Investigation Of Posterior Architecture Establishment During Cell Migration

Cell migration plays an important role in many biological processes and contribute to pathological conditions including inflammation and cancer metastasis.Migrating cells adopt a polarized state with morphologically and functionally distinct leading and trailing edges,which relies on the asymmetric distribution of signaling and cytoskeleton molecules.In addition to migration,these polarly distributed molecules display characteristic behaviors in cells responding to chemoattractant stimuli or undergoing morphological changes in general.Intriguingly,some of the so called "front" or "back"molecules are encoded by oncogenes and tumor suppressor genes,respectively.To date,studies on cell polarity highlight the role of PIP3 as one of the master regulators of leading-edge activities.By recruiting downstream effectors,PIP3 signaling organizes actin rearrangement to promote front protrusions and cell migration.However,less is known about the regulation of trailing edge activities in highly motile cells.In the first part of the thesis,we systematically screened PH domain-containing proteins in the model system Dictyostelium to identify novel back proteins and elucidate the mechanism regulating their localization.By fluorescence microscopy imaging,we demonstrated that an uncharacterized protein we named trailing edge enriched protein 1(Teep1)localizes to the rear of randomly moving cells or cells migrating along a chemical gradient,and falls off transiently from the plasma membrane in an actin cytoskeleton independent manner in response to uniform chemoattractant stimulation.Truncation and mutation analyses indicated that the trailing edge localization of Teepl depends on conserved positively charged residues within the N-terminal PH domains.Furthermore,protein-lipid interaction assays revealed that Teepl preferentially interacts with PI(3,5)P2 and PI(4,5)P2.Decreasing the levels of the two lipids simultaneously in cells blocked the membrane association of Teep1 and caused the cells to bleb continuously.Further experiments revealed that a back-to-front gradient of PI(3,5)P2 exists on the plasma membrane and is generated,at least in part,by PIP3mediated recruitment of a PI3P/PI(3,5)P2 phosphatase to the leading edge.Consistently,deleting the phosphatase enhanced the plasma membrane targeting efficiency of Teep1.These experiments indicate that a reverse PI(3,5)P2 gradient on the plasma membrane modulates the localization of specific back proteins.The establishment of polarity also relies on a coordination between signaling proteins and cortical F-actin.However,how trailing edge-localized signaling proteins instruct cortical actin assembly is not fully understood.Since no candidates responsible for actin polymerization has been identified in the first project,we directly analyzed and F-actin regulators with the purpose of cloning novel players for cortical actin assembly.In the second part of the thesis,we discovered that a transducer of Cdc42dependent actin assembly(Toca)family protein we named Fbp17 plays an important role in promoting branched actin assembly at the rear cortex.Biochemistry experiments revealed that Fbp17 reinforces cortical integrity by interacting with and activating WASP family nucleation-promoting factors(NPFs),which in turn promote Arp2/3mediated actin assembly.Cells deleted of fbp17 exhibit decreased cortical F-actin and impaired cortical rigidity,as measured by micropipette aspiration assays.The mutant cells are also defective in cytokinesis and migration,two events heavily relied on cortical integrity.Furthermore,we found that Fbp17 is recruited by a RhoGEF protein,GxcM,to the rear of migrating cells.Expression of GxcM in wild-type cells is sufficient to target Fbp17 and induce over-assembly of F-actin at the rear cortex.This phenotype can be blocked by deleting fbp17 or mutating the conserved catalytic residues in the GEF domain of GxcM.Therefore,GxcM,Fbp17,NPFs,and Arp2/3 constitute a signaling cascade participating in the formation of the rear cortical actin subcompartment.In conclusion,by focusing on the regulation of trailing edge activities in highly motile cells,this thesis uncovers the existence of a phosphoinositide gradient on the plasma membrane regulating the localization of specific back proteins and a signaling cascade governing the formation of rear actin cortex.These results expand our understanding of the polarized architecture of cell and the communication between signaling proteins and the underlying cortical actin network.