MCBL: Cytoskeleton, Part 1 Flashcards
1
Q
What is the cytoskeleton?
A
It is the structural framework, or lattice, of the cytoplasmic matrix in eukaryotic cells.
2
Q
True or false: Prokaryotes have a cytoskeleton.
A
False
3
Q
What does the structural support role of the cytoskeleton refer to?
A
The cytoskeleton provides structural support that determines & maintains the shape of the cell as well as the position of the various cellular organelles.
4
Q
What does the trafficking function of the cytoskeleton refer to?
A
The cytoskeleton is part of the machinery that is required to transport vesicles and organelles to specific sites within the cell.
It is also responsible for the invagination of the membrane during endocytosis.
It helps with the separation of chromosomes during cell division.
5
Q
What does “force generating” refer to in the context of the cytoskeleton?
A
The cytoskeleton is responsible for the movement of cells from one place to another.
It allows fibroblasts to crawl on surfaces and it is also responsible for the construction and motions of cilia and flagella.
6
Q
How does the cytoskeleton facilitate translation?
A
It serves as a site for anchoring mRNA and helps to facilitate mRNA translation.
7
Q
How does the cytoskeleton play a role in signal transduction?
A
The deformation of the cytoskeleton, when it comes into contact with another cell / object, allows it to play a role in signal transduction.
8
Q
What proteins are responsible for allowing the cytoskeleton to accomplish its functions?
A
Accessory proteins which associate with the cytoskeleton, are necessary for the controlled assembly/disassembly of the cytoskeletal filaments.
Motor proteins move filaments as well as vesicles/organelles along the cytoskeletal filaments.
9
Q
What are the major functions of the cytoskeleton?
A
Scaffold
Cellular trafficking
Movement
Translation
Signal transduction
10
Q
What does the cytoskeleton consist of?
A
The cytoskeleton consists of filamentous structures made of different types of proteins.
11
Q
What are the elements of the cytoskeleton held together by?
A
All cytoskeletal filaments are composed of multiple protein subunits that are held together by non-covalent bonds.
12
Q
What are protofilaments?
A
Protofilaments are linear arrangements of cytoskeletal monomers.
13
Q
What do multiple protofilaments come together to form?
A
Multiple protofilaments come together to form the final cytoskeletal structure.
14
Q
What are some of the key features of the individual protein subunits that make up the cytoskeleton?
A
Individual, small subunits
These subunits can rapidly move to regions of the cell where cytoskeletal elements are being built
The non-covalent interactions between subunits allows rapid growth/breakdown
15
Q
What is an advantage of using small protein subunits to build cytoskeletal filaments?
A
These small subunits can move rapidly to wher ethey are needed and filaments can be rapidly broken down and rebuilt as needed.
16
Q
Why are multiple protofilaments used as opposed to only one?
A
Multiple protofilaments provide stability to the entire cytoskeletal network.
17
Q
How do cytoskeletal monomer protein s ubunits interact?
A
They bind end-to-end to form protofilaments as well as binding laterally, side-to-side to hold the protofilaments together.
This conformation stabilizes the filament in the middle while allowing the ends to polymerize/depolymerize.
18
Q
Why is it important to have multiple protofilaments be part of a cytoskeletal network?
A
Multiple protofilaments impart strength and bending resistance, especially in intermediate filaments.
19
Q
How many types of cytoskeletal filaments are there and what are their names?
A
Three:
Intermediate filaments
Microtubules
Microfilaments
20
Q
What are the characteristics of intermediate filaments?
A
Intermediate filaments are tough, rope-like structures
Composed of several different proteins that have similar structures
About 10 nm in diameter
21
Q
What are the characteristics of microtubules?
A
Microtubules are hollow, cylindrical structures
Make a single type of structure (a 13 protofilament tube)
Also make cilia, flagella, centriole, basal body
Composed of different proteins called tubulin (at least 3 different types)
25 nm in diameter
22
Q
What are the characteristics of microfilaments?
