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Am J Physiol Regul Integr Comp Physiol 292: R2020-R2027, 2007. First published February 22, 2007; doi:10.1152/ajpregu.00649.2006
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COMPARATIVE AND EVOLUTIONARY PHYSIOLOGY

Evidence for the role of G-proteins in flow stimulation of dinoflagellate bioluminescence

Antony K. Chen,1 Michael I. Latz,2,* Peter Sobolewski,1 and John A. Frangos1,*

1La Jolla Bioengineering Institute, La Jolla, California; and 2Scripps Institution of Oceanography, University of California San Diego, La Jolla, California

Submitted 14 September 2006 ; accepted in final form 14 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Luminescent dinoflagellates respond to flow by the production of light. The primary mechanotransduction event is unknown, although downstream events include a calcium flux in the cytoplasm, a self-propagating action potential across the vacuole membrane, and a proton flux into the cytoplasm that activates the luminescent chemistry. Given the role of GTP-binding (G) proteins in the mechanotransduction of flow by nonmarine cells and the presence of G-proteins in dinoflagellates, it was hypothesized that flow-stimulated dinoflagellate bioluminescence involves mechanotransduction by G-proteins. In the present study, osmotic swelling of cells of the dinoflagellate Lingulodinium polyedrum was used as a drug delivery system to introduce GDPbetaS, an inhibitor of G-protein activation. Osmotically swollen cells produced higher levels of flow-stimulated bioluminescence at a lower threshold of shear stress, indicating they were more flow sensitive. GDPbetaS inhibited flow-stimulated bioluminescence in osmotically swollen cells and in cells that were restored to the isosmotic condition following hypoosmotic treatment with GDPbetaS. These results provide evidence that G-proteins are involved in the mechanotransduction of flow in dinoflagellates and suggest that G-protein involvement in mechanotransduction may be a fundamental evolutionary adaptation.

osmotic drug delivery; shear stress; Lingulodinium

ALL CELLS LIVE IN A FLUID environment where the mass transfer of nutrients, other chemical agents, and dissolved gases to cells is mediated by flow. In aquatic systems, flow also affects the spatial distribution of planktonic organisms with respect to their physical, chemical, and biological environment. Both mammalian (17) and nonmammalian eukaryotic cells (26–28, 31, 40, 58) exhibit a range of adaptive responses to flow, including changes in morphology, growth, metabolism, and viability.

A dramatic expression of flow response is the bioluminescence of dinoflagellates, protists that are common members of the plankton. Flow-stimulated dinoflagellate bioluminescence is associated with high shear conditions (31, 33–36) such as the boundaries and wakes of swimming animals (44) and ships (45), breaking waves (50), and agitated bioreactors (9). This flow response is shear-stress dependent (Maldonado EM and Latz MI, unpublished data).

The flow-stimulated intracellular signaling pathways associated with dinoflagellate bioluminescence are incompletely known. Mechanochemical signal transduction is mediated by changes in intracellular calcium (54), and light emission is mediated by an action potential propagated along the vacuole membrane (14, 56) that allows proton flux from the vacuole to cytoplasm. The resultant pH drop acidifies vesicles containing the luminescent chemistry, activating the luciferase that catalyzes the luminescent reaction (57). However, the primary signaling events involved in sensing flow remain to be identified.

By comparison, extensive studies of mammalian endothelial cells have provided some insight into how cells sense and respond to flow, and several mechanisms for the mechanotransduction process have been proposed (10). One proposed mechanism involves shear forces acting on the cell, increasing the fluidity of the plasma membrane (22), resulting in activation of heterotrimeric GTP-binding proteins (G-proteins) (21). According to this scheme, the cell membrane and G-proteins act as flow sensor and transducers, respectively.

A similar mechanism may function in dinoflagellates. Flow induces changes in fluidity of the plasma membrane of dinoflagellates (38) in similar fashion as mammalian endothelial cells (22), and dinoflagellates are known to contain G-proteins (52). However, the role of G-proteins in the mechanotransduction of flow by dinoflagellates has not been investigated. We propose here that in dinoflagellates flow may modulate membrane fluidity and G-protein activation leading to changes in the calcium-mediated bioluminescence pathway. In this study, we present the first evidence for the involvement of G-proteins in flow-stimulated dinoflagellate bioluminescence. As dinoflagellates are some of the most basal eukaryotes, G-protein involvement in mechanotransduction may be a fundamental evolutionary adaptation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and preparation. Cultures of the dinoflagellate Lingulodinium polyedrum strain HJ were grown in half-strength f/2 medium minus silicate in an environmental chamber maintained at 20 ± 0.5°C on a 12:12-h light-dark cycle. On each day of testing, cell abundance was calculated by counting samples under a dissecting microscope. Experiments were performed using cultures in the exponential phase of growth (6–15 days after inoculation; concentrations of 1,500–12,000 cells/ml).

