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Molecular Biology of the Cell Chapter 16 Part 1

Assembly and structure of cytoskeletal filaments including actin, microtubules and intermediate filaments
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1. Microtubules
2. Actin
3. Intermediate filaments
What are the 3 main types of cytoskeletal elements in eukaryotic cells?
Microtubule filaments
What cytoskeletal elements are made from alpha and beta tubulin subunits?
Actin filaments
What cytoskeletal elements are made up of actin monomers?
Microtubules
What cytokeletal elements emanate from a single organizing center within interphase cells?
1. Positioning organelles within the cell
2. Transport of vesicles for intracellular trafficking
3. Partioning of chromosome during division
What are the functions of microtubules?
Actin
What cytoskeletal elements determines cell shape and ae involved in whole cell movement?
Intermediate filaments
What cytoskeletal element is a large and heterogenous family of proteins and provides mechanical strength?
Shape
The cytoskeleton provides the cell its what?
Environmental changes/stress
The cytoskeleton allows for cellular remodeling during periods of what?
Cell motility
The cytoskeleton is used for what? (It includes swimming and crawling)
Chromosome segregation
What is the cytoskeleton used for during mitosis?
Intracellular trafficking
What kind of trafficking does the cytoskeleton drive?
Mechanical support
The cytoskeleton provides what kind of support to the plasma membrane?
Muscle contraction and axon/dendrite extension in neurons
The cytoskeleton provides the machinery used for what?
Generates and/or maintains organelle structure
The cytoskeleton does what for organelle structure?
Dynamic and adaptable
Are cytoskeletal elements static and unchangeable or dynamic and adaptable?
The needs of the cell
Changes to the cytoskeleton depend on what?
Flux
Individual subunits of the cytoskeleton in a constant state of what?
Just beneath the plasma membrane
Where are actin filaments found?
Strength and shape
Actin filaments provide what to a cell?
Lamellipodia or filopodia which are essential for cell motility
During interphase, the actin filaments beneath the plasma membrane form dynamic structures called what?
Cell motility
Lamellipodia or filopodia are essential for what?
Crawling cells
Actin has a polarized organization in what kind of cells?
Microtubules
What exists as long filaments that project from the microtuble organizing center which is located adjacent to the nucleus?
Division
The structural organization of microtubule and actin filaments undergo dramatic changes when the cell undergoes what?
The mitotic spindle
After chromosomes duplication the single microtubule array will form a bipolar structure called what?
Duplicated chromosomes
The mitotic spindle is essential for the segregation of what?
Disassemble
Actin structures formed for motility will do what to prevent cell movement?
The contractile ring
Actin will reform, with the help of associated motor proteins, to form a 'belt' around the middle of cell called the what?
Microtubule-dependent chromosome segregation
The contractile ring will 'pinch' the cell in half after what?
The cytoskeletal filaments
What will reassemble to form the structures first described in interphase?
Microvilli
Bundles of actin filaments form what to increase the cell surface area thereby increasing nutrient uptake?
Stable structures
Are actin filaments stable structures or instable structures?
Constant
Do microvilli have a constant location, length and diameter or variable location, length and diameter?
The contents of the intestinal lumen from leaking into the body
Actin filaments also form below the microvilli; these cell-cell junctions prevent what?
Polarity
Actin and microtubule filaments have what? It enables cells to
differentiate between top and bottom and left from right.
Vertically
In polarized epithelial cells, microtubules run vertically (apical to the basal) or horizontally which is important for the trafficking of newly synthesized material?
Desmosomes
Intermediate filaments, attached to what, link adjacent epithelial cells together to form a sturdy sheet of cells
A sturdy sheet of cells
Intermediate filaments, attached to desmosomes, link adjacent epithelial cells together to form what?
Hemidesosomes
Intermediate filaments, attached via what, link epithelial cells to the underlying extracellular matrix on the basal lateral side of the cell?
The underlying extracellular matrix on the basal lateral side of the cell
Intermediate filaments, attached via hemidesosomes, link epithelial cells to what?
