FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2113146146> ?p ?o ?g. }

• W2113146146 endingPage "1121" @default.
• W2113146146 startingPage "1110" @default.
• W2113146146 abstract "•Polarized sensitivity to chemoattractants is independent of cytoskeletal dynamics•Threshold for response is correlated with static F-actin distribution•Immobilized cells retain characteristic responses to spatial and temporal stimuli•Overall directional response depends on gradient and internal polarity Polarized migrating cells display signal transduction events, such as activation of phosphatidylinositol 3-kinase (PI3K) and Scar/Wave, and respond more readily to chemotactic stimuli at the leading edge. We sought to determine the basis of this polarized sensitivity. Inhibiting actin polymerization leads to uniform sensitivity. However, when human neutrophils were “stalled” by simultaneously blocking actin and myosin dynamics, they maintained the gradient of responsiveness to chemoattractant and also displayed noise-driven PIP3 flashes on the basal membrane, localized toward the front. Thus, polarized sensitivity does not require migration or cytoskeletal dynamics. The threshold for response is correlated with the static F-actin distribution, but not cell shape or volume changes, membrane fluidity, or the preexisting distribution of PI3K. The kinetics of responses to temporal and spatial stimuli were consistent with the local excitation global inhibition model, but the overall direction of the response was biased by the internal axis of polarity. Polarized migrating cells display signal transduction events, such as activation of phosphatidylinositol 3-kinase (PI3K) and Scar/Wave, and respond more readily to chemotactic stimuli at the leading edge. We sought to determine the basis of this polarized sensitivity. Inhibiting actin polymerization leads to uniform sensitivity. However, when human neutrophils were “stalled” by simultaneously blocking actin and myosin dynamics, they maintained the gradient of responsiveness to chemoattractant and also displayed noise-driven PIP3 flashes on the basal membrane, localized toward the front. Thus, polarized sensitivity does not require migration or cytoskeletal dynamics. The threshold for response is correlated with the static F-actin distribution, but not cell shape or volume changes, membrane fluidity, or the preexisting distribution of PI3K. The kinetics of responses to temporal and spatial stimuli were consistent with the local excitation global inhibition model, but the overall direction of the response was biased by the internal axis of polarity. IntroductionDirected cell migration is a fundamental cell biological process that plays an important role in embryogenesis and wiring of the nervous system, immune cell trafficking, wound healing, and a host of other critical physiological events. It also contributes to the pathogenesis of inflammatory diseases and cancer metastasis. Neutrophils exhibit a robust chemotactic response to chemical gradients and serve as a model for understanding this process. The similarity in the behavior of neutrophils to that of simple amoeboid cells, such as Dictyostelium, indicates that chemotaxis is a highly conserved fundamental cell biological process.The mechanisms cells use to sense and migrate toward external cues are beginning to be elucidated. Directed migration depends on the seamless integration of motility, directional sensing, and polarity. Motility relies on spontaneous activation of signaling and cytoskeletal events and extension of pseudopods. Directional sensing is independent of the cytoskeleton, since external gradients can localize signaling activities in immobilized cells. Chemotactic cells are also often polarized, which increases the efficiency of the response.Even without directional information, cells can establish an elongated polarized state with differential sensitivity between the front and the back. This cellular state is semistable and can be reset. Neutrophil-like HL-60 cells often make a “U-turn” rather than generate a new front when a chemoattractant is delivered by a micropipette toward the back of the cell (Gerisch and Keller, 1981Gerisch G. Keller H.U. Chemotactic reorientation of granulocytes stimulated with micropipettes containing fMet-Leu-Phe.J. Cell Sci. 1981; 52: 1-10PubMed Google Scholar). Elongated Dictyostelium cells, late in their