The Cytoskeleton Flashcards
Why does a cell need a cytoskeleton?
- To keep its shape and modify it in response to environmental cues
What is the cytoskeleton?
- A group of proteins that are able to organise themselves into filaments in order to carry out various functions within the cell such as:
- Establishing cell shape
- Providing mechanical strength
- Cell movement
- Chromosome separation
- Intracellular transport of organelles
What are the main components of the cytoskeleton?
- Actin filaments (microfilaments)
- Intermediate filaments
- Microtubules
What are the other components of the cytoskeleton?
- Accessory proteins such as:
- Cytoskeleton binding proteins
- Cytoskeleton associated proteins
- Motor proteins
- These are needed to maintain and regulate the properties associated with each of the filaments
How do the cytoskeleton filaments differ in terms of the amount of force needed for tem to deform?
- Microtubules - Only require a small force for them to deform
- Intermediate filaments - Require a very large force for them to deform - very flexible
- Actin filaments - Require more force than microtubules to deform but still require way less than intermediate filaments
Describe each of the following characteristics for each cytoskeleton filament
Why is the cytokskeleton considered dynamic?
- Because it has the ability to polymerise/de-polymerise rapidly in response to external stimuli
What characteristics of the cytoskeleton allow it to be dynamic?
- Monomers that form the polymers are very abundant
- Monomers aren’t covalently linked when they form polymers so it makes it easier for them to disassemble
What processes do the accessory proteins regulate?
- Site and rate of filament formation (nucleation)
- Polymerization / depolymerization
- Function
Describe the structure of the intermediate filaments
- They’re made up of a large family of intermediate filament proteins
- Each filament contains the following:
- N-terminal domain
- C-terminal domain
- Central domain
- α-helical region
- 2 filaments join together to form a coiled-coil dimer
- These dimers associate with each other to form a staggered tetramer
- Finally, 8 tetramers orientate themselves to form a rope-like filament
How do intermediate filaments differ from actin filaments and microtubules?
- Intermediate filaments don’t have defined polarity (no + or - end)
- Intermediate filamemts don’t have associated motor proteins
- Intermediate filaments don’t bind to nucleotides (ATP or GTP)
- Intermediate filaments are much more stable compared to actin filaments and mcirotubules
Explain why tissue-specific expression of intermediate filaments is useful in diagnostics
- Because identification of intermediate filament proteins from tumour biopsises using antibodies can be used to locate the origin of the cancer
What are the 4 types of intermediate filament?
-
Cytoplasmic
- Keratins: In epithelial cells
- Vimentin and vimentin-related: In connective tissue, muscle cells and neuroglial cells
- Neurofilaments: In nerve cells
-
Nuclear
- Nuclear lamins: In all animal cells
Name some of the proteins that make up each type of intermediate filament and name the location of these intermediate filament proteins
What are the functions of the intermediate filaments in the cytoplasm?
- To provide tensile strength - enables cells to withstand mechanical strength and stretch
-
To provide structural support by:
- Creating a deformable 3D structural framework
- Reinforcing cell shape and fixing organelle localisation
State some characteristics of keratin
- Hard
- Waterproof
- Resistant to abraisions
How do keratins indirectly link epithelial cells?
- Neighbouring epithelial cells are connected by structures called desmosomes
- The structure of these desmosomes are maintained by keratins ensuring that the connection between epithelail cells remains strong
Apart from desmosomes, what other structure are keratins a part of?
- Hemidesmosomes
What are hemidesmosomes?
- Structures that mediate adhesion between the basal lamina and epithelial cells
- Integrins α6β4 binds to proteins in the plaques and to laminin in the extracellular matrix.
What happens if the keratin network in the skin is disrupted?
- Causes blistering
What intermediate filament protein is required for endothelail transmigration?
- Vimentin
- Process is impaired in vimentin mutant mice
What is transendothelial migration?
- When leukocytes leave the bloodstream to combat infection
How does desmin maintain muscle structural integrity?
