TBL 3 Cytoskeleton Flashcards
3 types of filaments form the cytoskeleton of the cell.
Actin filaments; intermediate filaments and microtubules
Microtubules are made up of _______.
tubulin
Microfilaments are made up of _______, ______ and ________.
actin, troponin and tropomyosin
The synthesis of filaments occurs via the _________ of subunits.
polymerisation
The _________ provides and defines the shape of the cell, which confers cell motility.
cytoskeleton
Free monomer molecule must be in ________ with the polymer (formed filaments).
equilibrium
Intermediate filaments are stable and durable filaments with a diameter of __ to __ nm.
8 to 12 nm
intermediate in size compared to thin filaments and microtubules
________ filaments form the most insoluble part of the cell.
Intermediate filaments
Intermediate filaments are very developed in cells which need to withstand mechanical stress.
E.g. epithelia
There are __ main types of intermediate filaments.
5
Types I and II intermediate filaments (e.g. ______) are found in _______ cells.
These confer resilience and mechanical strength by making cytoplasmic networks throughout the cell.
Keratin
Found in epithelial cells
Type ___ intermediate filaments include Vimentin, desmin, glial fibrillary acidic protein (GFAP) and peripherin.
These may form homo or heteropolymeric proteins.
Type III
________ (intermediate filament) is found in fibroblasts, endothelial cells and leukocytes.
It functions to support cellular membranes, keep organelles in a fixed location and transmit membrane receptor signals to the nucleus.
Vimentin (Type III intermediate filament)
________ (intermediate filament) is found as part of the desmosomes linking cells like cardiac and skeletal muscle fibres together.
Desmin (Type III intermediate filament)
[recall: desmosomes are intracellular junctions that provide strong adhesion between cells]
_________ (intermediate filament) is found in astrocytes and other glial cells.
It functions to maintain astrocyte mechanical strength in CNS and is important in repair and formation of glial scars after CNS injury.
Glial fibrillary acidic protein (GFAP) [Type III intermediate filament]
_______ (intermediate filament) is found in peripheral nerve fibres.
Peripherin (Type III intermediate filament)
Type IV intermediate filaments include __________ which are mainly found in axonal cells, internexins, filensin and phakinin which are mainly found in the ________.
neurofilaments H, M and L (mainly found in axonal cells)
internexins, filensins and phakinins are mainly found in the lens fibres of the eye.
Type V intermediate filaments include ______, which possess nuclear signal sequences to form filamentous support inside the inner nuclear membrane. It is vital for the reformation of the nuclear envelope after cell division, and therefore helps to protect the DNA.
Lamins
recall nuclear lamina
In intermediate filaments, different types of cells have (different/the same) monomer(s).
different monomer proteins
(different types of intermediate filaments
e.g. keratin in epithelial cells; vimentin in leukocytes, etc.)
A typical intermediate filament subunit has a -COOH end and -NH end (protein), with various ________ regions separated by spacer regions.
a-helical regions separated by spacer regions
Intermediate filaments begin forming via _______ assembly of monomers into ______.
The a-helical regions from two subunits wind together to form a parallel coiled-coil.
Parallel assembly of monomers into dimers
one end has two amino ends, the other has two carboxyl ends
Formation of intermediate filaments:
After formation of parallel dimer, 2 of these dimers bind to each other side-by-side to form a staggered (anti-parallel/parallel) tetramer.
anti-parallel tetramer
Why do intermediate filament polymers not have any polarity?
Parallel dimers –> Anti-parallel tetramers (Dimers are oriented in opposite ways, so each end of the tetramer has both COOH and NH ends –> no polarity)
4 tetramers are then elongated from end to end to form protofilaments, which also have no polarity due to the side-by-side assembly.
Formation of intermediate filament:
Parallel dimer –> Anti-parallel ______ –> _________ (4 tetramers)
Parallel dimer —> Anti-parallel tetramers —> Protofilaments (4 tetramers)
There are two types of tubulin molecules () which make up __________.
a-tubulin and B-tubulin (make up microtubules)
Microtubules are hollow cylinders with circumference made of a ring of ___ tubulin monomers.