Because dinoflagellate bioluminescence exhibits a circadian rhythm, with maximum expression at night, all experiments began 3–6 h into the dark phase, when stimulated bioluminescence is maximal (4, 32). At the end of the light phase, when cells are mechanically inexcitable, subsamples of the cultures were prepared for testing. Before each experiment, the appropriate amount of testing solution and cells was transferred under dim light conditions into the testing apparatus to obtain a final concentration of 1,000 cells/ml. All experiments were performed at room temperature.

Media and chemicals. Full-strength seawater (SW) filtered to remove particles ≥5 µm was originally obtained at the Scripps Pier, University of California, San Diego. MilliQ water (MQ) was used to prepare 10% and 20% diluted seawater (90% SW and 80% SW). Concentrated seawater (CSW) was prepared by dissolving 52 g/l of Instant Ocean (Aquarium System) in MQ. Reconstituted seawater (RSW) was prepared from 80% SW and CSW at a 1:1 ratio. GDPbetaS, a nonhydrolyzable GTP analog that inhibits G-protein activation (15), was purchased from Calbiochem. MQ or SW working stocks of 5 mM GDPbetaS were used within 4 h after preparation.

Effect of osmolality on dinoflagellate size. Changes in dinoflagellate size upon osmotic manipulation were determined using a Coulter Multisizer II cell counter. Each experiment was performed with appropriate amount of cells in a testing medium at 20-ml final volume. Cells were incubated in either SW, 90% SW, 80% SW, or RSW. Dinoflagellates were incubated for 5 min in one of the solutions, except for the RSW treatments, in which dinoflagellates were first exposed to 80% SW for 5 min followed by another 5 min incubation after the addition of CSW at 1:1 ratio. Each experiment was replicated 3 or 4 times with a fresh dinoflagellate suspension. Each replicate measured ~6,000 cells and expressed cell size as equivalent spherical diameter.

The osmolarity of each solution was measured in triplicate with an osmometer (Advanced Micro-osmometer model 3MO plus, Advanced Instruments) using freshly prepared solutions and was expressed as the mean (SD).

Flow device and agitation protocol. A cone-plate viscometer (model DVII+Pro, Brookfield Engineering) was used with a stainless-steel cone (model CPE-40, Brookfield Engineering) and a custom manufactured cup containing 0.5-ml working volume. The cone was 2.4 cm in diameter with a shallow angle of 0.8°. The bottom of the cup was manufactured using a translucent polycarbonate of 0.5 cm thickness to allow bioluminescence measurements from underneath, while the rest of the cup was made of black Delrin. The maximum and minimum gaps between the cone and plate were 348 and 13 µm, respectively.

Cone-plate viscometers have previously been used to study cellular response to shear stress (5, 13, 24, 53). Provided that the fluid between the cone and the plate is Newtonian and the flow is laminar, the shear stress ({tau}) is identical at every point within the fluid, as well as on the cone and plate surfaces and is expressed as {tau} = (µ{omega}/{theta}), where µ is the fluid dynamic viscosity, {omega} is the angular velocity, and {theta} is the cone angle. The time (t) for diffusion of the boundary layer in which a new shear stress distribution is established in the gap is t = ({delta}2/{nu}), where {delta} is the gap size and {nu} is the fluid kinematic viscosity (46). On the basis of the maximum gap size in the viscometer of 348 µm, the shear stress distribution across the gap became fully developed 0.12 s after the onset of flow. Rotational speed of the cone was computer controlled using the manufacturer's software (Rheocalc ver. 2.4, Brookfield Engineering). All flow protocols started with a step increase in rotational speed to a steady level for 20 s. All experiments were performed at rotational speeds of 5, 10, 15, 20, and 30 rpm corresponding to shear stress levels of 0.5, 1, 1.5, 2, and 3 dyn/cm2, respectively. The Reynolds number (Re) of the cone-plate flow was calculated as Re = (r2{omega}{theta}2/12{nu}), where r = cone radius. For the rotational speeds used in this study, Re was <0.5, which confirmed that the flow field was laminar and secondary flows were negligible (47).

