Lecture 14 Flashcards
It was known for many years that material could move _______________ in cells?
Bi-directionally
What had already been discovered by the time kinesis were being examined?
Myosins had already been discovered; they used actin filaments as their cystokeletal framework
What had also already been discovered by the time kinesis were being examined?
Axonemal Dynein, a microtubule-associated motor had been shown to move flagella using microtubules
How did kinesin discovery start?
The discovery of kinesis really started with work from competitors/colleagues Robert Allen and Shinya Inoue
What did they use to greatly enchance the resolution during LIVE imaging?
The both used video cameras attached to microscopes that used polarized light to greatly enhance the resolution during LIVE imaging.
What was said in Inoue’s paper?
In this paper where he used polarized light to more clearly identify cellular components. This is of a Diatom
What was Inoue able to capture using his video capture technique?
He was able to capture fine detailed structures in real time…live! This is a 50nm acrosomal extension from Thyone sperm.
What does it mean to “optically” copy a video monitor image with another video camera?
This enabled them to ultimately substract one image (that was out of focus) from another image (that had the material they were interested in imaging). This enables the fine detail to be documented without the additional “junk” in the sample.
What technique did Robert Allen event?
Robert Allen invented a technique called “allen Video enchanced Contrast, Differential Intereference Contrast (AVEC-DIC) Microscopy”.
What does this technique show?
This technique could document microtubule-based motility of material in living things.
What kind of marine worms did Allen use?
Allen used the marine worm called Allogromia laticollaris for his studies.
When did Allen and Brady come together?
Allen and Brady came together when Brady worked with Ray Lesak as a postdoctoral fellow.
What did Brady use AVEC-DIC on?
There he used AVEC-DIC on Axoplasm from squid giant Axons
What is Axoplasm?
Axoplasm is essentially all of the stuff in the axon that is squished out onto a glass slide.
What did Robert Allen and Scott Brady study?
Robert Allen and Scott Brady had been studying the movement of material in squid giant axons for years. They documented the movement of material the sizes of small vesicales (30nm) all the way up to 5000nm (the sizes of organelles).
Who was Ron Vale?
Ron Vale was a graduate student at the time and went to Brady’s lab to learn his techniques of imaging axoplasm from squid giant axons. Discovered Kinesin in back to back papers in Cell
How did Vale acknowledge Brady, Allen and Lasek?
Barely acknowledged Brady, Aleen and Lasek
What did Brady do in return?
Brady (on his own) published 2 papers in Nature on the identification and partial characterization of a 130kDa ATPase (it was kinesis) that had properties expexted for fast axonal transport and caused organelles to bind to microtubules. But it was too late. They were submitted when Vale’s were in press
What do Kinesins use to move?
Use ATP hydrolysis to move primarily towards the + end of the microtubules
What is anterograde movement?
Moving towards the + ends of microtubules
What is retrograde movement?
Movement towards the - ends of the microtubules
What is Kinesin-1?
It is also known as Conventional Kinesis. Moves processively (they can do continuous movement without falling off of the microtubule). It moves cargo (like organelles, vesicles, etc…)
What is the structure of Kinesin-1?
Head(s): ~10nm in size (less than half the size of myosin heads), bind microtubules, catalyzes ATP hydrolysis.
There is a neck linker (between the head and tail).
Stalk (Tail): is a coiled-coil, binds cargo, has light chains (green) attached to it.
How do myosin heads compare to kinesin heads?
Myosin heads are bigger than kinesin heads, one binds actin and one microtubules. As a whole they are structurally very similar and are thought to have a common origin
What is the movement of kinesins?
It walks… takes 8nm steps usually on the SAME microtubule protofilament. That is the exact spacing of tubulin dimers. But, kinesin heads are only 10nm each. It can take these steps because of its neck. Moves at ~0.8um/s (so it steps once every 10 milliseconds)
Do the kinesin heads cooperate?
The 2 heads cooperate. One binds the microtubule while the other releases. All governed through ATP hydrolysis.
Are kinesins constitutively active?
Yes
What happens when Kinesin is not needed?
Tail to head interactions lock the heads in place. Thereby regulating (stopping) kinesin when not needed.
How many families of Kinesins are there?
At least 14 families
What is the general architechture of Kinesins?
General architecture of a head, coiled-coil, tail
Where do N-terminal motors naturally walk towards?
N-terminal motors naturally walk to the + ends of microtubules
What is Kinesin 3 (Kif1a,b)?