developmental program, behave similarly when presented with a new gradient (Futrelle et al., 1982Futrelle R.P. Traut J. McKee W.G. Cell behavior in Dictyostelium discoideum: preaggregation response to localized cyclic AMP pulses.J. Cell Biol. 1982; 92: 807-821Crossref PubMed Scopus (86) Google Scholar, Swanson and Taylor, 1982Swanson J.A. Taylor D.L. Local and spatially coordinated movements in Dictyostelium discoideum amoebae during chemotaxis.Cell. 1982; 28: 225-232Abstract Full Text PDF PubMed Scopus (84) Google Scholar). Thus, when the gradient is shifted, polarized cells maintain their original direction and then gradually reorient toward the gradient. This suggests that cell polarity and gradient sensing might be separate, interacting phenomena. Although the established leading edge is relatively more sensitive to chemoattractants, cells can be forced to repolarize by increasing the steepness of the reverse gradient, indicating that some sensitivity is maintained around the entire perimeter. It is unclear what determines this dynamic “polarized sensitivity” and how it is related to gradient sensing.It has been assumed that cell movement and cytoskeletal dynamics play a critical role in establishing and maintaining polarity. Most schemes for polarity couple positive feedback at the anterior with global inhibitory mechanisms to prevent additional fronts (Howell et al., 2009Howell A.S. Savage N.S. Johnson S.A. Bose I. Wagner A.W. Zyla T.R. Nijhout H.F. Reed M.C. Goryachev A.B. Lew D.J. Singularity in polarization: rewiring yeast cells to make two buds.Cell. 2009; 139: 731-743Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, Meinhardt, 1999Meinhardt H. Orientation of chemotactic cells and growth cones: models and mechanisms.J. Cell Sci. 1999; 112: 2867-2874PubMed Google Scholar, Neilson et al., 2011Neilson M.P. Veltman D.M. van Haastert P.J. Webb S.D. Mackenzie J.A. Insall R.H. Chemotaxis: a feedback-based computational model robustly predicts multiple aspects of real cell behaviour.PLoS Biol. 2011; 9: e1000618Crossref PubMed Scopus (120) Google Scholar, Orchard et al., 2012Orchard R.C. Kittisopikul M. Altschuler S.J. Wu L.F. Süel G.M. Alto N.M. Identification of F-actin as the dynamic hub in a microbial-induced GTPase polarity circuit.Cell. 2012; 148: 803-815Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Recent models, for example, suggest that protrusions at the front alter membrane properties, such as membrane tension or curvature, which affects cytoskeletal activity at secondary sites (Frost et al., 2009Frost A. Unger V.M. De Camilli P. The BAR domain superfamily: membrane-molding macromolecules.Cell. 2009; 137: 191-196Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, Houk et al., 2012Houk A.R. Jilkine A. Mejean C.O. Boltyanskiy R. Dufresne E.R. Angenent S.B. Altschuler S.J. Wu L.F. Weiner O.D. Membrane tension maintains cell polarity by confining signals to the leading edge during neutrophil migration.Cell. 2012; 148: 175-188Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). There is general agreement that pharmacological perturbations of F-actin abolish cell polarity (Casella et al., 1981Casella J.F. Flanagan M.D. Lin S. Cytochalasin D inhibits actin polymerization and induces depolymerization of actin filaments formed during platelet shape change.Nature. 1981; 293: 302-305Crossref PubMed Scopus (329) Google Scholar, Spector et al., 1983Spector I. Shochet N.R. Kashman Y. Groweiss A. Latrunculins: novel marine toxins that disrupt microfilament organization in cultured cells.Science. 1983; 219: 493-495Crossref PubMed Scopus (604) Google Scholar). Signal transduction responses can still be elicited by chemoattractant in such immobilized cells, but the cells are equally sensitive around their perimeter. These observations support the belief that cytoskeletal dynamics and migration are essential for maintenance of the polarized state (Wang et al., 2002Wang F. Herzmark P. Weiner O.D. Srinivasan S. Servant G. Bourne H.R. Lipid products of PI(3)Ks maintain persistent cell polarity and directed motility in neutrophils.Nat. Cell Biol. 2002; 4: 513-518Crossref PubMed Scopus (395) Google Scholar).Studies with a pharmacological cocktail (JLY) containing actin disassembly inhibitor jasplakinolide (J), actin polymerization inhibitor latrunculin B (L), and ROCK inhibitor Y27632 (Y), which preserves the existing actin cytoskeleton while blocking assembly, disassembly, and rearrangement of the actin network, also suggest that cytoskeletal dynamics are important for aspects of polarity. JLY-treated HL-60 neutrophils stop migrating and maintain their shape, although Rac activity seen at the leading edge of moving cells disappears (Dandekar et al., 2013Dandekar S.N. Park J.S. Peng G.E. Onuffer J.J. Lim W.A. Weiner O.D. Actin dynamics rapidly reset chemoattractant receptor sensitivity following adaptation in neutrophils.