scholarly journals Type I phosphatidylinositol 4-phosphate 5-kinase controls neutrophil polarity and directional movement

2007 ◽  
Vol 179 (7) ◽  
pp. 1539-1553 ◽  
Author(s):  
Rosa Ana Lacalle ◽  
Rosa M. Peregil ◽  
Juan Pablo Albar ◽  
Ernesto Merino ◽  
Carlos Martínez-A ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Cell Movement ◽  
Leading Edge ◽  
Cell Polarization ◽  
Type I ◽  
Lipid Kinase ◽  
Erm Proteins ◽  
Chemical Gradients ◽  
C Terminus ◽  
Phosphatidylinositol 4

Directional cell movement in response to external chemical gradients requires establishment of front–rear asymmetry, which distinguishes an up-gradient protrusive leading edge, where Rac-induced F-actin polymerization takes place, and a down-gradient retractile tail (uropod in leukocytes), where RhoA-mediated actomyosin contraction occurs. The signals that govern this spatial and functional asymmetry are not entirely understood. We show that the human type I phosphatidylinositol 4-phosphate 5-kinase isoform β (PIPKIβ) has a role in organizing signaling at the cell rear. We found that PIPKIβ polarized at the uropod of neutrophil-differentiated HL60 cells. PIPKIβ localization was independent of its lipid kinase activity, but required the 83 C-terminal amino acids, which are not homologous to other PIPKI isoforms. The PIPKIβ C terminus interacted with EBP50 (4.1-ezrin-radixin-moesin (ERM)-binding phosphoprotein 50), which enabled further interactions with ERM proteins and the Rho-GDP dissociation inhibitor (RhoGDI). Knockdown of PIPKIβ with siRNA inhibited cell polarization and impaired cell directionality during dHL60 chemotaxis, suggesting a role for PIPKIβ in these processes.

scholarly journals Characterization of a Rac1- and RhoGDI-Associated Lipid Kinase Signaling Complex

1998 ◽  
Vol 18 (2) ◽  
pp. 762-770 ◽  
Author(s):  
Kimberley F. Tolias ◽  
Anthony D. Couvillon ◽  
Lewis C. Cantley ◽  
Christopher L. Carpenter
Keyword(s):  
Nadph Oxidase ◽  
Rho Gtpases ◽  
Actin Polymerization ◽  
Cellular Proliferation ◽  
Type I ◽  
Lipid Kinase ◽  
C Terminus ◽  
Lipid Kinases

ABSTRACT Rho family GTPases regulate a number of cellular processes, including actin cytoskeletal organization, cellular proliferation, and NADPH oxidase activation. The mechanisms by which these G proteins mediate their effects are unclear, although a number of downstream targets have been identified. The interaction of most of these target proteins with Rho GTPases is GTP dependent and requires the effector domain. The activation of the NADPH oxidase also depends on the C terminus of Rac, but no effector molecules that bind to this region have yet been identified. We previously showed that Rac interacts with a type I phosphatidylinositol-4-phosphate (PtdInsP) 5-kinase, independent of GTP. Here we report the identification of a diacylglycerol kinase (DGK) which also associates with both GTP- and GDP-bound Rac1. In vitro binding analysis using chimeric proteins, peptides, and a truncation mutant demonstrated that the C terminus of Rac is necessary and sufficient for binding to both lipid kinases. The Rac-associated PtdInsP 5-kinase and DGK copurify by liquid chromatography, suggesting that they bind as a complex to Rac. RhoGDI also associates with this lipid kinase complex both in vivo and in vitro, primarily via its interaction with Rac. The interaction between Rac and the lipid kinases was enhanced by specific phospholipids, indicating a possible mechanism of regulation in vivo. Given that the products of the PtdInsP 5-kinase and the DGK have been implicated in several Rac-regulated processes, and they bind to the Rac C terminus, these lipid kinases may play important roles in Rac activation of the NADPH oxidase, actin polymerization, and other signaling pathways.

scholarly journals Interaction of the Endocytic Scaffold Protein Pan1 with the Type I Myosins Contributes to the Late Stages of Endocytosis

2007 ◽  
Vol 18 (8) ◽  
pp. 2893-2903 ◽  
Author(s):  
Sarah L. Barker ◽  
Linda Lee ◽  
B. Daniel Pierce ◽  
Lymarie Maldonado-Báez ◽  
David G. Drubin ◽  
...  
Keyword(s):  
Late Stage ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Temperature Sensitive ◽  
Type I ◽  
Reading Frame ◽  
Rich Domain ◽  
C Terminus

