Cytoskeleton Flashcards
What do eukaryotic cells contain? (8)
What do prokaryotic cells contain? (4)
Eukaryotic:
- Cytosol
- DNA within the nucleus
- Cell wall
- Cell membrane
- Ribosomes
- Mitochondria
- Golgi
- ER
Prokaryotic:
- Cytosol
- DNA
- Cell wall
- Ribosomes
See jotter for diagram and practice drawing out
What are the 6 main differences between eukaryotic and prokaryotic cells?
Eukaryotic = membrane-bound nucleus
Prokaryotic = No distinct nucleus
Eukaryotic = Big genomes (contains lots of non-coding regions)
Prokaryotic = small genomes
Eukaryotes have internal membranes, whereas prokaryotes don’t (no ER or Golgi)
Eukaryotes have membrane-bound organelles, whereas prokaryotes don’t
Eukaryotes have an internal cytoskeleton whereas prokaryotes don’t
Eukaryotes = Structural diversity
Prokaryotes = Metabolic diversity (can live in extreme environments)
What is the ultimate and proximate cause of differences in eukaryotic cell structure?
Ultimate = due to changes in gene expression (different proteins, different shapes)
Proximate = changes in internal organisation due to changes in the cytoskeleton
What are the 3 main filaments in the cytoskeleton (just names)?
- Actin filaments
- Microtubules
- Intermediate filaments
How are cytoskeleton polymers formed and what does this allow the cytoskeleton to do?
Formed via non-covalent protein-protein interactions
Allows the cytoskeleton to be dynamic as the polymers can break apart and reform easily (unlike covalent bonds)
How do the cytoskeleton polymers bind to each other and what determines which reactions will form macromolecules in the cytoskeleton?
Due to random collisions which allows them to self-assemble
Molecules need to meet with the correct orientation and with a good match (high affinity for each other) in order to have strong interactions and be kinetically stable
Lots of kinetically stable interactions leads to the formation of cytoskeleton macromolecules
What is a Homodimer?
What is a Heterodimer?
How do protein polymer chains form in the cytoskeleton?
Homodimer = 2 of the same molecule binding together
Heterodimer = 2 different molecules binding together
Via head-to-head protein-protein interactions stacked together in a chain
What are the 3 techniques that are used to study the cytoskeleton? (just names)
- Immunofluorescence microscopy
- GFP fluorescence
- Electron microscopy
How does immunofluorescence microscopy work (3 steps)
What is the limitation of using this?
- Primary antibody is specific to a cytoskeleton protein which it binds to
- A secondary antibody, which is labelled with a fluorescent marker, binds to the primary antibody to make the complex visible
- This complex can then be viewed under a fluorescent microscope
Technique kills the cell, so no dynamic movement can be viewed
How is GFP fluorescence used to visualise the cytoskeleton and how is this better than immunofluorescence microscopy?
GFP is naturally fluorescent
The GFP gene is fused to the gene of interest, and the fluorescent gene can then be followed through the protein making process
Allows dynamics of the cytoskeleton to be seen, such as chromosomes separating
How is electron microscopy used to visualise cytoskeleton proteins? (2)
Why is a negative stain used in conjunction with this and how does it work?
Cells are “fixed” - cut into thin sections so the details of the cells can be visualised.
Individual sections placed on a grid, which is then placed under a microscope to image the cell
Negative staining - uses heavy metal salts on the filament we want to visualise. Metal salts are electron dense, and so will appear dark on the image, making the filament seem lighter, making the filament easier to visualise
What are the 3 main roles of actin filaments in the cell? (just names)
- Cell crawling and migration
- Cell division (cytokinesis)
- Muscle cell contraction
How are actin filament polymers formed? (5)
(include how this involves the hydrolysis of ATP)
Through head-to-tail assembly of actin proteins, proteins add onto the plus end and leave from the minus end
- ADP filaments are usually found at the minus end and ATP filaments are usually found at the plus end
- ADP filaments are less stable so will fall off the actin filament more easily (fall off from the minus end)
- ATP-bound actin filaments will be attracted to other ATP-bound actin filaments at the plus end, and so will add onto the plus end of the polymer
- Once the ATP filament has bound and moved along, it will be hydrolysed to form an ADP-actin filament, which will eventually fall off at the minus end
(constantly occurring process + requires ATP)
(see jotter for diagram)
What are actin filaments made of, how do the subunits interact, and how does this mean that exchange only occurs at the ends?
