Week 10: Microtubules/intermediate filaments Flashcards
what are microtubules composed of?
alpha and beta tubulin globular proteins
- these stay as a unit and form a hollow space in the middle
protofilament
stacked microtubule heterodimers
- Stacked going down and as a tubular shape ⇒ asymmetry applies just as in actin filaments
- The top unit has 1 unit of the heterodimer and the bottom of the tubule has the other
microtubules and their strength
largest and most rigid filaments ⇒ actin are easier to bend
- You cannot bend a tube as easily ⇒ the further you spread the same mass away the more you can resist bending
what properties do microtubules have? (2)
- They form strong structures that resist bending forces
- They form hollow structures with multiple lateral interactions
do microtubules treadmill?
treadmilling length stays the same just as actin filaments
- Length will increase at the plus end and then fluxes out on the minus end
microtubule organizing center (MTOC)
microtubule origination comes from here
- nucleus is in the center and spindles emerge from the region around the nucleus (MTOC)
where do microtubule minus ends anchor?
at centrosomes
centrosome definition
the blob in the middle where microtubules emerge from the site where the minus ends are anchored here to gamma tubulin ring complexes
what are microtubule nucleation sites?
They anchor from the gamma tubulin ring complexes off the centrosome
- Many are localized around the centrosome and act as capping proteins for the minus end of the microtubules
- If you stabilize the minus end then the plus end is free to grow
- This allows microtubules to go in all directions
centrioles
a pair of short microtubules at right angles to one another in the pericentriolar material ⇒ on the outside the gamma tubulin lets the free tubulin polymerize out
what are gamma tubulin ring complex?
- the gamma tubulin look like a spring washer and has a slightly offset appearance that stabilizes the alpha and beta on it so the microtubule can grow off
- this provides a template (like ARP2/3 complex) for stabilizing the minus end
- the alpha tubulin is what is directly on top of the game and the beta is on top of the alpha unit
is the beta or alpha of the microtubule the plus end side?
the beta end is plus side and the alpha end is the minus side but they add on in a heterodimer
are plus ends of microtubules stable?
no
EB-1 protein
tracks microtubule tips via GFP tagging (+ end)
- Looks like comets all coming from the microtubule organizing center at the centrosome
- If you zoom in to the plus ends they are very dynamic
how did scientists demonstrate dynamic instability?
they took a seed that mimics the centrosome and they added in rhodamine labeled tubulin to observe what happens with just tubulin and a seed stabilizing the minus end
dynamic instability
the microtubules grow and then shrink constantly
- they go out and fall back rapidly
GTP in microtubules?
GTP is the nucleotide backbone allowing new dimer units to be added on
- the GTP is inside of the Beta dimer and GDP is exchanged for GTP when it gets added on to the plus end
- the older units will hydrolyze just like in actin
what does GTP hydrolysis do to microtubule filaments?
hydrolysis curves the filaments and makes them unstable ⇒ the microtubule tends to curve instead of staying straight
- When GTP is hydrolyzed it fits in slightly differently ⇒ GTP lines straight but GDP angles a little bit
catastrophe
when the microtubule peels apart after too much strain is placed on the filaments and most of the filament collapses again
- Eventually the GDP caused strain gets so large a catastrophe happens
what is needed for the microtubule catastrophe to happen?
to start peeling back it needs a charge
- As long as you have a GTP bound microtubule cap it will keep the entire microtubule from exploding and catastrophizing
what happens when you cannot add GTP fast enough to microtubules?
the GTP cap currently on the end will explode because a new dimer unit isn’t placed on fast enough which causes shrinkage
- technically if there is nothing holding it back it will catastrophe back to the gamma tubulin
how does catastrophization stop?
As you start losing filaments, these subunits will increase the monomer concentration which exchange GDP for GTP so they will add back on to the filament and stabilize the cap again
- As long as the cell is alive it will regenerate GTP
what 4 things are true about shrinking microtubules?
- they have a GDP cap at their plus end
- they have weak lateral interactions at the plus ends
- they have curved protofilaments at the plus ends
- they alternate with growing microtubules leading to dynamic instability
T/F the plus ends of the microtubules are capped which stabilizes the,?
True and dynamic instability allows the cell to figure out how to polymerize the microtubule network
- does not have a brain so it will go all over
emergent behavior
cells can strategically position your capping proteins where the cell wants to grow outward
- Often capping proteins are in one area of the cell
- Often capping proteins are controlled by other signaling proteins
- the cell can easily relocalize its network this way to change the shape of the network on demand
how can the microtubule capping protein influence cell shape?
based on where the capping protein is localized you will get very different shapes of cells
nerve axons
carry electrical impulses which include intermediate filaments and microtubules to keep the axon structure stable without falling apart
anterograde motor
kinesin
kinesin
anterograde (forward) motor protein and similar to myosin it has a head that engages with a microtubule
how does kinesin work?