Bioluminescence measurements. Bioluminescence was measured in a custom-designed light-tight chamber using a photon-counting photomultiplier detector (Electron Tubes model P30232 [GenBank] ) positioned 10 cm below the bottom of the cup. The field of view of the detector encompassed the entire diameter of the polycarbonate plate. The light signal was attenuated to 10% transmission with a 1.0 neutral density filter (Kodak Wratten #96) to avoid saturation of the detector. Light readings were acquired based on 10-ms integrations. On each day of testing, background light levels in the absence of cells were measured. In addition, measurements were performed with cells in the absence of flow to establish the baseline response. Analysis of bioluminescence responses was based on the 20-s total integrated bioluminescence after flow onset.

Effect of cell size on shear-stimulated bioluminescence. Flow tests were performed after dinoflagellates were incubated in SW, 90% SW, or 80% SW for 5 min. Bioluminescence responses were acquired at shear stress levels of 0.5, 1.0, 1.5, 2, and 3 dyn/cm2 with 10–13 replicates. For both 90% SW and 80% SW treatments, bioluminescence responses were acquired at shear stress levels of 0.5, 1.0, and 1.5 dyn/cm2 with 6–24 replicates.

Effect of GDPbetaS on shear-stimulated bioluminescence. Flow tests were performed after dinoflagellates were incubated in SW or 80% SW, with or without GDPbetaS, for 5 min. The following six solutions were tested: 1) SW, 2) SW + 200 µM GDPbetaS, 3) SW + 1 mM GDPbetaS, 4) 80% SW, 5) 80% SW + 200 µM GDPbetaS, and 6) 80% SW + 1 mM GDPbetaS. Bioluminescence responses were acquired at shear stress levels of 0.5, 1.0, or 1.5 dyn/cm2 with 6–24 replicates. Comparisons between bioluminescence responses in different media were made at the same shear stress levels.

Effect of GDPbetaS on osmotically reconstituted cells. Viscometer experiments were performed after dinoflagellates were first incubated in 80% SW with different GDPbetaS concentrations for 5 min, followed by another 5 min incubation after the addition of CSW at a 1:1 ratio. Dinoflagellates were exposed to one of the following media: 80% RSW, 80% RSW + 200 µM GDPbetaS, and 80% RSW + 1 mM GDPbetaS. Bioluminescence was measured at a shear stress of 2.0 dyn/cm2 with 11–17 replicates.

Assay for total luminescent capacity. The capacity of cells to produce light, that is, the total luminescent capacity (TLC), was assayed by acidification, in which pH < 6 activates the luminescent chemistry in dinoflagellates (16). A Sirius luminometer (Berthold Detection Systems) was used to add a 250-µl volume of 1 M acetic acid at pH 4.7 to samples while light emission was measured for 120 s. TLC was measured in cells treated with SW, 80% SW, and 80% SW + GDPbetaS with 4 or 5 replicates and expressed in relative light units per unit time per cell.

Statistical analysis. Unless otherwise stated, all values are expressed as means ± SE. All replicates are of independent samples. Statistical analyses were performed using StatView 5.0.1 (SAS Institute). ANOVA followed by Fisher's paired least significant difference post hoc test for pairwise comparisons were used to determine the differences in osmolarity, cell size, TLC, and shear-stimulated bioluminescence. Student's t-test was used to determine whether responses to shear stresses were significantly different from the baseline bioluminescence. Results from experiments performed on separate days were pooled. Statistical significance was taken at the P < 0.05 level.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Dinoflagellates are impermeable to GDPbetaS. If flow induces G-protein activation in dinoflagellates, leading to bioluminescence stimulation, then treatment with the nonhydrolyzable GTP analog GDPbetaS may inhibit bioluminescence. However, there was no effect of 200 µM and 1 mM GDPbetaS treatment on the shear-stimulated bioluminescence of SW cells (Fig. 1A); at a shear stress of 1.5 dyn/cm2 (Fig. 1B), neither 200 µM nor 1 mM GDPbetaS resulted in a significant change in bioluminescence (ANOVA, F = 1.488, P = 0.242). These results indicate that dinoflagellates are largely impermeable to GDPbetaS and proceeded to use osmotic loading to facilitate GDPbetaS uptake.


