6: Cytoskeleton Flashcards
What are the key difference between eukaryotes and prokaryotes?
Eukaryotes have membrane bound nuclei and organelles, prokaryotes do not
Eukaryotes generally have bigger genomes compared to prokaryotes
Eukaryotes have internal membranes (e.g. endoplasmic reticulum), prokaryotes do not
Eukaryotes have an internal cytoskeleton, prokaryotes do not
Eukaryotes are structurally diverse, prokaryotes are metaboilcally diverse
What function do introns have in gene expression?
Introns provide regulation of gene expression so that genes can be turned on in one cell type and off in a different cell type
This facilitates evolution
What evidence is there for the metabolic diversity of prokaryotes?
Prokaryotes can survive in extreme environments
E.g. deep sea thermal vents
What is the ultimate cause of major changes in cell structure and shape?
Changes in gene expression
What is the proximate cause of major changes in cell structure and shape?
The changes in internal organisation due to changes in the cytoskeleton
Is the cytoskeleton dynamic or static?
Very dynamic
Why is the cytoskeleton dynamic?
Because generally, cytoskeleton polymers are formed by non-covalent protein-protein interactions
This allows them to be assembled and disassembled easily
Give examples of non-covalent interactions.
Van der Waals
Hydrogen bonds
Charge interactions
Hydrophobic interactions
How does cytoskeleton polymer assembly happen?
No dedicated machinery/factory as there would be for covalent interaction formation
Random diffusion
Requires correct orientation, in the cases of strong surface complementarity
Why does consumption of ethanol lead to inebriation?
Because ethanol partitions into neurone membranes and changes their properties, leading to inebriation
This diffusion can happen very rapidly
How does poly-proline peptide bind to the SH3 protein domain?
The positively charged arginine region of poly-proline is attracted to the negatively charged region on SH3
When brought into contact, there are positive and negative charge interactions
Allows the relatively hydrophobic poly-proline to bind to SH3, finding good surface complementarity
What is the association rate equal to?
Association rate = kon[A][B]
Where [A] is concentration of molecule A, and [B] is B
kon is the association rate constant
What is dissociation rate equal to?
Dissociation rate = koff[AB]
Where [A] is concentration of molecule A, and [B] is B
koff is the dissociation RATE constant
What happens to association and dissociation rates at equilibrium?
Association rate and dissociation rate are equal at equilibrium
Therefore:
kon[A][B] = koff[AB]
koff/kon = [AB]/[A][B]
koff/kon = kd
kd is dissociation constant, not to be confused with koff, the dissociation RATE constant
kd is expressed in molar units
How do polymers like actin and tubulin form?
Via head-to-tail interactions
This allows polymerisation due to exposed surfaces
So more than one molecule can join
Leads to formation of stacks/chains
What are the three main imaging techniques of cytoskeletal polymers?
1) Immunofluorescence microscopy
2) GFP tagging
3) Electron microscopy
Describe immunofluorescence microscopy.
1) Retain antibodies to the proteins of interest (can be designed in labs)
2) Stale interactions of protein to antigen can be visualised when a secondary antibody (labelled with fluorescent dye) is coupled to the primary antibody
3) Can be visualised against a very dark background using technology to isolate the fluorescent light
*This depends on dead fixed cells, so cannot visualise dynamic behaviour
Describe GFP tagging.
Allows to look at protein localisation in living cells.
Naturally fluorescent protein so can be fused to desired genes and expressed in vivo.
This allows localisation and dynamics to be followed in live cells.
What are the two forms of electron microscopy that can be used to image cytoskeletal polymers?
1) Thin section
2) Negative stain
Describe thin section electron microscopy.
1) Use chemical fixatives to maintain a very strong structure that can undergo a vacuum in an EM to be visualised
2) Section cells and mount onto plastic - helps to isolate sections that can be visualised more easily due to high amounts of detail in EM microscopy
Describe negative stain microscopy.
1) Mount the protein to an EM grid
2) Incubate with heavy metal salt solution (e.g. uranyl acetate)
3) When solution is washed away, you’re left with electron dense areas in the ‘holes’ or gaps in the protein
4) The protein therefore shows up as a light image where the dye is not present
What are actin filaments?
