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?