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High-Speed Imaging of Sustained Metabolic Target Patterns in Living Neutrophils during Adherence Howard R. Petty* and Andrei L. Kindzelskii Department of Biological Sciences, Wayne State UniVersity, Detroit, Michigan 48202 ReceiVed: July 19, 2000
Propagation of circular NAD(P)H autofluorescence and proton waves (target patterns) was detected in individual living human neutrophils using high-speed fluorescence microscopy. Waves were initiated at the point where a cell attached to the substrate followed by propagation to the cell’s periphery. The time-dependence of wave propagation and critical radius are in reasonable agreement with theoretical expectations. We suggest that physical-chemical self-organization of metabolism is a fundamental aspect of cell function.
Introduction Chemical systems maintained far from equilibrium can spontaneously develop spatial and temporal chemical concentration patterns, known as dissipative structures.1,2 These spatial patterns often occur in excitable media due to the nonlinear coupling of reaction and transport processes, such as diffusion or convection. The Belousov-Zhabotinskii reaction is one of the most thoroughly studied oscillatory chemical systems. It displays both temporal oscillations and spatial waves.1,2 The Belousov-Zhabotinskii reaction is generally considered as a model for biological systems. Several biological systems display temporal and spatial oscillations as well. Traveling calicum waves have been observed in very large cells such as oocytes and myocytes.3-4 Traveling NADH and proton waves have been observed during oscillatory glycolysis of cell extracts.5-8 We have recently detected traveling NAD(P)H waves and proton waves in neutrophils.9 Neutrophils are an especially good biological model system as they rely predominantly on glycolysis for their energy requirements.10 In the present study we identify conditions that elicit expanding spherical chemical wave fronts, or target patterns, in living cells. We suggest that these chemical dissipative structures are of metabolic origin since they are observed using both NAD(P)H autofluorescence and the emission of an intracellular pH indicator. Using the known diffusion coefficient of NADH and an estimate of wave propagation velocity, we obtain rough agreement between the calculated and measured critical radii for traveling wave initiation. The velocity and amplitude of these self-organized dissipative structures suggest that they may participate in intracellular signaling. Experimental Section described.11,12
Human neutrophils were purified as previously Cells were observed microscopically at 37 °C. To observe cell attachment, cells were seeded onto a substrate on the microscope stage. High-speed image acquisition was performed using an axiovert fluorescence microscope with a quartz condenser, quartz objectives, quartz coverslips, and an AttoArc HBO 100 W mercury lamp (Zeiss Inc., New York, NY). To excite * To whom correspondence should be addressed. E-mail: hpetty@ biology.biosci.wayne.edu.
NAD(P)H autofluorescence, cells were illuminated using a 365 nmWB50 excitation filter, and fluorescence emission was collected using a 400 nm long-pass dichroic mirror and a 450AF58 emission filter (Omega Optical, Brattleboro, VT). The pH sensitive probe 5-(and 6)-carboxy SNARF-1, acetoxymethyl ester, acetate (SNARF-1) (Molecular Probes, Eugene, OR) was detected using a 485/22 nm excitation filter, a 510 nm longpass dichroic mirror, and a 530/30 nm emission filter. To minimize the number of optical components and thereby increase efficiency in light collection, the microscope’s bottom port was employed. The image was collected by a liquid N2cooled intensifier (Gen-II for better response in the blue-violet spectral region) attached to a Peltier-cooled I-MAX-512 camera (approx. -20 °C) (Princeton Instruments Inc., Trenton, NJ). The camera was controlled by a high-speed Princeton ST-133 interface and a Stanford Res. Systems (Sunnyvale, CA) DG535 delay gate generator.9 To improve computer acquisition times, 100 × 100 pixel arrays were employed. A