The yeast endocytic scaffold Pan1 contains an uncharacterized proline-rich domain (PRD) at its carboxy (C)-terminus. We report that the pan1-20 temperature-sensitive allele has a disrupted PRD due to a frame-shift mutation in the open reading frame of the domain. To reveal redundantly masked functions of the PRD, synthetic genetic array screens with a pan1ΔPRD strain found genetic interactions with alleles of ACT1, LAS17 and a deletion of SLA1. Through a yeast two-hybrid screen, the Src homology 3 domains of the type I myosins, Myo3 and Myo5, were identified as binding partners for the C-terminus of Pan1. In vitro and in vivo assays validated this interaction. The relative timing of recruitment of Pan1-green fluorescent protein (GFP) and Myo3/5-red fluorescent protein (RFP) at nascent endocytic sites was revealed by two-color real-time fluorescence microscopy; the type I myosins join Pan1 at cortical patches at a late stage of internalization, preceding the inward movement of Pan1 and its disassembly. In cells lacking the Pan1 PRD, we observed an increased lifetime of Myo5-GFP at the cortex. Finally, Pan1 PRD enhanced the actin polymerization activity of Myo5–Vrp1 complexes in vitro. We propose that Pan1 and the type I myosins interactions promote an actin activity important at a late stage in endocytic internalization.

scholarly journals Phosphatidylinositol 4-kinase III beta regulates cell shape, migration, and focal adhesion number

2020 ◽  
Vol 31 (17) ◽  
pp. 1904-1916
Author(s):  
Patricia Bilodeau ◽  
Daniel Jacobsen ◽  
Denise Law-Vinh ◽  
Jonathan M. Lee
Keyword(s):  
Cell Motility ◽  
Focal Adhesion ◽  
Cell Shape ◽  
Leading Edge ◽  
Lipid Kinase ◽  
Fibroblast Migration ◽  
Phosphatidylinositol 4

This work describes a role for the lipid phosphatidylinositol 4-phosphate (PI4P) and lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIβ) in cell motility, cell shape, and focal adhesion (FA) formation. During fibroblast migration, PI4P vesicles move to the leading edge and fuse with FA there. Deletion of PI4KIIIB impairs fibroblast migration, increases the number of FA, and alters cell shape.

scholarly journals Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement

2004 ◽  
Vol 167 (3) ◽  
pp. 505-518 ◽  
Author(s):  
Atsuo T. Sasaki ◽  
Cheryl Chun ◽  
Kosuke Takeda ◽  
Richard A. Firtel
Keyword(s):  
Actin Polymerization ◽  
Cell Movement ◽  
Leading Edge ◽  
Ras Signaling ◽  
Phosphatidylinositol 3 Kinase ◽  
Ras Activation ◽  
Directional Movement ◽  
Ras Effectors ◽  
Chemotaxis Receptors ◽  
Intermediate Event

During chemotaxis, receptors and heterotrimeric G-protein subunits are distributed and activated almost uniformly along the cell membrane, whereas PI(3,4,5)P3, the product of phosphatidylinositol 3-kinase (PI3K), accumulates locally at the leading edge. The key intermediate event that creates this strong PI(3,4,5)P3 asymmetry remains unclear. Here, we show that Ras is rapidly and transiently activated in response to chemoattractant stimulation and regulates PI3K activity. Ras activation occurs at the leading edge of chemotaxing cells, and this local activation is independent of the F-actin cytoskeleton, whereas PI3K localization is dependent on F-actin polymerization. Inhibition of Ras results in severe defects in directional movement, indicating that Ras is an upstream component of the cell's compass. These results support a mechanism by which localized Ras activation mediates leading edge formation through activation of basal PI3K present on the plasma membrane and other Ras effectors required for chemotaxis. A feedback loop, mediated through localized F-actin polymerization, recruits cytosolic PI3K to the leading edge to amplify the signal.