13 tubulin monomers
__-tubulin is found only in centrosomes, acting as nucleating sites for microtubule assembly.
y-tubulin (centrosomes)
Microtubule polymerisation occurs in two phases:
_______ and _______.
nucleation and elongation
_________: when an a and B tubulin molecule join to form a heterodimer (aB)
Nucleation
__-tubulin is at the plus end where polymerisation is favoured; whereas __-tubulin is at the minus end.
B-tubulin: plus end;
a-tubulin: minus end
Tubulin subunits are added to (GTP/GDP)-capped microtubules more efficiently.
GTP-capped microtubules
After polymerisation, the GTP bound to tubulin is slowly hydrolysed to GDP.
-
The switch between filament growth (polymerisation) and depolymerisation is __________.
dynamic instability
In interphase cells, _____ ends of the microtubules become capped by the centrosome.
minus ends
During ______ of mitosis, the tubulin subunits are added to the ‘plus’ end of the kinetochore microtubules and removed from the ‘minus’ end at the spindle pole. => microtubules maintain a constant length.
Metaphase
_________ refers to the growth of microtubule at one end, with depolymerisation at the other end.
Treadmilling
The polymerisation and depolymerisation of microtubules is controlled by ___.
They bind to microtubules to stabilise, cross-link and attach them to other cellular components.
MAPs (microtubule-associated proteins)
______ are motor proteins that move towards the periphery i.e. anterograde.
Kinesins
_______ are motor proteins that move towards the centre i.e. retrograde.
Dyenins/Ncd
____ protein results in plaques characteristic of Alzheimer’s disease.
Tau protein
Both cilia and flagella are composed of ________, which are thus important in locomotion.
microtubules
Centrosome consists of:
2 centrioles arranged at right angles to each other, from which 9 triplets of microtubules radiate
Cilia is made up of 9 doublets of microtubules and 2 in the centre (forming the ______), with (protein) associated with it, where its movement causes oscillation that travels down the cilium to provide force)
9 doublets and 2 in the centre: axoneme
associated with dyenin proteins
Actin exists in two forms:
G-actin (globular actin monomers) and F-actin (filamentous actin filaments)
The actin filament system is made up of the polymerisation of ______ (monomer).
G-actin (globular)
_____ is involved in cell surface shape changes including those which lead to cell migration.
Actin filaments
Actin filaments are able to form bundles known as ______, which are cables of actin found in fibroblasts and other cells where cell-to-cell adhesion is important.
stress fibres
Polymerisation of actin occurs in 2 steps:
1) _________: A high concentration of ATP-actin monomers would allow for the formation of _____ of ATP-actin.
2) _______: subsequent addition of actin monomers to the trimer
1) Nucleation: Formation of trimers of ATP-actin
2) Elongation
The F-filament has two ends.
Barbed ends (for elongation)
Pointed ends
______ motors require protein rails (usually cytoskeletal filaments) along which they can move, and are predominantly responsible for the motile activity in eukaryotic cells (cytoskeletal motors).
Linear motors
There are three families of linear motors:
1) Kinesin (microtubules)
2) Dyenin (microtubules)
3) Myosin (actin)
Other special molecular motors like helicases and topoisomerases move along DNA or RNA (nucleic acid motors).
_______ motors are complex multi-protein assemblies which require stators (static portion) for their action.
Rotary motors
embedded in the membranes of organelles
Movement of cells driven by molecular motors requires the generation of __________ force.
mechanical force
_________ is the movement of cells along a chemical gradient from a region of low concentration to the region of highest concentration in response to a chemical stimuli.
Chemotaxis
______ moves towards the minus end of the microtubule; whereas ________ moves towards the plus end.
Dynein - towards minus end
Kinesin - towards plus end
Some linear motors rely on __________ for their movement instead of moving along the rails. These are known as polymerisation motors.
filament polymerisation
e.g. actin polymerisation generates force which can be used for propulsion of entire cells with the hydrolysis of ATP
(Type of tissue) is rich in ECM, with a much sparser cellular component.
connective tissue (very few cells but a lot of ECM)
=> ECM carries mechanical load in connective tissues.
