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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? - PowerPoint PPT Presentation
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Lecture 13 : Lecture 13 : MechanoreceptionMechanoreception
10/14/09
Science / Nature papersScience / Nature papers
Broadly important phenomena Short format (4 pgs) Very dense
Multipanel figuresLong figure legendsEquivalent to several “regular” papers
Clear writing
QuestionsQuestions
1. What are the mechanoreceptive molecules?
How do you know if you found the right molecule?
2. Are the molecules common through all organisms?
3. How is the signal transduced?
How do you prove a protein’s How do you prove a protein’s function?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
macrochaete microchaete Proboscus bristles
Chordotonal organs of halteres
In situ expression of nompC in sensory organs
Walker et al fig 7
Halteres also beat and are used as gyroscopes during flight
Chordotonal organs are used to sense beating and provide feedback during flying
Skin connected to Skin connected to cytoskeleton by receptorcytoskeleton by receptor
MEC - mechanosensory proteins identified from mutants
Ion channel
Tubulins
NOMPCNOMPC
Textbook example - nompCTextbook example - nompC
NompC is new member of the TRP NompC is new member of the TRP family of ion channelsfamily of ion channels
GFP expression of nompC in C. elegans
Li et al 2006 : Sixth sense in wormsLi et al 2006 : Sixth sense in worms
Trp-4 = CeNOMPCTrp-4 = CeNOMPC
How is it working in the worm? What are the function of the cells
where it is present?
Li Figure 1Li Figure 1
Li et al 2006Li et al 2006
Trp-4 mutants move differentlyBend more Bend more frequently
Putting trp-4 back in returns to wild typeRescue
TRP-4 expressed in the TRP-4 expressed in the dopamine neuronsdopamine neurons
Dopamine neurons Interneurons
Wormbase
Expression of TRP4 in Expression of TRP4 in dopamine neuronsdopamine neurons
CEP neuronSupp Fig S1a,c
Li Figure 2 - What do these cells sense?Li Figure 2 - What do these cells sense?
Li Figure 2 - What do these cells sense?Li Figure 2 - What do these cells sense?
Cat-2
DVA neuron - expresses trp-4DVA neuron - expresses trp-4
Expression of TRP4 in DVA Expression of TRP4 in DVA and DVCand DVC
Supp Fig 1b
Fig 3Fig 3
Shimomura isolated fluorescent Shimomura isolated fluorescent protein from jellyfishprotein from jellyfish
Crystal structureCrystal 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
GFP naturally fluorescesGFP naturally fluoresces
Absorbs light at UV or blue wavelengths Emits light in green
GFP can be used to monitor when GFP can be used to monitor when and where a gene is expressedand where a gene is expressed
GFP expressed in mechanoreceptors GFP expressed in mechanoreceptors of of C. elegansC. elegans
G-CaMPG-CaMP
M13
M13 chain of myosin light chain kinase
CaM -
Ca+2 binding protein
cpEGFP
Ca+2
GCaMP [CaGCaMP [Ca+2]+2] dependent dependent fluorescencefluorescence
100 M Ca+2
Nakai et al 2001
Inc in Ca+2
Express GCaMP in DVA cellExpress GCaMP in DVA cell
Tie down end of worm
Trp-4Trp-4 mutants - no Ca mutants - no Ca+2+2 signalsignal
Li Fig 4Li Fig 4
Are you convinced?Are you convinced?
Does trp-4 transduce bending motion?
Is this the whole story? What other experiments could
they do?