FINAL Flashcards
Cytoskeleton
3 main types:
Intermediate filaments
Microtubules
Microfilaments (actin filaments)
Intermediate filaments
Main function:
size:
Main function: Mechanical support/resisting
Intermediate filaments to relieve the pressure and provide some resistance
Important for Cell shape
10nm Rope like fibers
Intermediate in size
Different types of cell types express different intermediate filaments
Share common structural properties
Function to absorb mechanical strain within cells and tissue
IFs form a strong network in the cytosol
Intermediate filaments structure
2 Monomers- long alpha helix- assemble together to form coiled structure
8 stacks of tetramers come together to form a rope like IF
Staggered tetramer of two coiled-coil dimers- opposite direction tetramers
Two tetramers packed together end to end
Intermediate filaments are made of long twisted strands of fibrous proteins
Intermediate filament structure depends on the lateral bundling and twisting of coiled coils
Desmosomes
Desmosomes connect IF of neighboring cells
joins intermediate filaments in one cell to those in a neighbor. Transmembrane proteins that are anchored to the ends if IF’s on the cytosolic side.
Desmosomes join IFs of adjacent epithelial cells while Hemidesmosomes anchor IFs in a cell to the basal lamina
Thicker looking PM due to all the membrane proteins (desmosomes) lines up to connect the IF
IFs end at the cell membrane but they are connected to desmosomes that act as an adapter to connect neighboring cell’s IFs.
IFs in epithelia form a strong network in the cytosol that links indirectly to neighboring cells with desmosome proteins
why cant motor proteins use IF as substrate
Motor proteins cannot use IF as substrate- motor proteins can not use IF as they lack intrinsic structural polarity- tetramers are coiled in opposite orientation, removing directionality of IF
Different types of IF in different cell types
Cytoplasmic:
Cytoplasmic:
keratin- found in epithelial tissue, and their derivatives- hair, feathers, nails, claws, horns
Skin epithelial cells have the highest density of IF- skin is subject to forces
neurofilaments- in neurons which give structure to the neuronal processes
In axons lined up in parallel and other proteins that form cross connections between neurofilaments
Vimentin and vimentin-related- in connective tissue, muscle cells, and glial cells
Different types of IF in different cell types
Nuclear:
Nuclear:
Nuclear lamins- in all animal cells, add structure to nucleus
Makes scaffolding structure underneath nuclear membrane
Assembly of nuclear lamins is regulated by phosphorylation
Phosphorylation of lamin disassemble in prophase
Dephosphorylation of lamins rebuild nucleus
Hemidesmosomes
looks like half a desmosomes
not connected to neighboring cells, it is connected to the basal lamina(layer just outside the cell- extracellular structure). Anchors intermediate filaments
Cells can be grown on flexible plates and stretched to…
gives elasticity and resistance to tissues
Description: in WT cells, keratin looks rope-like, continuous, uniformly distributed throughout the cell. In mutant cell, keratins not as distinct; strands broken into smaller pieces (compared to WT)
Explanation: the mutation decreases the tensile strength of the keratin. We know this because we see tht keratin has been broken up into small pieces after stretching, but it remains in rope-like structures in the WT cell. Mutations in intermediate filaments affect the cell’s ability to resist externally applied force.
Mutation in keratin 5 causes a form of epidermolysis Bullosa Simplex are generally autosomal dominant- you only need to inherit one copy of this mutated gene to have this genetic disorder
Both WT and mutant alleles are transcribed and translated to make proteins
WT monomer encoded by WT allele
Mutant monomer- encoded by mutant allele, recall that this mutant has frameshift +delayed stop codon
Mis of WT and mutant monomers affect overall structure, strength, and function of keratin IFs
SUMMARY (IFs)
Provide mechanical stability to animal cells
Interactions with accessory proteins enhance the strength of IFs, and help link IFs to other skeletal proteins
The only non-polarized and fibrous component of the skeleton
The only cytoskeletal filaments that do not have associated motor proteins
Built from coiled-coil alpha helical dimers that associate in an antiparallel fashion into tetramers
Microtubules
25nm thick sturdy tubes- thickest of the 3 cytoskeletons
Made of tubulin dimers
Mitotic Spindle formation- pulling apart chromosomes
Organelle positioning
Vesicle trafficking-motor tubule
Microtubule all along axon- carrying neurotransmitters in secretory vesicles
Microtubules In vitro-
monomers in a tube; initiating cytoskeletal polymerization (nucleation) to build microtubule or actin polymers is a slow process in vitro
Microtubules are hollow tubes of ɑβ-tubulin heterodimer (2 different types of tubulin stuck together) subunits
GTP bound by β-tubulin subunit. GTP has a role in MT dynamics; can be hydrolyzed and exchanged
Attached end to end to form a protofilament
Protofilament roll up into microtubule
Microtubules have a structural polarity
ɑ-tubulin exposed on (-) end
Β-tubulin exposed on (+) end
Tubulin dimers can be added and removed at BOTH ENDS but at different rates
Mechanism of spontaneous microtubule assembly
no enzymes required
Pool of free tubulin dimers to microtubules
Energetically favourable to form microtubules in the aqueous environment of the cell
Tubulin dimers -> oligomers -> protofilaments -> sheet of protofilaments -> closing microtubule -> elongating microtubule
dynamic instability
The easiest (and hardest) way to make sense of dynamic instability (microtubules can constantly grow and shrink) is to think of it in terms of chemical reaction equilibrium, with the monomers on one side and the polymers on the other
microtubulin polymerization process
- Nucleation (lag phase)- seeding period, slow process
- Elongation- rapid growth, can have addition and loss but more likely to add dimers than lose dimers
- Plateau phase: ”treadmilling” - rate of addition = loss of dimers; equilibrium. Constantly moving but length stays the same
when concentration increases, elongation increases.