Made of 2 intertwined filament strands
Each internal subunit interacts with 4 other adjacent subunits
The end subunits only interact with 2 adjacent subunits, and so are less stable which is why exchange of actin proteins only occurs at the end and not in the middle of the chain
(diagram in jotter)
What are the 5 actin-regulatory proteins? (just names)
- Nucleating protein
- Cross-linking protein
- Capping protein
- Motor protein
- Bundling protein
What is the role of actin filaments during cell migration?
What is the lamellipodium and the filopodium of the protruding cell?
To protect the cell and allow the cell to move forward by adding on filaments to the protrude the cell forward and contracting the filaments at the back of the cell so that the cell can keep moving forward
Lamellipodium = flat feet at plus end
Filopodium = spikes at plus end
What is the role of cross-linking protein?
Cross-linking protein = found in actin, stabilises the actin filament and gives it elasticity (holds multiple actin chains together to form a bundle)
What does the nucleating ARP protein do?
Binds to actin filament subunits, resulting in a chain reaction of filament growth, allowing the cell to move forward
What do capping proteins do?
What happens as actin filaments migrate towards the interior of the the migrating cell? (filaments that are capped)
Cap at plus end of newly formed filament chains, which stabilises them and prevents further growth of the chain occurring
Become depolymerised at the minus end, eventually destroying the whole filament chain (as can’t add any more filaments onto the plus end as they are capped)
(Diagram in jotter)
Where in the cell does filament nucleation and degradation occur and how does this allow it to protrude forward during cell migration?
Nucleation occurs at the front of the leading edge
Degradation occurs at the rear of the leading edge
Newly nucleated filaments create a transient meshwork. The cell body protrudes into this area, even as the meshwork disassembles.
How do poisons work? (to do with cell migration and actin filaments)
Poisons either prevent polymerisation of actin filaments occurring (acts as a cap)
OR
stabilises actin filaments, preventing depolymerisation
So both actin polymerisation and depolymerisation are needed for cell movement to occur?
What do filopodia do within protruding cells?
Pathfinders - sense the chemical environment in different directions and decide which direction the cell should move in
What are Formins and how do they help to nucleate actin filaments?
Nucleation proteins that bind to the plus end of the actin filament and nucleate the filament (grow it) (type of ARP)
Change conformation to allow another actin subunit to join onto the plus end of the chain. Their membrane is pushed forward every time a subunit is added on to change conformation to allow another subunit to bind (see diagram in jotter)
What is Fascin protein and what does it do? What does this allow?
Bundling protein that gives structural stability to actin filaments on filopodia
This allows Formin-induced polymerisation at the membrane to occur at filopodia, allowing the filopodium to extend
What are myosin’s?
What do they does Myosin I do in actin filaments and where in the cell is it mainly found? (4 steps)
Myosin’s = motor proteins
Myosin I “walks” along actin filaments to push the cell forward, mainly found at the front of the cell
Mechanism: (diagram in jotter)
- Starts off in a tight binding “rigor” state of myosin (strongly attached to the actin filament)
- ATP binds to the myosin head, which reduced the affinity of the myosin for the actin filament (becomes detached from the actin filament)
- ATP hydrolysis occurs, changing the conformation of the myosin head which binds it to the actin filament next to the actin filament it was originally bound to, but in a strained or “cocked” state
- The phosphate and ADP releases from the myosin head, causing a power stroke, which changes the myosin back to its original “rigor” conformation
What happens in muscle fibres to allow them to contract in terms of their internal structure? (3)
- Made of multiple myofibrils
- Myofibrils are made of tens of thousands of sarcomeres
- Each sarcomere contracts a very small amount, but total of all contraction of all sarcomeres allows the muscle fibres to have big contractions
What do Myosin II do in relation to contraction of muscle cells? (2)
Where are Myosin II mainly found in migrating cells?
Myosin II filaments are found within sarcomeres, allowing them to contract:
- The myosin thick filaments interdigitate (slot in between) thin polarised actin filaments
- The myosin filaments generate a force on the actin filaments in both directions, simultaneously, pulling in the actin filaments (contracting them)
Mainly found at the back of migrating cells to contract the actin filament mesh at the back of the cell
What are are microtubules made of?
What shape are microtubules and how is this different to actin filaments?
What are the 2 ends called and what is found at each end + why?
Dimers of alpha and beta tubulin
Dense cylinders instead of a single row of monomers (cylinder made of multiple rows all joined together in a circle)
The Beta-tubulin of the dimer points towards the plus end and the alpha-tubulin of the dimer points towards the minus end - because beta and alpha tubulin both contain a molecule of GTP, but only beta-tubulin associated GTP is hydrolysed to GDP (see diagram in jotter)
(Both growth and shrinking occurs at the plus end)
What does the centrosome do and what 2 things does it contain to help with its function?