Figure 1
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  		Fig. 1. Effect of GDPbetaS on the flow-stimulated bioluminescence of Lingulodinium polyedrum. A: cells were exposed to fluid shear stresses of 0.5, 1.0, or 1.5 dyn/cm2 in one of the following conditions: seawater (SW; bullet), SW + 200 µM GDPbetaS ({blacksquare}), SW + 1 mM GDPbetaS ({blacktriangleup}). Each value represents the averaged 20-s integrated bioluminescence as a result of 5–24 separate experiments. Lines represent the least squares power regression. The regression equations are SW, y = 3,308 x1.008, R2 = 1.000; SW + 200 µM GDPbetaS, y = 2,719 x1.260, R2 = 0.803; SW+ 1 mM GDPbetaS, y = 3,488 x1.531, R2 = 0.978. Bioluminescence at the lowest shear stress level was similar to the background light levels. B: summary for cells exposed to a shear stress of 1.5 dyn/cm2 in one of the following conditions: SW, SW + 200 µM GDPbetaS, and SW + 1 mM GDPbetaS. Each point represents the mean ± SE of 20-s integrated bioluminescence as a result of 5–24 separate experiments. The treatments did not result in any significant differences in bioluminescence response (ANOVA, F = 1.488, P = 0.242).

 
80% SW treatment affects dinoflagellate size and shear sensitivity. The osmolality of the 90% SW and 80% SW solutions was decreased as expected by dilution, while that of the CSW was ~17% higher than for SW (Table 1). The osmolality of RSW was not significantly different from that of SW.


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  		Table 1. Osmolality of the test solutions

 
Dinoflagellate size increased with decreasing osmolality as expected due to cell swelling (Fig. 2A); this change was reversible upon osmolarity reconstitution (Fig. 2B). On the basis of the size distributions, the equivalent spherical diameter was 36.7 ± 0.2 µm for SW, 37.3 ± 0.3 µm for 90% SW, 39.2 ± 0.9 µm for 80% SW, and 36.9 ± 0.2 µm for RSW. The differences in size were statistically significant (ANOVA, F = 14.195, P = 0.0009). The size of dinoflagellates treated in 80% SW was significantly different and 5% larger than those in 90% SW (P = 0.002), 7% larger than for SW (P = 0.003), and 6% larger than for RSW (P < 0.001). There was no significant difference in size between cells in 90% SW and SW (P = 0.294) and between cells in SW and RSW (P = 0.714). Similar trends in size were also observed based on microscope and flow cytometer measurements (data not shown). Overall, dinoflagellate size was altered by the osmolality of the medium.


Figure 2
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  		Fig. 2. Effect of osmolality changes on the cell size of L. polyedrum. A: distribution of cell size. Results of four representative experiments are shown. Approximately 6,000 cells were measured for each experiment. There was a shift in cell size distribution upon osmolality modulation. B: summary of cell size. Values represent the mean (SD) of 3 or 4 experiments. *P < 0.05 compared with the 90% SW, SW, and RSW groups.

 
In addition to altering dinoflagellate size, the osmolarity of the medium altered shear sensitivity. As expected for isotonic conditions (31), bioluminescence in the SW treatments increased according to the 1.8 power of shear stress (Fig. 3). A similar relationship was present for the 90% SW treatment, with an identical regression slope and elevation to that for the SW condition. However, for the 80% SW treatment, the bioluminescence was markedly increased. The minimum shear stress required to elicit bioluminescence significantly different from background levels in the absence of flow was 2.0 dyn/cm2 for the SW treatment, 1.5 dyn/cm2 for the 90% SW treatment, and 0.5 dyn/cm2 for the 80% SW treatment. Thus flow sensitivity was enhanced by the hypotonic treatment.


Figure 3
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  		Fig. 3. Effect of osmotic swelling on shear-induced bioluminescence of L. polyedrum. Cells were sheared in one of the following conditions: SW (bullet), 90% SW ({blacksquare}), and 80% SW ({blacktriangleup}) for 20 s. Shear stress levels were 0.5, 1.0, 1.5, 2.0, and 3.0 dyn/cm2. Each value represents the average and SE of 20 s integrated bioluminescence based on 6–24 separate experiments, while the lines represent the least squares power regression. The regression equations are SW, y = 4,201 x1.786, R2 = 0.912; 90% SW, y = 4,163 x1.849, R2 = 0.977; 80% SW, y = 17,890 x1.112, R2 = 0.969. *P < 0.05; at shear stress levels of 0.5, 1.0, and 1.5 dyn/cm2, the bioluminescence of cells exposed to the 80% SW treatment was higher and significantly different than that from the 90% SW and SW treatments, which were similar to each other.

