The Role of Calcium and Phospholypase C Signaling in Flagellum Regeneration in Chlamydomonas
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The Role of Calcium and Phospholypase C Signaling in Flagellum Regeneration in ChlamydomonasTahirah BakshSection 2PS2Introduction All organisms discovered on planet earth have been classified into three domains based on scientific observations of the organisms’ characteristics. These three domains are Eukaryota, Prokaryota (eubacteria), and Archaea. Each domain consists of organisms that may have similar features to organisms in another domain, but largely possess unique characteristics of their own. For instance, both eukaryotic cells and prokaryotic cells possess flagella, but the structure and mode of action of eukaryotic flagella differs significantly from that of prokaryotic flagella. Prominent scientists such as Lynn Margulis, who proposed the serial endosymbiotic theory (SET), have speculated upon the emergence of these differences but have not come to a definite conclusion. However, scientists can agree on the growth of flagella because flagellar regeneration can be easily observed under a microscope. While some eukaryotic organelles are inherited maternally and divide to reproduce, other organelles like flagella are built up and assembled from their component parts. (Lab Manual 2016). Eukaryotic organisms like Chlamydomonas can be experimentally induced to shed its flagella. Chlamydomonas are unicellular, biflagellate eukaryotes; they are excellent laboratory models because they can be manipulated and are known for their photosynthetic ability, motility and simple genetics. This simple cell removes its flagella in response to environmental stress, such as an acid shock or mechanical shear (Sineshchekov, 1999). Chemicals can be introduced to mechanically shear off the flagella; however, this lab uses the pH shock method for flagellar detachment. In this method, the pH level of the Chlamydomonas cell drops with the introduction of an acid. After approximately a minute, the pH is returned to neutral when a base is introduced. This method allows for flagellar regeneration after flagellar excision. Flagellar growth can be assayed by easily observing living cells with a phase contrast microscope on a 40x objective lens. The purpose of these labs is to focus on the effects of colchicine, Ca2+ ions, and neomycin on Chlamydomonas. Colchicine is an alkoid that prevents microtubule assembly; in turn, this inhibits reflagellation. It binds itself to tubulin monomers, preventing them from assembling into microtubules. In addition, colchicine promotes the disassembly of existing microtubules through a tubulin-colchicine complex, which can add to the growing end of the microtubule, but prevents the further addition of tubulin molecules. This destabilizes the microtubule structure and causes it to disassemble. In both experiments, colchicine treatment was used as a negative control. Therefore, when counting the Chlamydomonas cells under the microscope, colchicine treated tubes should show no change until more than 30-40 minutes after; numbers should be initially high and constant, and then somewhat dwindle down towards the end to show reflagellation starting to happen.
Calcium is a largely abundant mineral in many organisms. One of its main functions is to regulate intracellular signals between tissues and to regulate certain physiological functions. According to the lab manual, Chlamydomonas needs extracellular calcium ions in order to swim again. Calcium ions act as second messengers in IP3 signaling; IP3 binds to InsP3 receptor to open the calcium ion channel and allowing for calcium ions to enter the cytoplasm. Cultures suspended in a TAP medium with calcium should exhibit Chlamydomonas regenerating their flagella and swimming, ultimately causing a decrease in the number of non-motile cells. However, EGTA is a calcium chelator. A chelator is a substance whose molecules can form multiple bonds to a single metal ion. Because it binds so readily to free calcium ions, it can be used as a buffer and causes the number of calcium ions in the cytoplasm to decrease. When added to a TAP medium, it binds to calcium, making the TAP calcium-free. Since calcium promotes flagella regeneration, cells treated with EGTA will not swim. Lastly, neomycin is an amino glycoside antibiotic that inhibits deflagellation of Chlamydomonas by interacting with the enzyme-substrate complex. A deflagellated cell has increased levels of inositol 1, 4, 5- triphosphate; this compound activates phospholipase C, which helps the cell regenerate its flagella. (Quarmby, 1992) Neomycin prevents the hydrolysis of phosphatidylinositol 4,5-bisophosphate, and therefore, inhibiting the production of inositol 1,4,5-triphosphate and subsequent deflagellation. Ultimately, neomycin inhibits the role of phospholipase C, which should result in a higher number of non-motile cells. Materials and Methods For these experiments, initial protocols had to be followed in order to ensure consistency and proper technique. Three processes were followed throughout the experiments in certain microcentrifuge tubes: acid shock, neutralization, and washes. Acid shocks were used to deflagellate the Chlamydomonas cells. For the acid shock, 24µL of sodium acetate (1 M, pH 4) was added to designated microcentrifuge tubes. Chlamydomonas are capable of quick regeneration of their flagella after acid-induced flagellar abscission. The sodium acetate would effectively cause an abrupt decrease in the pH level of the culture medium, causing the Chlamydomonas cells to shed their flagella within seconds. After waiting 70 seconds, neutralization was done to designated centrifuge tubes. During this process, 48µL of potassium hydroxide (0.5M) was added to the acid. Lastly, three cell washes were done to every centrifuge tube prior to placing the tubes under the high-intensity lights and the hemocytometer slide for counting. To wash the cell, the top solution was removed after centrifugation and the cells were re-suspended in a certain medium depending on the treatment that the centrifuge tube was undergoing. In experiment 1, the effect of calcium was measured. We had 4 test tubes in total. The first three test tubes received acid shock treatment, neutralization, and cell washings. Test tube 4 didn’t receive any acid treatment. All 4 tubes are centrifuged for 10 seconds, followed by a supernatant removal and addition of 1 mL of distilled water. Centrifugation and washes were done and the tubes received their designated treatments: Tube 1: Distilled water; Tube 2: 1mL of 3mg/mL solution of colchicine; Tube 3 and 4: 1mL of mixed solution of TAP and 100µM of EGTA (calcium-free TAP medium). After receiving their designated solutions, the tubes were placed under a lamp to promote regeneration. All plants need to optimize their exposure to light by growing toward it. In this case, Chlamydomonas swim towards the light. During 10-minute intervals for 1 hour, 10μL of cell suspensions of every tube were mounted onto the hemocytometer and the number of non-motile cells were counted.