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. Proprioception - sense of where one’s own body is

6. TEP - transepithelial potential
Wild type responses - the layout

7. Wild type responses - the actual currents occuring in response to a displacement during voltage clamping
Response time is 200 us - 100x faster than 2nd messenger system
Displacement of 100 nm at tip = 2 nm at base or 1/2 thickness of cell membrane

8. Adaptation produces 85% of normal response
Adaptation produces 85% of normal response. This is same as in vert hair cells suggesting bristles and hair cells have similar response.

9. Difference in response current, response time and action potentials between controls (cn bw) and 4 different NompC mutants

10. Deficiency mapping in drosophila - have raised lines which are missing parts of chromosomes. Each line is missing a different chunk. They know what part of the chromosome is missing (as shown by the thin line). If they cross that deletion strain with mutant and rescue so no longer clumsy adults, then that strain has the part of the chromosome containing the mutant gene. If they don’t rescue, then the gene is in the missing part of that strain.

11. nompC1,2 and 3 have premature stops, nompC4 has missense
29 ANK repeats - maybe these bind to cytoskeleton??

12. Wikepedia - from huma erythrocytic ankyrin
Important for protein-protein interactions
Each ankyrin repeat is 33 AA which makes 2 alpha helices separated by a loop - so this picture shows 12 repeats

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

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

15. Skin connected to cytoskeleton by receptor
Ion channel
Schematic diagram of the proposed mechanotransduction complex in C. elegans body touch receptors. At its centre is an ion channel composed of MEC-4, 6 and 10, which interacts with the intracellular protein MEC-2. MEC-7 and 12 are microtubule proteins required for normal mechanosensation (they may be important for localisation or gating of the complex). MEC-1, 5 and 9 are extracellular proteins whose functions await further characterisation. (Figure adapted from Ref. 4.)Drew et al. Molecular Pain :8 ﾊ doi: /
Ion channel flux is order of magnitude higher with MEC2 than without.
Tubulins
MEC - mechanosensory proteins identified from mutants

16. NOMPC
Gillespie fig 3 Shows two genes identified from drosophila mutants - NompA (extracellular protein) and NompC (ion channel)
This system has both non adapting and adapting channels. These are just like MRO1 and MRO2 in crayfish which do not and do adapt, respectively.

17. Textbook example - nompC
Figure 3.5 Drosophila mutants identified as being very clumsy and uncoordinated Found mutation in gene NompC = No mechanoreceptor potential C Turned out to be different mutations in same gene NompC
So identified just like Shaker channel from Shaker mutants

18. NompC is new member of the TRP family of ion channels
Christensen and Corey 2007
These are transient receptor potential channels which come in different families. Here they are shown from many organisms from inverts to verts.

19. GFP expression of nompC in C. elegans
ADE - anterior deirid
CEPD/CEPV = cephalic dorsal / ventral (neurons in the head)
DVA/DVC

20. Li et al 2006 : Sixth sense in worms

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

22. Li Figure 1 Mutant worms bend more, bend faster and move faster.
Expressing TRP4 gene rescues mutants and recreates WT behavior.

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

24. TRP-4 expressed in the dopamine neurons
Interneurons
CEP = cephalic neurons
ADE = anterior deirid neuron
PDE = posterior deirid neuron
DVA/DVC = ring interneurons
Wormbase

25. Expression of TRP4 in dopamine neurons
TRP4 is enriched in cilia
Arrows imply force applied to surface of worm
CEP neuron
Supp Fig S1a,c

26. Li Figure 2 - What do these cells sense?
WT worms move slower when bacteria are present (basal slowing repsonse). This may be so they can eat them. Mutant worms move at faster rate as if they can’t sense that bacteria are there. TRP4 rescue (in dopaminergic neurons) again returns to WT behavior.
Interesting that extent of body bending is always more in mutants than WT, independent of presence of bacteria.
Cat-2 is a dopamine deficient mutant. It has same behavior as trp-4 mutants. Since trp4 is expressed in dopaminergic neurons, can knock out these neurons with either of these genes and affect frequency of moving.

27. Li Figure 2 - What do these cells sense?
Cat-2
However, cat-2 mutants have same extent of bending as WT. So dopamine neurons mediate fast bending but not extent of bending. So likely the primary effect is not in dopaminergic neurons but in neurons that talk to these neurons.

28. DVA neuron - expresses trp-4

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

30. Fig 3
Predict DVA/C send signal that worm is bending so bend less. So if ablate, should bend more. Surprise. Ablate and it bends less. So propose in cell trp-4 is negative regulator but also a positive regulator. If this is true, when ablate DVA/C in trp-4 mutant should make bend less. It does!
Most of this comes from DVA (and not DVC - it’s ablation has very small effect). So DVA is key.
Prove this because if express trp-4 in trp-4 mutant only in DVA cells, it rescues WT bending extent!! So DVA is stretch sensitive neuron.

31. Shimomura first to isolate GFP from jellyfish
Martin Chalfie showed it could be used in biological system
Roger Tsien developed variants for multicolor work

32. Shimomura isolated fluorescent protein from jellyfish
Jelly fish emits green light when it is agitated
Shimomura and Johnson collected 10,000 jelly fish, cut off edges and isolated few mg of green protein - fluoresces green when excite with UV light

33. 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
From Nobel 2008 chem science info
When protein forms, there is a sharp turn in alpha helix such that S65 and G67 come in close proximity and a cyclic structure forms
Comes from reaction of backbone CO from S65 reacts with NH from G67 to form 5 membered ring. Then reacts with O2 to form double bond in 5 ring
This is what gives protein its fluorescence

34. GFP naturally fluoresces
Can attach AA sequence for GFP gene and it will function as a tail which follows other protein around
Because GFP naturally fluoresces, just have to hit with UV or blue light and get green light out
Absorbs light at UV or blue wavelengths
Emits light in green

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

36. GFP expressed in mechanoreceptors of C. elegans
GFP expressed under control of promoter for mec-7 which is a beta tubulin expressed in mechanoreceptor cells
Not toxic. Allows to follow gene expression in living tissue - don’t have to add any substrates - fluoresces all by itself

38. G-CaMP Ca+2 M13 M13 chain of myosin light chain kinase CaM -
Ca+2 binding protein
cpEGFP
Nakai et al Engineered new structure that fluoresces in response to Ca+2. When Ca+2 binds to calmodulin, the kinase interacts and the whole loop modifies the structure of the GFP and enhances fluorescence.
Since neurons typically change Ca+2 when respond, use fluorescence to follow neural response.

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

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

41. Trp-4 mutants - no Ca+2 signal
Trp-4 mutant worms did not show fluorescence in response to motion.
If ablate cells upstream of DVA or if shut off synaptic input to DVA (unc-13 mutant) stil get fluorescence. Still have body bending too.
So DVA is the cell responding to bending and showing Ca fluorescence

42. Li Fig 4
If move head with pipette, can cause Ca release only when bending occurs. Seems to turn on when bending gets to 50 deg as if it is a limiter of bending beyond that.

43. Are you convinced? Does trp-4 transduce bending motion?
Is this the whole story?
What other experiments could they do?
There must be other channels which detect smaller bending angles.