Critical concentration = equilibrium point, treadmilling phase
The graph shows that:
MT elongation rate is directly proportional to [tubulin]
MT elongation rate increases with increasing [tubulin]
The concentration at which the length is stable is the critical concentration (Cc)
Factors that affect microtubule growth rates
- Tubulin concentration
- location
- GTP cap and MT growth
Factors that affect microtubule growth rates; Tubulin concentration:
Increasing the concentration of monomers will increase the rate of polymerization
Decreasing the concentration of monomers will decrease the rate of polymerization
Factors that affect microtubule growth rates; location
Tubulin dimers can be added and removed at BOTH ENDS but at different rates
Higher critical concentration- does not easily bind β-tubulin in an incoming dimer - not the right conformation= slow growing end; at the (-) ɑ-tubulin
Higher concentration of tubulin needed to maintain net growth on (-) end than (+) end
Lower critical concentration- adding new subunits causes a conformational change in β-tubulin that increase binding for more subunits (binds ɑ-tubulin of an incoming dimer) = fast growing end; (+) end
Critical concentration: the concentration of tubulin subunits when growth is at an equilibrium
Growth rate = disassembly rate
Factors that affect microtubule growth rates; GTP cap
Tubulins are GTP binding proteins, which work as molecular switches
GTP-binding proteins are active when they bind to GTP
GTP-bound and GTP-bound proteins have slightly different conformations. This affects how these proteins function
E.g rab proteins in vesicle formation
rab-GTP is ACTIVE and can be recognized by the tethering protein
Rab-GDP dissociates from the tethering protein
GTP-bound tubulin (at β subunit): has higher affinity with microtubules; added to the + end of existing MTs
Tend to move towards polymerization
GDP-bound tubulin: has lower affinity with microtubules
Tend to lose tubulins in polymer
The affinity of GTP tubulin dimers for tubulin in MTs is greater than that of GDP tubulin dimers
As GTP is in excess in the cytosol, most free tubulin monomers are in the activated (GTP-bound) form. But technically BOTH types have the capacity to form microtubules, if the concentration is high enough
Factors that affect microtubule growth rates; GTP influences microtubule growth/shrinkage
GTP influences microtubule growth/shrinkage
Formation of the GTP cap: when MT assembly happens faster than the rate of GTP hydrolysis
Happens in conditions with a high enough free tubulin concentration
Not cap, it is just a region where tubulin gets added on
Shrinkage: If polymerization slows down, the GTP hydrolysis catches up and is now converted to GDP- so the GTP cap disappears
tubulin- GDP has a lower affinity for the tubulin polymer or microtubule
Result = rapid shrinking
GTP hydrolysis changes subunit conformation forcing the protofilament into a curved shape, leading protofilaments to lose nonpolar interactions which is more likely that tubulin dissociate
Destabilization of MT through GTP hydrolysis results in instability in MT structure
Factors that affect microtubule growth rates; MT catastrophe (period of rapid shrinkage) & MT rescue:
MT catastrophe (period of rapid shrinkage) & MT rescue: the alternating phases of MT growth & shrinkage are what makes up dynamic instability
Dynamic equilibrium between tubulin at the end of the MT and free dimers in the tubulin pool (soluble tubulin)
Increased active tubulin pool leads to:
Increased active tubulin pool leads to: increased deposition leading to depletion of activated tubulin pool
Decreased activated tubulin pool means:
Decreased deposition leading to increased disassembly
Increased free tubulin
Increased tubulin monomer pool will then result in build up of the activated tubulin pool if GTP is present to drive activation
Concentration of tubulin dimers is critical
Above a critical concentration assembly exceeds
Below critical concentration disassembly exceeds
Note that there is built in feedback. Assembly reduces the pool concentration, and disassembly increases it
In vivo - inside the cell
Microtubule
Microtubule organizing centres (MTOCs) in cells provide the right conditions for rapid nucleation of microtubules
Cells control & promote MT assembly by adding nucleating sites known as Microtubule Organizing Centres (MTOC)
Special kind of tubulin used here (y-tubulin) to form ring complex
Y-tubulin rings hold minus end
Plant cells (interphase): non obvious central MTOC (microtubule organizing centre)
Animal cells: MTs nucleated by y-tubulin rings within the centrosome (house tubulin rings)- an organelle that organizes MTs in animal cells
Different Kinds of cellular MTOCs
- Animal cells (& many others) have centralized MTOC in interphase