A
Make a two-stranded helical fragment
Composed of actin (globular or g-actin)
Also known as actin filaments (f-actin)
5 to 7 nm in diameter
23
Q
True or false: Each type of cytoskeletal filament localizes to a specific region of the cell.
A
True
24
Q
Where do intermediate filaments loclaize within the cell?
A
The location of intermediate filaments is dependent upon the specific protein that makes up the intermediate filament in question
For example, vimentin associates with the inner nuclear membrane
Keratins radiate out from the plasma membrane in epithelial cells and help attach it to its neighbor
25
Where do microtubules localize within cells?
Microtubules form tracks throughout the cell
They also radiate out from the centrosome near the center of the cell (not always however)
26
Where do actin microfilaments localize within a cell?
Actin microfilaments are often found in the cell cortex around the periphery of the cell, just below the plasma membrane
They can also be found dispersed throughout the cell
27
What is the major role of intermediate filaments?
Their major role is to resist mechanical stress when stretched
Intermediate filament proteins have high tensile strength
Have the ability to withstand pulling without stretching/breaking due to their arrangement
These are the toughest & most durable of the cytoskeletal elements
28
How many genes encode intermediate filament proteins?
At least 60 different genes code for at least 60 different intermediate filament proteins
29
How many different classes are the intermediate filament proteins grouped into?
Six different classes based on tissue type they are found in
30
What is the structure of an intermediate filament protein?
They typically have a central, rod-shaped helical domain and globular domains of variable size at both their n and c terminus regions.
31
Which portion of the intermediate filament protein gives it its unique binding properties?
The globular regions of the N and C termini give intermediate filament proteins their unique properties.
The globular domains also serve as the binding sites between adjacent intermediate filaments.
32
Where are the binding sites between adjacent intermediate filaments?
There are binding sites BETWEEN adjacent intermediate filament monomers.
Non covalent interactions hold them together and depending on the type of intermediate filament, the dimers my be homo or heterodimers.
33
True or false: Homodimers of intermediate filaments are made of two different intermediate filament proteins.
False
34
True or false: Heterodimers of intermediate filament proteins are made up of two different proteins.
True
35
Picture showing how intermediate filaments come together.
36
2nd picture showing how intermediate filaments come together.
37
What are homodimer intermediate filaments composed of?
Homodimers are composed of the same intermediate filament protein.
They adopt a 'coiled-coil' conformation that shows structural polarity because the proteins line up in the same direction.
They also have a head and a tail that corresponds to the N-terminus and the C-terminus of the individual protein subunits.
38
True or false: The ends of the intermediate filament subunits are chemically distinct.
True
39
How do intermediate filament dimers come together to form an assembled intermediate filament?
Two IF dimers line up in an antiparallel orientation. This forms a tetramer.
NOTE: Because of the antiparallel conformation, the tetramer lacks structural polarity. That is, the N terminus and C-terminus cannot be distingusshed.
Tetramers line up in a staggered fashion end-to-end.
40
What does the arrangement of intermediate filament tetramers form and what does it ressemble?
The tetramers form the protofilament and it assumes a rope-like structure.
41
How do intermediate filament protofilaments line up?
IF protofilaments line up side-by-side to produce the long, rope-like IF structure.
The number of protofilaments required to interact to form the final IF is variable.
Most IF's form this rope-like structure but some form a 2D mesh.
Again, the variabilit between the different globular head regions (which are exposed along the sides of the IF's) give the different IF's their specificity in how they interact with other proteins.
42
True or false: Intermediate filaments are capable of self-assembely *in vitro*.
True; The interactions between IF subunits occurs spontaneously and they do not need instructions from the cell to form.
43
What are the major classes of intermediate filaments?
Nuclear lamins
Cytoplasmic Intermediate filaments
- Keratins
- Vimentin-like
- Neurofilaments
44
What do nuclear lamins do?
Nuclear lamins are a class of intermediate filaments that line and strengthen the inside of the nuclear envelope.
They organize into a 2D mesh and must disassemble and reassemble with each nuclear division.
Protein kinase phosphorylation initiates the breakdown of nuclear lamins.
