Lecture 23: Microtubule Motors

What kind of motors are kinesins?

plus end directed motors

What kind of motors are dyneins?

minus end directed motors

True or False: Many vesicles have both kinesin and dynein motors attached to position organelles halfway.

true

What is anterograde axonal transport?

newly synthesized proteins are moved from the cell body to the tip of the axon

What is retrograde axonal transport?

old membranes are transported back to the cell body

True or False: Axons can be up to 1 meter long.

true

True or False: Different materials move at different rates along axons.

true

How long does it take for a vesicle to move from the spinal cord to foot and then back?

3 weeks to 3 months

What species is known to have a giant axon?

Loligo (squid)

How many vesicles can be moved on 1 MT?

2 vesicles

Why can 2 vesicles be moved on 1 MT at the same time?

the protofilaments in the MT is straight

What type of motor is an anterograde motor?

plus-end directed motor

Will there be movement if vesicles, ATP, and MTs are the only materials present?

no movement

Will there be movement if vesicles, ATP, MTs, and squid cytoplasm extract is present?

yes, there will be movement

Will there be movement if ATP is substituted with a non-hydrolysable analog?

no movement, vesicles bind tightly to MTs

How was kinesin identified?

• Incubate MTs, cell extract, and ATP analog

• Motors bind tightly to MTs

• Collect MTs

• Release bound protein with ATP

What is the structure of kinesin?

• 2 heavy chains and 2 light chains

• central stalk/coilied coil

• tail

What do kinesin heads do?

binds ATP and MTs

What do kinesin tails do?

binds cargos

What is the linker region of kinesin also called?

the neck region

How are myosin and kinesin related?

distantly through their head regions and have similar shapes

What is a conventional kinesin?

kinesin that heads an N-terminal head

What is an unconventional kinesin?

a kinesin with a non N-terminal head

True or False: Most kinesins are dimers, so the proteins are able to walk processively.

true

What structure are kinesin in when inactive?

a folded-up structure

How is a kinesin activated?

by binding cargo

What is the length of a step for a kinesin?

8 nm or 1 dimer/step

True or False: Kinesin walks along 1 protofilament.

true

How is movement in kinesins generated?

by reversible binding of the neck to the head

What throws one kinesin head forward?

ATP hydrolysis

Do kinesin heads influence each other?

yes, they influence the nucleotide binding of each other

True or False: Kinesin heads must be in different phases of the ATPase cycle.

true

If nucleotide is not bound to head, then what is the state of MT binding and interaction with the neck?

• tight binding to MT

• no interaction with the neck

If ATP is bound to head, then what is the state of MT binding and interaction with the neck?

• tight binding to MT

• interaction with neck

If ADP-Pi is bound to head, then what is the state of MT binding and interaction with the neck?

• weak binding to MT

• interaction with neck

If ADP is bound to head, then what is the state of MT binding and interaction with the neck?

• weak binding to MT

• no interaction with neck

What is the kinesin ATP cycle?

1. The leading head is strongly bound to the MT, and the trailing head is weakly bound (in ADP state).

2. The leading head binds ATP and remains strongly bound.

3. Binding of ATP causes the linker to swing forward and dock into the head. Trailing head moves forward (power struck).

4. New leading head finds a finding site 16 nm ahead of previous site.

5. Leading head releases ADP, and trailing head hydrolyzes ATP to ADP and Pi. Linker becomes undocked and head becomes weakly bound to the MT.

What is kinesin-1 and kinesin-2 invovled with?

organelle transport

What is kinesin-5 invovled with?

• binds 2 MTs

• sliding

• forms tetramers

• mitosis

What is kinesin-13 involved with?

depolymerization

True or False: Dyneins were purified based on similar ideas and assays used to study myosins and kinesins.

true

What did cloning of genes encoding dyneins reveal?

• dyneins are substantially different from kinesins and myosins

• dyneins have a domain that is part of the AAA ATPase superfamily

What does AAA stand for?

ATPases associated with many activities

What is important to know about the progenitor of kinesins and myosins?

the progenitor predated the 3 domains of life

What is the common ancestor of the 3 domains of life?

LUCA

What does LUCA stand for?

last universal common ancestor

How long ago did LUCA exist?

3.6-4.1 billion years ago

How old is the Earth?

4.5 billion years old

True or False: AAAs are found in all 3 domains.

true

What is the typical structure of AAA ATPases?

hexameric disks that contain 6 AAA ATPase domains

What does the 26 S proteasome do?

degrades polyubiquitinated sequences

What does the cap of the 26 S proteasome contain?

an AAA ATPase

What does the AAA ATPase do in the cap?

unfolds the ubiquitinated protein and threads it through the chamber

What is a common feature of AAA ATPases?

threading activity

Where can “threading activity” be seen?

• protein degradation

• recombination

• DNA replication (helicase)

What materials can be “threaded”?

protein, dsDNA, and ssDNA

True or False: The AAA ATPase moves in the opposite direction of the threaded material.

true

True or False: DNA helicase is a hexameric AAA ATPase.

true

What is important to know about Spastin?

• Katanin-like protein

• AAA ATPase

• severs MTs

What is the sliding clamp important for?

helps move DNA Pol quicker along the sequence and allows it to not away from DNA

True or False: The sliding clamp is not an AAA but the clamp loader is.

true

What is the structure of the clamp loader?

5 subunits not 6

How does the clamp loader adopt a hexameric shape?

subunits bind to the clamp loader and ATP is hydrolyzed

What are the features of AAA?

• threading

• complex assembly or disassembly

What is the structure of dynein?

• stem (cargo binding)

• head (ATPase)

• stalk (MT binding)

What part of dynein is an AAA?

the head

True or False: Dynein is one giant polypeptide. There are different dynein family members in monomers, dimers, and trimers.

true

What conformation would dynein ADP-Vi be in?

pre-powerstroke conformation

What conformation would dynein Apo be in?

post-powerstroke conformation

How does the structure of dynein change from pre-powerstroke to post?

the head is rotating—changing angle from and stalk

What was the first use of Spectrin and Ankyrin?

to move organelles/vesicles

How does dynein contact vesicles?

indirectly through dynactin

True or False: Arp 1 is an actin-related protein.

true

How many monomers does Arp 1 have?

8 monomers

What are 2 modifiers of dynein processivity?

NudE and LIS1

What does NudE and LIS1 do?

forms a complex that bins to and lets go of the head—acting like a low gear for motors to allow for a heavier load

What does Lissencephaly result from and in?

results from mutations in LISI and results in no ridges or furrows in the brain

True or False: Researchers are still identifying motors and sorting out which family members move which vesicles.

true

True or False: There are approximately 45 kinesin genes in the human genome, and 16 genes that code for dynein heavy chains in humans.

true

What allows for a high density of organelles and vesicles within a cell?

the organization by MTs and motor proteins

What are melanosomes?

pigment vesicles

What are melanosomes moved by?

dynein and kinesin

What helps to change color in fish scales?

motor proteins

Where is the post-translational modification of MTs found?

the leading edge of cells

What is plus detyrosylation?

removing/replacing tyrosine at the C-terminal

What can post-translational modifications of MTs affect?

MT stability and motor association/activity