Polymers in which individual molecules of the protein join end to end
Two strands of protein rope joined end to end
Major protein in muscle cells, involved in motility (specifically muscle contraction)
What are five key ways that actin filaments can be arranged to serve different functions?
1) Microvilli in intestinal epithelial cells
2) Contractile fibres linked to adhesive contacts with the extracellular environment
3) Cell crawling and migration
4) Cytokinesis (actin/myosin interactions to split the cell)
5) Muscle cell contraction
How does ATP interact with actin?
Actin binds a molecule of ATP in its ATP-binding cleft
The ATP is then hydrolysed during actin filament assembly
Describe how actin filaments are assembled.
Head-to-tail assembly
The entire filament is polar with + ends and - ends (not due to electrical charge)
Looks like two intertwined strands
Multiple interactisns between subunits (each subunit directly contacts four others)
Internal subunits more stable than end subunits because of more bonds
What role does ATP hydrolysis have in actin filament assembly?
This is because taking away a phosphate group from ATP (to form ADP) changes the chemical environment, and therefore leads to ADP-actin having a different conformation
There is a higher Kd at the ADP-actin end than the ATP-actin end
Ultimately, ATP hydrolysis promotes instability of the filament and allows the cytoskeleton to be dynamic
What type of protein allows variety of actin cytoskeleton function?
Accessory proteins
Leads to different types of actin cytoskeleton: e.g. motor proteins, bundling proteins, etc.
What is the actin region underneath the plasma membrane that is related to cell migration?
The actin cortex
This is a dense actin meshwork under the plasma membrane
This region is under tension, keeping it anchored to the substrate
What is the lamellipodium and what is its role in cell migration?
A flat, sheet-like projection at the leading edge of a migrating cell
The “front wheel” of cell migration
Actin polymerisation occurs within this projection, causes forward protrusion
Allows attachment to extracellular matrix via focal contacts
The rear contracts via myosin-based contractions, also contributing to migration
What is the filopodium and what is its role in cell migration?
Filopodium is slender, spike-like projections extending from the cell surface often found in combination with lamellipodium
Allows pathfinding, filopodia can act like antennae for the cell to sense the extracellular matrix
Stabilises the protrusive activity of the lamellipodium, ensuring more precise movement
What role do cross-linking proteins have in the actin cortex?
They ‘staple’ together actin filaments at random locations, creating a dense meshwork with gel-like properties, anchored to the plasma membrane
Water molecules are bound to the filaments via hydrogen bonds, causes elastic deformation
What is the ARP complex and what is its role in cell migration?
Multi protein complex
Binds to actin filaments at their minus ends
Nucleates the formation of new actin filaments
Promotes the polymerisation of actin to create a branched framework
Polymerisation at the front causes protrusive force needed for cell migration
What are capping proteins and what is their role in cell migration?
Proteins that bind to the plus ends of actin filaments and prevent further polymerisation (promote depolarisation)
The oldest assembled filaments are less stable, so they release free actin monomers
They ensure that actin polymerisation is focused at the leading edge of the cell in structures like the lamellipodium, ensuring directionality of migration
This also allows actin to be recycled
What is the net filament activity at the front of the leading edge compared to the rear of the leading edge of the migrating cell?
Net assembly at the leading edge
Net disassembly at the rear of the leading edge
“Front-wheel drive”
What are formins and what is their role in cell migration?
Actin polymerisation factors that promote nucleation and elongation of actin filaments
Nucleate new actin filaments at the plus end
Formins remain at the plus end and help elongate unbranched filaments
Critical for filopodia formation
What are bundling proteins and what is their role in cell migration?
Bundling proteins (e.g. fascin) help stabilise and organise filaments into tightly packed bundles in structures like filopodia
Works alongside forming to ensure that actin filaments are structured properly for migration
Compare the structures of myosin I and myosin II.
Myosin I: singular globular head and short tail
Typically involved in intracellular transport and membrane association
Moves along actin filaments using ATP hydrolysis
Tail can bind to membrane or cargo
Myosin II: two globular heads and a long coiled tail (dimer)
Responsible for muscle contraction and generating contractile forces
Forms bipolar filaments that slide actin filaments past each other
Tail forms thick filaments and interacts with other myosin II molecules
Describe the mechano-chemical cycle of myosin proteins from ATP hydrolysis.
