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 Chlamydomonas

The Role of Calcium and Phospholypase C Signaling in Flagellum Regeneration in Chlamydomonas

Tahirah Baksh

Section 2PS2

Introduction

            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.