MOLECULES INVOLVED IN THE SPIDER, CUPIENNIUS SALEI, MECHANOTRANSDUCTION by Jessica Johnson Submitted in partial fulfilment of the requirements for the degree of Master of Science at Dalhousie University Halifax, Nova Scotia July 2019 © Copyright by Jessica Johnson, 2019 TABLE OF CONTENTES LIST OF TABLES .........................................................................................................v LIST OF FIGURES ..................................................................................................... vi ABSTRACT ................................................................................................................ vii LIST OF ABBREVIATIONS AND SYMBOLS USED ........................................... viii ACKNOWLEDGMENTS ........................................................................................... xi CHAPTER 1. INTRODUCTION..................................................................................1 1.1. Mechanotransduction ............................................................................................1 1.2. Mechanotransduction channels ..............................................................................2 1.2.1. DEG/ENAC/ASIC channels ...........................................................................2 1.2.2. TRP channels .................................................................................................4 1.2.3. TMC proteins .................................................................................................7 1.2.4. Piezo proteins .................................................................................................8 1.3. Arthropod mechanosensilla ...................................................................................9 1.4. Cupiennius salei .................................................................................................. 11 1.4.1. C. salei central nervous system ..................................................................... 12 1.4.2. C. salei mechanosensilla .............................................................................. 14 1.4.3. C. salei VS-3 slit sensillum .......................................................................... 15 1.4.4. C. salei transcriptome ................................................................................... 17 1.4.5. C. salei Piezo protein.................................................................................... 18 1.5. Objectives ........................................................................................................... 21 CHAPTER 2. MATERIALS AND METHODS ......................................................... 23 2.1. Experimental animals and dissection ................................................................... 23 2.1.1. Dissection of the patella and VS-3 organ ...................................................... 23 2.1.2. Central nervous system dissection ................................................................ 24 2.2. RNA probe construction for in-situ hybridization ................................................ 25 2.3. In-situ hybridization protocol for patellar samples ............................................... 26 2.3.1 Post hybridization washes ............................................................................. 29 2.3.2 Immunohistochemical detection .................................................................... 29 ii 2.4. Central nervous system embedding, sectioning and in-situ hybridization ............. 30 2.5. Electrophysiology ............................................................................................... 32 2.5.1. Experimental preparation ............................................................................. 32 2.5.2. Intracellular recording and mechanical stimulation ....................................... 34 2.5.3. Ion channel blockers and an activator ........................................................... 35 2.6. RNA interference ................................................................................................ 36 2.6.1. Construction of double stranded RNA .......................................................... 36 2.6.2. Tissue culture and processing ....................................................................... 37 2.7. Statistical analysis ............................................................................................... 38 CHAPTER 3. RESULTS ............................................................................................. 39 3.1. Putative mechanotransduction channels in C. salei transcriptomes ...................... 39 3.2. In-situ hybridization ............................................................................................ 40 3.2.1. Expression of four genes in the DEG/ENAC/ASIC channel family .............. 40 3.2.2. Expression of two genes coding TRP channels ............................................. 43 3.2.3. Expression of two genes coding TMC channels ............................................ 44 3.2.4. Expression of Piezo in the C. salei hypodermis and CNS ............................. 45 3.2.5. In-situ hybridization summary ...................................................................... 48 3.3. Mechanotransduction channel inhibitors and an activator .................................... 49 3.3.1. Ruthenium Red had no effect on VS-3 neurons receptor current ................... 51 3.3.2. GsMTx4 had no effect on VS-3 neuron receptor current ............................... 53 3.3.3. Yoda1 had an excitatory effect on VS-3 neurons .......................................... 54 3.3.4. Amiloride effects on VS-3 neuron excitability .............................................. 58 3.4. Knockdown of C. salei Piezo by RNA interference ............................................ 63 CHAPTER 4. DISCUSSION ....................................................................................... 65 4.1. Expression of putative mechanotransduction channels in C. salei ........................ 65 4.2. Mechanotransduction channel pharmacology ...................................................... 67 4.3. Knockdown of Piezo gene ................................................................................... 71 4.4. Conclusions ........................................................................................................ 72 4.5. Future directions ................................................................................................. 73 REFERENCES ............................................................................................................ 74 ii i Appendix 1. Primer sequences used for generation of RNA probes .......................... 81 Appendix 2. Copyright Permission for Figure 1.2. ..................................................... 83 iv LIST OF TABLES Table 2.1. In-situ hybridization conditions .................................................................... 27 Table 2.2. Hybridization solution .................................................................................. 28 Table 2.3. Post hybridization washing buffers ............................................................... 29 Table 3.1. Parameters for all successful experiments under control conditions. ............. 50 v LIST OF FIGURES Figure 1.1. Photograph of a male C. salei ...................................................................... 12 Figure 1.2. A schematic diagram showing the C. salei CNS and the VS-3 organ. .......... 13 Figure 1.3. C. salei VS-3 slit sesillum ........................................................................... 17 Figure 1.4. Cartoon model of the trimeric mouse Piezo1 ............................................... 19 Figure 1.5. Western blot of the C. salei brain homogenate with Piezo antibody ............. 20 Figure 1.6. Immunocytochemistry with the C. salei Piezo antibody .............................. 21 Figure 2.1. The VS-3 organ preparation for electrophysiology ...................................... 33 Figure 2.2. Experimental setup for the VS-3 organ ........................................................ 33 Figure 3.1. Relative abundances of putative mechanotransduction channel transcripts .. 39 Figure 3.2. In situ hybridization using the Amil-1 probes. .............................................. 41 Figure 3.3. In situ hybridization using probes for three ENaC channels ......................... 42 Figure 3.4. In situ hybridization using the Trp probes ................................................... 44 Figure 3.5. In situ hybridization results for Tmc channels .............................................. 45 Figure 3.6. In situ hybridization of VS-3 organ for Piezo .............................................. 46 Figure 3.7. In-situ hybridization of subesophageal ganglion with Piezo probes ............. 47 Figure 3.8. Typical responses of a Type B VS-3 neuron to mechanical stimulation ....... 50 Figure 3.9. Ruthenium Red had no effect on VS-3 neuron receptor current ................... 52 Figure 3.10. GsMTx4 had no effect on VS-3 neuron receptor current ............................ 54 Figure 3.11. Effect of Yoda1 application on a VS-3 neuron .......................................... 55 Figure 3.12. Yoda1 effects on VS-3 neuron receptor potentials and excitability ............ 56 Figure 3.13 Yoda effects on VS-3 neuron action potentials ........................................... 58 Figure 3.14. Amiloride effect of VS-3 neurons receptor potential.................................