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2013; 368: 20130008Crossref PubMed Scopus (12) Google Scholar, Peng et al., 2011Peng G.E. Wilson S.R. Weiner O.D. A pharmacological cocktail for arresting actin dynamics in living cells.Mol. Biol. Cell. 2011; 22: 3986-3994Crossref PubMed Scopus (55) Google Scholar). Thus a dynamic cytoskeleton appears to be necessary to maintain polarization in the signal transduction system. However, polarized sensitivity to chemoattractants was not examined in these experiments.By manipulating the polarity of HL-60 neutrophils and examining the responses of moving and immobilized cells to uniform increases and gradients of the chemoattractant, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), we were able to distinguish the contributions of motility, directional sensing, and polarity to the overall response. As previously shown for latrunculin-treated cells, we find that JLY-treated, “stalled” cells respond and adapt to uniform stimuli and respond persistently to applied gradients. Furthermore, we find that polarized sensitivity depends strongly on cytoskeletal architecture and does not depend on cell shape, volume, membrane curvature, or membrane fluidity. Thus, the overall directional response of the cell depends on the balance between the external gradient and the polarized architecture of the cytoskeleton. These conclusions are consistent with the turning behaviors of cells exposed to shifting gradients.ResultsResponses to Chemoattractant and Spontaneous Activities Are Polarized in JLY-Treated, “Stalled” NeutrophilsWe first compared the morphology and migration behavior of cells before and after JLY treatment. As previously reported, cells kept migrating in the presence of a ROCK inhibitor Y27632 but stopped as soon as latrunculin B and jasplakinolide were added (Peng et al., 2011Peng G.E. Wilson S.R. Weiner O.D. A pharmacological cocktail for arresting actin dynamics in living cells.Mol. Biol. Cell. 2011; 22: 3986-3994Crossref PubMed Scopus (55) Google Scholar, Xu et al., 2003Xu J. Wang F. Van Keymeulen A. Herzmark P. Straight A. Kelly K. Takuwa Y. Sugimoto N. Mitchison T. Bourne H.R. Divergent signals and cytoskeletal assemblies regulate self-organizing polarity in neutrophils.Cell. 2003; 114: 201-214Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar). Cell shape, judged by phase microscopy, and actin cytoskeletal architecture examined by the F-actin biosensor, Lifeact, was maintained for at least 2 hr. We also verified that the rate of fluorescence recovery after photobleaching (FRAP) of actin-mCherry was negligible (see below). Latrunculin B treatment also immobilized cells, but the cell shape and cytoskeletal architecture was not maintained (Figure 1A) (Riedl et al., 2008Riedl J. Crevenna A.H. Kessenbrock K. Yu J.H. Neukirchen D. Bista M. Bradke F. Jenne D. Holak T.A. Werb Z. et al.Lifeact: a versatile marker to visualize F-actin.Nat. Methods. 2008; 5: 605-607Crossref PubMed Scopus (1486) Google Scholar). As observed in Dictyostelium, the PIP3 biosensor pleckstrin homology domain of serine-threonine protein kinase AKT1 (PH-AKT), typically observed at the front of migrating cells, decreased following latrunculin B treatment (Figure S1A). Furthermore, PIP3 rapidly decreased following JLY treatment of cells migrating in uniform fMLP (Figure S1A). This suggests that even the JLY-stabilized cytoskeleton cannot maintain activated phosphatidylinositol 3-kinase (PI3K).Previous studies have suggested that there is feedback from actin polymerization to activation of PI3K, although the conclusions have been based principally on the ability of latrunculin B to block the process (Inoue and Meyer, 2008Inoue T. Meyer T. Synthetic activation of endogenous PI3K and Rac identifies an AND-gate switch for cell polarization and migration.PLoS ONE. 2008; 3: e3068Crossref PubMed Scopus (102) Google Scholar, Srinivasan et al., 2003Srinivasan S. Wang F. Glavas S. Ott A. Hofmann F. Aktories K. Kalman D. Bourne H.R. Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis.J. Cell Biol. 2003; 160: 375-385Crossref PubMed Scopus (363) Google Scholar, Wang et al., 2002Wang F. Herzmark P. Weiner O.D. Srinivasan S. Servant G. Bourne H.R. Lipid products of PI(3)Ks maintain persistent cell polarity and directed motility in neutrophils.Nat. Cell Biol. 2002; 4: 513-518Crossref PubMed Scopus (395) Google Scholar). When we transiently photoactivated Rac in differentiated HL-60 cells expressing PH-AKT-GFP, we observed a 14.4% ± 1.5% increase in PIP3 (Figure S1B). However, the increase was negligible in cells treated with JLY or latrunculin B plus nocodazole. These observations suggest that actin dynamics and/or movement are required for feedback regulation from Rac activation to PI3K activity in neutrophils.Surprisingly, when we exposed the JLY-treated, stalled cells to a uniform, low concentration (1nM) of fMLP, not only did they generate PIP3, but it was accumulated in a gradient (Figures 1B and 1C; Figure S1C; Movie S1). The levels were higher at the front and sides than the back, indicating that the anterior is more sensitive to PI3K activation. When we increased the global fMLP concentration (100 nM) applied to the same cell, there was no significant difference between back and front maximum membrane PH-AKT accumulation (Figure 1C). The same results were obtained whether cells were pretreated with JLY for several minutes or 2 hr. Cells with obviously polarized morphology were used for quantification (Figure S1E). Using the C5a receptor as a membrane marker, we verified that the graded signal was not due to an uneven membrane distribution from front to back (Figure S1A). In contrast to the JLY cells, rounded cells treated with nocodazole and latrunculin B responded equally along the entire membrane at all concentrations of fMLP (Figures 1B and 1C; Movie S2). The responses at the back of the JLY-treated cells peaked at a lower level and were briefer than those at the front and sides (Figure 1C; Figures S1C and S1D). Both the amplitude and duration contribute to a smaller integrated response seen at the back. PH-AKT translocated to a broader membrane region with increasing doses of fMLP (Figure 1D). Together, these observations suggest there is an anterior-posterior gradient of sensitivity for chemoattractant-mediated activation of PI3K and PIP3 accumulation in the JLY-treated, stalled neutrophils.Previous studies have shown that Hem1 is spontaneously recruited in waves to the basal surface of migrating neutrophils and accumulates at the edge of the basal surface in response to fMLP (Weiner et al., 2007Weiner O.D. Marganski W.A. Wu L.F. Altschuler S.J. Kirschner M.W. An actin-based wave generator organizes cell motility.PLoS Biol. 2007; 5: e221Crossref PubMed Scopus (279) Google Scholar). The spontaneous waves are silenced by latrunculin. Similarly, we found that Hem1 waves disappeared in JLY-treated cells. Although JLY-treated cells were less sensitive than untreated cells, Hem1 was recruited transiently to the boundary of the basal membrane after stimulation with 100 nM fMLP, and the response was more persistent at the front than at the back (Figure 1E), as was the case for PIP3 production (Figure S1D).Recent studies showing there are independent excitable signal transduction and oscillatory cytoskeletal networks in Dictyostelium cells and the fact that cytoskeletal component Hem-1 displays propagating waves in neutrophils (Huang et al., 2013Huang C.H. Tang M. Shi C. Iglesias P.A. Devreotes P.N. An excitable signal integrator couples to an idling cytoskeletal oscillator to drive cell migration.Nat. Cell Biol. 2013; 15: 1307-1316Crossref PubMed Scopus (131) Google Scholar, Weiner et al., 2007Weiner O.D. Marganski W.A. Wu L.F. Altschuler S.J. Kirschner M.W. An actin-based wave generator organizes cell motility.PLoS Biol. 2007; 5: e221Crossref PubMed Scopus (279) Google Scholar) prompted us to further explore the similarities in the chemotactic systems in the two cell types. To ask whether there are similar separable networks in neutrophils, we monitored by total internal reflection fluorescence (TIRF) microscopy the spontaneous recruitment of PH-AKT to the membrane on the basal surface of randomly migrating or stalled JLY-treated neutrophils. We observed spontaneously