4/14/15%1%Determina.on%by%cytoplasmic%localiza.on:%specifica.on%of%the%germ%line%fate%in%C.#elegans%%Learning%goals:%1. Understand%that%early%embryonic%cell%fates%in%C.#elegans%are%determined%both%cell@autonomously%and%condi.onally.%%2. Be%able%to%describe%how%P%granules%and%PIE@1%proteins%are%localized%to%the%germ%line%cells%in%the%early%embryo,%including%the%roles%of%PAR%proteins%and%MEX@5/6%proteins.%3. Be%able%to%explain%how%P%granules%and%PIE@1%contribute%to%germ%line%development.%%%4. Understand%the%gene.c,%cell%biological%and%molecular%evidence%that%led%to%our%current%understanding%of%the%system.%O%How%do%cells%remember?%4/14/15%2%1. Introduc.on%to%the%C.#elegans#germline%and%test%of%the%hypothesis%that%germline%fate%is%determined%cell%autonomously.%2. P%granules%are%localized%to%the%germline%lineage.%3. Are%P%granules%a%cytoplasmic%determinant?%4. PIE@1%has%a%role%in%specifying%the%germ%line.%5. PAR%proteins%and%MEX@5/6%proteins%localize%PIE@1%and%P%granules.%4/14/15%3%C.#elegans#cell$lineage$(abbreviated)#AB#P1#P2#EMS#neurons#skin#pharynx#other#E#MS#C#pharynx#muscle#neurons#other#intesBne#muscle#skin#neurons#(zygote)#P0#P3#P4#D#muscle# germ$line#Cells$in$blue$are$founder$cells$Molecular%Biology%of%the%Cell,%Figure%22@18%AB%ABa%ABp%MS%E%P1%P2%P3%P4%Soma%vs.%germ$line$4/14/15%4%How%is%the%fate%of%the%germ%line%founder%cell%(P4)%determined%in%C.#elegans?%4/14/15%5%Hypothesis:%%germ%line%fate%is%determined%cell@autonomously.%How%could%one%test%this%hypothesis?%Isolated%P4%cell%?????%Experiment:%%use%laser%beam%to%isolate%the%P4%cell%%If%it%is%determined%cell%autonomously,%it%will%behave%like%a%germ%cell.%Behaves%like%a%germ%line%cell%4/14/15%6%1. Introduc.on%to%the%C.#elegans#germline%and%demonstra.on%that%germline%fate%is%determined%cell%autonomously.%2. P%granules%are%localized%to%the%germline%lineage.%3. Are%P%granules%a%cytoplasmic%determinant?%4. PIE@1%has%a%role%in%specifying%the%germ%line.%5. PAR%proteins%and%MEX@5/6%proteins%localize%PIE@1%and%P%granules.%P$granules%are%segregated%%to%the%germ%line%cells%by%a%series%of%asymmetric%divisions.%Figure%22@19,%MBoC%4/14/15%7%P%granules%are%composed%of%many%proteins%and%mRNAs:%Ribonucleoprotein%par.cles%(that%behave%with%the%physical%proper.es%of%liquid%droplets)%What%are%P%granules?%Germline%P%granules%are%liquid%droplets%that%localize%by%controlled%dissolu.on/condensa.on.%Brangwynne%CP,%Eckmann%CR,%Courson%DS,%Rybarska%A,%Hoege%C,%Gharakhani%J,%Jülicher%F,%Hyman%AA.%Science.%2009%Jun%26;324(5935):1729@32.%4/14/15%8%1. Introduc.on%to%the%C.#elegans#germline%and%demonstra.on%that%germline%fate%is%determined%cell%autonomously.%2. P%granules%are%localized%to%the%germline%lineage.%3. Are%P%granules%a%cytoplasmic%determinant?%4. PIE@1%has%a%role%in%specifying%the%germ%line.%5. PAR%proteins%and%MEX@5/6%proteins%localize%PIE@1%and%P%granules.%Are%P%granules%necessary%for%germ%line%determina.on?%%%%%Are%P%granules%sufficient%for%germ%line%determina.on?%4/14/15%9%pptr-1#(phosphatase#PP2A#regulatory#subunit)#Wild#type#Gallo%et%al.