45
Where are cytoplasmic intermediate filaments found and what do they do?
Cytoplasmic intermediate filaments are found in some metazoans (vertebrates, nematodes, and mollusks)
They are prevelant in cells that are subject to mechanical stress (skin, neurons for example)
They act to stretch and distribute the effects of locally applied forces
This property is due to their staggered subunit composition
46
Describe keratins.
Keratins are a family of cytoplasmic intermediate filaments.
They are comprised of two (2) subfamilies that are made up of about 15 members each
These are found in epithelial cells, hair, horns, wool, cornea, feathers
Connect neighboring cells intermediate filaments via cell junctions called desmosomes
47
Describe vimentin-like intermediate filaments.
Vimentine-like IFs are a family of cytoplasmic intermediate filaments that are found in connective tissues, muscles, glial cells, etc.
They help to distribute the effects of mechanical stress on the cell.
48
Describe neurofilaments.
Neurofilaments are a category of cytoplasmic intermediate filaments that are found in neurons.
The form long, parallel intermediate filaments that form the structural framework that supports the axon
Lou Gehrig's disease (amyotrophic lateral sclerosis) results from the inappropriate expression and abnormal assembly of axonal IF's
49
What is the major purpose of microtubules?
Microtubules major role is to organize.
They form a cytoplasmic scaffold and have molecular motors that associate with them to help move organelles within the cell.
They form the mitotic spindle
Also function in movement as they are part of the cilia and flagella
50
What are the two types of microtuble proteins?
Alpha and Beta tubulin
They are very abundant and make up about 2.5% of the protein in non-neuronal cells
51
Describe the structure of microtubules?
Microtubules are made up of tubulin subunits that are arranged in protofilaments.
The walls are made of heterodimers of tubulin subunits stacked together (alpha and beta tubulin)
Alpha and beta tubulin line up head-to-tail to form protofilaments
They show structural polarity since they always line up head to tail (+ and - end)
13 protofilaments line up to form the walls of the hollow microtubule walls
52
Picture of microtubule structure.
53
Describe the + and - end of microtubules.
The + end of the microtubule is the end that polymerizes and depolymerizes the fastest.
The - end polymerizes and depolymerizes the slowest.
NOTE: *in vivo* (in the organism), the - end of the microtubule us stablized by association with other proteins and does not depolymerize.
54
What molecule is used to power assembly and disassembly of microtubules?
GTP. Tubulin is a GTP-binding proteinand both alpha and beta tubulin are bound to guanine nucleotides.
55
Where does GTP bind the alpha tubulin?
GTP binds the alpha tubulin in the middle of the dimer and is stuck.
It can't be hydrolyzed or exchanged.
Alpha tubulin is ALWAYS bound to GTP.
56
Where does GTP bind on beta tubulin?
GTP binds to a cleft in the beta tubulin.
It is hydrolyzed to GDP after its addition to the growing microtubule.
GDP or GTP affects the affinity of the tubulin dimer for other dimers and for the microtubule itself.
57
True or false: The GTP bound dimer has high affinity for other GTP tubulins and the + end of the microtubule.
True; alpha tubulin, which is always bound to GTP, has high affinity for other GTP bound tubulins and the + end of the MT.
Beta tubulin that has GTP bound to it also exhibits a similar binding affinity.
58
True or false: A GDP bound tubulin has low affinity for other tubulins and the MT.
True; Beta tubulin that has had its GTP hydrolyzed to GDP has a low affinity for other tubulins and the + end of the MT.
NOTE: A GDP bound tubulin in the middle of the MT is held in place by lateral interactions with the rest of the MT.
59
How do microtubules assemble *in vitro*?
In vitro, ab-tubulin dimers will slowly self-assemble.
Growth occurs in three phases:
Lag
Nucleation
Elongation/Growth
Dimers will self aggregate to a certain size but may fall apart so growth is slow (lag/nucleation phase). Once the oligodimer reaches a certain size, growth proceeds rapidly (elongation/growth)
Rapid growth will continue until the rate of subunit addition equals the rate of subunit removal. The number of free dimers becomes limiting so the rate of addition slows down while the rate of removal stays the same.