appearing PIP3 patches with a lifetime of a few minutes in the majority of randomly migrating cells. T-stacks (Huang et al., 2013Huang C.H. Tang M. Shi C. Iglesias P.A. Devreotes P.N. An excitable signal integrator couples to an idling cytoskeletal oscillator to drive cell migration.Nat. Cell Biol. 2013; 15: 1307-1316Crossref PubMed Scopus (131) Google Scholar) of coexpressed PH-AKT and Lifeact showed the two overlapped but were not precisely colocalized (Figure S1F; Movie S3). Interestingly, we observed similar dynamic PIP3 patches in JLY-treated cells; however, unlike in randomly migrating cells, the F-actin signal was static (Figure 2A). There was more PIP3 activity at the anterior of the cell, where static F-actin was enriched, than at the back (68.1% versus 31.9% of total intensity of PIP3 fluctuations, p < 0.001, n = 7) (Figure 2A; Movie S4). Coexpressed membrane marker C5aR showed that the PH-AKT flashes were not due to membrane deformations or rearrangements (Figure 2B; Movie S5). The addition of 20 μM PI3Kγ inhibitor AS605240 abolished the PH-AKT flashes (Figure 2C). Taken together, these observations show the chemotactic signal transduction network in neutrophils displays spontaneous activity in the absence of cytoskeletal activity or movement and, furthermore, that the activity is biased toward the front.Figure 2TIRF Images of Spontaneous Recruitment of PH-AKT to the Membrane on the Basal Surface of JLY-Treated NeutrophilsShow full caption(A) Dual-view TIRF images of coexpressed PH-AKT and Lifeact signals in JLY-treated neutrophils. T-stacks of both panels in the same cross-section show the signal dynamics. Vertical line scans at the point indicated by the yellow arrows are used to highlight dynamic changes in gray value over time. Detailed description of vertical line scans is described in Supplemental Experimental Procedures. See also Movie S4.(B) Dual-view TIRF images of coexpressed PH-AKT and C5aR signals in JLY-treated neutrophils. T-stacks and vertical line scans of both panels are also shown. See also Movie S5.(C) TIRF images of PH-AKT in JLY-treated neutrophils treated with PI3K inhibitor AS605240. T-stack and a vertical line scan are also shown.View Large Image Figure ViewerDownload (PPT)Polarized Sensitivity Is Correlated with the Arrested F-actin Distribution and Is Independent of Cell Shape, Volume, Membrane Curvature, or FluiditySince polarized migrating neutrophils usually have more F-actin at the front (Nishikimi et al., 2009Nishikimi A. Fukuhara H. Su W. Hongu T. Takasuga S. Mihara H. Cao Q. Sanematsu F. Kanai M. Hasegawa H. et al.Sequential regulation of DOCK2 dynamics by two phospholipids during neutrophil chemotaxis.Science. 2009; 324: 384-387Crossref PubMed Scopus (215) Google Scholar), we speculated that the polarized sensitivity might be related to the arrested F-actin distribution. Examples of cells with broader or narrower Lifeact regions appear to support this (Figure 3A). Quantification showed that when exposed to a uniform, low concentration of fMLP, JLY-treated cells with narrower regions of F-actin, as indicated by a higher ratio of Lifeact fluorescence intensity at the front versus the back, also had narrower zones of PIP3 production on the membrane. The asymmetric response was nearly absent when we increased the fMLP concentration applied to the same cell (Figure 3A; Movies S6 and S7).Figure 3Polarized Sensitivity Is Correlated with the Arrested F-actin Distribution and Is Independent of Other FactorsShow full caption(A) Fluorescence images of coexpressed PH-AKT and Lifeact signals in JLY-treated neutrophils exposed to uniform fMLP. The upper two panels or lower panel show cells with asymmetric or uniform Lifeact distributions, respectively. Merged images show the extent of co-localization. Graph on the right shows the relationship between length of PH-AKT as a fraction of the perimeter and front to back Lifeact fluorescence ratio. Trend lines (second degree polynomial) are also shown. Scale bar, 10 μm. See also Movies S6 and S7.(B) Fluorescence images of PH-AKT in neutrophils treated with JLY for 10 min followed by 1:3 volume of hypotonic buffer for 20 min. Cells were exposed to uniform fMLP. The arrowheads indicate PIP3 accumulation after fMLP stimulation. Scale bar, 10 μm.