Myosin II-dependent cylindrical protrusions induced by quinine inDictyostelium: antagonizing effects of actin polymerization at the leading edge

2001 ◽  
Vol 114 (11) ◽  
pp. 2155-2165
Author(s):  
Kunito Yoshida ◽  
Kei Inouye
Keyword(s):  
Cell Membrane ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Cell Movement ◽  
Leading Edge ◽  
Contractile Vacuole ◽  
Cell Periphery ◽  
Slowing Down ◽  
On Line ◽  
Cylindrical Protrusion

We found that amoeboid cells of Dictyostelium are induced by a millimolar concentration of quinine to form a rapidly elongating, cylindrical protrusion, which often led to sustained locomotion of the cells. Formation of the protrusion was initiated by fusion of a contractile vacuole with the cell membrane. During protrusion extension, a patch of the contractile vacuole membrane stayed undiffused on the leading edge of the protrusion for over 30 seconds. Protrusion formation was not inhibited by high osmolarity of the external medium (at least up to 400 mosM). By contrast, mutant cells lacking myosin II (mhc− cells) failed to extend protrusions upon exposure to quinine. When GFP-myosin-expressing cells were exposed to quinine, GFP-myosin was accumulated in the cell periphery forming a layer under the cell membrane, but a newly formed protrusion was initially devoid of a GFP-myosin layer, which gradually formed and extended from the base of the protrusion. F-actin was absent in the leading front of the protrusion during the period of its rapid elongation, and the formation of a layer of F-actin in the front was closely correlated with its slowing-down or retraction. Periodical or continuous detachment of the F-actin layer from the apical membrane of the protrusion, accompanied by a transient increase in the elongation speed at the site of detachment, was observed in some of the protrusions. The detached F-actin layers, which formed a spiral layer of F-actin in the case of continuous detachment, moved in the opposite direction of protrusion elongation. In the presence of both cytochalasin A and quinine, the protrusions formed were not cylindrical but spherical, which swallowed up the entire cellular contents. The estimated bulk flux into the expanding spherical protrusions of such cells was four-times higher than the flux into the elongating cylindrical protrusions of the cells treated with quinine alone. These results indicate that the force responsible for the quinine-induced protrusion is mainly due to contraction of the cell body, which requires normal myosin II functions, while actin polymerization is important in restricting the direction of its expansion. We will discuss the possible significance of tail contraction in cell movement in the multicellular phase of Dictyostelium development, where cell locomotion similar to that induced by quinine is often observed without quinine treatment, and in protrusion elongation in general.Movies available on-line

scholarly journals Essential and unique roles of PIP5K-γ and -α in Fcγ receptor-mediated phagocytosis

2009 ◽  
Vol 184 (2) ◽  
pp. 281-296 ◽  
Author(s):  
Yuntao S. Mao ◽  
Masaki Yamaga ◽  
Xiaohui Zhu ◽  
Yongjie Wei ◽  
Hui-Qiao Sun ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Type I ◽  
Dependent Manner ◽  
Fcγ Receptor ◽  
Protein Activation ◽  
Phosphatidylinositol 4 ◽  
Syndrome Protein ◽  
Pharmacological Manipulations

The actin cytoskeleton is dynamically remodeled during Fcγ receptor (FcγR)-mediated phagocytosis in a phosphatidylinositol (4,5)-bisphosphate (PIP2)-dependent manner. We investigated the role of type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) γ and α isoforms, which synthesize PIP2, during phagocytosis. PIP5K-γ−/− bone marrow–derived macrophages (BMM) have a highly polymerized actin cytoskeleton and are defective in attachment to IgG-opsonized particles and FcγR clustering. Delivery of exogenous PIP2 rescued these defects. PIP5K-γ knockout BMM also have more RhoA and less Rac1 activation, and pharmacological manipulations establish that they contribute to the abnormal phenotype. Likewise, depletion of PIP5K-γ by RNA interference inhibits particle attachment. In contrast, PIP5K-α knockout or silencing has no effect on attachment but inhibits ingestion by decreasing Wiskott-Aldrich syndrome protein activation, and hence actin polymerization, in the nascent phagocytic cup. In addition, PIP5K-γ but not PIP5K-α is transiently activated by spleen tyrosine kinase–mediated phosphorylation. We propose that PIP5K-γ acts upstream of Rac/Rho and that the differential regulation of PIP5K-γ and -α allows them to work in tandem to modulate the actin cytoskeleton during the attachment and ingestion phases of phagocytosis.