- Desmin filaments are tethered to the Z disc/Z line of the musckle fibres which keeps them in a uniform/constant shape
How do neurofilaments differ from other intermediate filaments?
- They have side arms that project from the core filament
What disease occurs when neurofilament proteins aren’t expressed?
- Charcot-Marie-tooth disease: Neuropathy where the peripheral nervous system degenerates
What are the functions of the intermediate filaments in the nucleus (nuclear lamins)?
-
Line the inner face of the nuclear envelope in order to:
- Strengthen it
- Provide attachement sites for chromatin
-
Disassemble and reform during each cell division as the nuclear envelope disintegrates
- This is controlled by post-translational modifications (mainly phosphorylation and dephosphorylation)
Briefly describe the structure of nuclear lamins
- Form a basket-like structure on inner side of nuclear envelope
How are nuclear lamins disassembled during mitosis?
- Nuclear filaments disassemble to form lamin tetramers
- These tetramers get phosphorylated by MPF (maturation/mitosis-promoting factor) to form phosphorylated lamin dimers
- These get disassembled further to form lamin monomers
Name some intermediate filament binding proteins (IFBPs) and state their functions
- Fillagrin - binds keratin filaments into bundles
-
Synamin and Plectin - bind desmin and vimentin
- Link IF to the other cytoskeleton compounds (i.e. actin and microtubules) as well as to cell-cell contact structures (desmosomes).
- Plakins - Keep the contact between desmosomes of epithelial cells.
What are the functions of the microtubules?
- Establish internal polarity to structures within the interphase cell allowing those structures to move around the cell
- Participate in chromosome segregation during cell division
- Establish cell polarity which allows for cellular movement to occur
- Produce extracellular movement via beating of cilia and flagella
Describe the structure of the microtubules
- Microtubules are made up of α and β tubulin.
- α and β tubulin form a tubulin heterodimer or a microtubule subunit
- These microtubule subunits organise themselves to form a hollow tube with a lumen in the centre called a microtubule
- The Microtubule has a + end and a - end
At what end of the microtubule does tubulin subunit addition and tubulin subunit substration take place?
- Both take place at the + end of the microtubule
With regards to microtubules what do each of the following terms means:
- Elongation
- Shrinkage
- Catastrophe
- Rescue
- Elongation - Growth of the microtubule via addition of GTP-tubulin subunits
- Shrinkage - Shortening of the microtubule via release of GDP-tubulin subunits
- Catastrophe - Occurs when a microtubule goes from elongation to shrinkage
- Rescue - Occurs when a microtubule goes from shrinkage to elongation
Explain how microtubule elongation occurs
- GTP-tubulin subunits add to + end of microtuule
- GTP-subunit addition occurs faster than GTP hydrolysis so more GTP-subunits are added than released
- This results in a GTP cap being formed at the end of the microtubule as it grows
Explain how microtubule shrinkage occurs
- Protofilaments containing GDP-tubulin peel away from microtubule
- This allows GDP-tubulin to be released from the microtubule resulting in it shrinking
How do microtubules exhibit dynamic instability?
- Each microtubule is able to grow and shrink quite rapidly, they’re dynamic
- However, the total mass of polymerized tubulin remains constant
- The loss/gain of the GTP defines whether the microtubule grows or shrinks
- This is because once the cap is lost GTP hydrolysis of the protofilaments occurs forming GDP-tubulin subunits which can then be released
- Regain of the GTP cap allows for growth as the GTP-tubulin dimers can’t be hydolysed
What is the centrosome?
- It is an organelle that serves as the primary microtubule nucleation site in most cells (where the microtubules grow from)
Describe the structure of the centrosome
- A pair of centrioles surronded by a centrosome matrix
- Thera are also nucleating sites (γ-tubulin ring complexes) on the surface of the centrosome matrix
Why is the centrosome so important?
- Cell polarity, including the organization of cell organelles, direction of membrane trafficking, and orientation of microtubules is determined by the centrosome a.k.a microtubule-organizing centers (MTOCs).
What happens to centrosomes during the cell cycle?
- During each round of the cell cycle the centrosome is duplicated
What disease often results in centrosome abnormalities?
