Cytoskeleton (Unit 6) Flashcards
What do the following chemicals do?:
-Latrunculin
-CytochalasinB
-Phalloidin
-Taxol (Paclitaxel)
-Nocodazole
-Colchicine
-Binds actin subunits to depolymerize
-Caps filament + ends to depolymerize
-Phalloidin: Binds Actin filaments to stabilize
-Binds MT to stabilize
-Binds tubulin subunits to depolymerize
-Caps both ends of MT to depolymerize
What does Critical Concentration mean?
Subunit Concentration where monomeric subunits and the polymer are at equilibrium
Thus, rate of addition equals the rate of loss.
What is the axoneme?
The core of cillia and flagella
Contains:
-9 MT doublets in a circle that have dyenin arms
-2 MT singlets in the middle
-Associated proteins
When it bends, it creates movement
What are cillia and flagella made of?
MT and dynein
What is the difference in cillia and flagellar motion?
Flaggella: Undulating motion
Cilia: Whip-like motion
Where would you find cilia vs flagella?
Flagella: Protozoa, sperm (not the same as bacterial flagella)
Cilia: Protozoa, respiratory system & oviduct cell surfaces
Are cillia motile or non-motile?
Can be either
Compare the structures of cilia and flagella
Cilia is short and hairlike while flagella is long and thread-like
What roots cilia and flagella to the cell surface?
Basal Bodies
-sturcutre with 9 MT triplets like a centriole
What does Dynein do in the axoneme?
Forms bridges between doublets on the outer circumenference
Moves towards - end once activated which (instead of having the MTs slide past eachother) creates a bend due to the tubules between cross-linked
Why would a cell want to move?
Immune cells must find bacteria infection
Nervous system cells need to form neural network during development
Chemotaxins direct cells based on chemical gradient
Amoeba need to eat
Cancer Cells
Why is cell movement unidirectional?
Driven by actin which treadmills in one direction due to coflin dissembly at ADP g-actins
What regulates actin by helping us determine the front vs back of a cell?
Cell surface receptors
What do cell surface receptors work through to control actin
The Rho Protein Family
-Rho
-Rac
-Cdc42
What do Rho, Rac and Cdc 42 do?
Rho: Bundles actin and myosin II to make stress fibers and brings integrin to focal contacts
Rac: Creates lamellipodia around the cell circumference
Cdc42: Nucleates actin at the + end to make lots of filopodeia
What are the steps of Cell movement?
Protrusion:
Bits of PM pushed outwards and filled by filamentous actin (contains no membrane-bound organelles)
Attachment:
Focal Adhesions (where integrin links cell to ECM)
*Form at the front and disengage at the back
Traction:
Myosin contracts to pull actin to new orientation
What is the cytoskeleton?
What does it do?
A network of filamentous proteins involved in the shape, strength and movement of the cell(SSM)
What do accesory cytoskelton proteins do?
-Act as motors
-Help assembly/dissembly
-regulate connections
What are the 3 main cytoskleton proteins
Actin: Shape and movement
MT: Intracellular movement (Transport, organelle positioning + mitotic spindle during cytokenesis)
IF: Mechanical Strength
What is meant by filament polarity?
The two ends act differently
Tends to polymerize at + end (where mostly likley T form) and depolymerize at - end (where most likely D form)
How does polarity work in actin
Actin monomers all face the same direction thus te filament will always have a plus and minus end
These ends have different kon/koff ratios such that the plus end will always polymerize faster than the minus end
This means that both ends have different Critical concentrations
*Note: If in same T/D state both ends still have the same monomer affinity they just have different kinetics !
why does treadmilling happen in actin?
If the [subunit] is above the Cc for the + end (such that it has net growth) but below the Cc for the minus end (such that it shrinks) then there will be an overall steady state where loss = gain
This requires ATP so that the difference in polymerization speed is great enough between the ends (ATP makes polymerization go way faster)
What is the subunit for actin?
A monomer called G-actin (globular actin) which binds ATP
What is the full structure of actin?
G-actin polymerize to F-actin
2 F-actins polymerize to right-handed helix to make actin molecule
What is the difference in structure between + and - actin?
Plus end is barbed
Minus end is pointed
How strong is actin
it is a flexible molecule whose rigidity can be INC by accessory proteins
where on actin does ATP bind?
minus end
what is the point of actin nucleation?
When there is only a small number of monomers are bound to each other, their a few bonds and so the molecule is unstable and there is a (lag phase- it will literally take forever)
Nucleation helps us by-pass the kinetic barrier by helping us to reach the critical # of monomers required for filament formation
In vivo, what form will most G-actin be in?
T form!
Why is delta G the same for both ends of actin?
Because the same interaction is being broken/formed
Why is the + end of actin more likely to be in T form than the - end?
Because its polymerization is faster, it can catch-up to and go faster than ATP hydrolysis unlike at the minus end (hence why both ends have different critical concentrations)
What if polyermization and hydrolysis rates are around the same for a filament?
