Avsievich T.I., Abdulkarim S.N., Proskurin S.G. Tambov State Technical University, Russia

1. Tambov State Technical University Biomedical engineering department Spectral characteristics of P.polycephalum plasmodium endoplasmic shuttle motility using sing-sensitive velocity registration Avsievich T.I., Abdulkarim S.N., Proskurin S.G. Tambov State Technical University, Russia tavsievich@tamb.ru Saratov Fall Meeting 2015

2. Amoeboid motility is a typical example of self-oscillating waves, which are inherent for a wide class of cells from single-celled amoeboid to cancer cells in multicellular organisms. Examples: • Belousov - Zhabotinsky reaction; • mechanisms in cardiac muscle contraction; • nerve impulse propagation; • activity in cells from single-celled amoeboid to fibroblasts, leukocytes, and cancer in multicellular organisms; • amoeboid motility Model organism to study amoeboid motility, nonmuscle motility. Nature of oscillators generating plasmodium self-oscillating motility are still unknown INTRODUCTION P. Polycephalum plasmodium P. Polycephalum

3. To analyse self-oscillatingmotility in the isolated plasmodium strand of plasmodium PhysarumPolycephalumuponexposure to inhibitors of cellular respiration by sign-sensitive velocity registration. 1. Registration of self-oscillating activity in plasmodium strand in normal conditions (buffer solution) and after inhibitors treatment; 2. Spectral analysis of the time dependences obtained using sign-sensitive laser Doppler microscope; 3. Mathematical model building describing the oscillations in the strand plasmodium. OBJECTIVES PURPOSE OF RESEARCH

4. P. Polycephalum • potassium cyanide (KCN): • cytochrome pathway inhibitor • salicylhydroxamicacid (SHAM): • alternative oxidase inhibitor • Slime mold • Network of cylindrical bands 100-500 µm in diameter and length up to 2 cm • Model organism for many studies involving amoeboid movement and cell motility 1)inhibition of aerobic ATP synthesis 2) cessation of breathing 3)cessation the movement of protoplasm • Strand of plasmodiumwas cut out of plasmodium and placed in the buffer solution pH=7.2, velocity time dependence V (t) was registered; • Strand was treated by KCN and SHAM (5 and 7 µM respectively) which leads to a cessation the endoplasm streaming; • Further inhibitors were removed and strand was placed in buffer. Resuming endoplasm motility through 10 min was recorded. MATERIALS and METHODS

5. P. Polycephalum • potassium cyanide (KCN) • salicylhydroxamic acid (SHAM) Laser Doppler spectroscopy MATERIALS and METHODS Detector He-Ne Laser λ=638.2 nm, P=1-15 mW Strand of plasmodium with a stream of particles inside Results of measurements - Velocity time dependence (20 min) Biophysics (2014), p. 928

6. Sign-sensitive registrationprinciple Doppler spectra obtained from a moving endoplasmin a strand of PhysarumPolycephalum plasmodium. The spectra correspond to the motion at different velocities and motionless endoplasm (V=0 µm/s). Solid lines - Gaussian approximation (r >0.98). Optics & Spectroscopy (2015), in press

7. Velocity module registration Sign-sensitive registration Sign-sensitive registrationprinciple SPIE Proc. (2015) Vol. 9448

8. Short-time Fourier Transform - STFTof time dependencies(length 600 s) 0-600 s 600-1200 s Sign-sensitive registrationprinciple Frequency spectra of the time dependencies of endoplasmic streaming obtained by STFT in case of velocity module (circles) registration and in an alternating mode registration (squares). Sign-sensitive registration SNR >7 Velocity module registration SNR ≈ 1 Two distinct peaks are clearly seen in case of sign-sensitive registration only Optics & Spectroscopy (2015), in press

9. Velocity module registration r≈0.46 Simulated (red solid line) time dependencies based on the spectral characteristics: frequencies, amplitudes and phases, obtained from STFT Sign-sensitive registration r≈0.95 RESULTS Optics & Spectroscopy (2015), in press

10. Normal conditions, buffersolution One half treated by KCN and SHAM 10 min after treatment by KCN and SHAM Solid lines - Gaussian approximation ω1=0.013±0.0004 Hz ω2=0.0254±0.0001 Hz ω2/ω1= 1.961, SD 1.7 % ω1=0.0082±0.0004 Hz ω2=0.0161±0.00009Hz ω2/ω1= 2.008, SD 2 % RESULTS ω1=0.0198±0.0004 Hz ω2=0.0388±0.0013 Hz ω2/ω1= 1.963, SD 1.8 % Spectral analysis reveals two distinct harmonic signals. Despite the frequencies are differs in first and second signals, the ratio ω2 /ω1=1.96±2% in each of themremains constant. Biophysics (2015), submitted

11. Analytical calculation of the self-oscillation frequencies of harmonics: where E - Young's modulus of ectoplasm, k1, k2, k4 - rate constants, n - harmonic number, l, r – length and radius of the strand, μ – endoplasm viscosity [2]. RESULTS Theoretical frequencies: ω1=0.0191 Hz, ω2=0.0123 Hz ω1/ω2=1.6

12. The correspondingsimulated (blue solid line) time dependencies based on the spectral characteristics: frequencies, amplitudes and phases, obtained from STFT. Significant correlation (r≈0.95) between experimental and simulated data is observed. Normal conditions, buffersolution RESULTS One half treated by KCN and SHAM Biophysics (2015), submitted

13. 10 min after treatment by KCN and SHAM - motion restoring appearance of a second harmonic ω2 1 harmonic ω1 RESULTS Harmonic oscillator with ω1 is still active after treatment – no movement is registered. Activity recovering of the second oscillatorwith ω2 means that endoplasm is moving again. Biophysics (2015), submitted

14. STFT of velocity time dependencies allows obtaining two distinct harmonic components in both signals. • The ratio of obtained frequencies with good accuracy equal to two and remains constant in all measurements, regardless the KCN+SHAM treatment. This indicates the presence of an internal oscillator, which frequency is doubled and shifted in phase, or the two types of oscillators whose frequencies differ by the factor of two. 3) Influence of KCN and SHAM leads to a complete cessation of endoplasmic motility. After removal of the inhibitors the respiratory system becomes normal, gradually restoring the activity of both harmonic oscillation sources. 4) Significant correlation between mathematical model and experimental data were demonstrated, no less than r=0.95. DISCUSSION and CONCLUSIONS

15. S.G. Proskurin, T.I. Avsievich. Spectral analysis of self-oscillating motility in an isolated plasmodial strand of Physarum polycephalum // Biophysics, 2014, Vol. 59, No. 6, pp. 1143–1150. • T. I. Avsievich, K. E. S. Ghaleb, S. V. Frolov, S. G. Proskurin Endoplasmic motility spectral characteristics in plasmodium of Physarum polycephalum // Proceedings of SPIE, 2015, Vol. 9448, p. 94480H. • Avsievich T.I., Frolov S.V., Proskurin S.G., Spectral characteristics of shuttle self-oscillating endoplasmic motility in slime mold plasmodium // Optics and Spectroscopy, 2015 (in press) • Avsievich T.I., Frolov S.V., Proskurin S.G., Influence of respiration inhibitors on self-oscillating motility of Physarum polycephalum plasmodium // Biophysics, 2015 (submitted) REFERNCES