Complex photoreceptor pathways exist in algae to exploit light as a sensory stimulus. Previous studies have implicated calcium in blue-light signaling in plants and algae. A photophobic response to high-intensity blue light was characterized in the marine benthic diatom Navicula perminuta (Grunow) in van Heurck. Calcium modulators were used to determine the involvement of calcium in the signaling of this response, and the fluorescent calcium indicator Calcium Crimson was used to image changes in intracellular [Ca²⁺] during a response. A localized, transient elevation of Calcium Crimson fluorescence was seen at the cell tip at the time of cell reversal. Intracellular calcium release inhibitors produced a significant decrease in the population photophobic response. Treatments known to decrease influx of extracellular calcium had no effect on the population photophobic response but did cause a significant decrease in average cell speed. As the increase in intracellular [Ca²⁺] at the cell tip corresponded to the time of direction change rather than the onset of the light stimulus, it would appear that Ca²⁺ constitutes a component of the switching mechanism that leads to reversal of the locomotion machinery. Our current evidence suggests that the source of this Ca²⁺ is intracellular.

Abbreviations:

CPA: cyclopiazoic acid
E.R.: endoplasmic reticulum
FDA: fluorescein diacetate
IP₃: inositol trisphosphate
IP₃R: inositol trisphosphate receptor
PLC: phospholipase C

Motile responses to light stimuli can yield physiological advantages for organisms, such as maximizing photosynthetic efficiency and rates of cell reproduction (Senger and Schmidt 1994, Underwood et al. 2005). Many types of motile microalgae have been observed to accumulate in areas irradiated by light of moderate fluence rates but disperse from areas of high fluence rates, which could cause photodamage (Underwood et al. 2005, Waring et al. 2007). An obvious macroscopic result of this is the light-dependent appearance and disappearance of thick mats of diatom-dominated biofilms on the surface of estuarine intertidal mudflats (e.g., Consalvey et al. 2004). These benthic diatoms inhabit an environment where physical and chemical conditions change constantly, yet very little is known about which environmental stimuli diatoms can perceive and how they respond and adapt to the changing conditions (Falciatore et al. 2000).

Motility in raphid, pennate diatoms involves the secretion of mucilage strands from the raphe, a slit running the length of each valve (Edgar and Pickett-Heaps 1983, Lind et al. 1997, Higgins et al. 2003), and cytoskeletal reorganization involving actin and myosin (Edgar and Zavortink 1983, Poulsen et al. 1999). When in contact with a surface, movement involves a series of forward and reverse gliding motions, with progression of the diatom across the substratum resulting when the glide in one direction lasts longer than the other (Apoya-Horton et al. 2006). Experiments examining timing of direction changes (Nultsch and Wenderoth 1973, Cohn et al. 2004) and angle of travel (McLachlan et al. 2009) in individual diatom cells show that phototaxis in diatoms is the result of a series of photophobic responses in which the time between direction changes is prolonged if the cell is moving in a beneficial direction and curtailed if the cell is moving in a unfavorable direction. This type of phototaxis, characterized by changes in the autonomous rhythm of reversal, contrasts sharply with the active steering exhibited by many flagellates (Nultsch 1985), does not allow orientation along the light vector, and only works if a steep light gradient is present (Jékely 2009). Similar responses have been seen with chemotaxis in some species of diatoms (Cooksey and Cooksey 1988).

We have recently shown that the raphid, benthic diatom N. perminuta will move down a light gradient away from areas of high illumination, and that blue light between wavelengths 430 and 510 nm produces directional responses (McLachlan et al. 2009). Blue light has previously been implicated in directed movements in diatoms—Craticula cuspidata showed maximal light sensitivity at ∼500 nm (Cohn and Weitzell 1996, Cohn et al. 1999), and Nitzschia communis exhibited phototaxis between 335 and 550 nm with wavelengths beyond 550 nm being ineffective (Nultsch 1971). Light quality experiments suggest that the same photoreceptor(s) are used for positive movement as for negative movement (Cohn et al. 1999, McLachlan 2007). The mechanisms by which changes in the light regime are relayed from the light receptor(s) to the cytoskeleton and translated into motile responses are unknown.

