Combinatorial Marking of Cells and Organelles with Split Fluorescent Proteins

1. Combinatorial Marking of Cells and Organelles with Split Fluorescent Proteins Shifang Zhang, Charles Ma, & Martin Chalfie Department of Biological Sciences, Columbia University

2. OUTLINE • Background • The principle of split GFP • The test of split GFP in C.elegans • 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 • Split GFP can identify cells that coexpress different promoters • Split GFP can be used demonstrate changes in gene expression • Split GFP can also be used to identify cells expressing a particular gene • Split GFP can be used to label cell constituents in a restricted set of cells

3. Background • Advantage: encoded and expressed in living cells and organisms • Limited: individual cell types cannot be labelled using single regulatory element

4. The split GFP 2000, Indraneel Ghosh NZGFP=NGFP + 6 amino acid linker + NZ CZGFP=CZ + 4 amino acid linker + CGFP NZ: ALKKELQANKKELAQLKWELQALKKELAQ CZ: EQLEKKLQALEKKLAQLEWKNQALEKKLAQ

5. The test of split GFP in C.elegans

6. Pmec-18 nzgfp + Pmec-18 czgfp Split GFP can be expressed in C.elegans • Pmec-18 :only expressed in the six touch receptor neurons When both parts of split GFP were expressed If only one part of the split GFP was expressed, there is no fluorescence can be observed.

7. Pmec-18 nzgfp + czgfp fluorescence • The fluorescence did not result from DNA rearrangement duringtransformation • The absence of CZ prevented the production of fluorescence

8. Split GFP was not promoter or tissue dependent Phsp16.2 : heat shock promoter Phsp16.2 nzgfp +Phsp16.2 czgfp

9. <2 hr 20 hr 40 hr Punc-4gfp Punc-4nzgfp& Punc-4czgfp 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

10. NZGFP+CZGFP NZYFP+CZYFP NZCFP+CZCFP NZGFP+CZCFP NZYFP+CZCFP CFP Filter Set YFP Filter Set Reconstitution was not restricted to split GFP

11. The use of split GFP

12. 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

13. Punc-24gfp Punc-24nzgfp& Pmec-2czgfp

14. Pmec-3 Pegl-44 HSN neurons touch receptors FLP neurons a member of the transcription enhancer factor family PVD neurons Pmec-3nzgfp& Pegl-44czgfp only the two FLP neurons fluoresced .

15. Split GFP can be used demonstrate changes in gene expression 1999, Winnier

16. Punc-4 nzgfp + Pacr-5 czgfp

17. VA, DA and possibly VC neurons Split GFP can also be used to identify cells expressing a particular gene Which motor neurons of the ventral cord VB and DB motor neurons VD and DD motor neurons In the excitatory motor neurons but not the inhibitory motor neurons

18. Pa NZGFP Px NZCFP X= Pb NZCFP Pc NZYFP A possible extension 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)

19. Punc-4nzgfp& Psto-6czgfp Punc-4nzgfp& Punc-4czgfp 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

20. 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

21. The usage of GFP should be aided by the use of split fluorescent proteins Thank you!