Dell (Round Rock, TX) Precision 410 workstation using Winspec (Princeton) software, 0.5 Gb of RAM, and a high-speed PCI interface (National Instruments, Austin, TX) was employed to capture data. To further enhance image acquisition repetition rates, Winspec calls on the CPU were given system priority and data were acquired without reporting to the monitor. Data were stored as TIFF files for later processing. Although the patterns reported below could be discerned without further computer processing, the images were corrected for the point spread function of the optics using rapid deconvolution (MicroTome software, VayTek, Fairfield, IA). Results and Discussion Cell metabolism is an excitable matrix, as shown experimentally by Hess and colleagues for cell extracts and subsequently extended by our group to living cells.5,9 To study spatiotemporal patterns in cell metabolism, we photographed the autofluorescence of NAD(P)H or the emission of a proton-sensitive dye, SNARF-1, as described.9 Greater than 95% of the neutrophil’s autofluorescence in this spectral region is due to cell metabolism. We use the term NAD(P)H (NADH + NADPH) because it is not possible to distinguish between the emission spectra of NADH and NADPH. The ICCD camera was electronically gated to give 100 ns exposure times. These high speeds overcome the effects of image blurring due to wave motion and blurring
10.1021/jp002551h CCC: $19.00 © 2000 American Chemical Society Published on Web 11/02/2000
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J. Phys. Chem. B, Vol. 104, No. 47, 2000 10953
Figure 1. Time sequence of NAD(P)H autofluorescence images of a neutrophil. Spherical NAD(P)H waves were observed during neutrophil adherence (100 ns shutter speed, 30 ms duty cycle). Cells were unlabeled. The bright NAD(P)H level lingers at the adherence site for some time before propagating throughout the cell’s cytoplasm, as would be expected for this phenomenon.15 In frames 49 to 80, the target pattern evolves into a series of longitudinal waves, which we have previously described for polarized neutrophils.9 The time covered by these images is 2.4 s. This experiment was repeated on six independent days. (X760)
due to molecular diffusion during image acquisition by the CCD chip. For example, a molecule with a diffusion coefficient of ∼10-6 cm2/s would be expected to have an rms displacement of ∼4 nm during a 100 ns image acquisition period. Figure 1 shows a representative series of NAD(P)H autofluorescence images. Unlabeled neutrophils were allowed to adhere to coverslips. A series of images was collected using a 100 ns shutter speed and a 10 ms duty cycle. NAD(P)H autofluorescence target patterns were found to originate from the adherence site. The increase in NAD(P)H brightness at the adherence site is likely due to the local reduction in ATP concentration (presumably due to local activation of signaling cascades, actin assembly, etc.). As illustrated by the first eight frames of Figure 1, waves do not expand at a uniform rate. The
disturbance lingers for ∼100 ms at the adherence site prior to rapid expansion from this site. Inspection of the micrographs reveals a wave velocity of approximately 5 × 10-3 cm/s, which is difficult to estimate due to the small size of these cells. Nonetheless, this estimate is consistent with other determinations of NAD(P)H wave velocity.6,9 Thus, when a metabolic perturbation reaches a critical size, a spherical metabolic wave then propagates throughout the cell. Following a series of NAD(P)H autofluorescence waves, cells spread uniformly on the substrate to form a circle at their perimeter. Several control experiments were performed to confirm the nature of these autofluorescence waves. In the presence of the metabolic inhibitor 2-deoxy-D-glucose (100 mM), cellular autofluorescence in this spectral region and autofluorescence waves
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Figure 2. Time sequence of fluorescence images of a SNARF-1-labeled neutrophil. Spherical pH waves were observed during neutrophil adherence (100 ns shutter speed, 15 ms duty cycle). SNARF-1 fluorescence varies in a spatiotemporal fashion analogous to that found for NAD(P)H autofluorescence described above. The time covered by these images is 1.0 s. Data are representative of those obtained on 4 independent days. (x 840)