60
How do microtubules assemble *in vivo*?
* In vivo* MT assembly takes place as so:
1) Self aggregation will take place much faster because 13 gamma tubulin monomers & accessory proteins forms a nucleation ring at the - end of the MT. Each gamma tubulin serves as an individual nucleation site.
2) - end does not grow!
3) One gamma tubulin gives rise to one MT until there are 13 protofilaments to form the MT
4) Alpha and beta dimers are added one at a time to the gamma ring; dimers only add at the + end
61
Picture showing microtubule dynamics.
62
What is the microtubule organizing center?
The MTOC is the area of the cell where MT formation starts.
Nucleation begins here and it allows rapid and location-specific growth of the MTs
Centrosomes are the MTOCs in animal cells and they are usually located near the cell nucleus
Centrosomes are composed of a pair of perpendicular centrioles that are surrounded by the pericentriolar material
63
Describe the make up and composition of the centrioles.
Centrioles are made up of nine (9) short MT triplets and their accessory proteins.
One triplet is made up of one complete tubule w/ 13 protofilaments connected to two (2) incomplete tubules.
A tubule is the innermost tubule while the B and C tubules are incomplete.
True function of centriole is not known as MT polymerization can occur without them
Plants and fungal cells do not have centrioles
64
What does the pericentriolar material have?
Large numbers of gamma tubulin rings that serve as nucleation sites for MT formation
65
Where do MT's start at and where do the grow towards?
MTs originate in the centrosome and radiate outwards towards the cell periphery.
The + ends move towards the plasma membrane and the - ends stay close to the nucleas.
66
What does the 'dynamic instability' of microtubules refer to?
MT's are dynamic and are constantly being built and broken down.
Individual tubules independently alternate between elongation and shortening.
This allows the cell to rapidly respond to any situation that requires a change in the cytoskeleton.
67
What is the ratio of free tubulin heterodimers to microtubules?
50 / 50 ratio of free tubulin dimers to tubulin bound to microtubules in the cell.
Low Ca2+ intracellularly favors MT growth/elongation
High Ca2+ (greater than 1x10-3 ) inhibits growth and allows the MT to be depolymerized
68
What is 'dynamic instability' due to?
Dynamic instability is due to the GTP binding and hydrolysis activity of beta tubulin.
GTP bound to a beta tubulin facilitates the addtion of an alpha beta tubulin GTP dimer to be added to the MT
Once this is done, the beta tubulin will hydrolyze its GTP
Adding GTP to the + end increases the binding affinity of other GTP bound tubulins
Eventually a GTP cap is formed at the plus end of the MT
As long as there is a GTP cap on the + end, the MT will not depolymerize and will continue to grow
69
What other activities does the beta tubulin posses and how do they relate to the 'dynamic instability' of the MT?
Beta tubulin has GTPase activity
Beta tubulin bound to GDP has low affinity for the other MT subunits
This beta tubulin will come off the + end *in vivo* and start the disassembly process
Once GDP beta tubulin is free in the cytoplasm, it exchanges the GDP for GTP and the process of MT formation starts over
70
What occurs when the concentration of alpha beta GTP tubulin in the cytoplasm decrease?
Addition of the GTP bound tubulin at the + end slows down
This allows GTP hydrolysis to 'catch up' at the + end (in other words, the GTP cap shrinks)
The MT becomes destabilized as GDP bound dimers are exposed at the + end and this starts the disassembely of MTs
71
Picture of dynamic instability of microtubules.
72
True or fales: Microtubules are involved in intra and extracellular movement.
True
73
What are 'motor proteins'?
Motor proteins are accessory proteins that carry intracellular cargo along the microtubule 'tracks'.
Two types
Kinesin
Dynein
Both hydrolyze ATP to provide the energy for movement
74
Describe kinesins.
