Biology

The mechanism and nature of cell motility in Euglena cells was examined through the use of varying intensities and wavelengths of light, as well as detergents, ions and energy substrates.  The degree of cell motility was determined through the percentage of rounded cells in the field of view of the microscope.  The results showed that cell motility in Euglena involved a change in its cell shape from elongated to round.  There was an increase in the number of rounded cells as light intensity increased.  The highest amount of motility was observed when light of 690 nm wavelength was applied.  The photoreceptor response was also deactivated when the cells were treated with detergents, yet re-established when Ca2 ions were introduced.  The addition of ATP to the cell suspension increased the rate of photoreceptor response in the cells.

Introduction
Cell motility is a biological phenomenon that involves a number of protein structures within a cell.  In the case of unicellular organisms, cell motility is achieved through the cytoskeleton, which is a network of structural proteins that interact with each other to generate a highly coordinated action.  Such molecular motors enable a unicellular organism to move, in relation to feeding, reproduction and survival.  This study focused on the unicellular alga Euglena, a flagellate that is commonly found in shallow aquatic ecosystems, such as ponds and pools.  This flagellate alga is also photosynthetic, capable of converting sunlight to a chemical form of energy that is utilized for other cellular processes.  Euglena is also photosensitive, allowing the organism to response to any light stimuli (Gualtieri et al., 1989).  These organisms are also equipped with chloroplasts, which are responsible for capturing energy from sunlight, as well as for detection of light in its immediate environment (Gualtieri et al., 1992).  The alga is also featured with two flagella that are positioned at the reservoir, or the invagination at the subapical region.  An eyespot, filled with rhodopsin pigments, is located at the cytoplasmic space adjacent to the apical flagella (Pasarelli et al., 2003).  In addition to the flagella, a photoreceptor is situated at the base of the flagellum (Gualtieri et al., 1989).    

The photoreceptor of Euglena is a crystal protein composed of approximately 100 layers of crystalline material organized in an intricate manner.  The layers have been determined to be of around 7 nm in thickness, with a consistency of a gel and capable of functioning as a fluid substrate.  The gel nature of the photoreceptor also decreases the chances of its dissolution into liquid environment that are associated with their aquatic ecosystem (Pasarelli et al., 2003).  This nature and structure of the photoreceptor has allowed Euglena to thrive in their common habitat, as well as detect any environmental changes that may influence its growth, development and survival.  This study will investigate the response of Euglena to various environmental stimuli, as well as investigate its nature and mechanism of action.

Materials and methods
All experiments in this study were performed according to the procedures presented in the laboratory manual.  Briefly, Euglena cells were exposed to varying microscope light intensities to determine pellicular movement based on cell shape.  In addition, the nature and location of the photoreceptors of Euglena through exposure of the cells to different wavelengths of light and observing its response based on cell shape.  The nature of the signal-transducing system of Euglena was also determined by permeabilizing the cells with detergent, subsequently exposing these to different dyes and checking for the extent of changes in cell shape.  The nature of the force-generating mechanism was also determined by introducing Euglena cells to ATP and Ca2 observing any changes in its cell shape.

Results

Effect of light intensity on pellicular movement
Exposure of Euglena cells to microscope light of varying intensities showed that the number of rounded cells increased as the light intensity increased (Q1).  This intensity-dependent response to light is graphically presented in Figure 1.  The graph shows that the light reaction is not saturable as the trend line shows a constant increase in the percentage of rounded cells as the light intensities increases (Q2).

2a.  The nature and location of the photoreceptor
Exposure of Euglena to various filters showed that the number of rounded cells increased as the wavelength increased.  The wavelength-dependent response to light is graphically presented in Figure 2.  From the graph, it is apparent that the absorption maximum of the photoreceptor is 690 nm (red) (Q3).

2b.  The colour of the active light signal (action spectrum)
Euglena cells that were excited with blue excitation light emitted a green fluorescence (Q4).  The green fluorescence was observed along the cell membrane (Q5).  This location represents the location of the photoreceptors in the cells.

The nature of the signal-transducing system
3a.  Permeability of the extracted cell model
Mixing the Euglena cell suspension with the extraction solution, containing detergents Triton X-100 and Nonidet P-40, increased the membrane permeability of the cells (Q6).  The control setup, which only involved mixing the cell suspension with the neutral red dye, did not show any staining of the cells, as these were examined under the microscope.  It is also highly likely that ATP and Ca2 ions would diffuse into the cells when the extraction solution is added to the cell suspension.  

3b.  Reaction of the extracted cell model to light
Treatment of cells with the extraction solution and 1 methylcellulose, as well as subsequent exposure to varying light intensity, resulted in the generation of rounded cells.  When the cells were exposed to a low light intensity, approximately 10 of the cells in the field of view of the microscope were rounded.  When the cells were exposed to a high light intensity, around 20 of the cells in the field of view of the microscope were rounded.
The results of this setup suggest that the photoreceptors in cells could have been destroyed or denatured by the detergents present in the extraction solution (Q7).

