Self-oscillating endoplasmic motility study in a strand of plasmodium P. Polycephalum

1. Biomedical engineering Tambov State Technical University Self-oscillating endoplasmic motility study in a strand of plasmodium P. Polycephalum T.I. Avsievich, S.N. Abdulkareem, S.G. Proskurin http://bmt.tstu.ru/ spros@tamb.ru

2. INTRODUCTION Endoplasmic motility is an example of auto oscillation, which is inherent to a wide class of cells from single-celled amoebas to cancer cells in multicellular organisms. Examples: • Belousov - Zhabotinsky reaction; • mechanisms in cardiac muscle contraction; • nervous impulse propagation; • activity in cells from single-celled amoeboid to fibroblasts, leukocytes and tumor cells in multicellular organisms; • amoeboid motility Autowaves  in Belousov - Zhabotinsky reaction P. Polycephalum P. Polycephalum

3. OBJECTIVES PURPOSE of the STUDY To analyse time-dependent velocity V(t) of self-oscillating motility of endoplasm in the isolated strand of plasmodium Physarum Polycephalum upon exposure to inhibitors of cellular respiration. 1. Registration of self-oscillating motility in the strand in normal conditions (buffer solution) and after the inhibitors’ treatment; 2. Fourier analysis of the time dependences V(t)of the endoplasm; 3. Mathematical model describing the registered oscillations.

4. MATERIALS and METHODS • Slime mold • Network of cylindrical bands 100-500 µm in diameter and length up to 2 cm • Model organism for many studies involving amoeboid motility • potassium cyanide (KCN): • cytochrome pathway inhibitor • salicylhydroxamic acid (SHAM): • alternative respiration inhibitor 1) inhibition of aerobic ATP synthesis 2) cessation of breathing 3) cessation of endoplasmic motility P. Polycephalum • 10 mm strand was isolated out of plasmodium and placed in buffer solution pH=7.2; • Strand was treated with KCN and SHAM (5 and 7 µM, respectively) which leads to a cessation of protoplasmic motility; • Further inhibitors were removed and strand was placed in the buffer solution. Resumption of endoplasmic motility, V(t) recording for 10 min after.

5. MATERIALS and METHODS P. Polycephalum • potassium cyanide (KCN) • salicylhydroxamic acid (SHAM) Laser Doppler anemometer Velocity time dependence Detector Laser Strand of plasmodium with shuttle endoplasmicmotility inside

6. RESULTS Spectral analysis (Fast Fourier Transform - FFT)(length 600 s) from 0 and 600 s Figure 1. Fourier spectra of the time dependence V(t) of protoplasmic motility from 0 to 600 s (blue) and 600 to 1200 s (pink) in the buffer solution. Figure 2. Similar spectra after KCN and SHAM treatment Solid lines - Gaussian approximations to the peaks Proskurin S. G., Avsievich T. I., Biophysics, 2014 (in press)

7. RESULTS Influence of inhibitors • After removal of respiratory inhibitors • During first 10 minincreases activity of the first peak and the complete suppression of the second one. • On 12th minute, system begins to return to the normal state with two harmonic components. ω1 ω2 Respiratory inhibitors affect on both sources of oscillation, but one of them (ω1) recovers faster. Proskurin S. G., Avsievich T. I., Biophysics, 2014 (in press)

8. RESULTS In buffer after treatment by KCN and SHAM Buffer solution ( pH=6,8) at first 10 min just one source of oscillations ω1 is present, in the next 10 min noted the appearance of a second frequency peak ω2: ω1=0.0198±0.0004 Hz ω2=0.0388±0.0013 Hz ω2/ω1= 1.963 SD 1.8 % ω1and ω2 remains constant during 20 min: ω1=0.013±0.0004 Hz ω2=0.0254±0.0001 Hz ω2/ω1= 1.961 SD 2.6 % Spectral analysis reveals two distinct harmonic signals. Despite the frequencies differ in first and second signals, the ratio ω2/ω1=1.96±2%in each of them remains constant.

9. RESULTS Analytical calculation of the self-oscillation frequencies of harmonics: where E - Young's modulus of ectoplasm elasticity, k1, k2, k4 - rate constants, n - harmonic number, l, r – length and radius of the strand, μ – endoplasm viscosity [2]. Theoretical frequencies: ω1=0.0191 Hz, ω2=0.0361 Hz ω2/ω1=1.89

10. RESULTS Time dependences of endoplasmic motility in plasmodium strand before (a) and after removing the respiration inhibitors (b) a) R2=0.943 Significant correlation between the model and experimentaldata is observed b) R2=0.971

11. RESULTS 1 harmonic ω1 appearance of a second harmonic ω2 R2=0.971 Significant correlation between mathematical model and experimentaldata up to 800 s is observed. Additional parameters are required to describe transition to the normal state.

12. RESULTS Directed motion of endoplasm Area under the graph is changed S+ - above X axis S– - under X axis S+ S+ /S - ≈0, 6 S- S+ S+ /S- ≈1, 6 S- Directed motion of the strand is different for the first and the second signal

13. DISCUSSION and CONCLUSIONS • Sign-sensitive laser Doppler velocimetry allows to register shuttle endoplasmic motility in plasmodium strands with diameter up to 0.5 mm; • Fourier analysis of velocity time dependences of the endoplasm allows obtaining two distinct harmonic components in both signals; • 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; • Significant correlation between mathematical model and experimental data were demonstrated.

14. REFERENCES • Proskurin S.G., Avsievich T.I., Spectral analysis of self-oscillation motility in isolated plasmodium strand of Physarum Polycephalum // Biophysics. 2014 (in press). 2. Teplov V.A., Mitrofanov V.V., Romanovsky Y.M., Synchronization of mechanochemical auto-oscillations within the Physarum polycephalum plasmodium by periodical external actions // Biophysics. 2005, T. 50, № 4. p. 704-712.

15. Biomedical engineering Tambov State Technical University Self-oscillating endoplasmic motility study in a strand of plasmodium P. Polycephalum T.I. Avsievich, S.N. Abdulkareem, S.G. Proskurin http://bmt.tstu.ru/ spros@tamb.ru