Kinesins are a large multi-subunit protein that walks TOWARDS the + end of the microtubule
10 different kinesin-related protein families
All share homology between the motor domains
Each walks along a MT protofilament at a rate proportional to ATP hydrolysis up to 3 um/sec
Each kinesin has a cargo binding domain that binds to vesicles/organelles, other MTs and chromosomes
Work by cAMP dependent phosphorylation/ dephosphorylation of ATP
dephosphorylation activates kinesin
75
Picture showing kinesin 'walking'.
76
Describe cytoplasmic dyneins.
Cytoplasmic dyneins are very large proteins with 9 to 10 subunits
Move rapidly along the MT towards the - end at up to 14 um/sec
Moves vesicles/organelles back from the plasma membrane
Work by cAMP dependent phosphorylation/ dephosphorylation of ATP
dephosphorylation inactivates dynein
77
Picture of kinesin and cytoplasmic dynein.
78
What is the other non-cytoskeletal role of microtubules?
Microtubules make up cilia and flagella and help cells move by the use of a special dynein motor protein.
79
Describe cilia and flagella.
Cilia are usually very numerous, short and beat in a coordinated fashion. Movement is a power-stroke movement.
Flagella (eucaryotic) are longer and usually 1-2 per cell. Move in multiple waves.
NOTE: Procaryotic MTs don't show dynamic instability in cilia and flagella
80
Describe the structure of cilia and flagella.
The core (axoneme) has a 9 + 2 arrangement of 9 outer doublet rings and 2 inner singlet rings.
The outer doublet ring has 1 complete (A) MT protofilament and 1 incomplete (B) MT filament made up of 9 protofilaments
Inner singlets are made up of two separate MTs made up of 13 protofilaments
Large number of Microtubule Associated Proteins associated with the axoneme
Dynein side arms on the complete A ring of outer doublet
Protein radial spikes connect central sheath surrounding the inner singlets w/ outer doublets
Nexin bridge connects the doublets to one another
Basal body or the part of the cilia/flagella that continues into the cell, has the same structure as the centriole w/ nine triplet rings. It can regenerate cilia/flagella if they are sheared off at the membrane
A and B tubules of basal body continue up to form the A and B tubules of the doublet in cilia/flagella
81
What is the mechanism that drives ciliary /flagellar movement?
The mechanims exist within the 9 + 2 structure
Movement is dependent upon ATP concentration
Cilary dynein is responsible for movement
Dynein arms on A subtubule have a head region where ATP hydrolysis takes place at the cross bridge that links to the B subtubule
cAMP and Ca2+ both regulate beating frequency (When calcium is high intracellularly, via voltage gates channels, motion slows)
Ca2+ - Calmodulin acts on cilia to reverse direction while the rate of forward movement is regulated by cAMP
82
What are actin microfilaments?
Actin microfilaments are found in all eukaryotic cells and are responsible for most cell motions
Intracellular contractions
Crawling
Skeletal muscle contractions
83
What are the characteristics of actin?
Weight: 42,000
Unpolymerized form: Globular dimer
Bound nucleotide: ATP
Factors needed for polymerization: Ca2+, Mg2+ , NaCL
Abundance: 5% of total protein
Polymer form: 2-stranded helix
Diameter of filament: 7 nm
84
What are the characteristics of tubulin?
Weight: 50,000 for alpha and beta
Unpolymerized form: Globular dimer (1 alpha and 1 beta)
Bound nucleotide: GTP
Factors needed for polymerization: Mg2+ , NaCL
Abudance in cell: 2.5% of protein
Form of polymer: Hollow tube made up of 13 protofilaments
Diameter of filament: 25 nm
85
What are all microfilaments composed of and what do they do?
All microfilaments are composed of the monomer G-actin
G-actin subunits come together to form an F-actin microfilament in an ATP-dependent manner
Microfilament is a flexible, 2 stranded wound helix
Microfilaments have a + and - end
Usually thinner, shorter and more flexible than MTs
They are usually found in groups/bundles in the cell and increase the cells strength and has a major role in muscle contraction and cell movement
86
Describe the process of microfilament polymerization.