could not be detected (data not shown). Similarly, cell spreading on the substrate was also abolished. Furthermore, if the cells were killed by chemical fixation with paraformaldehyde, waves of NAD(P)H autofluorescence could not be observed (data not shown). The enzyme phosphofructokinase plays a key role in the formation of spatiotemporal glycolytic patterns.13,14 We therefore sought to examine the spatial distribution of phosphofructokinase in these cells to control for the possibility that NAD(P)H patterns are due to an underlying pattern of enzymes. We found that phosphofructokinase was uniformly distributed within cells under all conditions. Thus, NAD(P)H patterns are linked to the metabolic activity of living cells, but not to the spatial distribution of the key enzyme involved in the feedback activation and inhibition of metabolism. To provide further evidence supporting the formation of metabolic target patterns, intracellular pH was studied. Proton generation is known to temporally oscillate during glycolysis.14 Cells were labeled with SNARF-1 at 50 ng/mL for 50 min at 37 °C in suspension. Cells were washed then transferred to microscope chambers. The imaging system was triggered to record data immediately prior to cell adherence to the substrate. Figure 2 shows a series of high-speed SNARF-1 images of a cell. As anticipated, fluorescent target patterns were again
visualized using high-speed CCD gating. The features of pHassociated fluorescence target patterns recapitulated those of NAD(P)H, including width, velocity and orientation with respect to an adherence site on a cell. Thus, cell adherence is characterized by both NAD(P)H and pH patterns. The eikonal equation for wave propagation in an excitable medium is
N ) c + DK
(1)
where N ) normal velocity, c ) plane wave velocity, D ) diffusion coefficient, and K ) curvature.15 The target patterns observed in this study, such as those illustrated in Figures 1 and 2 for NAD(P)H and SNARF-1 emission, can be analyzed by this equation. The effect of curvature on wave front velocity is noticeable only at small radii. After reaching a critical radius (rcr), the wave expands rapidly to reach the velocity c. The diffusion coefficient of NADH has been reported to be 7 × 10-6 cm2/s in phosphate buffer.16 This value certainly overestimates that of NADH in living cells because it neglects (1) the reversible binding of NADH to other intracellular components, (2) the viscosity of the cytoplasm, and (3) percolation through or around the numerous intracellular structures. In a
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similar vein, the diffusion coefficient of calcium in cells is ∼10fold smaller than that measured in salt solutions.17 Nonetheless, using this known value of D and an estimate of wave velocity from the micrographs, the value of rcr can be calculated as
rcr ) D/c
(2)
which gives a value of 14 µm. The observed size of rcr is approximately 1 to 1.5 µm. Given the assumptions listed above and the fact that eq 2 overestimates the critical radius in the Belousov-Zhabotinskii reaction at small radii,15 we feel that the order of magnitude agreement of these estimates constitutes reasonable agreement between current theory and experiments. Thus, the lag time during initiation and the size of the critical radius are not inconsistent with physical theory. In the present study we have observed intracellular biological redox waves (NAD(P)H and NAD(P)+) that bear a striking resemblance to the redox waves found during the BelousovZhabotinskii reaction. We have observed spherical waves propagating from an initiation core. We have previously noted the presence of longitudinal NAD(P)H waves in elongated neutrophils.9 Thus, cell shape (spherical vs elongated) correlates with the dissipative metabolic patterns observed (circular vs longitudinal). Theoretical analyses of dissipative structures in cell shape have