The ____________ is a complex network of proteins and carbohydrates secreted by cells to fill intercellular spaces, and comprises both fibrillar and non-fibrillar components.
Extracellular matrix (ECM)
4 components of connective tissues:
- Cellular component
- Collagens
- Glycoproteins
- Proteoglycans
Each collagen molecule comprises of 3 a-chains that form a _______, and can be composed of one or more different types of a-chains.
triple helix
Each a-chain of collagen is made up of a regular, repeating tripeptide sequence of ________.
glycine-X-Y, where X is commonly proline and Y commonly hydroxyproline
Three (left/right-) handed helical a-chains are supercoiled around each other to form a (right/left-) handed triple helical structure known as ________.
left-handed helical a-chains
right-handed triple helical structure
tropocollagen
Collagen:
Due to the tight packing of the a chains, there is very little space within the interior of the helix, so every third position must be occupied by ______ in a repeating fashion.
glycine
Tropocollagen molecules are associated with one another via ________ and ______ to form collagen ______.
Tropocollagen molecules are associated with one another via covalent cross-linking and hydrogen bonding to form collagen fibril.
Multiple collagen fibrils will then aggregate to form collagen _______ which are laid down within the ECM.
collagen fibres
Newly synthesised collagen (known as ________) have non-collagenous globular domains at their N and C terminals, which are removed after secretion from cells in the case of _______ collagens.
Propeptides are cleaved off by _______ in the ECM.
Procollagens
Removed AFTER secretion in the case of fibrillar collagens
Cleaved off by proteinases in the ECM
The addition of hydroxyl groups to lysine and proline residues occurs (before/after) the a-chain synthesis.
This is catalysed by (enzymes), which require ___ and _______ as coenzymes.
What is the function of hydroxylation?
Hydroxylation occurs after the a-chain synthesis.
Catalysed by prolyl and lysyl hydroxylases
Requires Fe2+ and Vitamin C as coenzymes
Function: Formation of interchain hydrogen bonding
Vitamin C deficiency results in under-hydroxylated collagens, causing severe damage to tissue stability in a condition known as ______.
Scurvy
What is the advantage of
(A) Parallel arrangement of tropocollagen molecules
(B) Collagen fibrils that are laid down in multiple directions?
(A) Parallel - greater tensile strength
B) Multiple directions - resist tensile force in all directions (perpendicular
Type ___ collagen is found in the basal lamina and it does not form fibrils.
It therefore has non-cleaved globular domains at the terminals (propeptides) to help regulate formation of aggregates in tissues.
Type IV collagen
__________ are important in conferring tissue elasticity and are interwoven with collagen to limit the extent of stretching.
Elastic fibres
Elastic fibres consist of
1)
2)
1) Protein elastin core
2) Fibrillin-rich microfibrils
Marfan’s syndrome arises from an autosomal (dominant/recessive) mutation in the gene coding for ________.
autosomal dominant
gene coding for fibrillin-1
Elastin is a protein consisting of two types of alternating segments on the polypeptide chain:
1) Hydrophobic region
2) a-helical region
The a-helical region of elastin is rich in ______ and ______ (AA), which are often covalently cross-linked.
alanine and lysine
Elastin is made by linking together many small soluble precursor _______ proteins to make an insoluble complex immediately after synthesis and during export into ECM.
tropoelastin (soluble)
=> elastin (Insoluble)
_______ are flexible, thin mats of ECM underlying epithelial sheets.
Basal lamina (basement membrane)
In the kidney glomerulus, the basement membrane of endothelial cells acts as a __________ which allows only some substances to pass into the Bowman’s capsule from the glomerular blood.
highly selective filter
GBM separates the endothelial cells of the capillaries and the epithelial cells of the urinary space
_________ is a consequence of diabetes, resulting in the thickening of the glomerulus basement membrane.
=> can no longer filter appropriately, resulting in the accumulation of ECM that impinges on capillaries, restricting renal filtration.