However, a membrane-associated microfilament system involving actin and a pair of actin cables just beneath the plasma membrane at the raphe have been identified, disruption of which prevents motility (Poulsen et al. 1999, see also Molino and Wetherbee 2008 for recent review of diatom motility). This model of diatom motility involving actin and myosin requires Ca²⁺ to generate motive force. Consistent with this, reduction of external Ca²⁺ has previously been shown to reduce population motility in a marine diatom (Cooksey and Cooksey 1980, Cooksey 1981).

Calcium (Ca²⁺) is a ubiquitous intracellular messenger in eukaryotic signal transduction and can regulate a diverse range of processes, including motility, secretion, and muscle contraction (Berridge et al. 1998, Gomperts et al. 2002, Bothwell and Ng 2005). Evidence is now also emerging for calcium playing a role in diatom signaling of environmental stimuli. Signaling systems based on changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) have been implicated in diatom responses to stress (Falciatore et al. 2000, Vardi et al. 2006), and there is also evidence that Ca²⁺ is involved in chloroplast motility in centric diatoms (Makita and Shihira-Ishikawa 1997) and that certain raphid diatoms require calcium to regulate their movement (Cooksey and Cooksey 1980, Cohn and Disparti 1994). As with all eukaryote cells, [Ca²⁺]_cyt is maintained at ∼100 nM (Brownlee et al. 1987), and elevations above this have been demonstrated in Phaeodactylum tricornutum in response to a variety of stimuli (Falciatore et al. 2000).

In this study, we have used the long-wavelength fluorescent Ca²⁺ indicator Calcium Crimson, which is excited by a longer wavelength than that which induces the photophobic response (Richter et al. 2001), to visualize fast, highly localized, transient elevations in [Ca²⁺]_cyt that correspond with the sudden changes in direction associated with the photophobic response to high irradiance blue light in N. perminuta.

Materials and Methods

Cultures. A unialgal, nonaxenic culture of the benthic, pennate diatom N. perminuta, from the University of Essex culture collection, was maintained at 15°C, 12:12 light:dark (L:D), and 50 μmol·m⁻²·s⁻¹ in 90 mm petri dishes with media consisting F/2 nutrients (http://ccmp.bigelow.org) in Milli-Q^® water (Millipore, Watford, UK). Salinity was adjusted to 20 ‰ using aquarium salts (Tropic Marin, Wartenberg, Germany; this also added 5.6 mM Ca²⁺ [Atkinson and Bingman 1998]). Experimental chambers were created by cutting a 1 cm diameter hole in the base of a small petri dish and fixing a glass coverslip across the underside with Sylgard (Dow Corning, Wiesbaden, Germany). This produced a small well into which 500 μL of cell suspension could be placed; sample dishes were then covered and returned to the culture room overnight to allow cells to fully establish themselves on the coverslip.

Fluorescence imaging. The simultaneous monitoring of [Ca²⁺]_cyt during a photophobic response required the use of a Ca²⁺ indicator that could be excited by light at a wavelength that did not elicit a photophobic response. This precludes the use of a range of calcium indicators (e.g., fura-2) that would allow ratiometric monitoring of [Ca²⁺]_cyt. Calcium Crimson was chosen in this study as it has excitation and emission maxima at 590 and 615 nm, respectively, well clear of the 430–510 nm range for a directional response in N. perminuta. Although the use of Calcium Crimson allowed rapid changes in [Ca²⁺]_cyt to be monitored with confidence during a photophobic response, the use of a single wavelength excitation dye can only provide estimates of resting [Ca²⁺]_cyt. Richter et al. (2001) correlated relative fluorescence changes with the published relative calcium-concentration-dependent fluorescence changes of Calcium Crimson. This method has been used here to relate in vivo fluorescence changes in N. perminuta cells loaded with Calcium Crimson to changes in [Ca²⁺]_cyt. Calcium Crimson was excited at 568 nm delivered from a krypton/argon laser, and emission was monitored at 605 nm ± 10. Fluorescent images were acquired with a Bio-Rad (Hemel Hempsted, UK) 1024 Confocal Scanning Laser Microscope using LaserSharp v3.2 (Bio-Rad) software and analyzed using ThumbsPlus v5.01-R (Cerious Software Inc., Charlotte, NC, USA) and Scion Image vBeta 4.0.2 (Scion Corporation, Frederick, MD, USA).