Organises and allows microtubules to grow from it (nucleate)
Contains:
- Pair of centrioles in the centre
- Gamma-tubulin ring structures (like holes) to facilitate nucleation of microtubule assembly
The minus ends of the microtubules are anchored into the Gamm-tubulin rings, and the plus ends point outwards, so that they grow outwards from the centrosome
How many interactions does each microtubule unit make with surrounding units?
Each dimer makes 4 interactions with surrounding dimers - 2 up and down (one up and one down) and 2 side to side (one on each side)
How do microtubules shrink and grow?
What does the loss of the GTP cap lead to?
Shrinking microtubule - GDP tubulin is unstable, and so protofilaments containing GDP tubulin will peel away from the microtubule
Growing microtubule - Contain a GTP cap, as all tubulin molecules being added on are GTP molecules, and since addition is happening at a faster rate than the GTP molecules are being hydrolysed, the microtubule grows. (see jotter for diagram)
(prevents the GDP molecules from peeling away)
Loss of this cap leads to self-unpeeling (hydrolysis will occur and the molecules will peel away)
How are microtubules “dynamically instable”, how is this specific, and what does this allow the microtubule to do?
Some proteins cap the microtubules to stabilise them and prevent their GDP molecules from peeling away
Other unstable proteins aren’t capped by proteins, and so their GDP molecules peel away
Selective mechanism, specific capping proteins are specific to certain microtubules, so only specific microtubules are stabilised
Allows the microtubules to “search” the cellular space
(see diagram in jotter)
What are the 2 drugs called that makes the microtubules stable and how do they do this?
How is this useful?
- Colchicine = bind to free tubulin dimers and prevents them from being incorporated into the microtubules
- Taxol = stabilises tubulin in the microtubule lattice (prevents peeling away), which leads to cell death
Useful to stop cell division in cancer cells
What are the 3 main roles of microtubules in cells?
- Internal organisation of the cell - moving vesicles and positioning organelles
- Chromosome segregation - via the mitotic spindle
- Moving fluids or moving cells in fluids (e.g. cilia/flagella)
How do microtubules help in internal organisation of the cell?
How are motor proteins involved with this? (roughly)
What are the tracks in neurons and epithelial cell? (direction)
Since microtubules are polar, they provide a track for polar molecules to transported along, which helps to organise intracellular membranes
Motor proteins use the microtubule tracks to transport different molecules around the cell and get them to the right place in the cell
Neurons = proximal vs distal (left to right) transportation (proximal = minus end and Distal = plus end)
Epithelial cells = Apical vs basolateral (top to bottom) transportation (Apical = minus end, bottom = plus end)
How do motor proteins transport molecules across microtubule tracks? (4)
Motor proteins have 2 heads
- Motor proteins start off bound to the microtubule track
- Hydrolysis of ATP releases one of the heads off of the microtubule track via a conformational change
- The same head rebinds further forward on the microtubule
- The rear motor (rear head) acts to move the whole motor protein forward
(diagram in jotter)
What are the 2 types of motor proteins that do this and in what direction does each transport molecules in?
Dynein = transports molecules towards the minus end of the microtubule (to the left)
Kinesin = transports molecules towards the plus end of the microtubule (to the right)
Can switch between Dynein and Kinesin to change the direction that the molecule is moving in
How are does the mitotic spindle result in chromosome segregation? (2)
- Microtubule motors at the kinetochore of the chromosome drives chromosome movement, as they push interpolar microtubules apart to elongate the spindle
- Selective stabilisation of only the kinetochore microtubules then occurs (the microtubules will then shrink to pull the chromosomes apart)
What do cilia/flagella do and how are microtubules involved in forming their structures?
Include how many microtubules are involved and the 2 different proteins which link the proteins together and what they do
Machines for moving fluids and moving in fluids.
They extend from the centrioles at the centre of centrosomes
Contain 9 outer doublet microtubules and 2 centre pair of microtubules
- Nexin proteins link the adjacent doublets together (see jotter for diagram)
- Cross-link proteins between adjacent doublets to allow for sliding and bending of microtubules to provide cilia/flagella movement (see diagram in jotter)
What is Kartagener’s syndrome and what does it result in?
What is situs inversus?
Immobility of cilia
Results in difficulty clearing bronchia and can cause situs inversus
Situs inversus = All organs are mirror-imaged and fluid moves in the body from right to left instead of left to right (Usually plus end will be on the right, in this case will be on the left)
What are intermediate filaments important for and what 3 types of organisms are they found in and what 2 types of organisms are they not found in?