%(2010)%Science%330:1685%Results:%%As%shown%in%the%movies,%P%granules%are%not%restricted%to%the%germ%line%lineage%and%are%present%in%smaller%aggregates%(droplets).%%Worms%homozygous%for%muta.ons%in%pptr-1%have%germ%cells%and%most%animals%are%fer.le%4/14/15%10%December%2010%Science%1. Introduc.on%to%the%C.#elegans#germline%and%demonstra.on%that%germline%fate%is%determined%cell%autonomously.%2. P%granules%are%localized%to%the%germline%lineage.%3. Are%P%granules%a%cytoplasmic%determinant?%4. PIE@1%has%a%role%in%determining%the%germ%line.%5. PAR%proteins%and%MEX@5/6%proteins%localize%PIE@1%and%P%granules.%4/14/15%11%PIEK1$mutant#Pharynx%and%Intes.ne%Excess%%pie,1#mutant$ABaABpEMSEMSwild#type%ABaABpEMSP2Gene.cs:%C.#elegans#cell$lineage$(abbreviated)#AB#P1#P2#neurons#skin#pharynx#other#C#muscle#skin#neurons#(zygote)#P0#EMS#E#MS#pharynx#muscle#neurons#other#intesBne#P3#P4#D#muscle# germ$line#Cells$in$blue$are$founder$cells$EMS#E#MS#pharynx#muscle#neurons#other#intesBne#4/14/15%12%PIE@1%protein%is%segregated%to%the%germ%line%cells%in%the%early%embryo%through%a%series%of%asymmetric%divisions%1. Introduc.on%to%the%C.#elegans#germline%and%demonstra.on%that%germline%fate%is%determined%cell%autonomously.%2. P%granules%are%localized%to%the%germline%lineage.%3. Are%P%granules%a%cytoplasmic%determinant?%4. PIE@1%has%a%role%in%specifying%the%germ%line.%5. PAR%proteins%and%MEX@5/6%proteins%localize%PIE@1%and%P%granules.%4/14/15%13%How%do%P%granules%and%PIE@1%become%localized?%%PAR$proteins$%MEXK5/6$Asymmetric%P%granule%segrega.on%depends%upon%a%number%of%proteins%including%PAR$proteins%%and$MexK5/6$Wild%type%par-1%mutant%Beginning%of%1st%cell%cycle%Early%prophase%Anaphase%Gallo%et%al.%(2010)%Science%330:1685%mex-5/6#(RNAi)%4/14/15%14%PAR@2::GFP%PAR@6::mCherry%PAR@2::GFP%Some%PAR%proteins%are%asymmetrically%distributed%in%the%early%embryo.%MEX@5/6%embryos%also%fail%to%specify%P2%%but%the%MEX@5%protein%localizes%to%the%anterior%of%the%one@cell%embryo.%4/14/15%15%What%is%the%rela.onship%among%MEX@%5/6,%PAR%proteins%and%PIE@1?%Image courtesy of C. Schubert"MEX@5%PIE@1%Posterior%PAR%proteins%4/14/15%16%If%PAR%proteins%are%not%present,%then%neither%MEX@5%nor%PIE@1%are%asymmetrically%distributed%Par%mutant% Par%mutant%Image courtesy of C. Schubert"MEX@5%PIE@1%The%PAR%proteins%are%required%for%asymmetric%accumula.on%of%MEX@5%and%PIE@1.%Posterior%PAR%proteins%4/14/15%17%MEX@5%restricts%the%localiza.on%of%PIE@1%PIE@1%in%wild%type% PIE@1%in%mex-5#MEX@5%in%wild%type%MEX@5%in%pie-1#PIE@1%does%NOT%restrict%the%localiza.on%of%MEX@5%MEX@5/6%PIE@1%Posterior%PAR%%proteins%MEX@5%PIE@1%Posterior%PAR%proteins%P%granules%MEX@5/6%PIE@1%P%granules%4/14/15%18%• Germ%line%in%C.#elegans#is%determined%cell%autonomously.%• PIE@1%and%P%granules%become%localized%to%the%germ%line%cell%lineage%in%the%early%embryo%through%the%ac.on%of%PAR%proteins%and%MEX@5/6.%• Enrichment%of%P%granules%in%the%germ%cell%lineage,%however,%is%not%required%for%determining%germ%line%fate,%nor%is%the%presence%of%P%granules%sufficient%to%specify%germ%line%fate.%•
View Full Document