 
There was no effect of osmolality treatment on bioluminescence capacity. TLC of cells treated in 80% SW was 87.4 ± 5.3 RLU·s–1·cell–1 (n = 4), not significantly different from that in SW of 93.2 ± 4.3 RLU·s–1·cell–1 (n = 5) (t7 = 1.749, P = 0.124).

GDPbetaS inhibits shear-stimulated bioluminescence in osmotically swollen cells. In cells swollen by 80% SW, GDPbetaS treatment inhibited shear-stimulated bioluminescence (Fig. 4A). At a shear stress of 1.5 dyn/cm2 (Fig. 4B) treatment with 50 µM GDPbetaS inhibited bioluminescence by 28%, but the change was not significant (P = 0.130). For the 200-µm GDPbetaS treatment, bioluminescence was inhibited 75% and was significantly different from the 80% SW treatment without GDPbetaS (ANOVA, F = 4.843, P = 0.006). Thus shear-stimulated bioluminescence was inhibited by GDPbetaS, but only when cells were osmotically swollen from the 80% SW treatment.


Figure 4
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  		Fig. 4. Effect of GDPbetaS on the flow-stimulated bioluminescence of osmotically swollen cells of L. polyedrum. A: cells were exposed to fluid shear stresses of 0.5, 1.0, or 1.5 dyn/cm2 in one of the following conditions: SW (bullet), 80% SW + 50 µM GDPbetaS ({blacksquare}), and 80% SW + 200 µM GDPbetaS ({blacktriangleup}). Each value represents the average and SE of 20-s integrated bioluminescence based on 5–24 separate experiments, with the lines representing the least squares power regression. The regression equations are 80% SW, y = 17,890 x1.112, R2 = 0.969; 80% SW + 50 µM GDPbetaS, y = 13,043 x1.368, R2 = 0.884; 80% SW + 200 µM GDPbetaS, y = 6,509 x0.356, R2 = 0.494. Bioluminescence at the lowest shear stress level was similar to the background light levels. *P < 0.05; at shear stresses of 1.0 and 1.5 dyn/cm2, the responses of the 200 µM GDPbetaS treatment were lower and significantly different from those for the other conditions, which were not significantly different from each other. B: summary for cells exposed to a shear stress of 1.5 dyn/cm2 in one of the following conditions: 80% SW, 80% SW + 50 µM GDPbetaS, and 80% SW + 200 µM GDPbetaS. Each point represents the mean ± SE of 20-s integrated bioluminescence as a result of 5–24 separate experiments. *P < 0.05 compared with the 80% SW group.

 
There was no significant difference in TLC for 80% SW cells (87.4 ± 5.3 RLU·s–1·cell–1, n = 4) and the 80% SW treatment with 1 mM GDPbetaS (86.9 ± 1.3 RLU·s–1·cell–1, n = 4) (t6 = 0.184, P = 0.860). Thus the differences in shear-stimulated bioluminescence were not due to changes in bioluminescence capacity.

GDPbetaS inhibits shear-stimulated bioluminescence in osmotically reconstituted cells. Because GDPbetaS decreased shear-stimulated bioluminescence in 80% SW-treated cells but did not have an apparent effect on SW-treated cells, it was hypothesized that osmotic swelling facilitated GDPbetaS entry that subsequently inhibited shear-stimulated bioluminescence. However, hypotonic treatment also increased cell size and enhanced flow sensitivity. Thus the goal of experiments with dinoflagellates that underwent osmotic reconstitution was to allow GDPbetaS entry into cells during hypotonic treatment but prior to reconstitution of isotonic conditions when cell size and presumably shear sensitivity would return to that for SW conditions. The effect of GDPbetaS on osmotically reconstituted cells was tested at a shear stress of 2 dyn/cm2 (Fig. 5). GDPbetaS treatment had a significant effect on shear-stimulated bioluminescence (ANOVA, F = 8.557, P = 0.009). For the 200-µM GDPbetaS treatment (n = 11), the 15% decrease in bioluminescence was not significantly different (P = 0.417) from SW controls (n = 17). For the 1-mM GDPbetaS treatment (n = 11), bioluminescence was decreased 72% from RSW controls; this difference was significant (P < 0.001). Shear-stimulated bioluminescence from the 1 mM GDPbetaS treatment was also significantly different from that of the 200-µM treatment (P = 0.006). Thus GDPbetaS that entered the cells during hypotonic treatment was retained in reconstituted cells and inhibited flow-stimulated bioluminescence.