Important in organisms and tissues that experience mechanical stress
Found in - vertebrates, nematodes, some molluscs
NOT found in - arthropods, echinoderms (organisms with a hard-shell/exoskeleton)
How are coiled-coil structures of intermediate filaments formed?
Explain why there is a slight mismatch?
Made from 2 amphipathic alpha helices.
Hydrophobic residues are at positions a and d. These hydrophobic residues make a hydrophobic slanted stripe down each helix.
The 2 helices twist together with their hydrophobic stripes stuck together to keep the hydrophobic residues away from water. This forms a coiled-coil left handed “supertwist”.
Slight mismatch as the helices are heptads (a,b,c,d,e,f,g residues) and an alpha helix turn is 3.6 residues so 2 turns = 7.2 residues and 7<7.2 so slight mismatch
(see jotter for diagram)
What do coiled-coils packing together form and how does this happen?
Forms tetramers via 2 coiled-coil dimers packing together via lateral interactions
2 tetramers can then pack together via lateral interactions and N- terminus and C-terminus binding (see jotter for diagram)
What are the 4 classes of intermediate filament and in what cells are they found?
- Keratins = found in epithelia cells
- Vimentin = found in connective tissue, muscle cells, and glial cells
- Neurofilaments = found in nerve cells
- Nuclear Lamins = found in the nucleus of all animal cells
How do the 4 classes of intermediate filament polymerise? (all the same)
Interactions occur via the head/tail regions
Different classes have different heads + tails, so won’t copolymerise (keratins will only polymerise with other keratins, can’t get keratins polymerising with Lamins etc.)
What is the neurofilaments functions and how does it’s structure relate to this?
Neurofilaments are stable and fairly rigid to stabilise fragile neuronal processes (stabilise the axon and dendrites)
What is the Keratin filaments function?
What role do Vimentins have?
What can mutations in keratin genes cause?
Keratin filaments allows sheets of cells to stretch without rupturing by linking neighbouring cells at the desmosomes (see jotter for diagram)
Vimentin does the same in muscle and connective tissue
Mutations disrupts the basal layer of the epidermis (upper layer), which can cause skin related diseases such as epidermolysis bullosa simplex (skin blistering)
What is Nuclear Lamins filaments function?
What happens to the nuclear Lamin during cell division? (2)
Links the chromatin (DNA which forms chromosomes) to the nuclear envelope (see diagram in jotter)
During cell division the Nuclear Lamin meshwork disassembles, as the Lamins get phosphorylated, which frees the Lamin proteins (during Prophase)
Later on in cell division the Lamins are dephosphorylated and the meshwork is re-established (during Telophase)
Why are Laminopathies so diverse?
What is an example of a Laminopathy, its symptoms and the mutation it is cause by?
Laminopathies cause defects in one gene which can lead to several different diseases with different symptoms (as it affects gene expression)
Example = Hutchinson-Gilford Progeria syndrome
Symptoms = premature ageing which leads to teenage mortality, mostly from cardiovascular disease
Mutation = point mutation in the gene encoding Lamin A
What do Actin filaments, Microtubules, and intermediate filaments all do in epithelia cells? (Overall cytoskeletal control)
Actin filaments:
- Make up the apical microvilli to increase surface area
- Actin belt present near the apical (top) surface
- Actin-rich cortex (actin rich core)
Microtubules - direct transport between apical and basolateral surfaces (between top and bottom of cell)
Intermediate filaments:
- Links the neighbouring cells (prevents rupture)
- Links to basal Lamina (bottom of cell)
What do Actin filaments, Microtubules, and intermediate filaments all do in neurons? (Overall cytoskeletal control)
Actin filaments - initiates a turn
Microtubules:
- Invade the new growth site
- Deliver new membrane vesicles, may also stimulate actin polymerisation
Neurofilaments - fill in from behind
What do Actin filaments, Microtubules, and intermediate filaments all do in during cell division? (Overall cytoskeletal control)
Lamin intermediate filaments - Lamin phosphorylation causes nuclear envelope breakdown
Microtubules form the spindle, allowing chromosomes to separate
Signals from the central spindle activate actin filament assembly and myosin recruitment in cell middle to form a contractile ring (see jotter for diagram)
Myosin and actin contractile ring divides the cell into 2
What is plectin?
What do mutations in plectin cause? (4)
What type of cytoskeleton proteins do prokaryotes contain?
Binds actin, microtubules, and intermediate filaments together
Mutations can cause skin blistering, muscle defects, neuronal defects, and death
Prokaryotes - contain proteins structurally similar to actin and tubulin
