Week 10: The Cytoskeleton Flashcards
Section 2 Week 4
What are the three components of the cytoskeleton?
actin filaments, microtubules, and intermediate filaments
What does it mean when it is said that the network of protein filaments is highly dynamic?
It means that you can assemble, disassemble, and change the shape
Which component(s) of the cytoskeleton have the function of structural support (cell shape)?
All (actin filamets, microtubules, intermediate filaments)
Which component(s) of the cytoskeleton are responsible for the internal organization of the cell (i.e. through organelles, vesicle transport)?
microtubules
Which component(s) of the cytokeleton play a role in chromosome segregation and dividing the cell in two (in cell division)?
actin filaments, microtubules
Which component(s) of the cytoskeleton play a role in crawling cell and muscle contraction?
actin filaments
True or false: the diameters cytoskeleton filaments are so small (7 - 25 nm) that most forms of light microscopy are unable to pick up on them
True!
What form of light microscopy can detect cytoskeletal filaments? How?
A flourescence microscope (which is a light microscope) can detect cytoskeletal filaments through fluorescent labels that are added to detect specific proteins
What type of microscope can detect cytoskeletal filaments with great detail? How does it do this?
A transmission electron microscope can detect cytoskeletal filaments with detail. It does this by using beams of electrons with very short wavelengths.
When viewing microtubules, which is better: fluorescence microscope or transmission electron microscope?
Transmission electron microscope because a fluorescence microscope would make it look 200 nm wide rather than their actual width of 25 nm
What is immunofluorescence microscopy used for? What has to be done to cells in order for this to work?
Immunofluorescence microscopy is used to determine the the location of proteins within a cell. In order for this to work, it cannot be live imaging and the cell must therfore be fixed.
What does a primary antibody bind to?
The protein of interest
What does a secondary antibody bind to? How is it different from a primary antibody?
It is bound to the primary antibody and is covalently tagged to a fluorescent marker.
What is a general reason for why you can’t have a fluorescent marker on the primary antibody?
It’s too expensive
Order the three components of the cytosleton in the order of increasing size
Actin filaments < intermediate filaments < microtubules
What are the different types of intermediate filaments?
cytoplasmic and nuclear
What are intermediate filaments mostly involved in?
Structural support
What are cytoplasmic intermediate filaments?
They are a type of IF that (in animal cells) are subjected to mechanical stress, and in doing so provide mechanical strength
What are nuclear intermediate filaments?
They are a type of IF that make up the nuclear lamina which formed by lamins
What is the importance of the nuclear lamina?
If you break this the nucleus will fall apart
What is the structure of a cytoplasmic IF monomer?
It has a conserved alpha helical central rod domain with N and C terminal, which means it is polar
Describe the dimer of a cytoplasmic IF
It is a coilded-coil with different ends, making it polar
What makes up a cytoplasmic IF tetramer? Is a tetramer polar or non polar?
Two dimers make a staggered antiparallel tetramer. Because it is antiparallel, the ends are the same making it non-polar
How many tetramers make up an cytoplasmic IF?
8 tetramers associate side by side and assemble into a filament
What kind of interactions happen between cytoplasmic IF tetramers?
Noncovalent
Why do cytoplasmic IFs have no filament polarity?
Because the tetramers which they are made up of have no polarity
Describe the strength of a cytoplasmic IF
Tough, flexible and high tensile strength
What does it mean when it is said that cytoplasmic IFs have high tensile strength?
They can take a lot of stress before breaking when pulled or stretched
What is keratin?
It is a type of cytoplasmic intermediate filament within epithelial cells. They form a network throughout the cytoplasm out to cell periphery. They are achored in each cell at cell-cell junctions (called desmosomes) which connect to neighboring cells.
While microtubules and actin filaments are found in all eukaryotes, what kind of eukaryotes are intermediate filaments found in?
Vertebrates and a number of other soft-bodied animals
What functions are microtubules involved in?
Cell organization, mitosis, structural support
How do microtibules participate in cell organization?
vesicle transport, organelle transport and positioning, and in animal cells it is involved with the centrosome
How are microtubules involved in structural support?
They support cell structures and assist with motile stuctures such as flagella and cilia
What are microtubules made of?
Tubulin: these are long, stiff, hollow tubes, that are inextensible. This means that they are not elastic, however you CAN assemble and disassemble (meaning they can get longer and shorter)
Describe the stucture of microtubules
Microtubule are made of individual subunits of two closely related globular proteins called alpha-tubulin and beta-tubulin. Both of these together form a tubulin heterodimer, and they are both bound to GTP
Since tubulin hetero dimers are polar, how do you classify each end?