Cells were incubated with 5 μM Calcium Crimson AM ester (Invitrogen, Paisley, UK) in F/2 media for 30 min prior to experiments. To image Calcium Crimson fluorescence during the photophobic response, a high-intensity light spot was produced in the field of view by focusing the beam from a KL200 cold light source with fiber optic light guide (Schott AG, Mainz, Germany) through the epifluorescence port of a Nikon Diaphot inverted microscope (Nikon Instruments Europe B.V., Badhoevedorp, the Netherlands) fitted with a 450–490 nm filter. The spot was 30–40 μm diameter at the coverslip (about two to three times the length of an N. perminuta cell) and was calculated to have an intensity of >1,000 μmol·m⁻²·s⁻¹ using the focusing power of the objective (×40/1.3 oil) and the intensity of the beam leaving the light guide. The blue-light spot was positioned to one side of the field of view and switched off. Cells were imaged as they approached the position of the light spot, which was then switched on for as long as the cell was in the field of view, exposing them to a steep gradient of high-intensity blue light.

To verify the cytoplasmic localisation in N. perminuta, cells were loaded with 2 μM fluorescein diacetate (FDA) for 5 min before fresh medium was added and intracellular FDA distribution was imaged by excitation at 488 nm and emission monitored at 522 nm ± 10.

Inhibitor treatments. Normal Ca²⁺ homeostasis was altered using the ionophore 4-bromo-A23187 (4 μM), the endoplasmic reticulum (E.R.) calcium pump inhibitors cyclopiazonic acid (CPA; 15 μM), and thapsigargin (4 μM), the phospholipase C (PLC) inhibitor U73122 (100 nM), the calcium channel blocker verapamil (20 μM; all from Sigma, Dorset, UK), and low-Ca²⁺ medium. Low-Ca²⁺ medium was prepared by adding F/2 nutrients to an artificial seawater preparation of 259 mM NaCl, 5.5 mM KCl, 14 mM MgCl₂.6H₂O, and 15.6 mM MgSO₄.7H₂O in autoclaved Milli-Q water. EGTA was not added as it has been shown to affect the ability of diatoms to attach to surfaces (Cooksey and Cooksey 1980). Free Ca²⁺ was estimated to be ∼25 μM. To determine the effects of the inhibitor treatments on the photophobic response, a blue-light spot was produced as mentioned above, and the microscope stage controls were used to move the sample until an actively moving cell could be positioned so that the spot was about three to five cell lengths ahead of the moving cell. The cell was then allowed to approach the spot. Fifty cells were scored for their behavior as either “reverse” or “through” (referring to cells that continued moving in the same direction regardless of the light field). Occasionally (less than once per 50-cell experimental run), a cell would stop but not reverse or continue in its original direction; this atypical behavior was disregarded, and an alternative cell selected. After scoring cells under control conditions, the medium was removed and replaced with a treatment medium for 5 min, after which 50 cells were again scored for their behavior. These experiments were repeated in triplicate. Student’s t-test was used to compare the number of cells that changed direction before and after treatment. To determine the effects of the inhibitor treatments on cell speed, a Hamamatsu Orca C4742-95 cooled CCD camera operated with Wasabi v1.5 software (Hamamatsu Photonics UK Ltd., Welwyn Garden City, UK) was used to record cell movements. Recordings were made for 1 min before and 10 min after the medium was changed. Each experiment was repeated three times. Image files were analyzed with Image ProPlus v5.0 (Media Cybernetics, Marlow, UK), as detailed previously (McLachlan et al. 2009).