(-) ends are anchored within the centrosomes; MTOC in the perinuclear space
(+) ends radiate out of towards the plasma membrane
Gives polarity - Spindle poles found in all cells during mitosis
Centrosomes radiate out to either ends of the cell which help form the mitotic spindles for division of cell - Cilia & flagella found in specialized cell types only
Have basal bodies that help to nucleate microtubules - Plant cell wall formation
Cellulose gives plant cells their shape
MTs have role help guide cellulose deposition
No obvious central MT organizing centre in interphase
Microtubules just below cell membrane to help bring structure to the cell
A capped microtubule will NOT grow or shrink. This ‘cap’ is made of protein (capping proteins) and has nothing to do with the GTP cap involved in dynamic instability of microtubules
MTs can be stabilized by binding to proteins called MAPs: Microtubule associated proteins
Some MAPs bind microtubules to neighbouring microtubules, but still grow, this helps establish a network of MT’s going in the same direction
Some of MAPs bind to sides of microtubules to make cross-connections
MTs can be stabilized by binding to proteins called MAPs: Microtubule associated protein
Eventually MTs can be capped at the growing end by different types of MAPs (now MT’s won’t grow or shrink) and are stabilized at the membrane, can polarize cells
Some microtubules reach out from (+) end and reach capping proteins to prevent growth or shrinkage
Motor proteins allow polarized movement along MTs
Molecular motors (dynein and kinesin) bind ATP in the two motor domains ATP hydrolysis causes conformational change to drive movement of the motor (ATP to ADP; ADP to ATP; powers creates conformational changes- which lead to walking motion of motor proteins) E.g. kinesins move towards (+) end; important for maintenance of the structure of ER Secretory vesicles E.g. dyneins move towards (-) end; important for maintenance of the structure of Golgi (in perinuclear region)
Motors help organelles to maintain their structure
Microtubules and their motor proteins help to position organelles within the cell
ER and golgi are attached to microtubules network via motor proteins
Nerve cell shape is maintained by MTs
The extreme case of polarized cells ar where MTs extend the full length of an axon (up to a meter or more; MTs help to shape it) and provide a substrate for motor proteins to move cargo (vesicles or other particles) down the length of the axon or back
Study microtubules by treating cells with agent that disrupt microtubules;
Vincristine- inhibiting polymerization; bind to dimers and prevent from polymers
Equilibrium shifts in favour of depolymerization as it is essentially preventing available monomers from polymerizing (removing monomers)
Without drug, polymerization starts again
Vincristine and taxol are used in
in chemotherapy for humans (and dogs)
Stunted mitosis as microtubules are not able to form spindle fibers for division
Taxol- binds to ends of microtubules and stabilizes them
Microfilaments (actin filaments)
5-9nm flexible helical polymers
Smallest filaments of 3 types
Coiled structure
Cell movement- pushing out cell membrane to make new attachments
Cell shape/structure
Cell contractility- works in muscles to cause contractions in muscles cells and other cells
E.g. actin at tips of neurons to pull out filopodia or lamellipodium- rounded sheet shape
Microfilaments (actin filaments);
different structures:
(A) Can form into bundles and create protrusions
(B) stress fibers throughout the cell
(C) Responsible for movement- actin filaments can cause protrusions which can move the cell by pushing membrane foreward to make new attachments to the surface
(D) actin and myosin filaments can come together in the middle of the cell in order to facilitate cytokinesis to pinch off daughter cells
Microfilaments (actin filaments);In vitro
monomers in a tube: in a test tube, actin and tubulin work in similar ways despite structural differences. Actin undergoes dynamic instability and is activated by the binding of ATP
Structure of the actin monomer and an actin filament; forms globular protein (g-actin; globular actin; monomer) and binds to ATP
When it forms a polymer, ATP with hydrolyzed into ADP in a filament (F-actin; filamentous actin)
Actin monomers and filaments exist in a dynamic equilibrium
Actin monomers (g-actins) assemble into actin microfilaments (f-actin)
Minus (-) end: more prone to disassembly and slower assembly: higher critical concentration of actin monomers is required
Binds with reduced affinity to the polymer
Plus (+) end: more prone to assembly: lower critical concentration of actin monomers is required- actin bound with ATP
Binds with greater affinity to the polymer
The kinetics of spontaneous actin filament assembly in vitro: same polymer assembly kinetics as microtubules