1. They provide dynamic structures for cellular reorganization,
2. They are stable structures for the maintanance of cellular structure
3. They establish polarity in a cell
Name three functions of cytoskeletal elements
α- and β-tubulin (γ tubulin also exists)
Microtubules are formed from two tubulin subunits (dimers); What are they?
A heterodimer
α- and β-tubulin subunits are tightly bound to each other as a what?
GTP
Each tubulin monomer (α- or β-tubulin) binds to one molecule of what?
Hydrolysis; (α-tubulin always has GTP bound)
Organization of the tubulin heterodimer is such that the GTP bound to the alpha subunit is protected from what?
Hydrolysis; (β-tubulin can have either GTP or GDP bound)
GTP bound to the beta subunit can undergo what? (This effects MT dynamics)
"Dynamic instability"
α-tubulin always has GTP bound and β-tubulin can have either GTP or GDP bound. This difference is important for the property known as what?
Microtubules
What are hollow tubes formed by 13 protofilaments containing alternating α- and β-tubulin heterdimers?
Lateral interactions
Microtubules are stiff and difficult to bend due to what kind of interactions?; This makes MT the straightest structural element in cells (up to millimeters in length!)
A protofilament
Tubulin subunit assemble into what in the same direction giving the microtubule a distinct polarity.
At the other end
Tubulin subunit assemble into a protofilament in the same direction giving the microtubule a distinct polarity; α-tubulin subunits exposed at one end and β-tubulin subunits exposed where?
The minus end
Microtubule end with the free alpha subunit is called the what end?
The plus end
Microtubule end with the free beta subunit is the what end?
Nucleus
Microtubules nucleate from the microtubule organizing center (MTOC) located near the what?
Plus-end distal
Microtubules nucleate from the microtubule organizing center located near the nucleus. This sets up polarity such that all microtubles are "what"?, that is the plus ends of the microtubule emanate outwards from the center of the cell
The microtubule organizing center
All minus ends are embedded within what?
All eucaryotic cells
What kind of cells is tubulin found?
Conserved
Tubulin is highly what?
75%
Yeast and human tubulin share what percentage of amino acid sequence identity?
6 isoforms (each of alpha and beta tubulin)
There are multiple isoforms of tubulin: how many isoforms are in humans?
Functions
Each isoform has a distinct location within the cell and performs subtly different what?
Actin monomer subunits
Actin filaments are composed of what kind of subunits?
ATP (or ADP)
Actin subunits bind what?
A head-to-tail fashion
Actin subunits assemble in what fashion to generate a filament with polarity?
To the "minus end" of the actin filament
ATP binds in a deep cleft in the center of an actin monomer which is open to what end of the actin filament?
The 'pointed end'
The "minus end" of the actin filament is what?
The 'barbed end'
The "plus end" of the actin filament is what?
Myosin heads
Pointed versus barbed nomenclature refers to the arrowhead
appearance of what heads when bound to actin filaments?
Righthanded helix
Actin filaments are a single helix of monomers that forms what looks like two parallel protofilaments that twist around each other in what kind of helix?
They are typically crosslinked by accessory proteins making actin structures stronger than an individual filament
Why are actin filaments flexible and easily bent, but strong?
Plasma membrane (cell cortex)
Actin filaments are nucleated at the what?
Eucaryotic cells
What kind of cells do you find actin filaments?
(Extraordinarily well) conserved
Actin is what?
~90%
The amino acids sequence of actin from different organisms are what percentage identical?
Alpha, beta, and gamma
What are the three different isoforms of actin in vertebrates?
1. They differ only slightly in their amino acid sequence
2. The alpha isoform is expressed only in muscle cells
3. The beta and gamma isoforms are found in nonmuscle cells
What are the differences between the three isoforms of actin?
Intermediate filaments
What kind of filaments are only found in some metazoans including vertebrates, nematodes, and mollusks but not in all cell types of these organisms
Cells subjected to mechanical stress
Intermediate filaments are prominent in the cytoplasm of what kind of cells?
