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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 1. 2. 3. 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 1. 2. 3. 4. 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 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 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. The fluorescence did not result from DNA rearrangement during transformation Pmec-18 nzgfp + czgfp 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