The cytoskeleton Flashcards
What is the skeleton comprised of ?
what are each of these components size ?
What are the components used for?
Actin microfilaments
- 7-9 nm
- dynamic, flexible, helps provide shape to plasma membrane
Microtubules
- 25 nm
- rigid, dynamic, helps form polymer
Intermediate filaments
- 10 nm
- heterogenous, tissue specific expression and strength, less dynamic
All have motor proteins associated with them
Whats the most abundant protein in the cell?
Actin
in muscle it comprises 10% by weight total protein
non-muscle cells 1-5% of total protein
How many actin genes do humans have?
Humans have 6 actin genes
How many different isoforms of actin exist in vertebrates?
What are these isoforms?
Where are the isoforms expressed?
There are 3 different isoforms of actin in vertebrates
- Alpha actin: only expressed in muscle cells
- beta actin: expressed in non-muscle cells
- gamma actin: expressed in non-muscle cells
Where is actin found in the following:
- epithelial cell
- migrating cell
- other specific functions of actin
Tell me about the structure of actin?
- polarised filament
- plus end and a negative end
- plus end is called the barbed end
- minus end is called the pointed end
How often do units repeat in actin ?
every 37 nm
What is added to both ends of the filament to extend the molecule?
G-actin
What are actin filaments also known as?
what are they made up of?
Actin filaments (F-actin)
Actin filaments are made up of individual globular actin monomers (G-actin)
What binds to G-actin in the presence of the cofactor Mg2+?
What effect does this cause?
ATP or ADP binds to G-actin within cleft
Hydrolyses ADP in filament and this is released at the minus end
What is added faster to the growing, plus end of actin filament?
ATP-G-actin
Tell me the steps to actin filament formation?
- Slow nucleus formation (initial log phase, rate-limiting)
- Rapid filament elongation (dependent on G-actin concentration)
- Preferred end of actin polymerisation at +ve end
- treadmilling of G-actin at steady state
- Rate modified by actin-binding proteins e.g. capping proteins
what occurs at the +ve and -ve ends of the actin ?
What does this result in?
ATP-actin adds faster at the +ve end
ADP-actin dissembles from the -ve end
This results in treadmilling of actin
Tell me about actin treadmilling ?
- When the filament grows- elongation is faster than hydrolysis at the plus end and therefore have more ATP bound
- However, hydrolysis is faster than elongation at the minus end, so terminal subunits are more predominantly in ADP form
What are actin dynamics influenced by?
capping proteins
Tell me about capping proteins at the +ve and -ve end of the actin filament and an example for each?
Capping proteins at the +ve end limits actin dynamics to the -ve end, thus influencing actin assembly
example: CapZ
Capping proteins at the -ve end helps stabilise actin filaments by blocking disassembly
example: Tropomodulin
Tell me the components of the microvilli (brush border)?
Where are epithelial cell microvilli found?
in the small intestine and the kidney
Whats Fimbrin and its role?
Its an actin cross-linking protein that cross-links filaments into bundles within the microvilli
Whats does myosin 1 do?
links bundles of actin to cell membrane
Whats Spectrin and its role?
A cytoskeleton protein that lines the intracellular side of the plasma membrane in eukaryotic cells; an actin binding protein
cross links bundles of microvilli rootlet
Where does the microvilli-actin complex sit?
On intermediate filament platform
Why does the RBC have a bioconcave shape?
The bioconcave shape is a result of interactions between plasma membrane proteins with underlying cytoskeleton
What is the cytoskeletal network rich in?
What does this help with?
short actin filaments (14 subunits in length)
Helping to support the structure and flexibility of the plasma membrane
Actin-based motor proteins are powered by what?
ATP hydrolysis
What do myosins contain?
- motor domain (head)
- lever-arm neck domain (facilitates movement)
- a cargo-building tail domain
What domain binds to the actin filament?
The motor domain head
What is the role of myosins and what is this linked to?
Myosins convert ATP hydrolysis to mechanical work
Linked to conformational change within head/neck region
Tell me how myosin heads move and in which direction?
Most myosins walk towards the plus end
BUT only one myosin, myosin 6, walks towards the negative end
Tell me the class of myosins
The size of the myosins
The function of the myosins
Tell me about Actin-myosin interaction?
- Myosin head binds ATP – releasing it from actin
- ATP hydrolysis pivots myosin head
- Myosin head then binds actin
- Pi released leads to power stroke - moves filament
- ADP released
- Myosin head binds ATP and cycle continues
- myosins are present in muscle and non-muscle cells
Tell me about skeletal muscle contraction?
How are actin filaments in epithelial cells attached to the plasma membrane ?
via cytosolic protein attachment to the transmembrane protein cadherin
What do actin filaments form in cell-cell junctions ?
They form contractile bundles around the cell (adherens belt)
What do adherens belt help to regulate?
epithelial cell structure and apical/basal polarity
What happens during cell division (mitosis)?
2 daughter cells need to seperate from each other- the final step is called cytokinesis
Tell me what happens in cytokinesis ?
- The contractile ring (cleavage furrow) made up of actin filament and myosin II filaments helps drive this process of cell separation
- comprises parallel arrays of linear, unbranched actin filaments
- These overlapping filaments of actin and myosin contract to generate forces that divides cytoplasm into 2
What are cell-substrate attachment sites called?
Focal adhesion
What are focal adhesions linked to and what does it allow?
These focal adhesions are linked to the actin cytoskeleton and allow the cell to pull itself forwards and migrate along a substrate
What are the different orientations of actin filaments within migrating cells?
- At leading edge- branched actin filaments push membrane forward
- Within cell body- contractile actin filament bundles attached to focal adhesions provide cell-substrate attachment
What are the basic steps to cell locomotion?
