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
