Combinatorial Marking of Cells and Organelles with Split Fluorescent

Proteins

Shifang Zhang, Charles Ma, & Martin Chalfie Department of Biological Sciences, Columbia University

OUTLINEOUTLINE BackgroundBackground The principle of split GFPThe principle of split GFP The test of split GFP in The test of split GFP in C.elegansC.elegans

1.1. The split GFP can be expressed in The split GFP can be expressed in C.elegansC.elegans with P with Pmec-18 mec-18 2. Split GFP was not promoter or tissue dependent3. Reconstitution was not restricted to split GFP

The use of split GFPThe use of split GFP1. Split GFP can identify cells that coexpress different promoters2. Split GFP can be used demonstrate changes in gene expression3. Split GFP can also be used to identify cells expressing a

particular gene4. Split GFP can be used to label cell constituents in a restricted set

of cells

BackgroundBackground

Advantage: encoded Advantage: encoded and expressed in and expressed in livingliving cells and cells and organismsorganisms

Limited: individual Limited: individual cell types cannot be cell types cannot be labelled using single labelled using single regulatory elementregulatory element

The split GFPThe split GFP

2000, 2000, Indraneel Ghosh

NZ: ALKKELQANKKELAQLKWELQALKKELAQCZ: EQLEKKLQALEKKLAQLEWKNQALEKKLAQ

NZGFP=NGFP + 6 amino acid linker + NZ

CZGFP=CZ + 4 amino acid linker + CGFP

The test of split GFP in The test of split GFP in C.elegansC.elegans

Split GFP can be expressed in Split GFP can be expressed in C.elegansC.elegans

PPmec-18 mec-18 :: only expressed in the six touch only expressed in the six touch receptor neuronsreceptor neurons

PPmec-18 nzgfp mec-18 nzgfp + P+ Pmec-18 mec-18

czgfp czgfp

When both parts of split GFP were expressed

If only one part of the split GFP was expressed, If only one part of the split GFP was expressed, there is no fluorescence can be observed.there is no fluorescence can be observed.

The fluorescence did not result from DNA rearrangement during transformation

PPmec-18 nzgfp mec-18 nzgfp + + czgfpczgfp

fluorescencfluorescencee

The absence of CZ prevented the production of fluorescence

PPhsp16.2 hsp16.2 : heat shock promoter: heat shock promoter

Split GFP was not promoter or tissue dependent

PPhsp16.2 nzgfp hsp16.2 nzgfp ++ PPhsp16.2 czgfphsp16.2 czgfp

The split GFP has a shorter half-life than GFP

Punc-4nzgfp& Punc-4czgfp

Punc-4gfp

<2 hr

20 hr

40 hr

PPunc-4 unc-4 : expressed in : expressed in four types of motor four types of motor neurons (SAB, VA, DA, neurons (SAB, VA, DA, and VC)and VC)

Masha et al., 2003

Reconstitution was not restricted to split GFP

CFP Filter SetYFP Filter Set

NZYFP+CZCFP

NZGFP+CZCFP

NZCFP+CZCFP

NZYFP+CZYFP

NZGFP+CZGFP

The use of split GFPThe use of split GFP

Punc-24 nzgfp+ Pmec-2 czgfpPunc-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

Split GFP can identify cells that coexpress different promoters

Punc-24gfp

Punc-24nzgfp& Pmec-2czgfp

Pmec-3nzgfp& Pegl-44czgfp

only the two FLP neurons

fluoresced

Pmec-3 Pegl-44

touch receptors

PVD neurons

FLPneurons

HSN 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

VD and DD motor neurons

VA, DA and possibly VC neurons

VB and DB motor neurons

In the excitatory motor neurons but not the inhibitory motor neurons

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)

Px NZCFP X=Pb NZCFP

Pc NZYFP

Pa NZGFP

A possible extension

The apparent short half life of split GFP raises an important caution about negative results

HSN neurons egl-44 : in the embryocat-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