Figure 5
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  		Fig. 5. Bioluminescence response to GDPbetaS by osmotically reconstituted cells of L. polyedrum. Cells were subjected to a shear stress of 2.0 dyn/cm2 for 20 s. Values represent the means ± SE of 11–17 replicates. *P < 0.001 compared with the RSW control group.

 
There was no significant difference in TLC for RSW cells compared with the SW control (t8 = 0.158, P = 0.158). Thus the inhibition of shear-stimulated bioluminescence by GDPbetaS was not due to the RSW treatment.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study is unique in exploring the role of G-proteins in shear-stimulated dinoflagellate bioluminescence. The strong inhibition of bioluminescence response by GDPbetaS, a G-protein inhibitor, suggests that G-proteins play a pivotal role in the mechanochemical signal transduction pathway of dinoflagellate bioluminescence. Furthermore, the observation that osmotic swelling increased bioluminescence in response to shear suggests that changes in membrane fluidity may play a role in shear sensitivity. These treatments had no significant effect on bioluminescence capacity, suggesting that the results were specific to the mechanosensory system and not due to general toxicity.

Although treatment of dinoflagellates with GDPbetaS alone was ineffective, this is likely to be due to the inability of the inhibitor to effectively enter the cell. The permeability of mammalian cells to guanine nucleotides is known to be low, requiring either long-duration exposures (30) or permeabilization (7) to facilitate uptake. The entry of GDPbetaS into dinoflagellates is likely to be even further stymied, because as members of the protist lineage Alveolata, dinoflagellates such as L. polyedrum possess multiple peripheral membrane layers, including a submembrane layer of cellulose-filled vesicles (thecae), which is likely to further impede the diffusion of nucleotides. In other alveolates, guanine nucleotides can be introduced only with permeabilization (3). To facilitate GDPbetaS entry into dinoflagellates, hypotonic loading was used. The uptake of ions and other charged molecules in dinoflagellates generally occurs via active transport mechanisms (11), and, in mammalian cells, similar transporters are volume sensitive (51). Hypotonic swelling has been shown to increase uptake of nucleotides in rat colonic cells (18) and to increase permeability to ions and mannitol in the alga Platymonas subcordiformis (29). Hypotonic swelling by 80% SW likely facilitates GDPbetaS uptake by L. polyedrum via a similar ion transporter mechanism, thereby allowing it to modulate intracellular G-protein function. Exposing L. polyedrum to GDPbetaS in 90% SW did not result in any inhibitory effect; however, this treatment did not swell the cells. This observation supports the hypothesis that volume-sensitive transporters may be facilitating GDPbetaS uptake. The ability of GDPbetaS, a G-protein inhibitor, to decrease shear-stimulated bioluminescence strongly suggests that G-proteins are involved in the signal transduction pathway of fluid shear stress in dinoflagellates.

An unexpected consequence of hypotonic loading of GDPbetaS was the observation that exposure to hypotonic solution alone had a marked effect on bioluminescence. Treatment of cells with 80% SW, which caused cell swelling, resulted in dramatically increased sensitivity to shear stress, leading to increasing levels of shear-stimulated bioluminescence. One possible mechanism to explain this phenomenon is an increase in membrane fluidity upon swelling. In both liposomes and plant protoplasts (6), as well as mammalian endothelial cells (23), membrane fluidity increases in response to hypotonic treatment. Fluid shear stress also increases membrane fluidity in both dinoflagellates (38) and mammalian endothelial cells (22), suggesting that it may be involved in the mechanochemical transduction of fluid shear stress. Further, the basal activity of G-proteins reconstituted in liposomes is modulated by membrane composition, with increased membrane fluidity resulting in increased G-protein activity in liposomes (21). Together, these observations suggest that hypotonic treatments of dinoflagellates increase shear sensitivity by increasing G-protein activity via an increase in membrane fluidity. An alternative explanation would be that hypotonic treatments result in the translocation of G-proteins from the Golgi or cytoplasm to the membrane, as observed in the alga Dunaliella salina (39). If this is the case, then the presence of additional G-proteins in the membrane may result in increased mechanotransduction in response to shear stress. Finally, intracellular calcium is involved in the signal transduction of shear-stimulated bioluminescence in L. polyedrum (54) and hypotonic treatments of mammalian cells can increase intracellular calcium by both calcium mobilization from intracellular storage (41), as well as influx of calcium (48). However, this is not the case in the alga P. subcordiformis, in which intracellular calcium levels are not significantly affected by hypotonic exposure (29). In this context, it is possible that swelling caused by 80% SW may have resulted in an increase in intracellular calcium levels, leading to increased bioluminescence; however, this is unlikely because swelling occurs over a much longer time scale than the calcium flux associated with luminescent flashes.

