Transmembrane Protein PAN-1 Regulates Membrane-to-Nucleus Localization Of Transcription Factor MYRF In Development

Developing neural circuits undergo refinement as they mature,including generation of new synapses and elimination of existing ones.These changes allow the nervous system to become an efficient functional network.These changes are referred to as synaptic remodeling.The molecular and cellular mechanisms of synaptic remodeling are poorly understood.C.elegans DD motor neurons exhibit a dramatic synaptic remodeling process.Synapses of DDs are localized on its ventral processes in L1 animals,releasing GABA onto ventral muscles.DD neurons' dorsal processed receive ACh,thus being dendritic.By the end of L1,new synapses are generated on the dorsal processes of DDs,while the early ventral synapses are being eliminated.This process of synaptic remodeling in DDs becomes valuable model to study the mechanism of synaptic remodeling.Previous laboratory studies have shown that MYRF(myelin regulatory transcription factors)family proteins myrf-1 and myrf-2,play a key role in promoting synaptic remodeling.Full-length MYRF is a transmembrane protein that can be cleaveded to release N-terminal fragment into nucleus.Via immunoprecipition and mass spectrometry analysis of MYRF::GFP,a transmembrane protein PAN-1 was identified to bind to MYRF,and was found to be essential for synaptic remodeling.Genetic interaction analysis showed that myrf acts downstream to pan-1.There remains intriguing questions what the functional role of the PAN-1-MYRF interaction in DD synaptic remodeling,and how inactivation of PAN-1 affects the activity of MYRF.I have explored these mechanistic questions in my thesis research.I analyzed the knock in strain of PAN-1::GFP constructed by CRISPR,and found that PAN-1::GFP signals are localized to cell membrane.However,it was previously observed that full-length MYRF::GFP was located on ER membrane,which is inconsistent with the direct interaction between PAN-1 and MYRF.After careful imaging analysis of MYRF::GFP,I found that MYRF-1::GFP signals were distributed on the cell membrane in early L1,while GFP signals appeared in the nucleus when the development proceeds,showing a trend of increased nuclear signals and decreased membrane signals.Importantly,in pan-1 mutant,MYRF::GFP signals on both the cell membrane and nucleus mostly disappeared,indicating that pan-1 plays an important role in the localizing MYRF::GFP on the cell membrane and in the nucleus.Based on the transcription reporter and quantitative PCR,I concluded that the transcription of myrf is not changed when pan-1 is lost,which support PAN-1's direct regulation on MYRF::GFP.In addition to its cell membrane localization,PAN-1 ::GFP exists as small puncta in the cytoplasm,which is not co-located with P granule proteins or endocytic organelles.The function of cytoplasmic PAN-1::GFP puncta as well as its connection with plasma membrane PAN-1::GFP are of interest for further studies.Moreover,I identified PAN-1 interacting proteins via immunoprecipitation and mass-spec analysis of PAN-1::GFP,which provides new clues for the future study of PAN-1 function.