- Cancer
- Centrosomes either have structural abnormalities or have defects in the way that they duplicate in cancer cells
Why is it important that microtubules are dynamic?
- Being dynamic allows the cell to quickly reorganise when for example, building mitotic spindle
- It also allows microtubules to probe the cytoplasm for specific objects and sites on the plasma membrane - this is known as search and capture
What are the main functions of the microtubule-associated proteins (MAPs)?
- Function as cross-bridges which connect microtubules together
- Affect microtubule rigidity and assembley rate
Name some specific MAPs and their specific function
- MAP-2: filament binding and cross-linking
- Plectin: links to intermediate filaments
- + TIPs: remain associated with growing + ends and link them to other structures
- XMAP215: Stablises + ends and accelerates assembly
- Katanin: Severs microtubules
Stathmin causes microtubule shrinkage. How does it do this?
- Stathmin sequesters GTP-tubulin preventing them from added to the microtubule
- This eventually leads to GTP-subunit addition stopping which allows GDP-hydrolysis to catch up
- This results in GDP-tubulin being released from the microtubule causing it to shrink
What are motor proteins?
- Enzymes that convert ATP hydrolysis directly into movement along cytoskeletal filaments
- They carry cargo e.g. organelles, protein complexes and RNA
- Some move towards the + end of the microtubule while others move towards - end.
What are the 2 types of microtubule motor protein and what are their functions?
-
Kinesins
- Move cargo to + end
- Participate in mitotic spindle dynamics during mitosis
-
Dyneins
- Move cargo to - end
- Participate in spindle dynamics during mitosis
- Power beating of cilia and flagella
What domain is conserved across all types of kinesin proteins?
- N-terminal conserved motor domain
Describe the structure of a kinesin
- 2 heavy chains and 2 light chains
- Head - contains microtubule and ATP binding sites
- Tail - Cargo-binding site
Explain how Kinesin moves along a microtubule
- The kinesin “walks” along the microtubule
- A molecule of ATP binds to the ATP-binding site on one the kinesin heads
- This causes the kinesin molecule to take a “step forward” resulting in the other kinesin head binding to the microtubule via the microtubule-binding site
- The ATP bound to the ATP-binding site is then hydrolysed leading to the release of ADP from the kinesin head that just “took the step”
- This frees up the ATP-binding site on this kinesin head allowing for cycle to continue.
Dynein is actually a complex of many proteins. What proteins are within the dynein complex?
- Dynein
- Ankyrin
- Spectrin
- Arp1 filament
- Dynactin complex
- membrane glycoprotein
What are the 2 classes of dynein?
-
Cytoplamsic dynein
- Carries cargo in the cytoplasm
-
Axonemal dynein
- Localised to flagella and cilila
- Motors that power the beating of flagella and cilia
What is the axoneme?
- It’s a microtubule-based structure that forms the core of both cilia and flagella
Describe the structure of the axoneme
- There are 9 doublet-rings of A and B tubules with a central pair of singlet microtubules
- The 9 doublet rings are linked by nexin
- They also each have a outer-arm dynein molecule and an inner-arm dynein molecule
How does dynein allow for flagella to bend?
- Dynein allows for microtubules in the flagellum to slide up and down
- There are also linking proteins between the microtubules that move as the microtubules slide up and down which causes the microtubules to bend
What diseases can occur due to cilia/flagella mutations?
