Cytoskeleton Flashcards
(123 cards)
1
Q
Describe the structure of an actin filament
A
Made up of monomeric actin protein subunits
Assembled into a twisted, two stranded polymer
2
Q
Describe the structure of a microfilament
A
Made up of monomeric actin protein subunits
Assembled into a twisted, two stranded polymer
3
Q
What are the general purposes of actin filaments?
A
Provide structural support (particularly to plasma membrane)
Important roles in certain types of cell mobility
4
Q
Describe the structure of a microtubule
A
Composed of alpha and Beta-tublin heterodimers.
Assembled into a hollow, tubelike cylinder.
5
Q
What are the general purposes of microtubules?
A
Provide structural support
Involved in certain types of cell motility
Help to generate cell polarity
6
Q
Describe the structure of intermediate filaments
A
Formed from a family of related proteins (such as keratin or lamin)
Subunits assembled into strong, ropelike polymer
7
Q
What are the general purposes of intermediate filaments?
A
(Depending on specific protein)
Provide support for nuclear membrane
Provide support for cell adhesion
8
Q
List microfilaments, microtubules and intermediate filaments in order of width size, smallest to largest
A
Microfilaments, intermediate filaments, microtubules
9
Q
What is the width of a microfilament?
A
7-9nm
10
Q
What is the width of a microtubule?
A
25nm
11
Q
What is the width of an intermediate filament?
A
10nm
12
Q
What is the subunit of a microfilament?
A
Actin
13
Q
What is the subunit of a microtubule?
A
alpha-beta Tubulin dimer
14
Q
What is the subunit of an intermediate filament?
A
Various
Formed from a family of related proteins such as keratin or lamin
15
Q
Where are microfilaments usually located in cells?
A
Around cell membrane, especially in microvilli and filopodia
Cytoplasm
16
Q
Where are microtubules usually located in cells?
A
Around edges of cell (sides and bottom)
Cytoplasm
17
Q
Where are intermediate filaments usually found in cells?
A
Around edges of cells at junctions to other cells
Surrounding nucleus
18
Q
What are the general functions the cytoskeleton performs?
A
Cell shape
Cell movement / migration
Cell contraction
Organisation and movement of organelles
19
Q
Briefly describe how external cell signalling can lead to cell migration, eg; after a cut in the skin
A
Other cells (eg; those adjacent to cut) release growth factor which attracts more cells (eg; epithelial cells)
Growth factor interacts with receptors on plasma membrane of other cells
Through signal transduction pathways, production of actin stimulated, cells migrate to target area (eg; a cut in the skin)
20
Q
What does G-actin stand for?
A
Globular actin
21
Q
What does F-actin stand for?
A
Filamentous actin
22
Q
Which end of F-actin is the ATP binding site?
A
The negative end
23
Q
How does G-actin assemble into F-actin?
A
ATP binds to G-actin, allowing assembly into F-actin
24
Q
If the ATP on G-actin is hydrolysed what happens?
A
G-actin does not assemble into F-actin / F-actin disassembles
25
Is energy required for G-actin to assemble into F-actin?
No.
ATP doesn't need to be hydrolysed for the G-actin to assemble into F-actin
26
Describe the process of G-actin assembling into F-actin.
Include what causes the microfilament to disassemble / continue growing
G-actin binds to ATP. This allows it to assemble into F-actin - no energy is required.
After ATP-G-actin assembles onto the actin filament, the ATP is hydrolysed to ADP and phosphate. The molecule becomes ADP-F-actin.
If ADP-F-actin is left exposed at the ends, it is unstable and the filament will disassemble from the ends.
If there is lots of G-ATP continuing assembly, the filament is stable and keeps growing.
27
What will happen if there is a mutation preventing G-actin from binding ATP?
G-actin won't assemble into F-actin, microfilaments will not form
28
What will happen if there is a mutation preventing the ATP of ATP-G-actin from being hydrolysed?
The actin microfilament will be made up of ATP-F-actin / ATP-G-actin which is more stable than ADP-F-actin, and so won't be able to disassemble normally once G-ATP stops being added
29
In a graph of the mass of microfilaments against time, describe the usual curve
Slow rise during "nucleation" phase ("lag phase"), then a faster increase during "elongation", then a plateau after reaching the "steady state" phase.
