Research ArticleChemotaxis

Extension of chemotactic pseudopods by nonadherent human neutrophils does not require or cause calcium bursts

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Science Signaling  13 Mar 2018:
Vol. 11, Issue 521, eaal4289
DOI: 10.1126/scisignal.aal4289
  • Fig. 1 Complement-mediated, pure chemotaxis, and Ca2+ bursts.

    (A to C) Examples of single-live-cell, pure-chemotaxis experiments (51, 52) in which human neutrophils (N) were placed near bacterial (B) or fungal (F) pathogens. The nonadherent neutrophils were held with gentle suction pressure at the tip of a micropipette and placed near a cluster of S. Typhimurium bacteria trapped with optical tweezers (A), a single C. albicans cell held with a micropipette (B) or a single C. posadasii endospore held with a micropipette (C). Each panel is a composite of three video images depicting the changes in neutrophil morphology in response to the placement of the pathogenic target at different sides of the cell. (D) The complement system in the serum of the host assembles complement complexes on the surface of foreign target particles (T). Some of these complexes, such as the C5 convertase, are enzymes that cleave other serum proteins and release anaphylatoxins, which are highly potent chemoattractants, such as C5a. The radial concentration profile of anaphylatoxins surrounding the target (color gradient) forms rapidly but has a limited spatial reach (37, 48). Neutrophils detect this anaphylatoxic cloud and respond by extending chemotactic pseudopods toward the target. (E) Example measurement of the time course of the mean fluorescence intensity (MFI) of the Ca2+ indicator Fluo-4 during pure chemotaxis of a human neutrophil toward a pipette-held zymosan particle (Z) and eventual phagocytosis of the particle. Included are bright-field and fluorescence images taken at the time points numbered (1) to (6) in the MFI graph. The MFI remained low and essentially flat throughout the chemotaxis stage but exhibited a steep increase caused by a Ca2+ burst after the zymosan particle was brought into physical contact with the neutrophil (see also movies S1 to S3). Scale bars, 10 μm. au, arbitrary units.

  • Fig. 2 Absence of Ca2+ bursts during pure chemotaxis.

    Three representative examples show experiments that examined the response of individual pipette-held neutrophils to various targets using the dual-micropipette assay (Fig. 1E). (A to C) Filmstrips show both bright-field and fluorescence images of experiments in which the target (held in the left pipette in each image) was first placed near and eventually in contact with a neutrophil (held at the right in each image). The targets were zymosan particles (A), β-glucan particles (B), and an immunoglobulin G (IgG)–coated bead (C). The test with β-glucan (B) depicts an experiment using a cluster of β-glucan particles but is representative of experiments with both clusters and individual β-glucan particles. (D to F) The graphs show the time course of the MFI of Fluo-4 corresponding to the intracellular Ca2+ concentration during each experiment. The numbered points on the graphs mark the times at which the respective images (A to C) were taken. During single-cell experiments with the fungal model particles, the shown neutrophil behavior—chemotaxis without a Ca2+ burst followed by phagocytosis with a Ca2+ burst—was observed in 34 (of n = 36) experiments with zymosan (A and D) and in 16 (of n =19) experiments with β-glucan particles (B and E). The shown neutrophil response to an antibody-coated bead—no chemotaxis but phagocytosis that then triggered a Ca2+ burst (C and F)—was observed in all experiments with these beads (n = 43). See also movies S1 to S3. Scale bars, 10 μm.

  • Fig. 3 Supraphysiological concentrations of C5a or costimulation by shear flow can trigger Ca2+ bursts in nonadherent neutrophils.

    Three representative examples show experiments in which jets of C5a solutions were ejected from the left micropipette toward neutrophils held with the right pipette. Filmstrips of bright-field and fluorescence images show snapshots of the neutrophil response at the time points marked by numbers in the included graphs of the MFI of Fluo-4. (A) The neutrophil responded to a jet of a 0.1 nM C5a solution by extending a chemotactic pseudopod for more than 25 min, during which time no Ca2+ burst occurred. This example is representative of n =19 single-cell experiments. (B) The neutrophil was subjected to a jet of a 10 nM C5a solution. The jet was initially applied with a low pressure over a distance of 7 μm, inducing the extension of a pseudopod (time points 1 and 2). After about 9 min, a fivefold increase of the pipette pressure triggered a Ca2+ burst, accompanied by a cell contraction in the horizontal direction (time points 3 and 4). After temporary removal of the left pipette, a low-pressure jet was applied once more (time point 5), causing the cell to resume the protrusion of a directed pseudopod. This example is representative of n =16 single-cell experiments in which C5a jets induced Ca2+ bursts in chemotaxing cells. (C) Application of a low-pressure, 0.1 μM C5a jet from a short distance triggered a Ca2+ burst in a pipette-held neutrophil without previous pseudopod extension. In response, the cell appeared to flatten against the pipette (time point 5). This example is representative of n =10 single-cell experiments. Movie S4 combines single-live-cell videos of the three example experiments included in this figure. Scale bars, 10 μm.