A rigid exoskeleton
Intermediate filaments are not found in animals that have what kind of exoskeleton?
Nuclear lamins
Cytoplasmic intermediate filaments are closely related to what?
Nuclear lamins
What form the meshwork beneath the inner membrane of the nuclear envelope and are the site of chromosome and nuclear pore complex attachment?
Parallel dimers
The individual polypeptides of intermediate filaments are elongated molecules with a central α-helical domain that assemble to form what?
Tetramers
The individual polypeptides of intermediate filaments are elongated molecules with a central α-helical domain that assemble to form parallel dimers. Dimers associate in a staggered antiparallel manner to form what?
Polarity
Since tetramers are composed of antiparallel dimers, intermediate filaments do not have what?
Eight tetramers
How many tetramers assemble in a laterally manner to form a rope like filament?
Break
Intermediate filaments have a rope-like characteristic making them easily bent but extremely difficult to what?
Vimentin
For what, phosphorylation may have a role in triggering their disassembly similar to what is seen for nuclear lamins during mitosis?
Actin or microtubules
The intermediate filament family is more diverse than what?
Keratin
What is the most diverse family of intermediate filaments?
50
How many different keratins are there?
~20
How many kinds of keratins are found in human epithelial cells?
~10
How many kinds of keratins are found in hair and nails?
Anchoring the intermediate filaments at sites of cell-cell contact sites
How do keratin filaments impart mechanical strength to epithelial tissues?
Human genetic disease
Keratin gene mutations have been linked to what?
Skin blistering
Defective keratin molecules can result in what in response to minor mechanical stress?
Mechanical trauma
Blisters are due to rupturing epithelial cells as a consequence of what?
along the axons of vertebrate neurons
Where are neurofilaments found in high concentrations?
NF-L, NF-M and NF-H
What are three types of neurofilaments?
NF-L plus either NF-M or NF-H
NF-L, NF-M and NF-H can coassemble in vivo to form what?
Axon
During axonal growth, new neurofilament subunits are incorporated along the what?
At the ends of neurofilament polymers and along the length of a filament
Filament addition occurs where?
Lateral addition
What kind of addition increases the diameter of the axon?
Neurofilament gene expression
Axonal diameter is controlled by what, which influences how fast an electrical signal travels down the axon?
ALS or Lou Gegrig's desease
Mutations in neurofilament genes that impact filament assembly have been linked to what disease?
Axonal degeneration
Abnormal assembly of neurofilaments in motor neurons and in the axon interferes with normal axonal transport leading to what?
The assembly and disassembly of actin and tubulin
Eukaryotic cell survival depends on the balance between what two things?
By plants, fungi or sponges
Toxins that specifically target actin and microtubule filaments are produced in self-defense by what (they can not run away from predators)?
Either filament formation or its disassembly
The toxins produced bind tightly to either the filament form or the free subunit forms to drive what opposing actions?
Latrunculin and swinholide
What two toxins (from different marine sponges) depolymerize actin filaments by binding to actin subunits to prevent their assembly or severing actin filaments?
Cytochalasins
What toxin (fungal metabolites) binds to the plus end of actin filaments to prevent actin polymerization?
Phalloidin
What toxin ("death cap" fungus) stabilizes actin filaments by binding at the interface between actin subunits?
1. Cholchicine (autumn crocus)
2. Vinblastine (Madagascar periwinkle plant)
3. Nocodazole
What three toxins all bind to tubulin subunits to prevent microtubule formation?
Taxol
What toxin from the yew tree binds to and stabilizes microtubules?
Growth and shrinkage
Microtubules switch between what two phases every few minutes??
Dynamic instability
The ends of microtubules exhibit what?
Dynamic instability
What is the rapid interconversion between a growing and shrinking state, in the presence of a uniform concentration of free subunits
'Catastrophe'
Changing from growth to shrinkage is what?
'Rescue'
Changing from shrinkage to growth is what?
Microtubules
Actin filaments undergo length flutuations, but they are on such a small scale that in eukaryotes, dynamic instability refers mainly to what?