Results

Evaluation of Calcium Crimson indicator. Cells loaded with Calcium Crimson exhibited a variety of fluorescence patterns (Fig. 1). Healthy, actively moving cells always had a similar appearance in the bright-field images and always showed the same characteristic pattern of fluorescence. Confocal sections acquired at the base of the cell (i.e., near the valve face that was in contact with the substratum) revealed a distinct, central line of fluorescence along the length (tip to tip) of the cells (Fig. 1b). This line was always visible in actively moving cells (n > 40). When the focus was in the middle of the cell, a rim of fluorescence could be seen around the perimeter of the cell with diffuse fluorescence in the center of the cell. When the focus was near the top of the cell, a central line of fluorescence was apparent along the length of the cell, although this was not as distinct as the one near the bottom of the cell. These central lines of fluorescence correspond to the position of the raphe. FDA allowed the overall distribution of the cytoplasm to be visualized (Fig. 1c). Fluorescence was seen at the tips of the cell and in the middle (corresponding to the perinuclear region in Navicula; e.g., Edgar and Pickett-Heaps 1982), with no increased fluorescence observed in the raphe region or around the cell periphery. This pattern suggests a central, cytoplasm-rich zone and also cytoplasm-rich areas at the tips of the cell. The pattern of FDA fluorescence was similar in all images, whether the focus was at the bottom, middle, or top of the cell.

Details are in the caption following the image — **Figure 1**
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Intracellular distribution of fluorescent dyes. Comparison of bright-field images with Calcium Crimson fluorescence images (a) showed that in healthy, actively moving cells (1) and stationary cells (2), there was diffuse fluorescence throughout the cell; whereas cells with altered intracellular contents (3) showed increased fluorescence and were often immobile, and cells with severely disrupted intracellular contents (4) showed extremely high fluorescence and were probably dead. Cells lying on their girdle (5) showed a line of increased fluorescence at the girdle. The three different patterns of fluorescence described in the text (b); (i) lower half of cell (i.e., nearest the substratum), (ii) middle, (iii) near top. (c) FDA fluorescence is seen in the interior of the cell, at the tips and middle, (i) near top, (ii) middle, (iii) lower half of cell. Scale bars = 5 μm.

The addition of 4-bromo-A23187 caused a gradual, but significant, increase in Calcium Crimson fluorescence throughout the cell, which saturated after ∼5 min. (Fig. 2a). The areas of highest fluorescence were at the cell tips and in the center. However, a similar relative 3-fold increase was also seen in the areas of lower fluorescence (Fig. 2b). This fits well with the published data (Haugland 2002) and suggests that [Ca²⁺]_cyt reached values >1.3 μM in response to 4-bromo-A23187. Treatment with 4-bromo-A23187 caused increased Calcium Crimson fluorescence at the tips and in the center, corresponding to areas that fluoresced with FDA, suggesting the dye was distributed throughout the cytoplasm and that changes in Calcium Crimson fluorescence represent a reliable indicator of changes in [Ca²⁺]_cyt in these experiments.

[Ca²⁺]_cyt changes during a photophobic response. Significant localized transient elevation of [Ca²⁺]_cyt coincident with a blue-light-induced direction reversal was seen in 5 of 15 responsive cells, when focus was near the top of the cell. Within a few seconds of exposure to the light stimulus, a discrete area of increased Calcium Crimson fluorescence could be seen at the leading tip of the cell, peaking at the time of direction change (Fig. 3 shows a representative example). When focus was near the bottom (n = 19) or near the middle of the cell (n = 12), no increases in fluorescence during a photophobic response were detected. This localized transient elevation of fluorescence appeared 4.0 ± 0.4 s after the onset of the light stimulus, with a mean 1.5-fold increase in fluorescence (±0.16) and was significantly different from the relative change in fluorescence at the other time points (analysis of variance with Tukey’s pair-wise comparison, P = 0.007, df = 6). Based on the published relative increase in dye fluorescence at saturating [Ca²⁺]_cyt (Haugland 2002; verified in Fig. 2), the elevation in Calcium Crimson fluorescence corresponded to [Ca²⁺]_cyt of 152–272 nM, with a mean of 179 nM (±23, Fig. 4).