The end with the beta-tubulin is referred to as the plus end, the end with the alpha-tubulin is referred to as the minus end
What is a line of tubulin heterodimers called?
Protofilament
How many protofilaments make up a hollow tube?
13
What is the middle of the hollow tube formed by protofilaments called?
lumen
All the bonds between the individual subunits (tubulin heterodimers) are ___________
noncovalent
Growth and disassembly of microtubules happen where on the microtubule? Where is it more rapid?
Growth and disassembly can happen at both ends of a microtubule but is more rapid at the plus end
Where are the weakest and strongest bonds within protofilaments?
The weakest bonds are in between individual protofilaments while the strongest bonds are between individual heterodimers within the same protofilament
What happens to the GTP on the plus end of a protofilament after it’s been there for a while?
The beta-tubuin cuts GTP which turns it into GDP
What’s the difference when you have a plus end beta-tubulin bound to GTP vs GDP?
A plus end beta-tubulin bound to GDP is more likely to undergo disassembly, while a plus end beta-tubulin bound to GTP is more likely to undergo assembly
What is the part between the minus end and the plus end of a protofilament called?
the axoneme
What does it mean when it is said that microtubules undergo dynamic instability?
Dynamic instability for microtubules means that the plus ends of microtubules grow and shrink which is needed for remodeling
What are Microtubule Organizing Centers? What is an example of an MTOC?
Microtubule Organizing Centers (MTOCs) are what stabalize microtubules at their minus end. In the cell, microtubules grow out of MTOCs. For example, the centrosome in animal cells are an MTOC where the minus ends are stabalized and the plus ends grow out.
How do microtubules grow?
- free alpha-beta-tubulin dimers that are bound to GTP add to the growing microtubule plus end (while the minus end is stabalized)
- however, as dimers are added, beta-tubulin hydrolyzes GTP to GDP
- to continue growing the microtubule, there is rapid addition of alpha-beta-tubulin dimers which is faster than the beta-tubulin that hydrolyzes GTP to GDP which stabalizes the plus end
- this creates a dynamic (always changing) GTP cap
What are the names for heterodimers bound to GTP and GDP?
GTP: GTP bound heterodimer or T form
GDP: GDP bound heterodimer or D form
How does a growing microtubules’ GTP cap compare to a 5’ cap on mRNA?
They are very different as the 5’ cap on mRNA is stationary while a microtubule’s GTP is not
How do microtubules shrink?
- free alpha-beta-tubulin dimers that are bound to GTP add to the growing microtubule plus end (while the minus end is stabalized)
- however, as dimers are added, beta-tubulin hydrolyzes GTP to GDP
- to shrink the microtubule, there is slow addition of alpha-beta-tubulin dimers which is slower than the beta-tubulin that hydrolyzes GTP to GDP which stabalizes the plus end
- this loses the dynamic (always changing) GTP cap
- in losing the GTP cap, the microtubule diassembles as GDP-tubulin dimer (D form) are released into the cytosol
True or false: GTP is tightly-bound to alpha-tubulin and does not get hydrolyzed
True
What is the difference between filaments with GTP caps vs filaments that are left with GTP hydrolysis
GTP cap: straight filaments, they have stronger binding and favors growth
GTP hydrolysis (GDP-tubulin dimers): there is a small conformational change, they have weaker binding and curved filaments, favors disassembly
What is the function of MTOCs?
They have nucleating sites for microtubule growth. For example, gamma(y)-tubulin ring complex (y-TuRC) acts as an attachment site for the minus end of aB-tubulin dimers and stabalizes the growth of the plus end.
In non dividing animal cells (in interphase) most microtubules ____ from ____ centrosome
radiate; one
In dividing animal cells, the centrosome ____________ to form ________ spindle poles (MTOCs)
duplicates; two
When an animal cell devides, what happens to the microtubules?
They are reorganized to form a bipolar mitotic spindle
What do microtubule-associated proteins do?
- Nucleate growth of new microtubules
- Promote microtubule polymerization
- Promote microtubule disassembly
- Stabilize microtubules (prevent disassembly) - the proteins bind to the sides or they’re plus-end linking proteins
How do neurotransmitters synthesized in the ER get to the axon terminals?
The ER and Golgi apparatus are located in the nerve cell body, the neurotransmitters are transported by by motor proteins on microtubules
What motor proteins use microtubules and in what direction do they move?