30
In a graph of the mass of microfilaments against time, describe the curve if nuclei are added at time = 0
Immediate fast increase during "elongation" phase, then a plateau after reaching the "steady state" phase.
No "nucleation" / "lag phase" at beginning.
31
Which end of a microfilament grows more rapidly?
The positive end grows more rapidly
32
Define the term critical concentration (Cc) when referring to microfilaments
Critical concentration (Cc) is the concentration of free ATP-G-actin at which assembly / disassembly are equal at one of the ends of the microfilament.
(Means that end is not growing or decreasing, it is stable)
(Not at both ends, as they have different Ccs)
33
When the critical concentration is exceeded at an end of a microfilament, the microfilament will _______ at that end.
When the critical concentration is exceeded at an end of a microfilament, the microfilament will GROW at that end.
34
When the critical concentration is not reached at an end of a microfilament, the microfilament will _______ at that end.
When the critical concentration not reached at an end of a microfilament, the microfilament will SHRINK at that end.
35
When the critical concentration is exceeded for one end of a microfilament but is not reached for the other, the microfilament will ____________________ and ____________________. This is called ___________.
When the critical concentration is exceeded for one end of a microfilament but is not reached for the other, the microfilament will grow at one end and shrink at the other end. This is called treadmilling.
36
What is treadmilling?
When actin subunit concentration exceeds critical concentration for positive end (0.12microM) of microfilament but is less than critical concentration for negative end (0.6microM).
Causes microfilament to grow at positive end and shrink at negative end at same rate, so filament length remains constant as subunits flow through filament.
37
What is the critical concentration for the positive end of a microfilament
0.12 microM
38
What is the critical concentration for the negative end of a microfilament
0.60 microM
39
At what concentration of actin subunits does treadmilling occur?
Any concentration between 0.12 microM and 0.6 microM
40
What happens to the length of the microfilament when treadmilling occurs?
It stays approximately the same length (but the subunits themselves are changing)
41
List four types of actin-binding proteins
Profilin
Cofilin
Thymosin beta4
(Various different) Capping Proteins
42
What does profilin do?
It is an actin-binding protein that enhances the exchange of ADP for ATP on G-actin
43
What does cofilin do?
It is an actin-binding protein that enhances the loss of ADP-actin from the negative end of the microfilament
44
What does thymosin beta4 do?
It is an actin-binding protein that binds G-actin to provide reserve actin when needed
45
What is the general function of capping proteins (actin-binding)
Bind to microfilament ends to stabilise them, preventing assembly and disassembly
46
What capping protein binds to the positive end of a microfilament to prevent assembly and disassembly?
CapZ
47
What capping protein binds to the negative end of a microfilament to prevent assembly and disassembly?
Tropomodulin
48
What end of the microfilament does the capping protein CapZ bind to?
The positive end
49
What end of the microfilament does the capping protein Tropomodulin bind to?
The negative end
50
When is it important to have capping proteins on microfilaments?
In muscle cell sarcomeres.
Muscle cells need their actin to be arranged properly at all times, stable and fixed ready to interact with myosin to perform their function for muscle contraction at any moment.
51
What are formins?
Group of proteins that, in response to external signals, facilitate growth of actin filaments near cell membrane, used to migrate cells
52
What is formin made up of?
RBD, FH1, FH2
53
How do formins work?
Formins nucleate the assembly of unbranched filaments
54
What state is formin maintained in usually?
Inactive state
(Due to association of its N and C termini)
55
How is formin activiated?
When Rho is in active GTP-bound form, it associates with formin to induce a conformational change to activate formin
56
What is Rho?
The protein activated by GTP that associates with formin's inactive form to induce a conformational change to activate formin
57
What is the shape of activated formin?
Semi circle
58
Once formin is activated, what happens?
Two activated formin molecules associate with each other to form a dimer of formin FH2 domain (makes a ring shape)
G-actin is then fed through the dimer, assembling into microfilaments
59
What does ARP2/3 stand for?
Actin related protein
60
What is the function of ARP2/3?
Actin related protein nucleates the assembly of branched actin filaments
61
What angle does an actin branch make with the microfilament?
70 degrees
62
What function does actin branching usually serve?
Filopodia creation to migrate cells
63
What does phalloidin bind to? What does this result in?
F-actin
Prevents disassembly
64
What can we use toxins like phalloidin for?
