Transcript Combinatorial Marking of Cells and Organelles with Split

Combinatorial Marking of Cells
and Organelles with Split
Fluorescent Proteins
Shifang Zhang, Charles Ma, & Martin Chalfie
Department of Biological Sciences, Columbia University
OUTLINE
Background
The principle of split GFP
The test of split GFP in C.elegans
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The split GFP can be expressed in C.elegans with Pmec-18
Split GFP was not promoter or tissue dependent
Reconstitution was not restricted to split GFP
The use of split GFP
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Split
Split
Split
Split
GFP
GFP
GFP
GFP
can
can
can
can
identify cells that coexpress different promoters
be used demonstrate changes in gene expression
also be used to identify cells expressing a particular gene
be used to label cell constituents in a restricted set of cells
Background
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Advantage: encoded
and expressed in
living cells and
organisms
Limited: individual cell
types cannot be
labelled using single
regulatory element
The split GFP
2000, Indraneel Ghosh
NZGFP=NGFP + 6 amino acid linker + NZ
CZGFP=CZ + 4 amino acid linker + CGFP
NZ: ALKKELQANKKELAQLKWELQALKKELAQ
CZ: EQLEKKLQALEKKLAQLEWKNQALEKKLAQ
The test of split GFP in C.elegans
Split GFP can be expressed in C.elegans
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Pmec-18 : only expressed in the six touch receptor
neurons
When both parts of split GFP were expressed
Pmec-18 nzgfp + Pmec-18 czgfp
If only one part of the split GFP was expressed,
there is no fluorescence can be observed.
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The fluorescence did not result from DNA
rearrangement during transformation
Pmec-18 nzgfp + czgfp
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fluorescence
The absence of CZ prevented the production
of fluorescence
Split GFP was not promoter or tissue dependent
Phsp16.2 :
heat shock promoter
Phsp16.2 nzgfp + Phsp16.2 czgfp
The split GFP has a shorter
half-life than GFP
Punc-4 : expressed in four
types of motor neurons
(SAB, VA, DA, and VC)
Masha et al., 2003
<2 hr
20 hr
40 hr
Punc-4gfp
Punc-4nzgfp& Punc-4czgfp
Reconstitution was not restricted to split GFP
NZGFP+CZGFP
NZYFP+CZYFP
NZCFP+CZCFP
NZGFP+CZCFP
NZYFP+CZCFP
YFP Filter Set
CFP Filter Set
The use of split GFP
Split GFP can identify cells that coexpress
different promoters
Punc-24 nzgfp+ Pmec-2 czgfp
Punc-24 :
expressed in C.elegans touch recetor neurons
and in many cells in the ventral cord
Pmec-2 :
expressed in the six touch receptor neurons
Punc-24gfp
Punc-24nzgfp& Pmec-2czgfp
only the two FLP neurons fluoresced
Pmec-3nzgfp& Pegl-44czgfp
Pmec-3
Pegl-44
HSN neurons
touch receptors
PVD neurons
FLP
neurons
a member of the
transcription enhancer
factor family
Split GFP can be used demonstrate
changes in gene expression
1999, Winnier
Punc-4 nzgfp + Pacr-5 czgfp
Split GFP can also be used to identify cells
expressing a particular gene
Which motor neurons of the
ventral cord
VA, DA and possibly VC neurons
VB and DB motor neurons
VD and DD motor neurons
In the excitatory motor neurons but not the inhibitory motor neurons
A possible extension
Pa NZGFP
Pb NZCFP
Px NZCFP
X=
Pc NZYFP
With the color coding provided by the different NZ
fluorescent proteins, relatively few (perhaps less
than thirty) strains could be used to characterize
gene expression in all of the 302 C. elegans
neurons (118 classes)
The apparent short half life of split GFP raises
an important caution about negative results
HSN neurons
egl-44 : in the embryo
cat-1 : in the late larval
HSN fluorescence was weak and
rarely seen when split GFP was
generated from these promoters
Punc-4nzgfp& Psto-6czgfp
Punc-4nzgfp& Punc-4czgfp
Split GFP can be used to label cell constituents in a
restricted set of cells
synaptobrevin::GFP (SNB-1::GFP) protein fusion localizes to presynaptic vesicles
Cell bodies and processes
of the B motor neurons in
ventral cord
Presynaptic regions
Nuclei
The usage of GFP should be aided by
the use of split fluorescent proteins