To verify that the hypotonic treatments were actually facilitating the loading GDPBbetaS into the cells, the inhibition experiments were repeated following reconstitution of solution osmolarity to that of SW. Because inhibition of shear-stimulated bioluminescence by GDPBbetaS persisted, despite the cell size returning to normal following osmotic reconstitution, we conclude that the hypotonic treatments served to facilitate GDPbetaS loading. This finding may be useful in the study of the effects of other large and/or charge molecules on dinoflagellates, which may otherwise be limited by uptake barriers.

Heterotrimeric G-proteins are involved in signal transduction of all eukaryotes, including unicellular organisms (43). There is growing evidence for the role of G-proteins in sensory transduction of members of the Alveolata, protists including dinoflagellates and ciliates that are some of the most basal eukaryotes. G-proteins are involved in phototransduction in ciliates, which contain a G-protein similar to that of mammalian transducin (49). The presence of G-proteins in ciliates and the action of guanine nucleotides on Ca2+ influx and motility suggests that voltage-dependent Ca2+ channels involved in swimming behavior are modulated by G-proteins (2, 3, 12). Furthermore, guanine nucleotides, such as GDPbetaS, affect mechanosensitivity in ciliates (37).

Here, we provide the first evidence of the involvement of G-proteins in the signal transduction of shear-stimulated bioluminescence of dinoflagellates. In mammalian endothelial cells, shear stress causes the rapid activation of G-proteins (20) and increase in fluidity of the plasma membrane (8, 22). Additionally, fluid shear stress can directly activate G-proteins reconstituted in liposomes (21), suggesting that G-proteins can act as the primary flow transducers. Our finding that G-proteins are important in the bioluminescence in dinoflagellates, some of the most basal eukaryotes, provides additional evidence that G-protein involvement in mechanotransduction is a fundamental evolutionary adaptation. In this context, the mechanism by which G-proteins are involved in shear-stimulated bioluminescence of dinoflagellates is still unclear. However, one possible explanation involves fluid shear stress causing changes in membrane fluidity, directly activating G-proteins embedded in the membrane and leading to changes in intracellular calcium, which would then mediate dinoflagellate bioluminescence (54). Bioluminescence is inhibited by BAPTA-AM, which chelates intracellular Ca2+, and Ruthenium Red, which prevents Ca2+ release from intracellular stores (54). In mammalian cells, G-proteins are known to modulate release of calcium from intracellular stores via inositol triphosphate (42). Further, both plants and animals are known to have G-protein-dependent calcium channels (25), and there is evidence of the existence of flow-sensitive calcium channels in endothelial cells (1). If shear stress were directly activating a calcium channel, it is not clear how G-protein inhibition would be able to inhibit bioluminescence; thus, our results support the notion that in dinoflagellates G-protein activation is one of the first, if not the primary, responses to shear stress, with intracellular calcium changes being a downstream event. However, the precise interplay between shear stress, G-proteins, and intracellular calcium remains to be fully elucidated in both dinoflagellates as well as mammalian cells.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a National Heart, Lung, and Blood Institute Grant HL-40696 (to J. A. Frangos) and a National Science Foundation Grant BES9730782 (to M. I. Latz and J. A. Frangos).


   ACKNOWLEDGMENTS
 
We wish to thank B. G. Mitchell for use of the Coulter counter, H. van der Heyde, J. Nolan, and H. Stevens for valuable assistance in flow cytometer and microscope usage, and H. Sahlin and R. Contreras for technical support and useful comments on the system design.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Frangos, La Jolla Bioengineering Institute, 505 Coast Blvd. South, La Jolla, CA 92037 (e-mail: frangos{at}ljbi.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* M. I. Latz and J. A. Frangos shared senior authorship. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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