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Lecture 13 : Mechanoreception

Lecture 13 : Mechanoreception. 10/14/09. Science / Nature papers. Broadly important phenomena Short format (4 pgs) Very dense Multipanel figures Long figure legends Equivalent to several “regular” papers Clear writing. Questions. What are the mechanoreceptive molecules?

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Lecture 13 : Mechanoreception

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  1. Lecture 13 : Mechanoreception 10/14/09

  2. Science / Nature papers • Broadly important phenomena • Short format (4 pgs) • Very dense • Multipanel figures • Long figure legends • Equivalent to several “regular” papers • Clear writing

  3. Questions • What are the mechanoreceptive molecules? How do you know if you found the right molecule? • Are the molecules common through all organisms? • How is the signal transduced?

  4. How do you prove a protein’s function? • Find a mutant or mutate gene - knock out • Knock gene in to rescue mutant • Show similarity to known sequences in other organisms (homologous) • Label and express - see if gene is turned on in right cells

  5. In situ expression of nompC in sensory organs macrochaete microchaete Proboscus bristles Chordotonal organs of halteres Walker et al fig 7

  6. Halteres also beat and are used as gyroscopes during flight Chordotonal organs are used to sense beating and provide feedback during flying

  7. Skin connected to cytoskeleton by receptor Ion channel Tubulins MEC - mechanosensory proteins identified from mutants

  8. NOMPC

  9. Textbook example - nompC

  10. NompC is new member of the TRP family of ion channels

  11. GFP expression of nompC in C. elegans

  12. Li et al 2006 : Sixth sense in worms

  13. Trp-4 = CeNOMPC • How is it working in the worm? • What are the function of the cells where it is present?

  14. Li Figure 1

  15. Li et al 2006 • Trp-4 mutants move differently • Bend more • Bend more frequently • Putting trp-4 back in returns to wild type • Rescue

  16. TRP-4 expressed in the dopamine neurons Dopamine neurons Interneurons Wormbase

  17. Expression of TRP4 in dopamine neurons CEP neuron Supp Fig S1a,c

  18. Li Figure 2 - What do these cells sense?

  19. Li Figure 2 - What do these cells sense? Cat-2

  20. DVA neuron - expresses trp-4

  21. Expression of TRP4 in DVA and DVC Supp Fig 1b

  22. Fig 3

  23. Shimomura isolated fluorescent protein from jellyfish

  24. Crystal structure Structure is made of barrel from 11 beta sheets and one alpha helix going up center S65 - Y66 - G67 react to form a chromophore which fluoresces

  25. GFP naturally fluoresces Absorbs light at UV or blue wavelengths Emits light in green

  26. GFP can be used to monitor when and where a gene is expressed

  27. GFP expressed in mechanoreceptors of C. elegans

  28. G-CaMP Ca+2 M13 M13 chain of myosin light chain kinase CaM - Ca+2 binding protein cpEGFP

  29. GCaMP [Ca+2] dependent fluorescence Inc in Ca+2 100 M Ca+2 Nakai et al 2001

  30. Express GCaMP in DVA cell Tie down end of worm

  31. Trp-4 mutants - no Ca+2 signal

  32. Li Fig 4

  33. Are you convinced? • Does trp-4 transduce bending motion? • Is this the whole story? • What other experiments could they do?

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