Kinesins: generally move towards the plus end of microtubules. E.g. kinesin I moves towards the plus end to axon terminus, it carries the cargo of organelles, vesicles, and macromolecules.
Dyneins: generally move towards the minus end of microtubules. E.g. cytoplasmic dynein moves towards the minus end to cell body, it carries the cargo of worn-out mitochondria and edocytosed material
Describe the structures of kinesin I and cytoplasmic dynein
Kinesin I and cytoplasmic dynein are dimers. Their heads move along the microtubules as they use ATP hydrolysis for movement. Their tails transport cargo. (meaning the walkies don’t actually walk…that’s their head)
How is the ER positioned in an animal cell in relation to microtubules?
The ER is positioned from the nuclear envelope to the cell periphery by kinesin-1, which makes it closer to the plus end of the microtubule.
How is the Golgi apparatus positioned in an animal cell in relation to microtubules?
The Golgi apparatus is positioned near the centrosome (which is pretty centrally located) by cytoplasmic dynein, which makes it closer to the minus end of the microtubule.
What are actin filaments also known as?
Microfilaments
True or false: Actin filaments are present in a select number of eukaryotes
False (they’re in all)
What are actin filaments made of?
They are made of actin monomers that are flexible and inextensible
What motor proteins use actin filaments?
mysosins
What are the functions of actin filaments?
- Provide stuff, stable sturctures (microvilli)
- Contractile activity
- Cell motility (crawling)
- Cytokinesis (contractile ring)
Describe the structure of actin filaments
It is a helical filament composed of a single type of globular protein that is made up of actin monomers that are held together via noncolvalent interactions. Two protofilements twisted in a right handed-helix is what makes up the helix.
Describe the polarity of an actin filament
Actin filaments have polarity. They have a plus end and a minus end, and growth is faster at the plus end.
How do actin monomers grow?
- Free monomers are bound to ATP which is bound to the center of the protein.
- Shortly after an actin monomer is added to the filament, the actin hydrolyzes ATP to ADP which reduces strength of of binding between monomers in filament.
- However, there is rapid addition of actin monomers which is faster than the ATP hydrolysis in newly addic actin monomers, this creates the ATP cap
Describe actin polymerization in a test tube
- First, actin subunits (monomers) and ATP are added to a test tube
- Nucleation (lag phase): small oligomers form but they are unstable
- Elongation (growth phase): some oligomers become more stable, which leads to rapid filament elongation (faster at plus end)
- Steady state (equilibrium phase): there is a decrease in actin subunits; rate of subunit addition = rate of subunit diassociation. This is called treadmilling
What happens at each end of the actin filament during actin gilament growth?
Plus end: ATP-actin - there is addition of actin monomers (polymerization) and shortly after actin hydrolyzes ATP to ADP
Minus end: ADP-actin - there is a loss of actin monomers (depolymerization)
What happens to actin filaments during treadmilling?
- During treadmilling, actin filaments look “stable” as they remain the same size
- But this “stable” size is possible though the continual addition of monomers at the plus end and loss of monomers at the minus end
- As monomers are added at the plus end, monomers are subtracted at the minus end, making monomers move through the filament before they are ultimately replaced
- This process requires a continuous supply of ATP
What is an example that uses the treadmilling of actin filaments?
Cell crawling uses the treadmilling of actin filaments to maneuver. This is done though an actin filament rapidly assembling at the leading edge and disassembling further back - this pushes the leading edge (and ultimately the cell) forward
True or false: actin filaments can only have treadmilling as microtubules only have dynamic instability
False! Both happen in either filaments
What are the functions of actin filaments that are regulated by actin-binding proteins?
- Sequester actin monomers (prevent polymerization)
- Promote nucleation to form filaments
- Stabilize actin filaments (capping)
- Organize: bundle, cross-link filaments
- Severe actin filaments
What are myosins?
Myosins are a type of motor protein that uses actin to move, they generally move towards the plus end of actin filaments. Their heads move along actin filaments, and they use ATP hydrolyis for movement.
What are the two types of Myosins?
Myosin I: has a tail domain that binds to cargo
Myosin II: is a dimer, their tails are organized in a coiled-coil which are used to assemble into a myosin II filament. E.g., Bipolar myosin II filaments slide two different actin filaments in the opposite directions from each other (making both ends not connected to the myosin II a plus end). This generated a contractile force
True or False: All motor proteins use ATP
True