(C) Phase and fluorescence images of Lifeact-expressing JLY-treated neutrophils exposed to sequential buffers of decreasing osmolarity as indicated. The times after each decrease are indicated. The duration of each treatment is approximately 5 min. Scale bar, 10 μm.(D) Immunofluorescent staining of the catalytic subunit of PI3Kγ in randomly migrating and JLY-treated neutrophils expressing PH-AKT with or without fMLP stimulation. Scale bar, 10 μm. Bar graphs show the front to back ratio of PH-AKT and PI3Kγ. Numbers in the bars show the number of cells quantified. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Error bars represent SEM.(E) C5aR fluorescent images before and after the FRAP assay in latrunculin B- and JLY-treated neutrophils. T-stack images show the fluorescence recovery over time. Scale bar, 10 μm. Line graph shows the kinetics of bleaching and recovery. n = 15 for JLY front membrane bleaching, n = 11 for JLY side membrane bleaching, n = 12 for JLY back membrane bleaching, and n = 8 for latrunculin B membrane bleaching. Error bars represent SEM.View Large Image Figure ViewerDownload (PPT)We assessed several other factors that could potentially underlie the polarized sensitivity of the JLY-treated cells. First, there is a marked difference in shape and curvature between fan-like fronts and elongated backs. Since polarized HL-60 neutrophils with broad backs were rare, we artificially produced wide tails in polarized cells by exposing JLY-treated cells to hypotonic buffer. At room temperature, the increased volume caused by hypotonic treatment lasts for at least 25 min (Ting-Beall et al., 1993Ting-Beall H.P. Needham D. Hochmuth R.M. Volume and osmotic properties of human neutrophils.Blood. 1993; 81: 2774-2780Crossref PubMed Google Scholar). In our experiments, some of the JLY-treated cells that had loosely attached backs formed large rounded backs that had even less curvature than the front. When we exposed these cells to low fMLP, they still did not respond at the back. The rounded backs did respond to higher concentrations of fMLP (Figure 3B). Thus, the polarized sensitivity was independent of cell shape, volume, or membrane curvature.Interestingly, in some of the JLY-treated cells that appeared to adhere strongly to the substrate at the rear, hypotonic treatment did not produce a rounded back but instead induced blebs from the sides in the zone where the F-actin content dramatically decreased (32 out of 52 cells form side blebs; Figure 3C). This observation suggested that the membrane was more loosely attached to the cortex at the sides and that polarized sensitivity therefore cannot be traced to a differential attachment of the membrane to the cortex. Curiously, these blebs formed in hypotonic conditions retracted spontaneously or after adding fMLP (Figure S2A).PI3K is found at the leading edge of chemotaxing Dictyostelium cells and several types of leukocytes (Funamoto et al., 2002Funamoto S. Meili R. Lee S. Parry L. Firtel R.A. Spatial and temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN mediates chemotaxis.Cell. 2002; 109: 611-623Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar, Gómez-Moutón et al., 2004Gómez-Moutón C. Lacalle R.A. Mira E. Jiménez-Baranda S. Barber D.F. Carrera A.C. Martínez-A C. Mañes S. Dynamic redistribution of raft domains as an organizing platform for signaling during cell chemotaxis.J. Cell Biol. 2004; 164: 759-768Crossref PubMed Scopus (189) Google Scholar), and if it were trapped at the fronts of the immobilized neutrophils by the JLY-treatment, the fronts would be expected to produce more PIP3 in response to uniform stimuli. Using PI3Kγ catalytic subunit antibody staining, we verified that the enzyme was colocalized with PH-AKT at the leading edge in fMLP-treated, migrating HL-60 cells. However, we found that in the JLY-treated cells, PI3Kγ was not localized at the fronts before stimulus addition. Statistical analysis showed that PI3Kγ translocated selectively to the membrane at the front of JLY-treated neutrophils after exposure to uniform low fMLP, while a high fMLP caused a broader translocation of this enzyme (Figure 3D). These observations indicate that the polarized accumulation of PIP3 in JLY-treated cells is due to specific recruitment of PI3Kγ to the front.Although the cytoskeleton is static in JLY-treated cells, the membrane may remain fluid. We used FRAP assays to assess components that reflect membrane fluidity. As noted above, actin dynamics were stalled in the JLY-treated cells, as