Diabetic nephropathy
________ are multi-adhesive proteins found in all basement membranes, and consist of 3 large chains, which form a cross-shaped molecule.
Laminins
____________ is an autosomal recessive disorder resulting from the absence of the __ chain in laminin-2.
Congenital muscular dystrophy - absence of a2 chain in laminin-2
Mutations in ______ proteins is associated with epidermolysis bullosa (EB).
Laminin protein
________ is a glycoprotein found in ECM, and can exist as an insoluble fibrillar matrix or soluble plasma protein.
Fibronectins
The entire fibronectin protein is derived from a SINGLE gene, with _________ generating diversity of fibronectin isoforms.
alternative splicing at mRNA level
_________ (glycoprotein in ECM) plays an important role in wound healing as it promotes blood clotting.
Fibronectin
Structure of fibronectin:
__-shaped, with basic unit being a _____ that is linked by disulphide bridge.
V-shaped; basic unit being a dimer linked by disulphide bridge
The cell-binding domain of fibronectin is where it interacts with ______ via ______.
interacts with actins via integrins
The _______ receptors at the cell surface provide the linkage between the extracellular fibronectin matrix and the intracellular actin cytoskeleton across the plasma membrane.
integrin receptors
(fibronectin bind to integrin receptors embedded on the plasma membrane which are in turn bound to adaptor proteins intracellularly that connect to actin filaments)
Many cell types adhere to fibronectin through integrin recognises the ____ motif (loop region) on the cell-binding site.
RGD motif
_________ are special types of glycoproteins with core proteins to which one or more glycosaminoglycan chains are covalently attached.
Proteoglycans
___________ are long, unbranched sugars consisting of repeating disaccharides. (i.e. the carbohydrate component of proteoglycans)
Glycosaminoglycans (GAGs)
______ is the proteoglycan that is found in basement membranes.
Perlecan
One of the two sugars in the repeating disaccharide chain in GAGs is always an ______ sugar.
amino sugar (i.e. sugar molecule in which OH group has been replaced with amine group)
Many GAGs are either sulphated or carboxylated, and thus are highly (negative/positive).
negative
GAG chains can be classified into 4 main groups according to the type of sugar present:
- Hyaluronan
- Chondroitin sulfate and dermatan sulfate
- Heparan sulfate
- Keratan sulfate
________ (GAG chain) is synthesised at the cell surface instead of the ER or GA.
It is unsulfated, unlike other types of GAGs.
It is a single long chain of repeated disaccharides.
Hyaluronan
Core proteins of proteoglycans have ______ (AA) residues which are linked to the GAG chains via a link __________.
serine residues
linked via link tetrasaccharide
__________ (GAG chain) has the repeating sugars iduronic acid and N-acetylgalactosamine-4-sulfate; whereas __________ (GAG chain) has the repeating sugars glucoronic acid and N-acetylgalactosamine-4-sulfate.
Both have alternating ______ and _______ groups.
Dermatan sulfate - iduronic acid
Chondroitin sulfate - glucuronic acid
Alternating carboxyl and sulfate groups
_______ is a small proteoglycan (with one dermatan sulfate chain) which binds to external surface of collagen fibres and is essential for their formation and organisation.
Decorin
decorate the collagen fibres
In cartilage, the fibrillar component is type __ collagen, which forms relatively thin fibrils.
Type II collagen
_______ cartilage is the most abundant type of cartilage in the body.
Hyaline cartilage
Articular cartilage is particularly rich in (proteoglycan), and functions to cushion the ends of long bones to prevent erosion.
Aggrecan
Aggrecan is a large proteoglycan with ________ and _______ (GAG chains) attached.
1) Keratan sulfate
2) Chondroitin sulfate
Aggrecan forms very large aggregates with _________. A link protein also forms part of the complex.
