Protein Kinase C Isoforms are Expressed During Intestinal Regeneration in Sea Cucumber Holothuria glaberrima

Defining the Relationship Between Vacuole Size and pHRoberto Carlos Segura1 and Yee-Hung M. Chan, Ph.D.2

1Skyline College2San Francisco State University

2016 SFSU NIH Bridges Summer Research Program

Acknowledgments

Data Collection and Interpretation

MethodsAbstract

Previous studies have shown that the vacuole of a cell is involved in the pH regulation for the

organism. This regulation is critical to the survival of the cell, as many functions in the cytoplasm must

be done at an optimal pH. However, the relationship between capacity for regulation and vacuole size

is still unexplored. Discoveries in this region would result in connections between vacuole size and cell

optimization. We hypothesize three possible correlations: that a large vacuole relates to a low pH, that

a small vacuole relates to a low pH, or that there is no correlation between the two. To identify the

vacuole, S. cerevisiae was transformed with a fluorescence dye that binds to the membrane of the

vacuole, and to observe pH regulation, a pH sensitive dye was localized to the vacuole by cell uptake

and used to determine the internal pH of the organelle. To determine the relationship of vacuole size

to pH, the internal pH of S. cerevisiae was placed under different conditions by placing cell cultures in

solutions of varying pH. These cultures were then incubated and imaged on a confocal microscope.

The resulting images were then processed with an algorithm to determine vacuole size and volume.

Currently, we are processing images and data to determine if a correlation is present or not.

The Vacuole and its Significance

Observing Trends in pH

Conclusions and Future Directions

The work of RCS was supported by the NIH Bridges to the Baccalaureate Program grant 5R25GM050078.

I would like to thank my PI and all the members of my lab. None of the work done would have gone through without their overwhelming support.

Expected Results and Analysis

vac8Δ

Wt

atg18Δ Wt

From these trials, the following can be concluded:● The CFDA uptake of S. cerevisiae is very specific and requires close observation and maintenance to

produce useable measurements.● The procedure described will allow us to determine if there is a relationship between vacuolar size

and pH.

To improve this experiment, the following changes are currently being done:● To simplify the dye uptake, a transformation that makes the vacuolar membrane fluorescence at the

same wavelength as the FM4-64 dye will be done. ● To enhance the significance of the data collected, more calibration will be done to improve the

accuracy of the calibration plot. ● Finally, routine data collection will be done to solidify any implications found in the preliminary data.

The Usage of Dyes

Collection of FM4-64 Images

Collection of CFDA Images Vacuolar pH

Vacuolar Size

+

Data

Cell cultures of type BY4741 yeast were grown overnight, then regrown to an optical density (OD) of 0.6. The yeast were grown in a pH buffered media to influence the conditions inside the cells.

pH Calibration

Once cells reached an OD of 0.6, they were incubated with 10 microliters of CFDA and 0.5 microliters of FM4-64 for one hour and 45 minutes with spinning. After incubation, the cells were resuspended in fresh, dye-free media.

4 microliters were mounted onto slides for observation under a Zeiss Scanning Confocal Microscope. Concanamycin was used to hold cells against the coverslip to aid in observation. Vertical Z-stacks were taken to collect data.

Shown to the right are sample images used in CFDA data collection. Image A correlates with Image B, and Image C correlates with Image D. Notice how in Images A and B, the interior of the cells are darker than their surroundings, while in C and D, there are clusters within the cells that are fluorescing. Images C and D are indicative of a successful dye uptake, while A and B are not. Once a consistent method of dye uptake was achieved, the project was able to move forward. The majority of this project was spent on devising a successful method of dye uptake; some conditions would show no fluorescing, while others would result in inaccurate data.

Sample Images from the Microscope

A.

B.

Once Z-stacks were collected, ImageJ and Matlab software was used to generate measurements about the size, area, and shape of the individual vacuoles.

Similar to the processing of the FM4-64 images, ImageJ was used to generate measurements of the intensities per pixel of the images. This was done by measuring different sections of the image with the same area, which was done to account for the area-based program used on Image J. A ratio between two wavelengths was used.

By measuring CFDA in media that was buffered at set pHs, a calibration plot was made to correlate fluorescence intensity to pH.

Currently, there are three possible hypotheses being tested right now. Data is actively being collected and analyzed. After significant data collection, we would expect to see one of the

following three trends.

While the function and activity of the vacuole have been well studied,1 there is little information about its relation to size. In regards to other organelles, much is known about how size affects function; for example, a large nuclei may be an indicator of disease3, and a liver cell has a large endoplasmic reticulum to account for the high amount of detoxification it must do4. By finding relations between function and size of the organelle, the path towards future discoveries can be set. This study, along with others, aims to set the foundation towards these goals.

1. Cell Cultures Grown 2. Cell Cultures Incubated with Dyes 3. Cells Observed Under a Microscope

1 hr 45 min

Sources1. Nelson, N. (1992). The vacuolar H(+)-ATPase--one of the most fundamental ion pumps in nature. 2. Vanda C., Santos H., Sa-Correia I. (1997). Effect of extracellular acidification on the activity of plasma membrane ATPase and on the

cytosolic and vacuolar pH of Saccharomyces cerevisiae. 3. Webster, M., Witkin, K., & Cohen-Fix, O. (2009). Sizing up the nucleus: nuclear shape, size and nuclear-envelope assembly.4. Endoplasmic Reticulum (Rough and Smooth) | British Society for Cell Biology. (2016).

The main dye used was 6-carboxyfluorescein diacetate, or CFDA (shown above). It expresses a property that is useful in observing vacuolar pH; when it is digested upon entering the vacuole, it enters a state where it can have a carboxylic acid group that is protonated or not, depending on the acidity of the environment. This change in structure affects its fluorescence properties, making it an indicator of pH. When excited, it shines green.

Along with this, a red dye called FM4-64 was used. This compound binds to the vacuoles surface, highlighting its shape and size.

C.

D.

At the cellular level, activities such as protein synthesis and sugar digestion are done under highly regulated conditions. One of these conditions is the pH inside the cell. One notable example of the importance of pH regulation is protein production and expression. If the correct pH is not maintained in the cell, proteins will not be synthesized correctly and may not function properly. Studies have shown that the pH of a cell’s environment can strongly influence the pH inside the cell.2 Other studies have also shown that the vacuole plays a critical role in maintaining the cell’s optimal pH when the cell is in extreme ranges of pH2. Saccharomyces cerevisiae is able to deal with these pressures by the use of its vacuole. Since the cell wouldn’t be optimal if the pH was out of range, the vacuole is used to control internal pH. It is able to do this through a series of proton pumps on its exterior, which are used to intake and expel protons to adjust pH. Along with this function, the vacuole is also used as a storage/digestion center.

Comparison of small vacuoles (left) to large, drug induced vacuoles (right)Credit to Chan and Marshall, Biophys J, 2014

Hypothesis 1:

A large vacuole correlates with a low pH value. This would imply that as a vacuoles volume and size increases, it intakes more protons from the cytoplasm. Conversely, this would imply that a small vacuole has a high pH.

Hypothesis 2:

A small vacuole correlates with a low pH value. This would imply that as a vacuoles volume and size decreases, the cytoplasm becomes more basic. It would also suggest that a large vacuole has a high pH.

Hypothesis 3:

To act as a null hypothesis, we also had to be open to the idea of finding no correlation between pH and size whatsoever. This would imply that pH regulation is controlled by a factor that was not monitored in this study.