1. Avsievich T.I., Abdulkarim S.N., Proskurin S.G. Tambov State Technical University, Russia tavsievich@tamb.ru Saratov Fall Meeting 2015 Tambov State Technical University Biomedical engineering department

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 P. Polycephalum P. Polycephalum plasmodium Model organism to study amoeboid motility, nonmuscle motility. Nature of oscillators generating plasmodium self-oscillating motility are still unknown

3. To analyse self-oscillating motility in the isolated plasmodium strand of plasmodium Physarum Polycephalum upon exposure 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.

4. potassium cyanide (KCN): cytochrome pathway inhibitor salicylhydroxamic acid (SHAM): alternative oxidase inhibitor 1)Strand of plasmodium was cut out of plasmodium and placed in the buffer solution pH=7.2, velocity time dependence V (t) was registered; 2)Strand was treated by KCN and SHAM (5 and 7 µM respectively) which leads to a cessation the endoplasm streaming; 3)Further inhibitors were removed and strand was placed in buffer. Resuming endoplasm motility through 10 min was recorded. 1)inhibition of aerobic ATP synthesis 2) cessation of breathing 3)cessation the movement of protoplasm P. Polycephalum 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

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

6. Doppler spectra obtained from a moving endoplasm in a strand of Physarum Polycephalum 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 SPIE Proc. (2015) Vol. 9448

8. Short-time Fourier Transform - STFT of time dependencies (length 600 s) 0-600 s600-1200 s Two distinct peaks are clearly seen in case of sign-sensitive registration only 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). Optics & Spectroscopy (2015), in press

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

10. 10 min after treatment by KCN and SHAM One half treated by KCN and SHAM Normal conditions, buffer solution ω 1 =0.013±0.0004 Hz ω 2 =0.0254±0.0001 Hz ω 2 /ω 1 = 1.961, SD 1.7 % ω 1 =0.0082±0.0004 Hz ω 2 =0.0161±0.00009 Hz ω 2 /ω 1 = 2.008, SD 2 % ω 1 =0.0198±0.0004 Hz ω 2 =0.0388±0.0013 Hz ω 2 /ω 1 = 1.963, SD 1.8 % Solid lines - Gaussian approximation Biophysics (2015), submitted

11. Analytical calculation of the self-oscillation frequencies of harmonics: where E - Young's modulus of ectoplasm, k 1, k 2, k 4 - rate constants, n - harmonic number, l, r – length and radius of the strand, μ – endoplasm viscosity [2]. Theoretical frequencies: ω 1 =0.0191 Hz, ω 2 =0.0123 Hz ω 1 /ω 2 =1.6

12. The corresponding simulated (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, buffer solution One half treated by KCN and SHAM Biophysics (2015), submitted

13. 1 harmonic ω 1 appearance of a second harmonic ω 2 Harmonic oscillator with ω 1 is still active after treatment – no movement is registered. Activity recovering of the second oscillator with ω 2 means that endoplasm is moving again. Biophysics (2015), submitted

14. 1)STFT of velocity time dependencies allows obtaining two distinct harmonic components in both signals. 2)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.

15. 1)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. 2)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. 3)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) 4)Avsievich T.I., Frolov S.V., Proskurin S.G., Influence of respiration inhibitors on self-oscillating motility of Physarum polycephalum plasmodium // Biophysics, 2015 (submitted)