Hyaluronan (GAG chain)
GAGs of aggrecan are highly sulfated, coupled with the presence of many carboxyl groups, causing the attraction of _______.
attraction of cations like Na+ which are osmotically active
Aggrecan functions to resist compressive forces by giving up and retaining ______ as buffer.
water
under compressive load, water is given up by the cartilage but is regained once the load has been reduced
__________ is characterised by the excessive loss of ECM.
Osteoarthritis
cartilage becomes thin and is eventually lost, resulting in the rubbing of bones against bones
An important early event in osteoarthritis progression is the loss of _______.
aggrecan (which makes up the bulk of cartilage matrix)
With age, the aggrecan can become cleaved by enzymes like __________ and __________ into fragments, which are lost into the ________.
=> loss of cushioning property of cartilage and thus leading to osteoarthritis.
cleaved by aggrecanase and metalloproteinase
lost into the synovial fluid
What are fibrotic disorders?
Excessive production of fibrous connective tissue, resulting in the overproduction of ECM (deposition of collagen)
____________ is the replacement of healthy tissue by collagenous connective tissue, leading to the formation of stiff scar tissue and resulting in organ dysfunction.
Alcoholic liver cirrhosis
________ is collagen fibre deposition that results in the replacement of healthy lung parenchyma with scar tissue.
Lung fibrosis
When cells are attached to each other, they do not move and are _____.
When cellular contacts become perturbed, they can move and are ______.
sessile (do not move)
motile (move)
In cancer cells, genetic alterations (mutations) can lead to __________ of cells forming a benign tumor.
hyperproliferation
Upon (A) hyperproliferation of cells, cancer cells can (B) __________ by losing their cell-cell contacts and polarity. This allows them to become (C) ______ by cleaving ECM proteins and detaching from the monolayer, becoming (D) motile.
Finally, they gain access to the blood and lymph and (E) ________ along these pathways, forming secondary tumors at distant sites.
A: Hyperproliferation B: De-differentiation C: Invasive D: Motile E: Metastasis
Scratch wound assays can be stimulated by growing __________.
Monolayer is scratched and ruptured and upon stimulation, the _____ cells begin to migrate to close up the gap left by the wound.
primary glial cells (found in the brain)
Invasive cells have an upregulation of genes involved in _________ and ________ compared to primary tumours.
upregulation of genes involved in cytoskeleton regulation and motility machinery
Cells are directed by their _______in their movement, and stop due to _________.
directed by their polarity;
stop due to contact inhibition of motility
_________ serve as mechanical linkages to the ECM, and exist at the ends of bundles of filamentous actin (F-actin) in the cell.
These attachments are mediated by _____ which recognise ECM proteins and connect them to a plaque of intracellular proteins that mediate interaction with actin filaments.
Focal adhesions;
mediated by integrins
_______ are finger-like protrusions rich in actin filaments, which help to form focal adhesions.
Filopodia
________ are sheet-like protrusions rich in actin filaments.
Lamellipodia
Mechanism of cell motility:
When the actin cortex of the cell is under tension, the cell uses its ________ to attach to the substratum via ________, anchoring the cell such that it can pull itself forward i.e. ________.
The back of the cell is then pulled forward by the tension of the actin cortex (_______), allowing the cell to inch forward.
When the actin cortex of the cell is under tension, the cell uses its lamellipodium to attach to the substratum via focal adhesions, anchoring the cell such that it can pull itself forward i.e. protrusion.
The back of the cell is then pulled forward by the tension of the actin cortex (retraction), allowing the cell to inch forward.
_________ motility refers to no direction movement and does not require attractant.
vs.
_________ motility that is movement with direction and requires a chemical attractant.
Hapoptatic motility (no direction)
vs.
Chemotatic motility (direction)
_______ can polymerise to form _______ in the direction that the cell wants to move (chemotaxis).
G-actin –> F-actin
i.e. disassembly of filaments and rapid diffusion of subunits; followed by the reassembly of filaments at the new site.
In filopodium, actin forms _______ bundles.
In cell cortex, actin is in a ______ arrangement of filaments, forming a gel-like network.
