Syracuse University SURFACE Physics - Dissertations College of Arts and Sciences 2011 Bioenergetics and dynamics of ciliary responses and systems biology of phototaxis in Chlamydomonas reinhardtii Suphatra Adulrattananuwat Syracuse University Follow this and additional works at: https://surface.syr.edu/phy_etd Part of the Physics Commons Recommended Citation Adulrattananuwat, Suphatra, "Bioenergetics and dynamics of ciliary responses and systems biology of phototaxis in Chlamydomonas reinhardtii" (2011). Physics - Dissertations. 111. https://surface.syr.edu/phy_etd/111 This Dissertation is brought to you for free and open access by the College of Arts and Sciences at SURFACE. It has been accepted for inclusion in Physics - Dissertations by an authorized administrator of SURFACE. For more information, please contact [email protected]. Abstract The goal of this dissertation is to understand how a eukaryotic cell makes decisions. Chlamydomonas reinhardtii, a biciliated unicellular green alga, is used as our model organism. This organism has the ability to track the light using its photoreceptor called rhodopsin, which overlays the eyespot. The organism makes decisions to swim toward, away from, perpendicular to or to ignore the light using its slender arm-like structures called cilia. It can integrate several external inputs such as ion concentration and light intensity, and then process this information to adjust the steering of its cilia corresponding to its environment. We investigated how red light (670 nm) influences cell behavior. Most studies were done with a single cell held on a micropipette making it possible to observe the cilia behavior over a long time. The cell is illuminated with near-infrared light (peak at 870 nm) to avoid photoreceptor excitation. Ciliary movement is monitored using a quadrant photodiode detector (Chapter 2). Interpreted ciliary behavior parameters are the beating frequency (BF) and the stroke velocity (SV). Pulse stimuli were used to stimulate the mutant strain 806 (agg1), a negatively phototactic cell, whose beating frequency is in the same range as wild type. The “step-up” red light from the dark increases the beating frequency as an exponential function, y(t) = a[1 − exp(−t / b)] , where y is a beating frequency, a is an amplitude and b is a time constant. On the other hand, the “step-down” red light drops the beating frequency transiently and recovers to its normal beating frequency of about 50 Hz in about 10 s. The 40 s duration pulse gave the maximum transient drop of the beating frequency. Using multi-sinusoidal red-light stimuli, I compared the behavior of the double mutant (cpc1-2) relative to the single mutant 806 (cpc1-2 was backcrossed to strain 806 so it is a single mutant with respect to 806). The mutant misses the part of the central-pair complex containing the enolase enzyme, one of the ciliary glycolytic enzymes that produces ATP in the cilia. Under a high constant- intensity of red light, the BF fluctuation is less than 2%. In the dark, BF of cpc1-2 is about 30 Hz which is lower than 806 probably due to less ATP being available. However, BF can be increased to the 806 level of 50 Hz by exposure to red light. A simple hypothesis is that red-light photosynthesis of the chloroplast makes ATP more available in the cell. In any case, sinusoidal red-light response of cpc1-2 shows that part of the early signal processing is approximately linear. In this case, cells respond to a decrease in light intensity by differentiating the red light signal. Our hypothesis is that the cell creates this signal to avoid futile usage of ATP. The transfer function describing this step is, G(s) = a ∗ s ∗e−τ s where τ = 0.40 sec. In addition to this linear part, both strains have non-linear or approximately full-wave rectified signal processing of another red light created signal with a simple delay in time described by the transfer function, −τ s G(s) = a ∗ e strain where τ806 = 1.18 sec and τcpc1-2= 0.37 sec. The longer delay time of 806 is likely due to the slow conversion of 3-phosphoglycerate (3PG) to adenosine triphosphate (ATP), in the glycolytic pathway, which is absent in the mutant. We hypothesize that the slow synthesis is due to the positive Gibbs free energy of two steps in the ciliary glycolytic pathway between 3PG and production of ATP and pyruvate. Furthermore, the beating frequency of red-light sinusoidal responses is stabilized by negative feedback. However, in the frequency range from 10 to 100 Hz in both strains that stabilizing negative feedback becomes positive and the BF jumps to a new state. In addition, I also studied how external ion concentration such as Ca2+, H+, and K+ and red light affect phototaxis of positively and negatively-phototactic cells (1117 and 806 respectively). I have tracked a cell population using the cell-tracking system for 10 s 2+ after stimulating them with green light (Chapter 4). Increasing [Ca ]ext with a red light background enhances the motion of cells in the same and the opposite direction