- Lag phase (nucleation) gathering of monomers
- Elongation phase (growing actin filaments)
- Plateau phase- actin filament with subunits coming on and off; treamilling; equilibrium: critical concentration
Critical concentration: neither growth or shrinkage- elongation rate=0
Concentration over Cc: tend to see growth- shift of equilibrium towards for F-actin, increase in elongation
Below Cc: shrinkage; shift to more monomers (g-actin)
Actin filaments would grow longer if actin monomers that bind a non-hydrolyzable form of ATP were incorporated into actin filaments; ATP bound monomers have a higher affinity and thus, it is less likely to dissociate and there will be more growth
actin filament In vivo
inside the cell; actin polymerization is heavily regulated by actin binding proteins
In plants, actin is important in vesicle traffic & organellar positioning with myosins
In animal cells, actin filaments have different arrangements in different parts of the cell that perform different functions
Role in motility: 3 actin arrangements:
Stress fibers form contractile bundles with motor proteins myosins
Throughout the cell
Connect to the surface and provide traction
Rear contraction helps the cell detach to move
Lamellipodium form branched actin network
Pushes membrane forward
Filopodium form parallel bundles- act as environment sensors
Parallel bundles push out finger like projections (filopodia) feeling ahead in the environment
Leading edge at lamellipodium
actin : Move in direction of filopodia
Actin polymerization in cell cortex results in cell movement
Branched actin polymerization at plus end protrudes lamellipodium
New regions of actin cortex are formed
Transmembrane proteins make attachments to the extracellular matrix
Increased tension at back end as the cell membrane is pushed forward
Contraction of stress fibres help detach the other end from the extracellular matrix
The leading edge makes new focal contacts between integral proteins and the extracellular matrix
Various actin-binding proteins (ABPs) control…
..control the behavior of f-actin polymers in cells
Various actin-binding proteins (ABPs):
Nucleating proteins Monomer sequestering proteins Capping and side binding proteins bundling proteins in filopofia motor proteins cross-linking proteins (in cell cortex) severing proteins
Nucleating proteins
Nucleating proteins help to see the polymerization of actin
ARP2/3 complex are nucleating proteins that nucleate branched actin arrays
ARP2/3 proteins bind to the side of other actin filaments so that it can nucleate the formation of new actin filaments- end up with branched structures that push outwards
Constantly pushing membrane forward by creating branched structures in one direction:
net filament assembly at leading edge
net disassembly behind leading edge
Monomer sequestering proteins
Monomer sequestering proteins- binding to actin monomers and keeping them from binding to each other; the actin starts to depolymerize and shifts equilibrium towards monomers
Capping and side binding proteins:
proteins that are bound to the ends and thus caps the filaments; reduces assembly- can no longer add or remove monomers
Side binding proteins- stabilize filaments
F-actin also uses nucleating sites in the cell
there is no equivalent to the MTOC to organize actin thus actin has to be nucleated at multiple sites
Actin-dependent motor proteins: myosin I
Myosin I family members help to manipulate the cell membrane and traffic organelles
Moves toward plius (+) ends of actin filaments
Myosin can walk for slide filaments around
Actin-dependent motor proteins: myosin II
Myosin II has a role in stress fibres and muscle contraction (myosin II-dimer)
Organized tail to tail so that they can make connections between 2 different actin filaments and slide the actin filaments towards each other and shorten the actin: contraction
Actin-dependent motor proteins: ATP dependent proteins
when a muscle is stimulated to contract, the myosin heads start to walk along the actin filament in repeated cycles of attachment and detachment
1. Attached: myosin head is attached to an actin filament without ATP
2. Released and shifts: binds ATP reduces affinity for actin, clamps around ATP and moves the head towards the plus end
3. Powerstroke: binds weakly to actin, hydrolyzes ATP, binds tight conformational change in myosin to regain its original shape. Results in myosin moving towards the plus end and pulling actin as it moves
1 ATP per powerstroke
Rigor mortis
after death, the muscles of the body become very stiff and inextensible; the corpse is said to into rigor
No more ATP is produced = no powerstroke
Body is frozen and myosin is stuck onto actin filament
We would die faster while exercising than sitting down because the ATP is used up faster