WASP and WAVE family proteins: key molecules for rapid rearrangement of cortical actin filaments and cell movement

2001 ◽  
Vol 114 (10) ◽  
pp. 1801-1809 ◽  
Author(s):  
T. Takenawa ◽  
H. Miki
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Cell Movement ◽  
Activation Mechanism ◽  
Cortical Actin ◽  
C Terminus ◽  
Inactive Form ◽  
Extracellular Stimuli ◽  
Proline Rich Region

Reorganization of cortical actin filaments plays critical roles in cell movement and pattern formation. Recently, the WASP and WAVE family proteins WASP and N-WASP, and WAVE1, WAVE2 and WAVE3 have been shown to regulate cortical actin filament reorganization in response to extracellular stimuli. These proteins each have a verprolin-homology (V) domain, cofilin-homology (C) domain and an acidic (A) region at the C-terminus, through which they activate the Arp2/3 complex, leading to rapid actin polymerization. N-WASP is usually present as an inactive form in which the VCA region is masked. Cooperative binding of Cdc42 and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) exposes the VCA region, activating N-WASP. In addition to this activation mechanism, WISH also activates N-WASP independently of Cdc42 and PtdIns(4,5)P(2), by binding to the proline-rich region of N-WASP. N-WASP activation induces formation of filopodia in vivo. In contrast, the ubiquitously expressed form of WAVE2 is activated downstream of Rac, leading to formation of lamellipodia. In this case, IRSp53 transmits a signal from Rac to WAVE2 through formation of a ternary Rac-IRSp53-WAVE2 complex. Thus, N-WASP, which is activated downstream of Cdc42 or independently by WISH, induces formation of filopodia and WAVE2, which is activated via IRSp53 downstream of Rac, induces formation of lamellipodia.

scholarly journals Cell migration requires both ion translocation and cytoskeletal anchoring by the Na-H exchanger NHE1

2002 ◽  
Vol 159 (6) ◽  
pp. 1087-1096 ◽  
Author(s):  
Sheryl P. Denker ◽  
Diane L. Barber
Keyword(s):  
Plasma Membrane ◽  
Ion Transport ◽  
Rho Gtpases ◽  
Actin Polymerization ◽  
Cell Movement ◽  
Focal Adhesions ◽  
Erm Proteins ◽  
Ion Translocation ◽  
Cell Front ◽  
Direct Binding

Directed cell movement is a multi-step process requiring an initial spatial polarization that is established by asymmetric stimulation of Rho GTPases, phosphoinositides (PIs), and actin polymerization. We report that the Na-H exchanger isoform 1 (NHE1), a ubiquitously expressed plasma membrane ion exchanger, is necessary for establishing polarity in migrating fibroblasts. In fibroblasts, NHE1 is predominantly localized in lamellipodia, where it functions as a plasma membrane anchor for actin filaments by its direct binding of ezrin/radixin/moesin (ERM) proteins. Migration in a wounding assay was impaired in fibroblasts expressing NHE1 with mutations that independently disrupt ERM binding and cytoskeletal anchoring or ion transport. Disrupting either function of NHE1 impaired polarity, as indicated by loss of directionality, mislocalization of the Golgi apparatus away from the orientation of the wound edge, and inhibition of PI signaling. Both functions of NHE1 were also required for remodeling of focal adhesions. Most notably, lack of ion transport inhibited de-adhesion, resulting in trailing edges that failed to retract. These findings indicate that by regulating asymmetric signals that establish polarity and by coordinating focal adhesion remodeling at the cell front and rear, cytoskeletal anchoring by NHE1 and its localized activity in lamellipodia act cooperatively to integrate cues for directed migration.

scholarly journals Phospholipase C and cofilin are required for carcinoma cell directionality in response to EGF stimulation

2004 ◽  
Vol 166 (5) ◽  
pp. 697-708 ◽  
Author(s):  
Ghassan Mouneimne ◽  
Lilian Soon ◽  
Vera DesMarais ◽  
Mazen Sidani ◽  
Xiaoyan Song ◽  
...  
Keyword(s):  
Rna Silencing ◽  
Actin Polymerization ◽  
Carcinoma Cell ◽  
Cell Movement ◽  
Leading Edge ◽  
Carcinoma Cells ◽  
Epidermal Growth ◽  
Phosphoinositide 3 Kinase ◽  
Blocking Antibodies ◽  
Interfering Rna

The epidermal growth factor (EGF)–induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF.

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