respectively according to cells’ phototactic behavior under normal condition (pCa4 and pH 6.8). Increasing the pH tends to induce cells to move away from the light while + increasing the [K ]ext gave the opposite results. Changing external ion concentration such as H+ and K+ affects the cell’s membrane potential. Changing Ca2+ concentration affects both membrane potential and likely triggers internal signaling proteins such as IP3 and cAMP. Therefore, we hypothesize that cells may integrate these and potentially other signals to decide its phototaxis. Finally, I developed a technique that can be used to measure changes of the electric field across the plasma membrane of the cell in response to rhodopsin excitation. Rhodopsin excitation is thought to cause transmembrane ion influxes resulting in changes in the electric field across the plasma membrane. These electric field signals are then sensed in the cilia to enable phototactic steering of the cell (Chapter 5). BIOENERGETICS AND DYNAMICS OF CILIARY RESPONSES AND SYSTEMS BIOLOGY OF PHOTOTAXIS IN CHLAMYDOMONAS REINHARDTII by Suphatra Adulrattananuwat M.Sc. Chulalongkorn University, 2003 B.Sc. Prince of Songkhla University, 1998 DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Physics in the Graduate School of Syracuse University May 2011 Approved by Professor Kenneth Foster Copyright 2011 by Suphatra Adulrattananuwat All rights reserved. TABLE OF CONTENTS List of Figures x List of Tables xv Acknowledgements xvi Chapter 1 Introduction 1 Chapter 2 The Opto-Electric Detector 6 2.1 Introduction…………………………………………………………… 6 2.2 Ciliary motility observation techniques………………………………. 7 2.2.1 Holding a single cell……………………………………………... 7 2.2.2 Illuminating the cilia…………………………………………….. 8 2.2.3 Observing ciliary motility and signal processing………………… 10 2.2.4 Stimulating the cell……………………………………………… 12 2.3 Designing the 2nd generation of electro-optical detector………………. 14 2.3.1 The 1st stage: the operation amplifier (op-amp) current to voltage converter……………………………………………………………….. 14 2.3.2 The 2nd stage: the 1st-order high-pass filter……………………… 16 2.3.3 The 3rd stage: the 2nd-order Butterworth low-pass filter………… 16 2.3.4 The last stage: the instrumentation Amplifier (INA101HP).…..... 17 2.3.5 Pin connections of the electro-optical detector system…………... 18 2.4 Data analysis…………………………………………………………... 20 2.4.1 Removal of low frequency and DC drift from recorded data…… 22 2.4.2 Beating-frequency analysis……………………………………… 23 vi 2.4.3 Stroke-velocity analysis…………………………………………. 24 2.5 Creating a stimulus file………………………………………………... 25 2.6 Future improvements and suggestions………………………………… 27 Chapter 3 Red light effects on ciliary motility 28 3.1 Introduction……………………………………………………………. 28 3.1.1 How does each compartment function?...................................... 30 3.1.2 The relationship between mutants (806 and cpc1-2 strain) and wild-type..................................................................................... 48 3.1.3 How does red light play a role in system function?.................... 50 3.1.4 How can we interpret a relationship between input and output?. 51 3.2 Results and Discussions……………………………………………….. 57 3.2.1 Continuous red and 2.5-second-duration pulse of green-light responses of 806 strain…………………………….…………... 57 3.2.2 Duration-pulse responses of 806 strain………………………... 59 • 5 second-duration pulse responses…………………………….. 59 • 40 second-duration pulse responses…………………………… 59 • Comparison of beating-frequency responses between 5 and 40 second-duration pulse…………………………………………. 64 • 5 to 160 second-duration pulse responses…..………………… 65 3.2.3 Besting frequency change in the dark and under DC red light… 67 3.2.4 Multi-sinusoidal responses at the low frequency range from 0.006 to 1 Hz of both 806 and cpc1-2 strain to red light…………….. 71 vii 3.2.5 Multi-sinusoidal responses at the high frequency range from 2 to 100 Hz of both 806 and cpc1-2 strain to red light at 15 W/m2… 88 3.3 Conclusions……………………………………………………………. 94 Chapter 4 Effects of external ion concentrations and red light on phototaxis 97 4.1 Introduction……………………………………………………………. 97 4.1.1 Change of extracellular ion concentration…………………….. 100 4.1.2 Red light as a background light……………………………….. 110 4.1.3 Signaling pathway……………………………………………... 110 4.2 Methods and materials…………………………………………………. 113 4.2.1 Cell preparation………………………………………………... 113 4.2.2 Free Calcium medium preparation…………………………….. 113 4.2.3 Free proton medium preparation………………………………. 114 4.2.4 Cell tracking machine…………………………………………. 115 4.2.5 Data collection and analysis…………………………………… 115 4.3 Results and discussions………………………………………………... 116 4.3.1 Varying external free calcium ion concentration……………… 117 4.3.2 Varying external free hydronium ion concentration…………… 122 4.3.3 Varying external free potassium ion concentration……………. 124 4.3.4 Varying red-light intensity 633 nm…………………………….. 125 4.4 Conclusions……………………………………………………………. 130 Chapter 5 The electric field technique 132 5.1 Introduction…………………………………………………………….