Effects of calcium antagonists on the photophobic response and cell speed. N. perminuta cultures showed a very strong reversal response to a focused spot of blue light (450–490 nm), with 85%–90% of cells reversing (18 experiments, n = 50 for each; Fig. 5a). Reducing external [Ca²⁺] (low-Ca²⁺ media) and blocking influx channels (verapamil) did not cause any significant change in the population photophobic response (P > 0.05 for both). In contrast, interfering with intracellular Ca²⁺ homeostasis (A23187, U73122, CPA, thapsigargin) caused a significant decrease in the photophobic response of the population (P < 0.05 for all).

Cells treated with low extracellular Ca²⁺ and verapamil showed significantly lower speeds than the control (P < 0.05; Fig. 5b). However, disrupting intracellular Ca²⁺ homeostasis with A23187, U73122, CPA, and thapsigargin did not significantly reduce mean cell speed.

It would seem safe to presume that, over the time course of our experiments, blocking influx of Ca²⁺ was not having a knock-on effect on internal stores as the response to the influx inhibitors was completely separate to that of the stores inhibitors. Submaximal concentrations of inhibitors were used such that cell speed was significantly reduced, but not prevented, as motility was required for the photophobic response assay.

Discussion

The results presented herein support the role of Ca²⁺ in maintaining motility in pennate diatoms and indicate further that Ca²⁺ influx rather than release from intracellular stores underlies this process. Moreover, elevated Calcium Crimson fluorescence observed in the raphe region provides additional support for localized, increased [Ca²⁺] associated with motility. The sensitivity of cell speed to external Ca²⁺ and verapamil suggests that voltage-activated Ca²⁺ permeable channels are present in N. perminuta. Experiments on chloroplast movement in diatoms also found verapamil treatment to have the same effect as lowering extracellular [Ca²⁺] (Makita and Shihira-Ishikawa 1997). Molecular evidence for Na⁺/Ca²⁺voltage-activated ion channel homologues in nonraphid diatoms has recently been reported (Taylor 2009, Verret et al. 2010), indicating key roles of plasma membrane Ca²⁺channels in diatom sensory and signaling mechanisms.

N. perminuta exhibited a strong, highly reproducible phobic response to high-intensity blue light. Established inhibitors of E.R. Ca²⁺ pumps (CPA and thapsigargin) significantly reduced the frequency in the number of cells exhibiting a photophobic response, suggesting that the presence of functional intracellular Ca²⁺ stores was necessary for blue-light-induced direction reversal. In addition, the PLC inhibitor U73122 reduced the frequency of response, suggesting that PLC is also necessary for the response and implying that inositol trisphosphate (IP₃) may be involved. Although IP₃ has been shown to be involved in Ca²⁺signaling in plant cells, there is no evidence of IP₃ receptor (IP₃R) orthologues in plants or most algae (Hetherington and Brownlee 2004, Nagata et al. 2004, Bothwell and Ng 2005), with the exceptions of Ectocarpus siliculosus and Chlamydomonas reinhardtii (Cock et al. 2010, Verret et al. 2010). The Thalassiosira pseudonana and P. tricornutum genomes do not appear to possess sequences homologous to IP₃R (Verret et al. 2010). However, IP₆ can also stimulate Ca²⁺release from endomembrane stores in plants, leading to the suggestion that IP₃ may not be generated or perceived in the same way as in animal cells (Bothwell and Ng 2005).