Dynamic instability!
(the rapid conversion between growth and shrinking at the same subunit concentration)
Involves catastrophe (Hydrolysis wins -> rapid deplymerization)
and rescue (ATP added -> rapid polymerization)
Why does growth happen with T subunits and shrinkage with D ones?
The end with T on it is more likely to be the faster-growing + plus.
Also ATP-bound G-actins are better at polymerizing.
Thus:
Growth: Growth outruns hydrolysis
Shrinkage: Hydrolysis outruns growth
What are the main structures involved in cell crawling?
Filopodium: Long and thin 1D bundled actin
Lamellipodium: Wide 2D sheet of cross-linked actin in a mesh that remains stationary with respect to the substrate. + ends point in migration direction while - ends attatch to other actin filaments via ARP
Invadopodium (podosome): Large 3D blob. Helps with crossing tissue barriers
Bleb: Actin-populated extension of PM that is detached from the cortex
How does Cdc42 work?
When bound to GTP it is active and can associate with WASp proteins.
These proteins bind and enhance Arp 2/3 complexes which then enhance actin nucleation activity
Why do we use keratocytes as a cell crawling model?
Their movement is locally regualted (thus even a sliced off fragement can keep crawling) and very fast.
They have a large, lamellipodium with a small trailing cell body
What are keratocytes?
Epithelial cells from the epidermis of frog and fish that are specialized for wound healing and contain a lot of keratin
What are septins?
GTPbinding proteins used to create cell polarity
They asseble into NP filaments that then create rings and cage-like structures for :
-membrane compartmentalization
-recruitment and activation of MTs and actin
Involved in:
trafficking (vesicles), cell migration and cell division
what do septins do?
Scaffolding for membrane compartmentalization
ex: assembles at the base of primary cillia to separate that part of the membrane
Other examples
-Cell Migration
-Vesicle trafficking
-Cell division
What does y-TURc do?
Caps the - end of MTs
What do cilial capping proteins do?
Help cilia keep uniform length and stability
What are MAPS
What regualtes them?
MT Associate proteins
(regualted by phosphoryalation)
what does kinesin-13 do?
Pulls apart MT ends to induce catastrophe (thus its a catastrophe factor)
What does XMAP215 do?
Brings free tubulin to + end to stabilize it and promote growth (thus its a rescue factor)
What are TIPs
+end trackers
They bind MTs that are growing and unbind those that aren’t
They:
regulate the shrinking and growth of the MTs
OR
stabilize MTs at specific positions in the cell to position them
What do kinesin-related proteins do to MTs?
Can you give an example of one?
Act as catrastrophe factors
ex: kinesin-13
What does Stathmin do?
INC the chance of catastrophe by binding tubilin subunits to keep them from polymerizing
What is stathmin?
a small protein that binds tubulin subunits to INC dynamic instability and DEC elongation
This process can be inhibited via phosphorylation
What is SUN-KASH bridge?
A protein Complex that attatches the Nuclear lamina to
-MTs (via motors or plakin) & actin and plectin (both directly) on the outside of the nucleus
-SUN binds Lamina and exists inside the nuclus and in the inner space
-KASH binds SUN and exists outside the nucleus and in the inner space
What is the structure of MTs?
Subunits: Heterodimers bound by tight non-covalent bonds where both bind ATP but only B acts as an ATPase (alpha needs ATP for structural reasons)
They stack to form protofilaments which combine in parallel groups of 13 to form an MT
*alphas and beta of diff protofilaments usually paired up just not at the seam
What are the stiffest and straightest structures in animal cells?
MTs.
Their structure makes them stiff and hard to break!
What happens when a protofilament has GDP at the end?
It starts to curve bc it needs GTP to caps to keep it straight
Where does nucleation tend to occur and what activates it for actin
A required first step when it comes to forming actin filaments
It often occurs at the PM and in response to external signals
What are the 2 Actin Nucleation factors?
-ARP 2/3 Complex: At negative end
-Formins: At + end
What does the ARP 2/3 Complex do?
What activates it?
Nucleates - end of actin to allow rapid growth at the + end
Can also bind to the side of another filament at the same time to form branches
If multiple branches form a web, a gel-like substance is made
Activated by the Nucleation promoting factor
What are formins
dimers where each monomer has a G-actin binding site
They hang out on the + end capturing G-actins (sometimes with whiskers)
They Form straight, unbranched actin that can be cross-linked into parallel bundles
What two motor proteins are associated with MTs?
Kinesins and Dyneins
What are kinesins
A superfamily of motor proteins that are all dimers of 2 heavy chains that create a coiled coil
They heads bind MT while their tails bind 2 light chains that mediate the binding of cargo
How do Kinesins move?
Lagging head (ATP-bound) hydrolyses ATP (losing Pi) so it can leap over the leading head (ADP-bound) (by a distance of ~one tubulin)
During this leap, the Leading head swaps ADP for ATP
What is the main structure of dyeneins?
