Microtubules

• Microtubules are hollow, unbranched, tubular structures made of tubulin
• • Microtubules have roles in cell support and movement of materials within a cell
  • They can extend across the length or breadth of a cell
  • The microtubule is composed of 13 protofilaments aligned side by side to form a tube through weak non-covalent interactions
• Protofilaments are assembled from dimers of one α-tubulin and one β-tubulin.

  Protofilament is asymmetric; the microtubule itself has polarity:

  • α-tubulin end: negative end
  • β-tubulin end: positive end

  The polarity helps direct motor proteins and the direction of microtubule growth
• Centrosome: a type of microtubule-organizing centre which initiates microtubule formation.

  • Composed of two centrioles surrounded by pericentriolar material (PCM)
  • Centrioles are cylinders made of microtubules
  • PCM: loosely organized fibrous lattice
• When centrosomes replicate, centrioles recruit PCM to form a new centrosome.

  Centrosomes usually remain at the centre of the cell's microtubular network.
• Centrosome diagram: (for the tubules, think of what they're made of)

  A) PCM

  B) Centrosome

  C) Centriole

  D) microtubules

  
• Centrosomes dictate:

  • number of microtubules
  • their (microtubule) polarity
  • the number of protofilaments
  • the time and location of microtubule assembly
  • Centrosomes do NOT dictate the rate of assembly nor the stability
• New microtubules do not make direct contact with centrioles, instead they are initiated in the PCM

  PCM contains ϒ-TURC (ϒ- tubulin ring complex)

  • ϒ-TURC contains:
  • ϒ-tubulin
  • Non-tubulin proteins in a ring
  • αβ-tubulin dimers assemble on the ϒ-tubulin, but only α-tubulin can bind to the ring of ϒ-tubulin
• Where the microtubules begin to form:

  A) Centrosome

  
• What is the polarity of each end of the microtubule and to which end does the microtubule grow?

  The end with the α-tubulin attached is negative while the end with the β-tubulin is positive.

  • Microtubule synthesis occurs at the PCM and grows outwards towards the positive end.

  A) Negative end

  B) Positive end

  
• At which end of the microtubule does growth/disassembly occur?

  Positive end
• ϒ-TURC complex also acts a cap to prevent disassembly and assembly at the negative end
• Stability of microtubules are determined by:

  • microtubule interacting proteins (MAPs)
  • +TIPS: proteins that bind at the + end of growing microtubules
  • Temperature: cold promotes disassembly
• MAPs:

  • increase stability and promotes assembly by linking tubulin dimers together
  • Activity of some MAPs is controlled by the presence of phosphate groups

  *Certain MAPs can decrease stability and promote disassembly
• GTP = guanosine triphosphate

  • Energy source that is analogous to ATP
• β- tubulin is a G-protein: Hydrolyzes GTP to GDP after the dimer is added to the microtubule. Dimers to be added to the microtubule are bound to GTP

  • GTP bound to the β-tubulin subunit is required for microtubule assembly
  • GTP hydrolysis affects microtubule structure
• Is α-tubulin a G-protein?

  No
• What type of tubulin is shown in each?

  Alpha or beta?

  A) beta

  B) alpha

  
• Why would adding ϒ-TURC help ensure that newly formed/elongated microtubule contains exactly 13 protofilaments in an in vitro experiment?

  Because the α and β tubulins are built off of the 13 ϒ-tubulins, there would be 13 protofilaments as well.
• Label the microtubule

  A) +

  B) -

  C) protofilament

  
• Microtubule growth/closure:

  1. In a growing microtubule, the tip consists of tubulin- GTP dimers in an open sheet
  2. Tube closure is associated with the hydrolysis of GTP
  3. GDP-tubulin has a different conformation (it has a weaker affinity for its neighbours) introducing mechanical strain.
  4. Thus, it needs MAPs to stabilize the microtubule
  5. In the absence of MAPs, protofilaments curl outward and undergo catastrophic shrinkage
• +Tips:

  • Bind to the + end of microtubule and regulate the rate of growth or shrinkage
  • Meditate the attachment to subcellular structures (eg: kinetochore of the mitotic chromosome)
• Microtubule polymerization (growth)/ disassembly (shrinkage) can effectively ' push ' and ' pull ' material within a cell
• Label the 'stage' of β-tubulin in the diagram w/ 1,2,3, or 4

  1. In a growing microtubule, the tip consists of tubulin- GTP dimers in an open sheet
  2. Tube closure is associated with the hydrolysis of GTP
  3. GDP-tubulin has a different conformation (it has a weaker affinity for its neighbours) introducing mechanical strain.
  4. Thus, it needs MAPs to stabilize the microtubule
  5. In the absence of MAPs, protofilaments curl outward and undergo catastrophic shrinkage