In stress fibres, ________ arrangement of actin filaments for sliding and shortening.
Filopodium: Tight, parallel bundles (same polarity)
Cell cortex: Relaxed arrangement => gel-like network
Stress fibres: Antiparallel arrangement for sliding and shortening
Cells have specialised proteins - ____ and ____ - to increase the efficiency of actin nucleation process.
(allows G-actin to form a trimer)
Arp2 and Arp3
The actin elongation process is regulated by the (A) _____ complex and (B) _____ complex.
Profilin complex
Thymosin complex
(Profilin competes with thymosin for binding to actin monomers.)
What does the profilin complex do in the regulation of elongation of actin?
It holds onto G-actin monomers and delivers them to the plus-end of F-actin.
What does the thymosin complex do in the regulation of elongation of actin?
Thymosin complex sequesters G-actin and prevents addition to F-actin.
_______ sequesters G-actin, but does not inhibit polymerisation.
ADF/cofilin
Cap Z, gelsolin, fragmin/severin cap the ____ ends of actin filaments, and therefore prevent addition of monomers.
plus ends
Tropomodulin and Arp complex cap the _____ ends of actin.
minus ends
Branch points in the actin filaments are formed by the ________, and binds to the sides of the filaments, allowing polymerisation and elongation of filaments at 70 degrees to each other.
Arp complex
________ (bundle protein) is involved in contractile bundle of actin with loose packing, allowing myosin to enter the bundle.
a-actinin
_______ is involved in parallel bundling of actin with tight packing, preventing myosin from entering, so no contraction can occur.
Fimbrin
Severing actin filaments will result in much (faster/slower) polymerisation and depolymerisation.
Much faster polymerisation as the number of plus ends increased
Severing proteins include ______, ______ and severin/fragmin.
gelsolin and ADF/cofilin
_____ transition: In the cortex, actin filaments are held together by various bundling and branch proteins, sustaining the gel-like matrix. When cells need to make protrusions to move, they clip the filaments to allow the membrane to bend and protrude (sol-state).
Gel-sol transition
required for the cell to change its shape
What are the functions of the Arp2/3 complex in actin?
- Catalyse nucleation of actin (for polymerisation)
- Branching
There are 3 signalling mechanisms that can regulate the dynamics of actin and actin-binding proteins.
- Ion flux changes (in particular ____ ion)
- Control by __________ signalling
- Signalling cascades via Rho family of _________
- Calcium ion flux changes
- Phosphoinositide signalling
- Signalling cascades via Rho family of small Ras-related GTPases
Gelsolin (which functions to _____ and _____ actin), is affected by the concentration of ______ ion.
functions to severe and cap (the plus ends) actin filaments
calcium ion
High intracellular calcium ion concentration results in (increased/decreased) binding of a-actinin to actin.
decreased binding
less cross-linking
Phosphoinositide will ALWAYS lead to actin-filament growth.
True or False?
True
How does phosphoinositide affect ADF/cofilin?
Phosphoinositide binding results in ADF/cofilin inhibition, which stabilises the minus end, promoting net growth of actin filaments.
How does phosphoinositide affect profilin?
Profilin functions to sequester G-actin monomers.
Phosphoinositide binding results in the release of G-actin from profilin, resulting in higher actin monomer concentration, allowing for actin filament growth.
How does phosphoinositide binding affect gelsolin as a capping protein?
Gelsolin has two functions - (1) Cap plus ends and (2) Severe actin filaments.
Therefore, phosphoinositide binding will cause uncapping and dissociation of gelsolin from the plus ends, increasing its ability to severe actin filaments, promoting net actin filament growth.
The 3 main proteins that are affected by phosphoinositide are:
1) Gelsolin
2) Profilin
3) ADF/cofilin
(G-protein) controls the formation and polymerisation of actin in filopodia.
Cdc42 protein
(G-protein) controls the formation of lamellipodia.
Rac protein
(G-protein) controls the formation of thick, parallel stress fibres within cells.
Rho protein