3c.  Nature of the signal-transducing molecule
The addition of the reactivation solution with Ca2 to the extracted cells showed that approximately 80 of the cells in the field of view of the microscope were rounded.  On the other hand, the addition of a reactivation solution with Mg2 only resulted in approximately 16.6 of cells in the field of view of the microscope as rounded.  Based on the difference in the percentage of rounded cells in the two setups, it is most likely that Ca2 is the messenger in the signal transducing system of Euglena cells (Q8).

Nature of the force-generating mechanism
The addition of ATP to the reactivation solution with Ca2 resulted in the generation of rounded cells in approximately 90 of the cells in the field of view of the microscope.  On the other hand, the addition of ATP to the reactivation solution without Ca2 only generated only 30 of rounded cells among the cells in the field of view of the microscope.

Discussion
The experiments performed in this study were aimed to determine the nature and mechanism of cell motility in Euglena cells.  The initial experiment involved the determination of the photoreceptor response of the cells to various light intensities.  The photosensitivity of the cells was measured by the counting the number of cells that have changed their cell shape from elongated to round.  Increasing the light intensity of the microscope bulb resulted in an increase in the percentage of rounded cells in the field of view of the microscope.  At minimum light intensity or setting 3, only 16.5 of the cells in the field of view of the microscope were rounded.  When the light intensity of the microscope was increased to setting 4, approximately 25.5 of the cells in the field of view of the microscope were rounded.  At setting 6, around 55 of the cells in the field of view of the microscope were observed to have a round shape.  Approximately 60 of the cells in the field of view of the microscope were rounded when the light intensity was increased to setting 8.  At setting 9, 66 of the cells in the field of view were rounded.  At the maximum setting of 10, 80 of the cells in the field of view were rounded.  This photoreceptor response is therefore positively correlated to the intensity of the light employed in the setup.

The nature of the photoreceptor in the cells was determined through the use of different filters that emitted various excitation wavelengths.  At a wavelength of 510 nm (green), approximately 20 of cells in the field of view of the microscope were rounded. The application of a 500 nm wavelength (yellow), on the other hand, resulted in rounded cells in approximately 40 of the cells in the field of view of the microscope.  Using a 620 nm wavelength (orange), 60 of the cells in the field of view showed a round shape.  Approximately 75 of the cells in the field of view of the microscope were round when a 690 nm wavelength (red) light was applied.  These observations are consistent with the fact that every type of photoreceptor has a specific wavelength that it is reactive to (Barsanti et al., 2009 Passarelli et al., 2003).  The exposure of the cells to various wavelengths facilitates in determining the nature of the photoreceptor present in the cells (Evangelista et al., 2003).  Based on the results of the experiment, the highest percentage (75) of rounded cells was observed under the 690 nm wavelength or red light.  This observation suggests that the electrons in the photoreceptors of the cells were excited by the red light and in turn, released an emission wavelength in order to use the energy that was created in this reaction (Frutos et al., 2007).

The emission of green fluorescence in the cells that were exposed to blue excitation light showed the actual location of the photoreceptors within the cell (Iseki et al., 2002).  Since the green fluorescence was observed along the cell membrane, this suggests that the photoreceptors are located within the phospholipid bilayer of the cells.  This location is a good strategic site for the cells, because it allows the photoreceptors to interact with its immediate environment and relay messages to the rest of the cell on whether there is a need to protect itself from harmful wavelengths or to allow a specific wavelength to continuously strike the cell.

The exposure of cells to the extraction solution showed that the photoreceptors were protein in nature.  The detergents present in the extraction solution are capable of denaturing the proteins involved in regulating the transport of nutrients, ions and other chemicals into and out of the cell.  This reaction is validated by the observation of cells staining with neutral red after mixing the cells with the extraction solution.  The control setup of mixing cells with neutral red dye alone showed that the cell membrane remained intact and thus the detergents in the extraction solution were responsible for the increased permeability of cells.  A parallel observation using methylcellulose dye showed that the percentage of rounded cells increased when the extraction solution was added.  This result showed that the photoreceptors are protein in nature and that these macromolecules are denatured when exposed to strong detergents.

The signal transduction mechanism of the photoreceptors was shown by the employment of Ca2 and ATP to the setup.  The addition of Ca2 resulted in the reactivation of the photoreceptors, thus indicating that this ion plays a major role in the photoreceptor response.  The addition of ATP, on the other hand, increases the rate of reaction of the photoreceptor (Mercatelli et al., 2009).  Our observations suggest that the photoreceptors are highly likely to be involved in the mechanism of signal transduction in the cells because the percentage of rounded cells is now very low despite the increase in light intensity.  In addition, the experiment shows that the photoreceptors of the cells are driven by energy, in the form of ATP.

Conclusions
Euglena cells have the capacity to respond to light through the use of photoreceptors.  These protein-bound macromolecules send signals to the cytoskeleton of the cell to move, resulting in a change in the cell shape from elongated to round.  The photoreceptors, as well as the cell membrane, of the cells are destroyed when detergents are added to the suspension.  The destruction of the photoreceptors resulted in the loss of sensitivity of the cells to light.  The addition of Ca2 to the cells reactivates the denatured photoreceptors and returns its capacity to sense any light stimuli in its immediate environment.  The addition of ATP to the cell suspension also increases the reaction speed of the photoreceptors to light sensitivity.