ATPase in motor head domain connects to two linker proteins in the tail domain
The linkers bind the unmoving MT, the MT binding domains (at the end of the ATPases’s stalk) fishes for the 2nd MT and pulls its - end closer
What are dyeneins?
What powers them?
The biggests and among the fastest molecular motors
Powered by ATP hydrolysis
What is the difference between cytoplasmic and axonemal dynein?
Cytoplasm: 2 heavy chains make a homodimer
Jobs: Transport in flaggella, transport of organnelles and mRNA in cell
Axonemal: Can be monomers, heterodimers or heterotrimers
How does the dyenein motor work?
1) Relaxed dyenine is bound to 2nd MT and has the linkers close to the head
2) ATP binds causing MT binding domain to release
3) ATP hydrloyzes causing stalk to swing 8 nm towards the - end of the 2nd MT and then rebind
4) ADP and Pi released and the motor (+ MT1) moves towards the - end of MT2 as the linkers go back to being close to ATPase
*Result is MTs slide past eachother but no net movement
What are IFs?
Rope-like structures made from paralell coiled dimers that form antiparallel tetramers that then associate in groups of 8 that laterally associate into a filament
What are some examples of IFs
kerain and Neurofilaments
What is keratin?
a heterodimer filament that’s been crosslinked into networks with disulphide bonds
One monomer is acidic and the other is basic/neutral
what does keratin do?
Gives mechanical strength to epithelial cells
What is epidermolysis bullosa?
Keratin is disorganized
Leads to blistering skin disease bc skin won’t be tough
What is flaggrin?
Bundles keratin
Can lead to dry skin if mutated
What are neurofilaments?
What disease are they associated with?
Heteropolymers found in neuron axons.
They can cause ALS if assembled abnormally
What are the 4 main things that actin accessories regulate?
-Length
-#
-Stability
-Geometry
What does Thymosin do?
Binds actin subunits to prevent growth
What does profilin do?
Concentrates monomers at actin assembly sites
How are MTs nucleated?
gamma-TuRC is made of gamma-tubulins and forms the template for building MTs with 2 other proteins
*Specifcally it nucleates the - end
What is the main MTOC of the cell?
The Centrosome (also called the center of the cell)
It has tons of gamma-Turcs along the circumference and two special MTs in the middle making an L shape (centrioles)
- MT ends embedded near its nucleus while + ends spread out towards periphery
What is an MTOC?
MT organizing center
-contains a lot of gamma-tubulin
-exists in a characteristic astral organization
How do cell fragments form a center
If they have MTs they will create a new MT that lacks centrioles
what does tropomyosin do?
Stabilizes + end of actin and controls the binding of other accessories
Fimbrin
forms tight actin cross-links with no myosin
Alpha-actin
forms loose actin cross-links that allow myosin in between them to create contractile filaments
Filamin
Forms a loose, high-viscosity gel for lamillipodia using right-angle clamps
Spectrin
Protein attatched to peripheral membrane proteins (which are attatched to integral ones) that
creates an actin web on the inner surface of the PM with its two short-actin binding sites
ERM family
Actin binding protein on the PM that:
-Regulates signal transduction
-bridges transmembrane proteins to cytoskeleton
-organizes membrane domains (main thing!)
Tropomodulin
Caps - end of actin to prevent assembly/dissembly there
found in muscle
-only at ends thus, in substoichiometric amounts
Colflin
Binds side of actin to create torsion (which weakens subunit interactions)
Binds D-form thus tends to only bind older filaments
Essential for polarized cell growth during cell crawling
Gelsolin
Binds + ends of actin and severs filaments
activated by high Ca+ in cytosol
Subdomains Binds 2 sites on actin side subunit and then waits for thermal fluctuations to create a space they can insert into
Does not need ATP
Capping protein
Binds + end of actin to stop it from polymerizing or depolymerizing
Augmin
Nucleates MT branching
Plectin
A type of Plakin thus:
Links IFs to other cytoskletal things like MTs or actin
Bundles IFs
Attatches IFs to adhesive structures at PM
MAP2
Binds side and then forms right angle for wider-spaced MT bundles
Katanin
Severs MTs
Dimer with a small subunit that can hydrolyzed ATP and a large subunit that directs it to the centrosome
Jobs:
Release MT from MTOC
Rapid MT depoly at spindle pole during mitosis
Depoly MT in proliferating cells during interphase and post-mitotic cells
tau
Binds side and then forms acute angle for more dense MT bundling
General role of MAPs
For MTs:
Stabilize and prevent dissembly
Cross-linking
Regulate tightness of bunding
What is the centromere?
The region where the spindle fibers attatch to a chromosome
What are microvilli?
Stable Actin-made protrusions found in epithelial cells that are turned over every few days
What determines the polarity of a cell?
Is polarity a stable structure?
Cytoskleton organization determines the polarity
Yes!
