Transcript Genomics of sensory systems

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
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
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
In situ expression of nompC in sensory organs
macrochaete
microchaete
Proboscus bristles
Chordotonal organs of halteres
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 cytoskeleton
by receptor
Ion
channel
Tubulins
MEC - mechanosensory proteins identified from mutants
NOMPC
Textbook example - nompC
NompC is new member of the TRP family
of ion channels
GFP expression
of nompC in C.
elegans
Li et al 2006 : Sixth sense in worms
Trp-4 = CeNOMPC
 How
is it working in the worm?
 What are the function of the cells where
it is present?
Li Figure 1
Li et al 2006
 Trp-4
mutants move differently
Bend more
Bend more frequently
 Putting
Rescue
trp-4 back in returns to wild type
TRP-4 expressed in the
dopamine neurons
Dopamine neurons
Interneurons
Wormbase
Expression of TRP4 in dopamine
neurons
Supp Fig S1a,c
CEP neuron
Li Figure 2 - What do these cells sense?
Li Figure 2 - What do these cells sense?
Cat-2
DVA neuron - expresses trp-4
Expression of TRP4 in DVA and
DVC
Supp Fig 1b
Fig 3
Shimomura isolated fluorescent protein
from jellyfish
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
GFP naturally fluoresces
Absorbs light at UV or
blue wavelengths
Emits light in green
GFP can be used to monitor when and
where a gene is expressed
GFP expressed in mechanoreceptors of
C. elegans
G-CaMP
Ca+2
M13
M13 chain of
myosin light
chain kinase
CaM -
cpEGFP
Ca+2 binding
protein
GCaMP [Ca+2] dependent
fluorescence
Inc in Ca+2
Nakai et al 2001
100 M Ca+2
Express GCaMP in DVA cell
Tie down end of worm
Trp-4 mutants - no Ca+2 signal
Li Fig 4
Are you convinced?
 Does
trp-4 transduce bending motion?
 Is this the whole story?
 What other experiments could they do?