Phalloidin binds to F-actin so if we attach a fluorescent tag to it, we can stain and view actin and microfilaments in cells
65
Give examples of cross linking proteins are involved in organising actin-based cell structures?
Fimbrin, for microvilli
Spectrin, for cell cortex
Filamin, for filopodia
Dystrophin, for muscle cell cortex
66
What is the function and location for fimbrin?
Function: cross-linking protein for actin, increases strength of actin scaffolding
Location: Microvilli, filopodia, focal adhesions
67
What is the function and location for spectrin?
Function: Combine with actin to form cell-shaping scaffolding
Location: Cell cortex, just below membrane
68
What is the function and location for filamin?
Function: Cross-link with actin to allow cells to migrate
Location: Leading edge, stress fibers, filopodia
69
What is the function and location for dystrophin?
Function: Cross-link with actin to support muscle contraction
Location: Linking membrane proteins to actin cortex in muscle
70
Where is the dystrophin gene located?
X chromosome
71
What gene is defective in Duchenne muscular dystrophy?
The dystrophin gene
72
What disease is caused by a defect in the dystrophin gene?
Duchenne muscular dystrophy
73
What is dystrophin, what does it bind to?
Adapter protein
Binds to cytoskeletal components like actin and to dystroglycan (the cell-adhesion molecule)
74
What is dystroglycan?
A cell-adhesion molecule that binds to dystrophin
75
Why is dystrophin important for muscle contraction?
It is the protein that connects the actin in muscle cells to membrane proteins that transmit the force of contraction to the tendon and bone
76
What is the name of actin's motor protein?
Myosin
77
Describe the general structure of all myosins
Composed of one / two heacy chains (motor subunit) and several light chains
78
What are the general functions of myosins?
Myosins use ATP-hydrolysis energy to walk along actin filaments.
Depending on specific myosin type, this movement is used for generating contraction or to transport specific cellular components
79
Where on a myosin is the actin binding site?
On the heavy chain head
80
Where on a myosin is the ATP binding site? (nucleotide binding site)
On the heavy chain head
81
Give a specific example of how a mutation in a myosin may cause a disease
Name and describe the disease, include the type of myosin mutated
Mutation in myosin II can cause Familial Hypertrophic Cardiomyopathy
(Enlarged heart, sudden death, often in young people with no previous indication of heart problems)
82
Outline the process of myosin movement
1. ATP binds to myosin, causing myosin head to release from actin
2. ATP hydrolysed, causing conformational change to myosin head, rotating head wrt neck of protein and storing the energy released by the hydrolysis (re-cocked state)
3. The now myosin-ADP-Pi complex binds to a new actin subunit
4. Pi is released, allowing myosin head to return to original conformation, moving bound actin filament along with the rotation (power stroke)
5. ADP released, allowing new ATP to bind
83
What is step size for myosin II?
5 - 10 nm
84
For most myosin, what does step size depend on?
Length of neck region (longer neck, longer step)
85
What is the step size for myosin I?
10 - 14 nm
86
What is the function of myosin II?
Contraction
87
What is the function of myosin V?
Organelle transport
88
What is the function of myosin I?
Membrane association, endocytosis
89
How do the differences in structure for myosin II and myosin V allow them to carry out their separate functions?
Myosin V has a longer neck (travels large distances across cell)
Myosin V has globular cargo binding domains at tail (to bind to organelles to transport them)
Myosin II can assemble into bipolar filaments (useful for contractions)
90
True or False:
The correct model for myosin V movement is called inchworm
False:
The correct model for myosin V movement is called hand over hand
91
True or False:
The correct model for myosin V movement is called hand over hand
True
92
How far does cargo for myosin V move for every step?
36 nm
93
How far does the myosin head move for myosin V for every step?
72 nm
94
Compare the inactive and active forms of myosin V
Inactive: tail domain tucked down near head domain
Active: tail extended out, allowing it to bind to cargo
95
Discuss the structure of myosin II and how this aids its function
Short duty cycle and cooperative action
Bipolar complexes of myosin II work together during contraction, binding only transiently, so hundreds of heads interact with actin filaments.
Allows fast movement and greater force.