By cycling ATP hydrolysis, it moves off the microtubule
- Unlike the lever arm in actin it has a neck linker
- These carry vesicle drops
-When ATP goes in it zips up on the kinesin side so it stays anchored while the next head anchors its spot
- The process gets repeated when ATP gets lost to ADP and the back foot releases
- There are mechanical gating techniques so one foot is held down at all time
retrograde motor
dynein
dynein
retrograde motor which is much bigger than kinesin
- It is a mega dalton protein
how doest dynein work?
dynein has a ring ATPase and function like a ratchet through the flower shaped structure
- Each subunit can bind ATP and act in a coordinated way to set off atp
- At the end of the ratchet there is a stalk which moves as the ratched turns
which motor protein can generate larger forces per motor?
kinesin over dynen even though dynein is bigger
why do we need dynein and kinesin in a nerve cell?
In both axons and dendrites you need movement of things toward the + and - end
- If you don’t have it one end will not receive certain factors which presents the synapse from happening
- It will fold up and you lose the synaptic connection
what does kinesin drive?
extension of the ER network
- the shape of the ER is formed on the back of the microtubules
how does dynein affect the Golgi?
it places it close to the centrosome
- Golgi is localized around the nucleus so this is being maintained by dynein
unconventional myosin
non primary myosin such as that in muscle that play a role in membrane transport
where do myosin VI bind with cargo binding domains?
in endocytic vesicles
what does myosin VI do? (3)
- The myosin can latch on to the clathrin coated pits and help transport on the actin network to move the vesicle
- They may also allow for filopodial membranes to generate force
- You can then position membrane organelles inside cells
filopodia
finger-like cellular protrusions that perform fundamental cellular functions during development and cell migration
what 3 things can myosin VI be involved in?
- endocytosis => import of clathrin vesicles
- filopodium => outstretches for cell movement
- Golgi morphology => moving vesicles from Golgi
what is a special property of myosin VI?
it is the only minus end directing myosin
T/f intermediate filaments are the most stable cytoskeletal filament?
True because they need to just be there and create the viscoelasticity of the cytosol
- these work by their own rules
viscoelasticity
you need stability for a network (cell) so it doesn’t fall apart
how do intermediate filaments assemble?
- One monomer is braided with another monomer protein
- They are not globular proteins ⇒ they are fibers you braid
- they are a lateral association of 8 tetramers of coiled-coil dimers
T/F intermediate filaments treadmill?
False they do not treadmill
which sides of the IF assemble together to make a staggered tetramer of 2 coil-coil dimers?
the COOH side on the inside and the remaining NH2 are on the outside
what is a coiled-coil dimer made up of?
2 alpha helical regions of monomer IF’s (48 nm long)
why are IF’s difficult to take apart?
because it 8 tetromers to form a filament which is much more stable than actin and microtubules
what is the purpose of IF’s?
to maintain the mechanical integrity of the cells
- axons need neurofilament proteins that create the actual mechanical rigidity for the axon
what are the 4 main classes of IF’s?
- keratins
- vimentins
- neurofilaments
- lamins
what are keratins?
junctions that protect epithelia from mechanical damage
- On your epithelial cells
- Have an acidic and basic keratin
what can you trace keratin to?
cancer
what are vimentins?
proteins found in connective tissue, muscle cells, and glial cells but not epithelial cells
- are a homodimer
what are neurofilaments?
support axons in nerve cells
what are lamins?
form nuclear lamina that support nuclear envelope in all animal cells
how do keratin intermediate connect cells together?
help epithelial cells form a mechanically continuous sheet
- Between the two cells you have a weld joint (spot weld) connecting the filaments to a plaque on either side
desmosomes
intercellular junctions that link the keratin filaments of one cell to another via cadherins
what prevents you from having an internal bruise if you pinch your skin?
the spot wells between keratin filaments across cells
- If the cells are displaced from the underlying basal lamina, this is a problem
hemidesmosomes
link the keratin filaments of epithelial cells to the basal lamina
- uses integrin and this protects cells against mechanical stress
- If any parts of this is not working right then your cells rupture
what is the cytoplasmic plaque made of between cells?
anchor proteins keeping the cell membrane/cadherins to the intermediate filaments
Epidermolysis bullosa simplex (EBS)
one type of skin blistering disease form a defect in keratin expressed in bottom layer of skin
- Blisters form between epidermis and connective tissues
- Until recently, no treatment beyond wound care
- Bone matter transplant is the only way to help fix this
how do EBS treatments work?
transplant donor stem cells into bone marrow
- Donor stem cells migrate to dermis (connective tissue layer)
- Express wild type protein and repair defective junction