  • Fig. 4 Coincidence of Ca2+ bursts and axial cell contraction.

    Representative examples of jet experiments in which C5a solutions were ejected from the left micropipette toward neutrophils held with the right pipette. The bright-field and fluorescence images show the changes of the neutrophil morphology that accompanied Ca2+ bursts during two different types of cell response, each illustrated with two examples. The images were taken at the time points marked by numbers in the graphs of the MFI of Fluo-4. (A) Initially chemotaxing neutrophils were subjected to a sudden increase of the jet pressure, which also increased the C5a concentration at their surface. The resulting Ca2+ bursts coincided with a contractile morphology change of the cells in the horizontal direction (time points 2 and 3). (B) Sudden exposure of neutrophils to jets of supraphysiological concentrations of C5a triggered Ca2+ bursts that coincided with a contractile morphology change of the cells. The cells did not extend chemotactic pseudopods in this case. We observed the illustrated behavior—Ca2+ bursts that were accompanied by a decrease or arrest of the total axial cell extension—in 24 (of n =26) single-cell experiments. Detailed time courses of such cell contractions are shown in fig. S3. Scale bars, 10 μm.

  • Fig. 5 Limited surface area expansion during pure chemotaxis.

    (A) The graph shows an example of the time-dependent surface area of a neutrophil during pure chemotaxis toward a pipette-held zymosan particle. Representative video snapshots were taken at the time points marked by numbers. (B) The column scatter plots show the values of the maximum cell surface area measured during pure chemotaxis in previous and current experiments with target particles (n =81) and during current jet experiments (n =23), respectively. The upper limit of these values is indicated by a dashed red line. Example videomicrographs of cells extending large chemotactic pseudopods are included. Our approach to measuring the cell surface area is illustrated in fig. S4. Scale bars, 10 μm.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/521/eaal4289/DC1

    Fig. S1. Dual-camera setup for simultaneous recording of bright-field and fluorescence images.

    Fig. S2. Supraphysiological concentrations of C5a trigger Ca2+ bursts in resting human neutrophils without inducing chemotaxis.

    Fig. S3. Concurrence of Ca2+ bursts and cellular contraction.

    Fig. S4. Estimation of the cell surface area.

    Fig. S5. Effective cortical tension during pure chemotaxis.

    Movie S1. A Ca2+ burst in a human neutrophil.

    Movie S2. Absence of Ca2+ bursts during complement-mediated, pure chemotaxis of a human neutrophil toward zymosan.

    Movie S3. Absence of Ca2+ bursts during complement-mediated, pure chemotaxis of a human neutrophil toward β-glucan.

    Movie S4. Supraphysiological concentrations of C5a or costimulation by shear flow can induce Ca2+ bursts in nonadherent human neutrophils.

  • Supplementary Materials for:

    Extension of chemotactic pseudopods by nonadherent human neutrophils does not require or cause calcium bursts

    Emmet A. Francis and Volkmar Heinrich*

    *Corresponding author. Email: vheinrich{at}ucdavis.edu

    This PDF file includes:

    • Fig. S1. Dual-camera setup for simultaneous recording of bright-field and fluorescence images.
    • Fig. S2. Supraphysiological concentrations of C5a trigger Ca2+ bursts in resting human neutrophils without inducing chemotaxis.
    • Fig. S3. Concurrence of Ca2+ bursts and cellular contraction.
    • Fig. S4. Estimation of the cell surface area.
    • Fig. S5. Effective cortical tension during pure chemotaxis.
    • Legends for movies S1 to S4

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). A Ca2+ burst in a human neutrophil.
    • Movie S2 (.mp4 format). Absence of Ca2+ bursts during complement-mediated, pure chemotaxis of a human neutrophil toward zymosan.
    • Movie S3 (.mp4 format). Absence of Ca2+ bursts during complement-mediated, pure chemotaxis of a human neutrophil toward β-glucan.
    • Movie S4 (.mp4 format). Supraphysiological concentrations of C5a or costimulation by shear flow can induce Ca2+ bursts in nonadherent human neutrophils.

    © 2018 American Association for the Advancement of Science

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