Plus end
The key factor influencing dynamic instability is the type of nucleotide present at what end of the filament?
GDP
β-tubuin subunits can hydrolyze its bound GTP to what?
GTP bound form
For free subunit dimers this rate of hydrolysis is so slow that the free subunits are primarily in what form?
GDP
After incorporation into a filament, the β-tubulin subunit hydrolyzes its GTP to what?
Polymerization
If what occurs faster than GTP hydrolysis, a microtubule filament will have GTP "cap" at its plus end?
Microtubule depolymerization
If polymerization slows down, the GTP cap will undergo hydrolysis to GDP causing what?
GTP cap
Depolymerization can be rescued if the filament regains its what?
Lateral bonds
GTP bound tubulin subunits form a linear microtubule protofilament that is stabilized by what kind of bonds?
A curved conformation
With GTP hydrolysis, the protofilament adopts what kind of conformation?
The curling at the ends of the shrinking microtubules
With GTP hydrolysis the protofilament adopts a curved conformation, this leads to the progressive disruption of the microtuble protofilament; evidenced by what?
The free subunit concentration and the rate constant
The rate of subunit addition to the ends of filaments is the product of what?
The structural differences between the two ends
The rate constant (kon) is much faster for the plus end of a filament than the minus end due to what?
At an intermediate concentration
At what concentration of free subunits will the rate of subunit addition be faster than nucleotide hydrolysis at the plus end, but slower than nucleotide hydrolysis at the minus end?
The subunit loss at this end
The minus end will therefore have a GDP/ADP bound nucleotide which promotes what?
Filament stability and subunit addition
The plus end will be in its GTP/ATP bound state which promotes what?
At the minus end
Thus the filament will add subunits at the plus end while simultaneously losing subunits where?
Treadmilling
The property of adding subunits at the plus end while simultaneously losing subunits at the minus send is referred to as what?
A filament whose net total length does not change
(Treadmilling) At a particular subunit concentration, filament growth at the plus end exactly balances filament shrinkage at the minus end generating what kind of filament?
Single actin filaments in vitro and may occur in live cells for microtubules
Treadmilling has been seen for what kind of filaments in vitro? Where has treadmilling been seen in live cells?
FtsZ
A tubulin homolog called what was identified that could assemble into ring-like structure at the site of septum formation during cell division?
Alpha or beta tubulin
The 3 dimensional structure of FtsZ is similar to what?
A conformation change in the filament structure
FtsZ polymerization causes GTP hydrolysis leading to what?
~30 seconds
Ftsz filaments are highly dynamic; What is its half life?
The FtsZ ring
What ring forms at the middle of a cell and becomes smaller as the cell pinches into two; similar to the actin based contractile ring in eukaryotes?
Septum
The FtsZ ring may act as a docking site for cell division enzymes required for building the what?
Rod-shaped or spiral shaped cells
Bacterial homologs Mbl and MreB are found in what kind of cells?
Abnormalities in cell shape and defects in DNA segregtion
Disruption of expression causes what kind of abnormalities and/or defects?
Cells grow into irregular twisted tubular shapes and eventually die
What changes in cell morphology occur due to loss of Mbl in Bacillus substilus? What is the eventual end result?
Certain bacterial plasmids (extrachromosomal circular DNA)
The ParM gene is found on what?
A plasmids origin of replication (shown in blue)
In vivo, ParM (shown in green) assembles into filaments that associate with another protein that binds to what?
The duplicated plasmids
After plasmid replication, ParM filaments form between what?
Opposite ends of the cell ensuring that each daughter cell gets a copy
ParM polymerization pushes the replicated plasmids where? For what purpose?
Caulobacter crescentin
Homologs to intermediate filament proteins have been identified in what bacterial species?
Sickle-shaped
What shape is Caulobacter crescentin?
Crescentin
What forms a fiber that runs down the inner side of the curving bacterial cell wall to influence cell shape?
Crescent shaped appearance
In the absence of crescentin, cells are viable but lose their what shaped appearance?