A single headed kinesin
Moves very quickly
Can move processively
How does Kinesin 3 not fall off the microtubule?
It can dimerize
Does Kinesin 3 move hand-over-hand?
Unknown
What gives rise to different cargo specificities?
One of the kinesins that showerd that alternative splicing can give rise to different cargo specificities.
What does the tail domain of Kif1b(alpha) target?
Targets to the mitochondria
What does the tail domain of Kif1b(beta) target?
Targets to synaptic vesicle precursors
What is Kinesin 7 (CENP-E)?
A very large kinesin
Where is Kinesin 7 found?
Found at the kinetechore with chromosomes
It is processive
It moves fast in vitro (~10X faster)
Slower in vivo
What is Kinesin 7 used for?
Used for initial stages of chromosome movement to the metaphase plate
How does CENP-E get phosphorylated?
CENP-E gets phosphorylated by Aurora kinases during mitosis. This enables CENP-E to move along kinetochore microtubules towards the psindle equator. As the chromosomes move, protein phosphotase 1 (PP1) starts to dephosphorylate CENP-E (other motors take over).
How many members of Kinesin 13 are there?
Four
What the 4 members of Kinesin 13?
Kif 2a
Kif 2b
Kif 2c (also known as the mitotic centromere-associated kinesin [MCAK]
Kif 24 (found in cilia and centrioles)
What does Kinesin 13 have?
Have internally located motor domains
Does Kinesin 13 use ATP?
They do NOT use ATO to move along microtubules. Instead they use ATP to depolymerize microtubules.
What is Kinesin 13 used for?
Used during anaphase to shorten the kinetochore microtubules
What can Kif 2c do?
Kif 2c can dissassemble microtubules very fast from either microtubule end
What is different about Kinesin 14 (Ncd)?
Minus-end directed kinesins (they walk the wrong way!)
Can dimerize
Move slowly as compared to the + end directed kinesins
Where are the motor domains located on Kinesin 14?
Have their motors domains at the C-termini. This is due to their central coiled-coil stalk (neck) region. It positions the stepping head to walk in the wrong direction. So, the neck dictates the direction kinesins will walk!
How are Kinesin 14 as single motors?
Non-processive as single motors, but you could have many bound to a single cargo
How far do Kinesin 14 move?
Only move 20-50nm (so,2 to 6, 8nm steps) then they fall off the microtubule
How do kinesins and dynein bind?
Kinesins and dynein (through dynactin) can bind directly through their cargo-binding regions to transmembrane proteins in a vesicle or organelle to transport them in the cytoplasm
How can additional specificity and selectivity be accomplished?
Additional specificity and selectivity can be accomplished by binding to additional adaptor proteins or protein complexes
What is thought about most proteins, organelles, etc?
It is thought that most proteins, organelles, etc… have developed ways to be recognized by both dynein and kinesin for transport in both directions
Developing ways to be recognized by both dynein and kinesin for transport in both directions, how is this accomplished?
This is accomplished through many mechanisms including:
1) The post-translational modification of the cargo protein
2) SUMOylation
3) Phosphorylation of adaptor proteins
4) ATP or calcium limitations
What can influence kinesin movement?
Microtubule modifications
Microtubule modifications can influence kinesin movement include:
1) Acetylation of alpha-tubulin
2) Lysine 40
3) Detyrosination of alpha-tubulin
4) Polyglutamination of the C-terminal tails of alpha- and beta-tubulin
What does Kinesin-1 bind with?
Kinesin-1 binds with higher affinity to acetylated and detyrosinated microtubules.
What do Kinesins-2 and -3 do?
Not really impacted by acetylation or detyrosination of microtubules
What is Kinesin-3 sensitive to?
Kinesin-3 is sensitive to polyglutamylation. A decrease of Kinesin-3 at synaptic vesicle cargoes at axon terminals and in mice mutated in tubulin polyglutamination sites.
What is KIF1B?
Loss of function mutations in the motor domain= Charcot-Marie Tooth type 2A disease
What is KIF5A mutation (only found in neurons)?
Hereditary spastic paraplegia
What is KIF5C mutation?
Amyloid precursor protein associates with the C-terminal tail (Alzheimer’s disease)
What is KIF3/Kinesin 2 disease?
These kinesins deliver material within cilia and flagella. So, there are many diseases associated with their mutations:
1) In kidney: polycystic kidney disease
2) In eyes: retinitis pigmentosa
3) Kartagener’s syndrome (sperm flagella, bronchial cilia defects)
