MCB 252 Topic 22 Intermediate FilamentsProf David Rivier MCB 252 Spring 2015 MCB 252 Topic 22 Cytoskeleton Intermediate Filaments Intermediate filaments IFs Reading Lodish section 18 7 Genetic Analysis Lodish pp 172 173 MCB 252 Topic 22 Cytoskeleton Intermediate Filaments Intermediate filaments IFs Classes Structure of monomer polymer Dynamics Functions Intermediate Filament Network in Epithelial Cells Keratin Cell boundaries Alberts et al Fig 16 18 IFs are Extremely Stable L V Cell extracted with detergent high salt stained for IF PAGE Pollard and Earnshaw Fig 35 4 Cytoplasmic vs Nuclear IFs Specificity of IF expression Vertebrates have over 50 genes encoding 6 classes of IF proteins Differentially expressed in nearly all cells of the body desmin muscle GFAP glia NFs neurons keratins epithelial cells Generally constitute 1 of cellular proteins in some cell types e g neurons and epidermal keratinocytes can be 85 of total protein Intermediate Filament Structure IFs do not bind nucleotides Pollard and Earnshaw Fig 35 2 Evolution of IF Superfamily Lamins were the progenitors Cytoplasmic IFs arose in animals after the branching off from plants Lamins lost NLS and prenylation region membrane attachment resulting in cytoplasmic localization Cytoplasmic Lamins formed several families each with specific functions skin muscles nerves Alpha Helical Coiled Coil Alberts et al Fig 3 11 Lodish Fig 3 9 How can one determine the orientation of monomers in dimers and tetramers If N and C termini are quite different in size Lodish 18 47 How can one determine the orientation of monomers in dimers and tetramers If N and C termini are similar in size dimer immunogold electron microscopy Lodish Fig 5 51 tetramer or tetramer Polymer Structure Proto filament Alberts et al Fig 16 16 Polymer Structure Pollard and Earnshaw Fig 35 3 Lodish 18 47 IF Dynamics Fluorescence recovery after photobleaching FRAP Photobleach GFP vimentin does it recover t 6 t 0 t 18 J Cell Biol 143 147 157 1998 IF FRAP T22M1 Neuron FRAP IF Dynamics Microinject biotin labeled keratin into cells Lodish 18 48 How is IF assembly regulated in vivo Not known if there are accessory proteins No additional proteins needed in vitro Most cells produce only 1 2 types Those with 2 types the 2 types can independently assemble in vitro Functional studies What do intermediate filaments do Microinject antibodies that block assembly into tissue culture cells Functional studies What do intermediate filaments do Microinject antibodies that block assembly into tissue culture cells Filaments disrupted but cells are OK Plus some animals e g insects don t have cytoplasmic IFs Conclusion How to test for a role of IFs in vivo in a cells within a living organism Keratin expression in skin epithelium Pollard and Earnshaw Fig 35 6 Thinking about Genetics Thinking about Genetics Lodish 5 2 How Does Ras Contribute to Cancer What is the mutant form of Ras that contributes to cancer Is the oncogenic form dominant or recessive to wild type What would be the phenotype of a cell that could not make any Ras protein Thinking about Genetics Genotype Phenotype RAS WT RAS WT Divides when signaled RAS CA RAS WT Divides without signal RAS CA RAS CA Divides without signal ras RAS WT Divides when signaled RAS WT Wild type RAS CA Constitutively active ras completely inactive gene null phenotype ras ras Cannot Divide Thinking about Genetics Genotype RAS WT RAS WT Divides Phenotype when signaled RAS CA RAS WT Divides without signal RAS CA RAS CA Divides without signal ras RAS WT Divides when signaled ras ras Cannot Divide Dominant mutations are typically Gain of Function Mutations here Ras has gained the ability to signal in the absence of the signaling molecule Recessive mutations are typically Loss of Function Mutations Here Ras has lost the ability to signal in the presence of the signaling molecule Thinking about Genetics Genotype Phenotype RAS WT RAS WT Divides when signalled RAS CA RAS WT Divides without signal RAS CA RAS CA Divides without signal ras RAS WT Divides when signalled ras ras Cannot Divide Gain of Function Mutations and Loss of Function Mutations typically have opposite phenotypes Here the Gain of Function Ras allele leads to constitutive cell division whereas Loss of Function of both alleles of Ras leads to the inability to divide Thinking about Genetics Genotype Phenotype RAS WT RAS WT Divides when signalled RAS CA RAS WT Divides without signal RAS CA RAS CA Divides without signal ras RAS CA Divides when signalled ras ras Cannot Divide The Null Phenotype is the phenotype that corresponds to the complete loss of function of both copies of the gene The null phenotype is typically the most extreme form of the loss of function phenotype The null phenotype is typically opposite to the dominant phenotype What About Genetics in Transgenic Organisms Transgenic organisms are no longer diploid they now have three copies of the gene the two endogenous wild type copies plus the mutant copy that we added Transgenic Genotype Transgene RAS WT RAS WT RAS CA RAS WT RAS WT ras Only Dominant Alleles have a phenotype in a transgenic animal that is wildtype for the endogenous copies of the gene Keratin expression in skin epithelium Pollard and Earnshaw Fig 35 6 How can we make a transgenic organism that doesn t make Keratin filaments How can we make a transgenic organism with a dominant allele that results in a loss of function phenotype Dominant Negative Mutations There is a relatively rare type of mutation known as a Dominant Negative Mutation It is Dominant so that only one copy is enough to display the mutant phenotype However the unusual property of dominant negatives is that the phenotype is similar to the null phenotype rather than being opposite to the null phenotype Dominant negative mutations Produce mutant phenotype in cells carrying a wildtype copy allele of the gene Dominant Negative Phenotype is similar to the loss offunction mutant phenotype Phenotype is the result of the dominant negative form of the protein interfering with the wild type version of the protein e g by preventing assembly of multimers Dominant negative mutations Wild type Wild type Transfected Dominant Negative Poisoned polymer model Dominant Negative Keratin Mutation in a Transgenic Animal Lodish Fig 19 37 K14 mutant blocks filament assembly in basal layer skin blisters due to weakening of basal cells Functional studies What do intermediate filaments do Microinject antibodies that block assembly