Lecture 4

• The cytoskeleton is relevant in a number of diseases, including cancer
• How scientists identified a new cancer therapeutic

  1. Chemists found a chemical in the Amazon rainforest

  2. The chemical halted cells in mitosis, specifically in metaphase

  3. The chemical stops microtubules from depolymerizing, stopping cell division

  4. This was useful for developing a new cancer drug
• The cytoskeleton is a network of protein filaments that is highly dynamic and has many important functions
• Components of the cytoskeleton

  • Actin filaments
  • Microtubules
  • Intermediate filaments
• Functions of the cytoskeleton

  • Structural support
  • Cell shape
  • Internal organization
  • Cell division
  • Large-scale movements
• Cytoskeletal filaments have diameters ranging from 7 nm to 25 nm, which is smaller than the resolution limit of light microscopes
• Fluorescence microscopy


  Can detect specific proteins by using fluorescent labels, but the filaments appear thicker than they actually are
• Transmission electron microscopy


  Can resolve the detailed structure of cytoskeletal filaments
• Immunofluorescence microscopy


  1. Cells are fixed

  2. Primary antibody binds to protein of interest

  3. Secondary antibody with fluorescent marker binds to primary antibody

  4. Fluorescent microscope is used to visualize the labeled proteins
• Secondary antibodies with fluorescent markers are used for amplification and cost reasons, rather than directly labeling the primary antibody
• Types of cytoskeletal filaments

  • Actin filaments
  • Intermediate filaments
  • Microtubules
• Actin filaments


  Made up of actin
• Intermediate filaments


  Made up of intermediate filament proteins
• Microtubules


  Made up of tubulin
• Cytoskeletal filaments are held together by non-covalent interactions
• Filaments


  Made up of actin
• Filaments are held together by non-covalent interactions
• Examples of cytoskeletal structures

  • Actin filaments (red/pink)
  • Intermediate filaments (blue)
  • Microtubules (green)
• The examples shown are typical arrangements, not the only possible arrangements
• Actin filaments, intermediate filaments, and microtubules are the three main types of cytoskeletal filaments
• Intermediate filaments


  • Involved in structural support
  • Cytoplasmic intermediate filaments provide mechanical strength in cells subjected to mechanical stress
  • Nuclear intermediate filaments (lamins) form a 2D meshwork that supports the nuclear membrane
• Types of cytoplasmic intermediate filaments

  • Keratin filaments (in epithelial cells)
  • Vimentin and vimentin-related filaments (in connective tissue, muscle, and glial cells)
  • Neurofilaments (in nerve cells)
• The nuclear lamina formed by nuclear intermediate filaments (lamins) is found in all animal cells with a nucleus
• Polarity


  The ends of a structure are different
• Structure of cytoplasmic intermediate filaments

  1. Monomers form coiled-coil dimers

  2. Dimers form staggered anti-parallel tetramers

  3. Tetramers associate laterally to form filaments

  4. Filaments have no overall polarity
• Cytoplasmic intermediate filaments


  • Conserved alpha-helical central rod domains
  • N-terminal and C-terminal domains differ
  • Assemble into rope-like filaments
  • Highly flexible and have high tensile strength
• Keratin filaments


  • Cytoplasmic intermediate filaments found in epithelial cells
  • Form a network anchored at cell-cell junctions (desmosomes)
  • Provide mechanical strength to epithelial sheets
• Microtubules


  • Play an organizing function in all eukaryotic cells
  • Involved in cell organization, transport, and mitosis
  • Provide structural support
  • Found in motile structures like flagella and cilia
  • Long, stiff, hollow tubes made of tubulin
  • Inextensible (not elastic)
• Structure of microtubules


  1. Alpha tubulin and beta tubulin form tubulin heterodimers

  2. Tubulin heterodimers assemble into protofilaments

  3. 13 protofilaments associate laterally to form a hollow microtubule

  4. Microtubules have polarity, with a plus end and a minus end
• Tubulin heterodimer


  • Alpha tubulin (light green)
  • Beta tubulin (dark green)
• Tubulin heterodimers bind to GTP
• Tubulin heterodimers bind to each other
• Tubulin heterodimer


  • Alpha end is the minus end
  • Beta end is the plus end
• Cartoon version of tubulin heterodimer shows Alpha (light green) and Beta (dark green) but does not show GTP
• Formation of protofilament


  1. Tubulin heterodimers bind to each other

  2. One end has Alpha, other end has Beta
• Protofilament


  • Polar structure with one end having Alpha, other end having Beta
• 13 parallel protofilaments form a hollow microtubule cylinder
• Bonds between individual tubulin subunits are non-covalent
• Bonds between protofilaments are weaker than bonds within each protofilament
• Microtubule growth and disassembly can occur at both ends