96
Recall gross muscle structure, largest to smallest
Muscles
Bundle of muscle fibers
Multinucleated muscle cell
Myofibril
Sarcomere
97
What is the source of phalloidin
Death cap mushroom,
Amanita phalloides
Toxic 50% mortality (affects kidney and liver)
98
What is the orientation of actin filaments in a sarcomere?
The positive end of the actin filaments attach to the Z disk
So within one sarcomere, the actin filaments will have the positive ends on the outside, at the Z disks, and the negative ends facing each other, inwards
99
How does mechanism of contraction in skeletal and smooth muscle differ?
Skeletal muscle: Binding of Ca2+ to troponin C leads to muscle contraction
Smooth muscle: Ca2+ - calmodulin activates myosin light-chain kinase
100
Outline how calcium ions regulate the interaction between myosin and actin
1. Action potential transmitted from cell surface via t-tubules triggers calcium ion release from sarcoplasmic reticulum
2. Calcium ions bind to troponin on actin filaments, causing conformational change in tropomyosin, exposing myosin binding sites on actin filaments, allowing muscle contraction to occur
3. After action potential finishes, calcium ions pumped back into sarcoplasmic reticulum and troponin and tropomyosin change and block myosin from binding to actin, so muscle returns to relaxed state
101
Compare troponin (TN) and tropomyosin (TM)
Troponin on actin filaments receives Ca2+ to cause conformational change in tropomyosin
Tropomyosin covers myosin binding sites on actin filaments
102
Why are actin and myosin important during mitosis?
After spindle pulls chromosomes apart, the contractile ring that pinches down the cell equator (cleavage furrow) to divide it in two (cytokinesis) is made of actin and type II myosin
103
Why is systemic application of Rho-kinase inhibitors to treat cytoskeletal related diseases problematic?
Not specific enough. Would inhibit all cell migration in body. Means epithelial cell replacement, wound healing etc; are affected throughout whole body.
104
Provide an example of approved topical application of Rho-kinase inhibitors to treat a cytoskeletal related disease?
Use Ripasudil a Rho-kinase inhibitor approved for treatment of glaucoma (can cause blindness).
Increases outflow of aqueous humour of eye by disrupting the actin skeleton (causes leaks) to decrease pressure.
105
How do Rho kinase inhibitors work?
They disrupt junctions.
Actin filaments are connected to Adherens and tight junctions, so can influence how leaky these junctions are.
106
Provide an example of semi-approved topical application of Rho-kinase inhibitors to treat a cytoskeletal related disease?
Fasudil, used for prevention of cerebral vasospasm, approved in Japan and China so far
107
What proteins are associated with Class I of intermediate filaments in mammals?
Acidic keratins
108
What proteins are associated with Class II of intermediate filaments in mammals?
Basic keratins
109
What proteins are associated with Class III of intermediate filaments in mammals?
Desmin, GFAP, vimentin
110
What proteins are associated with Class IV of intermediate filaments in mammals?
Neurofilaments (NFL, NFM, NFH)
111
What proteins are associated with Class V of intermediate filaments in mammals?
Lamins
112
Where are Class I intermediate filaments distributed in mammals?
Epithelial cells
113
Where are Class II intermediate filaments distributed in mammals?
Epithelial cells
114
Where are Class III intermediate filaments distributed in mammals?
Muscle, glial cells, mesenchymal cells
115
Where are Class IV intermediate filaments distributed in mammals?
Neurons
116
Where are Class V intermediate filaments distributed in mammals?
All cells, in nucleus and nuclear envelope
117
What is the proposed function of Class I intermediate filaments in mammals?
Tissue strength and integrity, for cells to resist stresses and strains they are subjected to every day
118
What is the proposed function of Class II intermediate filaments in mammals?
Tissue strength and integrity, for cells to resist stresses and strains they are subjected to every day
119
What is the proposed function of Class III intermediate filaments in mammals?
Sarcomere organisation
Integrity / strength to withstand contractions
120
What is the proposed function of Class IV intermediate filaments in mammals?
Axon organisation and integrity
121
What is the proposed function of Class V intermediate filaments in mammals?
Nuclear structure and organisation
122
What are plakins, what are their purpose?
Intermediate filament associated protein, that connects intermediate filaments to each other or to microtubules
123
Name a disease that results from mutation to intermediate filament genes
Epidermolysis bullosa simplex (causes skin to peel away at slightest trauma due to keratin mutation)