G-actin can be added at the + end or - end of the microfilament
Rate of growth is faster at the + end
Depolymerizes at both ends
Rate of depolymerization is fastest at the - end
GATP-Actin rate of addition at the + end ie enhanced when F-actin still has ATP bound to individual G-actin subunits
ATP cap forms at plus end of rapidly elongating microfilament
87
True or false; Polymerization and depolymerization can occur at both ends of the F-actin microfilament?
True; While ATP G-actin is being added at the + end (ATP cap), the minus end has hydrolyzed ATP to ADP and this destabizes microfilament
Thus, microfilament is undergoing depolymerization at the - end while undergoing polymerization at the + end
Individual G-actin molecules move down the length of the filament in a process called 'treadmilling'
Add one to + end and subtract one from - end
G-actin and F-actin ar are at equilibrium
Changes in local conditions can push equlibrium in either direction thus dynamic instability is the rule for microfilaments as well as microtubules
88
What is the purpose of actin binding proteins (ABPs)?
Actin can form filaments *in vitro* but can do no wok or interact with one another without APBs
In vivo actin filaments organize into a variety of patterns/shapes because of their interactions with APBs
89
What are the classes of APBs?
1) Monomer modyfying proteins
a) Thymosin Sequesters G-actin and prevents elongation and stimulates depolymerization
b) Profilin binds the actin binding site on G-actin and promotes the exchange of ADP for ATP thus activating G-actin. It may aid in polymerization.
2) Nucleating proteins
a) ARP 2/3 complex: Binds - end of microfilament and allows rapid growth at + end. Also promotes growth of web-like network of F-actin since it can attach to the sides of another actin filament.
b) Formin associates with the + end and promotes polymerization of straight, unbranched filaments that may link to other microfilaments in parallel bindles
3) Severing protiens break microfilament into large pieces to expose ADP-actin on + end to stimulate rapid dpolymerization
4) Filamin is a Cross-linking or gel proteins link microfilaments at angels to create a loose, gel-like network
90
What are the classes of APB's? (CONT.)
5) Fimbrin/villin, and alpha actinin are bundling proteins that hold actin in parallel bundles
6) Capping/end blocking proteins
a) CapZ binds the + end and prevents further growth and depolymerization
b) Tropomodulin binds the - end in muscle cells
7) Tropomyosin binds to the sides of microfilaments to stabilize. Wors with troponin in skeletal muscle to regulate actin interactions with myosin
8) Cofilin binds to the sides of actin and interacts with ADP-actin and acts to destabilize microfilament
91
What is the motor protein that is associated with actin?
Myosin
Walks along actin filament and is involved in contractile movements
92
What are actin membrane binding proteins?
Actin membrane binding proteins link actin filaments to the plasma membrane and allow the membrane to move with the movement of actin
Involved in phagocytosis and ameboid movements
Includes spectrins and ERM family of proteins
93
Where are actin filaments most concentrated?
Actin filaments are found throughout the cell but are most concentrated in the region just below the cell membrane called the cell cortex
They crosslink and provide strength and support; help give cells their shape
Crawling cells (fibroblasts, amobea, neutrophils) reorganize their cell cortex to move
94
What are the names of the structures located at the leading edges of moving cells?
Lamellipodia - Thin sheet-like structure w/ dense network of f-actin in a single orientation w/ the + end at the plasma membrane
Filopodia - Exploratory fibers, long and thin w/ paralel bundles of 10-20 F-actin microfilaments
Cell pushes out a leading edge. This edge adheres to the surface and forms an anchor. Rest of cell uses these anchoring points to pull itself forward.
95
What are the steps in cell movement?
Nucleating protein promotes actin polymerization.
Integrins adhere to surface when a favorable environment has been found. This anchors cell
Actin filament severing proteins break some microfilaments and this changes the cytoplasm from a thick, gel to more aqueous in nature. This allos the cytoplasm to more easily flow towards its leading edge.
Motor proteins (myosins) help to slide microfilaments in appropriate direction
96
How does cell retract trailing edge?
Internal contration hydrolyzes ATP
97
What do contractile actin microfilaments do?
They form a belt around dividing cells and pinch two daughter cells apart during cell division.