1) Rigor state: myosin globular head tightly binds to actin filament without ATP to form a stable rigor complex
2) ATP binds to myosin: causing a conformational change that weakens the interaction with actin and myosin releases from the actin filament
3) ATP hydrolysed: causes another conformational change, myosin head moves into a “cocked” state, ready to bind to adjacent actin filaments
4) Power stroke: release of Pi and ADP triggers the power stroke, where myosin reverts its original conformation, pulling the actin filament towards the centre of the sarcomere
5) One ATP molecule is consumed in each cycle, and the myosin head moves one step along the actin filament, generating physical force
Describe the structure of skeletal muscle.
Composed of muscle fibres, formed by the fusion of individual myoblast cells
Fibres contain multiple myofibrils, which are highly organised structures responsible for muscle contractoin
Myofibrils are made of sarcomeres
Sarcomeres are made up of actin and myosin filaments arranged in a highly organised manner
The Z-line anchors the plus ends of the actin filaments at each end of the sarcomere
Thick myosin II filaments are arranged in a bipolar configuration
How does actin and myosin move within sarcomeres during skeletal muscle contraction?
Myosin heads move towards the positive ends of the actin filaments, pulling the actin filaments inwards
–><–
This slides the actin filaments relative to the myosin, resulting in the shortening of the sarcomere (sliding filament model)
What signalling coordinates skeletal muscle contraction?
Calcium signalling
Calcium binds to enable exposure of actin binding sites to allow myosin to bind
What are the two ways in which myosin motors may move the cell body forward?
1) Myosin I bound to the plasma membrane can ‘walk’ along actin filaments
2) Myosin II bipolar mini-filaments contract the actin filament mesh at the rear of the cell, causing the back of the cell to contract and allowing the cell to move forward
What are microtubules?
Cytoskeletal systsems
Made of alpha and beta tubulin, a very stable dimer
Forms a long, hollow tube
More rigid than actin filaments
Dynamic structures
Describe the relationship of GTP to microtubules.
GTP-bound beta tubulin promotes polymerisation of microtubules
Beta tubulin hydrolyses GTP to GDP after polymerisation, reducing stability and promoting disassembly
Drives dynamic instability where microtubules can rapidly grow and shrink
Alpha tubulin keeps GTP bound in that state, it does not hydrolyse to GDP
What is GTP?
Guanine triphosphate
Related to dynamic microtubule structure
Describe the structure of microtubules and why this makes it dynamic.
Polar polymers with 13 protofilaments (linear chains of tubulin dimers)
Plus and minus ends (beta is at the plus end)
Each tubulin dimer forms two lateral and two longitudinal contacts
Tubulin dimers form heterodimers (subunits of microtubules)
Lateral exchange of dimers is difficult, longitudinal is easier
Assembly easier at the plus end than the minus end.
What is nucleation of microtubules?
Initial process by which microtubules begin to form from tubulin dimers
Involves assembly of a small number of tubulin dimers into a stable structure which serves as a ‘seed’ for further growth
Requires high concentrations of tubulin
Typically occurs at specialised regions in the cell like the centrosome
What are centrosomes and how do they facilitate MT nucleation?
Centrosomes are loose assemblies built around centrioles
Gamma-tubulin ring complexes are concentrated at the centrosome
Gamma-tubulin ring complexes facilitate the nucleation phase of microtubule asembly
Minus ends of MTs are anchored at the centrosome, as elongation is easier at the plus end
How does stability of microtubules change depending on binding of GTP or GDP?
GTP-bound tubules
Have a GTP-tubulin cap, so stabilises the plus end and allows dimers to be added longitudinally
GDP-bound tubules
Have no GTP-tubulin cap, so leads to ‘self-unpeeling’ and ‘catastrophe’ where the lateral interactions between dimers weaken, and the microtubule disassembles
Why is dynamic instability important for microtubules?