there was no recovery of actin-mCherry fluorescence within 12 min after photobleaching (Figure S2B). By contrast, Lifeact red fluorescent protein (RFP) fluorescence recovered quickly, presumably since it exchanged rapidly on the F-actin (Figure S2B). Similarly, the PH-AKT signal recovered 90% within 2 min. The C5aR signal recovered more slowly (50% within 3 min), and the recovery came from adjacent membrane, not the cytosol. Importantly, there was no significant difference in C5aR mobility in JLY- versus latrunculin B-treated cells, and furthermore, no positional difference was found in stalled neutrophils (Figure 3E). Thus, differences in membrane fluidity do not appear to contribute to polarized sensitivity.The Stimulus-Response Behavior of the Chemotactic System in Immobilized CellsPrevious studies have suggested that, like PI3K, Ras is activated by chemoattractants in neutrophils, although the localization of the activated form was not determined (Zheng et al., 1997Zheng L. Eckerdal J. Dimitrijevic I. Andersson T. Chemotactic peptide-induced activation of Ras in human neutrophils is associated with inhibition of p120-GAP activity.J. Biol. Chem. 1997; 272: 23448-23454Crossref PubMed Scopus (33) Google Scholar). To extend these studies, we stimulated cells with fMLP and measured activation of K Ras and N Ras using pull-down assays with subtype-specific antibodies. We found that both subtypes were transiently activated upon fMLP stimulation, peaking around 30 s to 1 min (Figure S2C). We then expressed a fragment of Raf-1 containing the Ras binding domain plus the cysteine-rich domain (Raf-1151–220-GFP) to study Ras activation in living cells. Raf-1151–220-GFP translocated to the membrane after 100 nM fMLP stimulation and then accumulated at the leading edge during migration (Figure S2D). Using this more sensitive biosensor, we observed responses to fMLP in 75% of the cells that were studied (33 out of 44 cells). The sites of Ras activation and PIP3 accumulation closely coincided, indicating that the polarized sensitivity we have described may involve multiple components of the signal transduction network (Figure S2E).Differences in the responses of control and JLY-treated neutrophils to chemoattractant allowed us to separate the direct effects of the stimulus from those due to feedback from movement and/or cytoskeletal dynamics. As noted above, a uniform increase in chemoattractant elicits a transient activation of Ras or PI3K, but when the cells “break symmetry” and migrate, they display persistent PIP3 at the leading edges (Servant et al., 2000Servant G. Weiner O.D. Herzmark P. Balla T. Sedat J.W. Bourne H.R. Polarization of chemoattractant receptor signaling during neutrophil chemotaxis.Science. 2000; 287: 1037-1040Crossref PubMed Scopus (728) Google Scholar). Unlike in migrating cells, the transient activations of PI3K and Ras triggered by uniform increases of fMLP in JLY-treated cells were not followed by persistent activity at the leading edge (Figures 4A and 4D ; Figures S1D and S2F). Thus, the response to chemoattractant adapts during continuous uniform stimulation and the persistent response observed at the front of migrating cells depends on feedback from movement and/or cytoskeletal dynamics.Figure 4Kinetics and Spatial Distribution of Chemoattractant-Stimulated Responses in JLY-Treated CellsShow full caption(A) Kineti" @default.
• W2113146146 created "2016-06-24" @default.
• W2113146146 creator A5004842912 @default.
• W2113146146 creator A5010737270 @default.
• W2113146146 creator A5030555149 @default.
• W2113146146 creator A5051355876 @default.
• W2113146146 creator A5091366547 @default.
• W2113146146 date "2014-11-01" @default.
• W2113146146 modified "2023-10-17" @default.
• W2113146146 title "The Directional Response of Chemotactic Cells Depends on a Balance between Cytoskeletal Architecture and the External Gradient" @default.
• W2113146146 cites W1804943159 @default.
• W2113146146 cites W1968272833 @default.
• W2113146146 cites W1968693703 @default.
• W2113146146 cites W1969410032 @default.
• W2113146146 cites W1973580688 @default.
• W2113146146 cites W1977885600 @default.
• W2113146146 cites W1984653721 @default.
• W2113146146 cites W1994370397 @default.
• W2113146146 cites W1999628770 @default.
• W2113146146 cites W2005088757 @default.
• W2113146146 cites W2009718333 @default.
• W2113146146 cites W2024765629 @default.