What are microtubules made of?
- Tubulin dimer- comprised of alpha and beta monomers
- -ve end is alpha tubulin
- +ve end is beta tubulin
In microtubules, what is attached to alpha tubulin and beta tubulin and whether it is exchangable?
GTP bound to alpha tubulin is non-exchangable
GDP bound to beta tubulin is exchangable with free GTP
What is the role of a centrosome?
Its a microtubule organising centre (MTOC) that nucleates the array of microtubules in non-mitotic animal cells
What are all microtubules nucleated from?
MTOC
What is the centrosome comprised of?
2 centrioles at right angles to each other, surrounded by pericentriolar material
A mother centriole and daughter centriole
Tell me about the arrangment of the tubulin in microtubules?
There is a 13, 10 and 10 triple arrangement
What do microtubules play a major role in?
transport and this is very fast
structure in axons along with other filaments such as intermediate filaments
The following shows microtubule organisation…
In cilia and flagells, the microtubules come out from the basal body
Tell me about the dynamic instability of microtubule turnover?
- Microtubule with GTP-β-tubulin on end of each protofilament is favoured to grow
- Microtubule with GDP-β-tubulin at the ends of protofilaments forms a curved structure, resulting in rapid disassembly as its very unstable
- Growing and shrinking phases, referred to as rescue and catastrophe (dynamic instability)
- If GTP hydrolysis occurs more rapidly than new subunit addition, then cap is lost, and the microtubule shrinks (catastrophe)
- But if enough free GTP bound β-tubulin is present, a new cap can form leading to rescue and regrowth
What can be used to transport cargo (proteins and vesicles) along microtubules?
- microtubule motor proteins
- kinesins
- dyneins
Whats required to transport cargo along microtubules?
ATP
What are kinesins and where do they transport cargo?
A protein belonging to a class of motor protiens found in eukaryotic cells from -ve end to +ve end of microtubules
What are Dyneins and where do they transport cargo?
Dyneins (a family of cytoskeletal motor proteins that move along microtubules in cells) move from (+) end to (-) end of microtubules.
Only 1 dynein in humans
Label whats attached to this stationary microtubule
When microtubules grow, where is the -ve end and where is the +ve end?
the -ve end is near the centrosome
in microtubules, what are the cilia and flagella assembled from?
The basal body which acts as a microtubule organising centre
Whats the cental bundle of microtubules called?
What does it consist of?
The central bundle of microtubules is called an Axoneme
An axoneme consists of 9 + 2 arrangement of nine doublet microtubules surrounding a central pair of singlet microtubules
Do all microtubules have the same polarity ?
tell me about its orientation ?
All microtubules have the same polarity
+ve ends orientated towards the distal tip
Whats the structure of an axoneme held together by?
protein cross-links
Whats the major motor protein?
What does it facilitate ?
Axonemal dynein
This facilitates cilia and flagella bending
Whats the role of Nexin?
Cross-links tubule bundles together
What does dynein interact with?
What does this allow?
It interacts with beta-tubule of neighbouring doublet microtubule
As fixed, via nexin, it creates a force which causes the movement of the microtubule
During mitosis, where do microtubules have their -ve ends?
At spindle pole
What are the 3 types of microtubules?
Where are they positioned/ help with?
1. Astral: project towards the cell cortex (help positions centrosome correctly)
2. Kinetochore: Attached to chromosomes (help with alignment of chromosomes for correct separation, regulatory role)
3. Polar: Project toward cell centre and +ve ends overlap (help push outwards to help separate cells and help chromosomes move to opposite ends correctly)
Are intermediate filaments heterogenous or homogenous?
Heterogenous
What forms a coiled-coil structure in intermediate filaments?
Elongated proteins with conserved central a helical rod domain containing heptad repeat motifs that form a coiled-coil structure
Whats the primary building block of the intermediate filaments?
Dimer held together through rod domains that associate as a coiled coil
How are tetramers intermediate filaments form?
The dimers then associate, offset from each other to form tetramers (the 2 dimers run in opposite directions)
How is a protofibril formed in intermediate filaments?
Dimers –> tetramers –> protofilaments –> protofibril
- Tetramers are then assembled end-to-end and interlocked into long protofilaments
- 4 protofilaments associate into a protofibril
How long is a usual intermediate filament?
10 nm
Tell me some key features of IF?
- less dynamic
- non-polarised
- no motors attached
- subunits dont bind to a nucleotide
What do IF provide to cells?
strength and organisation to cells and tissues
What does keratin extend from ?
The nucleus to plasma membrane in cytoplasm
What does the lamin in nucleus underlie?
the nuclear membrane
In epithelial cells, the keratin intermediate filament network links what?
Adjacent cells to each other and to the extracellular matrix (basal lamina)
Whats a desmosome?
a structure by which two adjacent cells are attached, formed from protein plaques in the cell membranes linked by filaments.
Whats a Hemidesmosome?
Hemidesmosomes are very small stud-like structures found in keratinocytes of the epidermis of skin that attach to the extracellular matrix. They are similar in form to desmosomes when visualized by electron microscopy, however, desmosomes attach to adjacent cells.
Desmosomes in heart cells are mediated by what?
Desmin intermediate filaments
Mutations in the keratin gene causes what?
many human genetic diseases
Expression in mice of a mutated form of keratin, which is non-functional results in what?
What is this caused by?
Expression in mice of a mutated form of keratin, which is non-functional, results in blistering of the skin (epidermolysis bullosa simplex)
This is caused by defective hemidesmosome formation – causing a disruption in epithelial cell attachment to the extracellular matrix (basal lamina)
Tell me the major classes of intermediate filaments in mammals
The proteins located there
The distribution of filaments
The proposed function of the filament
