Quiz 3 Material Cytoskeleton MFs Ac1n Polymeriza1on8Based Mo1lity MFs Ac1n Motor Protein8Based Mo1lity MTs Ac1n8Myosin Systems MTs MT Motors Mitosis Mitosis Cytokinesis Cilia IFs Mitochondria Structure Genome Origins Mitochondria Protein Import Cytoskeleton MFs Cytoskeleton network of protein laments highly dynamic i e laments change length exchange subunits important for cell shape cell polarity cell division mitosis cytokinesis cell mo1lity swimming crawling intracellular transport DATA CBIO3400 CBIO 3400 Microfilaments DATA Cytoskeleton CBIO3400 network of protein filaments in eukaryotic cells some filaments also present in prokaryotes highly dynamic filaments change length exchange subunits important for cell shape inside and outside cell polarity cell division mitosis cytokinesis cell motility swimming crawling etc intracellular transport MFs MTs IFs Subunits DATA CBIO3400 IFs more stable no polarity MFs MTs less stable have polarity all eukaryotes all eukaryotes metazoa MFs DATA monomer ac n ac1n monomers Microfilaments organized into helical lament monomer Filaments 8 nm wide actin 42 kD is a non8covalent bonds actin monomers are organized into a helical filament b tw subunits allow for rapid turnover of ac1n monomers Non covalent bonds between subunits allow for rapid turnover of actin monomers CBIO3400 Lodish et al Negative staining and electron microscopy uranium acetate DATA CBIO3400 Prokaryotes have actin Wickstead B Gull K J Cell Biol 2011 194 513 525 Bacterial proteins ParM and MreB are similar to actin assemble into non helical filaments MreB interacts with the plasma membrane and regulates cell shape ParM is required for segregation of plasmid DNA Note that none of these proteins participates in the contractile ring during cell division In prokaryotes the contractile ring is made of a tubulin like protein FtsZ DATA CBIO3400 Cytoskeletal filaments in bacteria FtsZ ParM MreB DATA CBIO3400 Examples of structures based on microfilaments DATA CBIO3400 DATA Actin MFs Ac n CBIO3400 Lodish et al globular protein 2 subdomains connected by cleR that binds ATP or ADP Mg2 polarity of lament determined by orienta1on of monomers within lament monomers associate head8to8tail pointed 8 end has cleR exposed to outside proks have proteins similar to ac1n MreB interacts w PM regulates cell shape ParM required for segrega1on of plasmid DNA FtsZ makes up contrac1le ring Globular protein two subdomains connected by a cleft that binds ATP or ADP and Mg2 Polarity of filaments is determined by orientation of monomers within filaments monomers associate head to tail The pointed end has the cleft side exposed to the outside MFs Ac n Exchange Hydrolysis of ATP Exchange and hydrolysis of ATP DATA CBIO3400 ADP ATP exchangeable in GI aRer polymeriza1on ATP hydrolyzed to ADP Pi inside lament only newly added subunits have ac n ATP ac1n8ATP has higher a nity for end than does ac1n8ADP hydrolysis of ATP destabilizes polymer hydrolysis not required for DATA polymeriza1on however hydrolysis is required for destabiliza1on of polymer allows depolymeriza1on DATA CBIO3400 Exchange and hydrolysis of ATP in G actin ADP is exchangeable for ATP after polymerization ATP is hydrolyzed to ADP Pi inside the filament Alberts et al CBIO3400 in G actin ADP is exchangeable for ATP DATA after polymerization ATP is hydrolyzed to ADP Pi inside the filament Only newly added subunits have ATP Hydrolysis of ATP destabilizes the polymer Alberts et al D D D D Pi T T D Pi CBIO3400 Only newly added subunits have ATP Hydrolysis of ATP destabilizes the polymer Hydrolysis of ATP is not required for polymerization However it is required for destabilization of the polymer to allow for depolymerization D D D D Pi T T D Pi Hydrolysis of ATP is not required for polymerization However it is required for destabilization of the polymer to allow for depolymerization DATA CBIO3400 Pure actin polymerizes in vitro DATA CBIO3400 Pure actin polymerizes in vitro G actin globular monomeric purified actin ATP monomers G actin globular monomeric F actin filamentous polymeric purified actin ATP Critical monomers concentration of monomers Critical concentration of monomers actin filaments have the actin filaments have the same structure as filaments same structure as filaments formed in vivo formed in vivo F actin filamentous polymeric DATA CBIO3400 Exchange and hydrolysis of ATP in G actin ADP is exchangeable for ATP after polymerization ATP is hydrolyzed to ADP Pi inside the filament Alberts et al DATA CBIO3400 Only newly added subunits have ATP Hydrolysis of ATP destabilizes the polymer D D D D Pi T T D Pi MFs Ac n Polymeriza on In Vitro Hydrolysis of ATP is not required for polymerization However it is required for destabilization of the polymer to allow for depolymerization pure ac1n polymerizes in vitro monomers reach Cc interact form laments monomeric ac1n CANNOT be present above Cc DATA CBIO3400 Pure actin polymerizes in vitro G actin globular monomeric F actin filamentous polymeric purified actin ATP monomers Critical concentration of monomers actin filaments have the same structure as filaments formed in vivo DATA CBIO3400 Pure actin polymerizes in vitro DATA CBIO3400 Three phases of polymerization of actin in vitro MFs Ac n 3 Phases of Polymeriza on trimer smallest stable polymer no net lament produc1on occurring rate of subunit addi1on rate of subunit loss THM ac1n polymeriza1on seeds polymer lament A trimer of actin subunits is the smallest stable polymer DATA CBIO3400 Decoration of actin filaments by myosin S1 heads reveals filament polarity Plus end barbed minus end pointed MFs Ac n Filament Polarity decora1on of ac1n laments w myosin8S1 heads reveals lament polarity isolate heads add to ac1n laments decorates monomers w myosin8S1 heads ac1n monomers preferen1ally add to end DATA CBIO3400 Monomers are added preferentially to the plus end short actin filaments decorated with myosin add G actin ATP Lodish et al Electron micrograph negative stain with uranyl acetate DATA CBIO3400 Capping proteins can be used to determine the Cc and rate of polymerization for each end DATA CBIO3400 Actin concentration DATA CBIO3400 Monomers are added preferentially to the plus end short actin filaments decorated with myosin add G actin ATP Lodish et al Electron micrograph negative stain with uranyl acetate MFs Ac n Capping Proteins DATA CBIO3400 Capping proteins can be used to determine the Cc and rate of polymerization for each end capping proteins