Decreasing the [Ca²⁺] in the external medium and using a Ca²⁺-channel blocker had no significant effect on the photophobic response, implying that any Ca²⁺ necessary for this response may come primarily from intracellular stores rather than via influx across the plasma membrane. It may seem counterintuitive that A23187 (an ionophore that increases [Ca²⁺]_cyt) caused a decrease in the photophobic response. However, if light intensity is sensed separately at each pole to determine the direction of motility (e.g., Nultsch and Wenderoth 1973, Cohn et al. 1999), and if this difference in light intensity is encoded by spatially distinct [Ca²⁺]_cyt changes, then the global increases in [Ca²⁺]_cyt induced by the ionophore would interfere with spatial patterns of [Ca²⁺] critical for phototransduction.

A transient but significant increase in [Ca²⁺]_cyt was seen near the cell apex when the cell changed direction in response to photophobically active light. Our current evidence suggests that the source of this Ca²⁺ is intracellular. Experiments involving partial illumination of freshwater diatom cells suggest that it is the tips of cells that are involved in light sensing (Nultsch and Wenderoth 1973, Cohn et al. 1999), although the exact mechanisms are unknown. In the present study, the peak-localized [Ca²⁺]_cyt elevation corresponded to the time of direction change, rather than the onset of the light stimulus. This finding suggests that [Ca²⁺]_cyt constitutes a component of the switching mechanism that leads to reversal of the locomotion machinery. It has previously been found that some motile diatom cells adhere to the substrate at the posterior terminal pore and that if the cell is plasmolyzed at the posterior pore movement ceases (Drum and Hopkins 1966). It has also been observed that the reversal of direction coincides with the reversal of streaming in the (formerly) anterior raphe (Edgar and Pickett-Heaps 1984). The elevation of [Ca²⁺]_cyt coincident with the switch from anterior to posterior pole suggests that the [Ca²⁺]_cyt transient could be involved in switching direction of streaming in the anterior raphe. In the current experiments, the area that showed the increase in [Ca²⁺] was about to become the most important area for maintaining motility. It is possible that light-stimulated Ca²⁺ dynamics at the anterior pole could cause differential activity in the two raphe-localized actin cables initiating a change in orientation. Alternatively, as Ca²⁺ can play a role in regulating secretion, then the observed increase could be to instigate or alter secretion into the raphe.

In conclusion, we have shown increased Calcium Crimson fluorescence in the raphe area and that decreasing influx of extracellular calcium causes a significant decrease in average cell speed. We have also shown that functional intracellular Ca²⁺ stores are necessary for blue-light-induced direction reversal and that during this photophobic response, a discrete, transient elevation of Calcium Crimson fluorescence can be seen at the cell tip. As the increase in intracellular [Ca²⁺] corresponds to the time of direction change rather than the onset of the light stimulus, it would appear that Ca²⁺ constitutes a component of the switching mechanism that leads to reversal of the actin–myosin–driven locomotion machinery found in the pennate diatoms.

Acknowledgments

We are grateful to Dr. John Bothwell (Queens University, Belfast) for helpful comments on this manuscript. This research was funded by a NERC CASE studentship (D. H. M., number NER/S/A/2002/10364) held at the University of Essex and the Marine Biological Association of the UK.

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Citing Literature

Volume48, Issue3

June 2012

Pages 675-681

CALCIUM RELEASE FROM INTRACELLULAR STORES IS NECESSARY FOR THE PHOTOPHOBIC RESPONSE IN THE BENTHIC DIATOM NAVICULA PERMINUTA (BACILLARIOPHYCEAE)¹

Abstract

Abbreviations:

Materials and Methods

Results

Discussion

Acknowledgments

References

Citing Literature

Figures

References

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CALCIUM RELEASE FROM INTRACELLULAR STORES IS NECESSARY FOR THE PHOTOPHOBIC RESPONSE IN THE BENTHIC DIATOM NAVICULA PERMINUTA (BACILLARIOPHYCEAE)1

Abstract

Abbreviations:

Materials and Methods

Results

Discussion

Acknowledgments

References

Citing Literature

Figures

References

Related

Information

CALCIUM RELEASE FROM INTRACELLULAR STORES IS NECESSARY FOR THE PHOTOPHOBIC RESPONSE IN THE BENTHIC DIATOM NAVICULA PERMINUTA (BACILLARIOPHYCEAE)¹