Enables microtubules to rapidly grow and shrink, allowing cells to respond to internal and external signals
Facilitates ‘search and capture’ of specific targets in the cell, helping to organise internal structures and guide transport
Allows cell polarity and directional organisation by stabilising certain MTs in response to spatial cues
Why is dynamic instability important during mitosis?
Allows microtubules to search the intracellular space to find and attach to kinetochores on chromosomes
Selective stabilisation of MTs helps to form the mitotic spindle, ensuring accurate chromosome alignment and segregation
Stabilised MT bundles can exert force on chromosomes, pulling them apart to opposite poles during anaphase
What are two examples of drugs that affect microtubule stability and dynamics?
1) Colchicine: binds to tubulin dimers and prevents incorporation into microtubules
2) Taxol: stabilises tubulin in the MT lattice leading to cell death - potent anti-cancer drug
What are the three major roles of microtubules in biology?
1) Internal organisation of the cell: moving vesicles and positioning organelles
2) Chromosome segregation: mitotic spindle
3) Moving fluids or moving cells in fluids: cillia and flagella
How are microtubules useful for cell organisation?
Microtubules form polarised arrays (plus and minus ends) creating an internal “road map” for cell organisation and directional transport
Intracelllular membranes and organelles are positioned and shaped by microtubules. E.g. the endoplasmic reticulum extends along microtubules, colocalising with them
Microtubules act as tracks for motor proteins (e.g. kinesins and dyneins) to move organelles, vesicles and other cargo
How do motor proteins use microtubules?
Motor proteins bind to MTs and use energy from ATP hydrolysis to generate conformational change (power strokes) that drive movement
Each motor protein will have a directional preference
What is the directional preference of kinesin and dynein?
Kinesin: moves towards the plus end (usually the cell periphery)
Dynein: moves towards the minus end (usually the cell centre, near the nucleus)
Describe the movement of motor proteins.
Movement often likened to a “walking” mechanism, with motor domains working in pairs for processive motion
Cargo binds to specific motors, determining direction of transport
Switching direction requires switching the motor protein attached to the cargo
Motors may work alone or in small teams depending on cargo
How are microtubules useful in cell division?
Centrosomes replicate and nucleate microtubules to form the mitotic spindle
Microtubules grow in radial arrays and are selectively stabilised to orient the spindle
Kinetochore microtubules attach to chromosomes and are stabilised for chromosome segregation
Interpolar microtubules are cross linked and stabilised by microtubule association proteins (MAPs), helping to structure the spindle
Motor proteins at kinetochores pull the chromosomes apart
Other motors push inter polar microtubules apart, elongating the spindle to separate daughter cells
Describe how microtubules are useful in moving fluids or cells in fluids?
Cilia and flagella are microtubule-based structures that move cells or fluids across cell surfaces
They are extended from centrioles, which act as basal bodies at the base of each structure
9+2 arrangement - 9 outer doublet microtubules and 2 central single microtubules
Movement is powered by dynein motors on the A tubule, walking along adjacent B tubules
Nexin links connected doublets, converting sliding into bending, creating a waving motion that propels fluids or the cell
Why are intermediate filaments important?
Intermediate filaments are important in organisms and tissues that experience mechanical stress
They enable cells to collect and form layers, which stabilises them
Helps to withstand mechanical stress
What type of organisms have intermediate filaments?
Vertebrates, nematodes and some mollusks have them Arthropods, echinoderms do not (they have hard shells and exoskeletons instead), because evolutionarily they don’t need them because their hard shells help minimise mechanical stress
Describe the key qualities of intermediate filaments.
Intermediate in diameter between actin and MTs
They have a very high tensile strength
They are resistant to many treatments that disrupt actin filaments and MTs
No nucleotide binding
Assemble by principle of ‘coiled-coil’ interactions
Describe the “coiled-coil” structure in proteins
A coiled-coil is made of two or more right-handed alpha helices wrapped around each other
Each helix is amphipathic, positions a and d in the 7-residue (heptad) repeat are hydrophobic, forming a stripe
These hydrophobic stripes align and twist around each other, creating a left-handed supercoil
This structure stabilises protein-protein interactions through a hydrophobic interface
Why are coiled-coil structures important for intermediate filaments?