been reported18 and may now be accessible to experimental analysis. The initiation center is identical to the adherence site or focal contact as determined by morphological criteria. Although the detailed chemistry at the initiation core is uncertain, it is true that this region is rich in adherence proteins such as integrins, signaling kinases, and related proteins. Hence, one would expect greater local turnover of ATP and, consequently, enhanced local levels of its conjugate metabolite NAD(P)H,14 thus causing bright autofluorescence at this site. The self-organization of cell metabolism described above may provide a physical mechanism explaining the coherence of biochemical reactions in living cells. In neutrophils, numerous biochemical reactions and physiological processes are known to oscillate at the same frequency as temporal metabolic oscillations.19 These include, for example, the enzymatic production of O2- and NO,12,19,20 the coherent interactions of cell surface proteins,11 the assembly of intracellular filaments, and the timing of kinase activation and surface proteolytic action.11,12 The spatiotemporal waves meet the requirements of an intracellular signaling mechanism. First, they occur faster than cell spreading on a surface (msec vs sec, respectively). Second, we have recently shown for polarized (elongated)
neutrophils that dissipative structures change in response to receptor ligation, suggesting a role in signaling.21 Third, these waves appear to be of sufficient magnitude to influence enzymatic reactions; thus, propagating waves of NAD(P)H, protons, ATP, etc. may switch enzyme activities and physiological functions on and off with specific patterns at specific times relative to other biochemical pathways. We are now developing a system to simultaneously monitor substrate (i.e., NAD(P)H) and product (i.e., O2-) levels at high speed to test the spatial and temporal coherence of these reactions in living cells. The unification of nonequilibrium thermodynamics, biochemistry, and cell physiology is likely to have important ramifications. Acknowledgment. This work has been supported by NIH grant CA74120. We thank Dr. R. Kemp for the generous gift of the anti-phosphofructokinase antibody. References and Notes (1) Nicolis, G.; Prigogine, I. Exploring Complexity; W. H. Freeman & Co.: New York, 1989. (2) Scott, S. K. Oscillations, WaVes, and Chaos in Chemical Kinetics; Oxford University Press: Oxford, U.K., 1994. (3) Wussling, H. P.; Salz, H. Biophys. J. 1996, 70, 1144. (4) Lechleiter, J. D.; Clapham, D. E. Cell 1992, 69, 283. (5) Boiteux, A.; Hess, B. Ber. Bunsen-Ges. Phys. Chem. 1980, 84, 392. (6) Mair, T.; Mu¨ller, S. C. J. Biol. Chem. 1996, 271, 627. (7) Mu¨ller, S. C.; Mair, T.; Steinbock, O. Biophysical Chem. 1998, 72, 37. (8) Shinjyo, T.; Nakagawa, Y.; Ueda, T. Physica D 1995, 84, 212. (9) Petty, H. R.; Worth, R. G.; Kindzelskii, A. L. Phys. ReV. Lett. 2000, 84, 2754. (10) Roos, D.; Balm, A. J. M. In The Reticuloendothelial System: A ComprehensiVe Treatise; Sbarra, A. J., Strauss, R. R., Eds.; Plenum Press: NY, 1980. (11) Kindzelskii, A. L.; Eszes, M. M.; Todd, R. F., III; Petty, H. R. Biophys. J. 1997, 73, 1777. (12) Kindzelskii, A. L.; Zhou, M. J.; Haugland, R. P.; Boxer, L. A.; Petty, H. R. Biophys. J. 1998, 74, 90. (13) Goldbeter, A. Biochemical Oscillations and Cellular Rhythms; Cambridge University Press: Cambridge, U.K., 1996. (14) Hess, B.; Boiteux, A. Annu. ReV. Biochem. 1971, 40, 237. (15) Tyson, J. J.; Keener, J. P. Physica D 1988, 32, 327. (16) Wu, Z.; Jing, W.; Wang, E. Electrochem. Comm. 1999, 1, 545. (17) Tillotson, D.; Nasi, E. Biophys. J. 1985, 47, 735. (18) Nicolis, G.; Prigogine, I. Self-Organization in Nonequilibrium Systems. John Wiley & Sons: New York, 1977. (19) Petty, H. R. In Self-Organized Biological Dynamics and Nonlinear Control by External Stimuli; Walleczek, J., Ed.; Cambridge University Press: Cambridge, U.K., 2000; Chapter 7. (20) Adachi, Y.; Kindzelskii, A. L.; Ohno, N.; Yadomae, T.; Petty, H. R. J. Immunol. 1999, 163, 4367. (21) Petty, H. R.; Kindzelskii, A. L. Proc. Natl. Acad. Sci., U.S.A. 2000, submitted for publication.