• W2113146146 cites W2024813407 @default.
• W2113146146 cites W2038867093 @default.
• W2113146146 cites W2043420241 @default.
• W2113146146 cites W2047433701 @default.
• W2113146146 cites W2049315117 @default.
• W2113146146 cites W2054808321 @default.
• W2113146146 cites W2063993190 @default.
• W2113146146 cites W2067917704 @default.
• W2113146146 cites W2078227548 @default.
• W2113146146 cites W2078423846 @default.
• W2113146146 cites W2086441678 @default.
• W2113146146 cites W2087538969 @default.
• W2113146146 cites W2098613050 @default.
• W2113146146 cites W2101679999 @default.
• W2113146146 cites W2102031413 @default.
• W2113146146 cites W2102337085 @default.
• W2113146146 cites W2122502269 @default.
• W2113146146 cites W2124028902 @default.
• W2113146146 cites W2149058887 @default.
• W2113146146 cites W2150939977 @default.
• W2113146146 cites W2151519283 @default.
• W2113146146 cites W2157886337 @default.
• W2113146146 cites W2162349490 @default.
• W2113146146 cites W2164318727 @default.
• W2113146146 cites W2171520735 @default.
• W2113146146 cites W2171857030 @default.
• W2113146146 cites W2188659719 @default.
• W2113146146 cites W2226774535 @default.
• W2113146146 cites W4256138746 @default.
• W2113146146 doi "https://doi.org/10.1016/j.celrep.2014.09.047" @default.
• W2113146146 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4250826" @default.
• W2113146146 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/25437564" @default.
• W2113146146 hasPublicationYear "2014" @default.
• W2113146146 type Work @default.
• W2113146146 sameAs 2113146146 @default.
• W2113146146 citedByCount "54" @default.
• W2113146146 countsByYear W21131461462015 @default.
• W2113146146 countsByYear W21131461462016 @default.
• W2113146146 countsByYear W21131461462017 @default.
• W2113146146 countsByYear W21131461462018 @default.
• W2113146146 countsByYear W21131461462019 @default.
• W2113146146 countsByYear W21131461462020 @default.
• W2113146146 countsByYear W21131461462021 @default.
• W2113146146 countsByYear W21131461462022 @default.
• W2113146146 countsByYear W21131461462023 @default.
• W2113146146 crossrefType "journal-article" @default.
• W2113146146 hasAuthorship W2113146146A5004842912 @default.
• W2113146146 hasAuthorship W2113146146A5010737270 @default.
• W2113146146 hasAuthorship W2113146146A5030555149 @default.
• W2113146146 hasAuthorship W2113146146A5051355876 @default.
• W2113146146 hasAuthorship W2113146146A5091366547 @default.
• W2113146146 hasBestOaLocation W21131461461 @default.
• W2113146146 hasConcept C12554922 @default.
• W2113146146 hasConcept C142669718 @default.
• W2113146146 hasConcept C1491633281 @default.
• W2113146146 hasConcept C168031717 @default.
• W2113146146 hasConcept C169760540 @default.
• W2113146146 hasConcept C170493617 @default.
• W2113146146 hasConcept C185592680 @default.
• W2113146146 hasConcept C54166955 @default.
• W2113146146 hasConcept C55493867 @default.
• W2113146146 hasConcept C86803240 @default.
• W2113146146 hasConcept C95444343 @default.
• W2113146146 hasConceptScore W2113146146C12554922 @default.
• W2113146146 hasConceptScore W2113146146C142669718 @default.
• W2113146146 hasConceptScore W2113146146C1491633281 @default.
• W2113146146 hasConceptScore W2113146146C168031717 @default.
• W2113146146 hasConceptScore W2113146146C169760540 @default.
• W2113146146 hasConceptScore W2113146146C170493617 @default.
• W2113146146 hasConceptScore W2113146146C185592680 @default.
• W2113146146 hasConceptScore W2113146146C54166955 @default.
• W2113146146 hasConceptScore W2113146146C55493867 @default.
• W2113146146 hasConceptScore W2113146146C86803240 @default.
• W2113146146 hasConceptScore W2113146146C95444343 @default.
• W2113146146 hasIssue "3" @default.
• W2113146146 hasLocation W21131461461 @default.

• next