Hydrophobic residues at positions a and d in the heptad drive tight helix packing
Side chains interlock to form strong, stable interactions
Coiled-coils are versatile motifs found in many unrelated proteins (e.g. myosin and kinesin)
Can be homo or heteromeric, forming dimers, trimers, or tetramers
Provide strength and flexibility ideal for forming rope-like intermediate filaments
How are intermediate filaments assembled?
Built from central alpha helical coiled-coil dimers
Dimers pair anti-parallel to form tetramers
Tetramers pack laterally and end-to-end into rope like filaments
N- and C-terminal extensions vary, enabling diverse functions
No polarity so motor proteins cannot act on them
Very stable due to many subunits but flexible from small breaks in coiled-coils
What are the different tpes of intermediate filaments?
Cytoplasmic:
Keratins (in epithelia)
Vimentin and vimentin-related (connective tissue, muscle cells, glial cells)
Neurofilaments (in neurones)
Nuclear:
Nuclear lamins (in all animal cells, sheet under nuclear envelope)
Why do different types of protein not copolymerise?
Interactions mostly via head/tail regions
Different proteins have different heads and tails so will not interact and copolymerise
What are neurofilaments?
Found in neurones
Stabilise fragile neuronal processes
Very long extensions
Keeps the axon as a stable structure
Full of neurofilament proteins and microtubules
What is keratin?
Found in epithelia
Allows sheets of cells to stretch without rupture
Most diverse class of intermediate filament proteins
Links to neighbouring cells at desmosomes
Like webbing in rip-stop fabrics
Other IF proteins (desmin, vimentin, etc.) have similar roles in muscle and connective tissue
What are nuclear lamins?
Present in nearly all animal cells
Found right beneath the nuclear envelope
Meshwork of filaments = nuclear lamina
Maintains integrity of the nucleus
Links chromatin to the nuclear envelope
Can be destabilised when cells divide
How is the nuclear lamina disassembled during cell division?
Through phosphorylation of nuclear pore proteins and lamin proteins
Disassembles the entire lamina network
Free lamins can then engage with microtubules in the cytoplasm
When division is complete, phosphate groups are removed, and lamina is reassembled to reform the meshwork
Give an example of a syndrome caused by a mutation in lamin A gene, and describe its symptoms.
Hutchinson-Gifford Progeria Syndrome
Extremely rare
Childhood onset of premature aging
Growth retardation, baldness, facial hypoplasia, aged skin, delayed tooth formation, osteoporosis, arthritis, teenage mortality, almost always from cardiovascular disease, caused by a point mutation in gene encoding lamin A
How do cytoskeletal systems integrate in epithelial cells?
Actin: forms apical microvilli, a contractile apical belt, supports the cell cortex
Microtubules: enables directed transport between apical and basolateral regions
Intermediate filaments: anchor cells to each other and the basal lamina giving mechanical strength
How do cytoskeletal systems integrate in neurones?
Actin initiates the turn by forming filopodia and lamellipodia at the growth cone
Microtubules invade these new protrusions, stabilising direction and delivering membrane vesicles
Microtubules can also stimulate local actin polymerisation
Neurofilaments fill in behind, providing structural support as the axon extends
How do cytoskeletal systems integrate during cell division?
Actin filaments assemble at the cell equator, recruiting myosin to form a contractile ring, contracts to pinch the cell in two (cytokinesis)
Microtubules form the mitotic spindle to segregate chromosomes
Intermediate filaments (lamins) are phosphorylated to break down the nuclear envelope
What is an example of a protein that binds all three cytoskeletal systems?
Plectin is a linker protein that physically binds actin, microtubules, and intermediate filaments, enabling mechanical integration e.g. if actin is pulled at one point, the force can be distributed throughout the whole network, keeping the cell structurally sound
E.g. in plectin-deficient mice, this loss of co-ordination can lead to skin blistering because cells can’t stay mechanically attached, muscle weakness, because forces from actin/myosin can’t be transmitted properly
Describe the prokaryotic cytoskeleton.
Small protein based structures in prokaryotes similar to actin and tubulin
Actin-like (MreB) and tubulin-like (FtsZ) proteins involved in bacterial cytokinesis
Evolutionary link indicates a common origin
