Molecular Cell Biology Intermediate Filaments Cooper Introduction n

Molecular Cell Biology Intermediate Filaments Cooper

Introduction n Filaments 10 nm wide => “intermediate” n Present in Metazoa / Animals • i. e. not Plants or Unicellular Organisms n Complex Gene Superfamily • 70 in Human Genome n Specific Expression at Different Times and Places

Intermediate Filament Biochemical Properties In Vitro n Very stable. Little subunit exchange. n Very strong. Filaments do not break. • MT’s strong but brittle • Actin weak

Intermediate Filament Potential Functions In Vivo n Mechanical Strength of Cytoplasm n Help a Layer of Epithelial Cells Resist Shear Stress - Filaments Connect to Cell-cell Junctions n Hold Nucleus in Center of Cell

Intermediate Filament Structure & Assembly

Intermediate Filaments by EM: Filament Unraveling

Classes of Intermediate Filaments

Regulation of IF Assembly n Notoriously Stable • No Nucleotide n Filaments Move Little • Precursors Move More n Disassemble Somewhat during Mitosis • Phosphorylation by Cyclin-depen Kinase

Vimentin Filaments in a Cultured Cell

Vimentin n All Cells in Early Development n Cage Around Nucleus n Interacts with Mt’s n Vimentin Knockout Mouse • Initially normal at gross inspection • Cultured cells have altered properties of uncertain significance

FRAP of Vimentin vs. Keratin in One Cell Left: Vimentin (Green) Right: Keratin (Red) 10 min time intervals

Dynamics of Keratin Particles in Periphery 11 micrometers over 10 minutes 18 micrometers over 10 minutes

Desmin n Expressed in Muscle n Elastic Elements to Prevent Over-stretching n Connects / Aligns Z lines n Knockout Mouse - Deranged Myofibril Architecture

Keratins n Expressed in Epithelia n Keratin Filaments Connect to Desmosome and Hemidesmosomes n Differentiation of Epidermis includes Production of Massive Amounts of Keratin n Provides Outer Protection of Skin n Composes Hair, Nails, Feathers, etc.

Density of Keratin Filaments in Outer Epidermis Layers

Keratin Mutations are Basis for Human Epidermal Diseases n Structure/Function Analysis of Keratin Assembly n Point Mutation in Terminal Domain Fails to Assemble n Mutant is Dominant, even in Low Amounts, in Cultured Cells and Mice

Epidermolysis Bullosa Simplex Wild-type Mutant

Keratins and EBS

Neurons n Neurofilament H, M, L Copolymer n Prevent Axon Breakage n Diseases with Clumps of Neurofilaments • Superoxide dismutase model for ALS • Clumps are secondary, not causative

Neurofilament Transport in Axons Photobleached Zone in the Middle

Neurofilament Transport in Axons Photobleached Zone in the Middle

Lamins n Square Lattice on Inner Surface of Nuclear Membrane n Present in Metazoans (Animals, not Plants or unicellular organisms) n Mitosis Breakdown • Phosphorylation of A & C by Cyclin-depen Kinase • B remains with Membrane n Mutations Cause Accelerated Aging Diseases • Progerias - Dominant Mutations

EM of Nuclear Lamina Nuclear Pores

End