- Infertility
- Polycystic kidney disease
- Respiratory infection
- Retinal degeneration
- Usher syndrome (hearing/balance loss)
Describe the structure of the actin filaments
- 7 nm in diameter
- Made up of G-actin (Globular actin)
- Plus end - fast growing
- Minus end - slow growing
- Monomers polymerize into a helical chain
Explain the process of actin polymerization
- ATP-actin binds to the actin filament
- Then actin ATPase activity hydrolyses ATP-actin into ADP-actin and Pi
- Older ADP-actin monomers are unstable and so are released from the filament and dissassemble
- This dissassembly releases ADP which can reform ATP which will bind to new actin monomers and continue the cycle
Explain what is meant by the phrase “Nucleation is the rate-limiting step in the formation of an actin filament polymer”
- Actin filaments can only grow to a certain length before the rate of actin monomers being added to the filament and the rate of actin monomers being released from the filament are equal
- This means the amount of actin monomers within that filament remain the same so the length of the filament will remain the same
- In other words, the rate of actin polymers being added to the filament limits its growth
Name some of the actin binding proteins (ABPs) and their functions
- Formin - Nucleates assembly and remains associated with + end
- Thymosin - Binds subunits and so prevents assembly
- Profilin - Binds subunits and so accelerates elongation
- Tropomyosin - Stabilizes filament
- Fimbrin - Cross-linking
Polymerization of actin filaments can produce “pushing” forces. Give some examples of cellular processes where this pushing force is important
- Cellular movement - Polymerization at the front of a cell pushes the leading edge forward
- Phagocytosis - formation of pseudopods
- Intracellular movement and cell-to-cell spreading of pathogens
What are the main actin binding proteins involved in actin polymerization at the cell membrane?
- Cofilin
- Capping protein
- ARP (Actin-related protein) complex
What are the functions of the ARP complex?
- ARP complex nucleates actin filament growth from the (-) end, allowing for rapid elongation from the (+) end
- It also can attach to the side of another actin filament while remaining bound to the (-) end of the filament that it has nucleated
Whar is the significance of the ARP complex being able to attach to the side of an actin filament other than the one that it nucleates?
- The ARP complex nucleates filaments more efficiently when it is bound to the side of a preexisting actin filament resulting in a filament branch that grows at a 70° angle relative to the original filament
Actin filaments can also form bundles as opposed to branches. How are these actin bundles produced?
- Formation of actin bundles is induced by formins
- They bind to the (+) end and nucleate the growth of straight, unbranched filaments by restricting the way they are added to the filament
- These unbranched filaments can be cross-linked by other proteins to form parallel bundles
Why do free actin subunits not polymerize into filaments if the concentration of free subunits is high (50-200 mM)?
- Because the free actin subunits are bound to special proteins, such as thymosin.
- Actin monomers bound to thymosin are locked and they cannot associate with either the (+) end or (-) end of the actin filament.
How do cells recruit actin monomers from the sequestered pool and use them for polymerization?
- Recruitment depends on another monomer-binding protein profilin
- Profilin binds to an actin monomer opposite its ATP-binding cleft and this binding increases its affinity for the (+) end of the filament eventually leading to that actin monomer binding to the (+) end
- Actin-profilin can bind to the plus end of the actin filament but is unable to bind to the minus end.
Which actin binding proteins are responsible for stabilizing and destabilizing the actin filaments?
- Tropomyosin - Stabilizes actin filaments by binding simultaneously to seven adjacent actin subunits in one filament; this prevents other proteins from binding to actin
- Cofilin - Destabilizes actin filaments by forcing them to twist a little more tightly
Cross-linking proteins are responsible for organising assemblies of actin filaments. What structures can they organise actin filaments into and where may these structures be found?
- Contractile bundles found in stress fibers
- Gel-like networks found in the cell cortex
- Tight parallel bundles found in filopodium
Explain how actin polymerization mediates engulfment during phagocytosis
- Actin cytoskeleton is reorganised to bend the membrane to allow for the release of capsids which engulf the pathogens
- The actin cytoskeleton is then reorganised again to allow for the capsids to be taken back up by the cell
Which bactrium uses actin for intracellular movement?
- Listeria monocytogenes
Which virus uses actin for intracellular movement?
- Vaccinia virus
What is the name of the group of actin-based motor proteins?
- Myosins
What are the functions of the myosins?
- Myosins convert ATP hydrolysis into movement along actin filaments
- Some myosins move cargoes, other myosins slide actin (muscle)
What is the specific function of myosisn I?
- Can carry organelles or slide actin filaments along the membrane
What is the specific function of myosin II?
- Slides actin filaments to produce contractile forces during muscle contraction