  A) 1

  B) GDP

  C) GTP

  
• Label the 'stage' of β-tubulin in the diagram

  1. In a growing microtubule, the tip consists of tubulin- GTP dimers in an open sheet
  2. Tube closure is associated with the hydrolysis of GTP
  3. GDP-tubulin has a different conformation (it has a weaker affinity for its neighbours) introducing mechanical strain.
  4. Thus, it needs MAPs to stabilize the microtubule
  5. In the absence of MAPs, protofilaments curl outward and undergo catastrophic shrinkage

  A) 2

  
• Label the 'stage' of β-tubulin in the diagram

  1. In a growing microtubule, the tip consists of tubulin- GTP dimers in an open sheet
  2. Tube closure is associated with the hydrolysis of GTP
  3. GDP-tubulin has a different conformation (it has a weaker affinity for its neighbours) introducing mechanical strain.
  4. Thus, it needs MAPs to stabilize the microtubule
  5. In the absence of MAPs, protofilaments curl outward and undergo catastrophic shrinkage

  A) 3

  
• Label the 'stage' of β-tubulin in the diagram

  1. In a growing microtubule, the tip consists of tubulin- GTP dimers in an open sheet
  2. Tube closure is associated with the hydrolysis of GTP
  3. GDP-tubulin has a different conformation (it has a weaker affinity for its neighbours) introducing mechanical strain.
  4. Thus, it needs MAPs to stabilize the microtubule
  5. In the absence of MAPs, protofilaments curl outward and undergo catastrophic shrinkage

  A) 4

  
• Microtubules as structural supports

  • microtubules help provide mechanical support as they are stiff enough to resist compression or bending forces
  • Help determine the shape of a cell
  • Maintains intracellular location of organelles

  makes animal cells spherical, while helps supports cellulose in plant cells
• Microtubules as agents of intracellular motility

  -Transports membranous vesicles from one membrane compartment to another.

  -Transports non-membrane bound cargo (RNAs, ribosomes, cytoskeletal elements)
• Microtubule motor proteins: how things actually get transported through the microtubule

  Motor proteins utilize ATP hydrolysis to generate a mechanical force that move the motor protein and attached cargo along the cytoskeleton.

  2 types of microtubule motor proteins:

  1. Kinesins
  2. Dyneins

  *Each type of motor protein moves unidirectional and move in a step-wise manner
• Kinesin-1 is a tetramer (2 heavy and 2 light chains) with a globular head and tail.

  Globular head:

  • binds to microtubules and does the actual walking along it
  • Binds to and hydrolyzes ATP
  • have highly conserved sequence

  Tail:

  • Binds to cargo
  • Diverse sequences
  • Requires an adaptor to link cargo to tail
• Label the kinesin

  A) Head

  B) Tail

  
• Kinesin movement:

  • kinesin moves towards the + end of the microtubule (towards growth / shrinkage)
  • Leading head binds ONE ATP → hydrolysis and release of ADP and phosphate result in power stroke that swings leading head forwards
  • Moves the motor 8nm (length of one tubulin dimer)
  • Kinesin heads primarily steps on the β-tubulin
• Kinesin walking!

  A) Leading head

  B) Cargo

  C) Adaptor

  D) +

  
• The step-wise or hand-over-hand mechanism means that at least one head of the motor protein is attached to the microtubule at all times.
• Kinesin movement is highly processive: capable of moving considerable distances without falling off.

  • Its speed is dependent on ATP concentration with a max speed of 1 micro meter/sec)
• Dynein structure

  • Dynein is much larger than kinesin
  • Its head is also faster than kinesin
  • Made up of 2 heavy chains + multiple intermediate and light chains

  Globular head:

  -Binds to ATP and hydrolyzes it (generates force for movement)

  Tail:

  -Binds to cargo via the adaptor protein dynactin

  Stalks:

  -Binds to microtubule
• Dynein structure diagram

  A) microtubule

  B) Tail

  C) Head

  
• Dynein movement

  Dynein moves towards the - end of the microtubule

  Roles in:

  • Positioning the spindle and moving chromosomes during mitosis
  • Positioning organelles and moving vesicles

  Kinesins and dynein move similar materials in opposite directions along the same microtubule
• Neuron directions...

  Moving retrograde = moving towards - end of microtubule (towards dendrites)

  Moving anterograde = moving towards + end of microtubule (towards terminal knob)