1
Q
General features
A
- cytoskeletal elements are not membrane bound
- all cytoskeletal elements are polymers
- MT= polymers of tubulin
- MF = polymers of actin
- IF = variable
- all non-covenant linkages
- i.e. dynamic elements
2
Q
Microtubules
A
- largest fibres (25nm)
- hollow tubes made of tubulin proteins
- a-tubulin + b-tubulin = heterodimers
- heterodimers —> protofilaments
- 13 protofilaments —> microtubule
-MT are POLAR
+ end (B-subunit) is where it grows (may have GTP or GDP cap)
- end (a-subunit) only has GTP cap
- both a and B can bind GTP
- B subunit can hydrolyze GTP to GDP
- a subunit ALWAYS has GTP
3
Q
Microtubule associated proteins
A
- structural MAPs
- increase stability of microtubules and promote assembly
-eg. MAP1, MAP2, MAP4, tau
4
Q
Tau
A
- stabilize MTS
- if mutated or hyperphosphorylated MTS become unstable, tangled climbs
- associated with frontotemporal dementia
5
Q
Dynamic instability model
A
- if there is a GTP cap, more aB dimers will join and MT will grow
- eventually GTP will be hydrolyzed
- if there is a low concentration of tubulin dimers in the cell, the GTP cap will disappear
- without GTP it is unstable and will disassemble leading to MT shrinkage (catastrophe)
6
Q
Microtubule organizing centres
A
- microtubules grow out of MTOCs
- nucleation = initiation of growth
- eg. Centrosome
7
Q
Centrosome
A
- composed of 2 centrioles
- each centriole is made up of 9 tripled MTS plus pericentriolar material (PCM)
8
Q
Pericentriolar matrix
A
- PCM is critical for microtubule nucleation
- gamma tubulin present in PCM
- gamma tubulin ring complex forms a base from which the microtubule can grow
9
Q
Microfilaments
A
- smallest fibres (6-8nm)
- made of actin
- important for movement (within cell and cell itself)
10
Q
Microfilaments functions
A
- Cell shape
- Cell migration
- Transport of vesicles and organelles
- Cytokinesis
- Muscle contraction
11
Q
Microfilament structure
A
- made of actin
- G-actin=globular actin (monomers)
- F-actin=filamentous actin (polymer/microfilament)
- G-actin binds ATP which is later hydrolyzed to ADP
- within a filament the actin monomers are oriented in the same direction
- filament is POLAR
12
Q
Myosin
A
- accessory protein for microfilaments
- attaches to filament
- had a head and neck
- S1 fragment sticks out to form “barbed wire” appearance
13
Q
Polarity of actin
A
- pointed end = minus end
- barbed end = plus end
- growth occurs at PLUS end
14
Q
Actin formation
A
- acting can be organized in bundles
- parallel assembly of filaments closely together
- often found in filopodia
- actin can be organized in networks
- meshwork of perpendicular fibres
- often found in lemllipodium
-microfilament organization is mediated by actin-binding proteins
15
Q
Actin binding proteins
A
- Proteins that regulate monomers and their polymerization
- Proteins that cap the ends
- Proteins that crosslink or bundle filament
- Proteins that sever microfilaments
- Proteins that link actin to membranes
- Proteins that promote branching
16
Q
Proteins that regulate monomers and their polymerization
A
- Thymosin B4
- controls amound of G-actin available for polymerization
- profilin
- adds ATP to monomers to increase filament growth rate
17
Q
Proteins that cap the ends
A
-a cap will prevent further addition or loss of subunits
- CapZ caps PLUS end
- no monomers can be added
- Tropomodulin caps MINUS end
- no monomers can fall off
18
Q
Proteins that crosslink or bundle filaments
A
- attach to more that 1 protein at a time
- holds filaments together
- Filamin
- holds filaments at right angles
- villin
- holds filaments parallel
19
Q
Proteins that sever microfilaments
A
- break filaments into smaller pieces
- allows more PLUS ends to be built upon
- Gelsolin
- cuts and caps minus end
20
Q
Proteins that link actin to membranes
A
-secures microfilament to membrane so that the membrane follows actin movement (cytokinesis)
- Band 4.1
- biggest family
21
Q
Proteins that promote branching
A
- formas a nucleation centre by mimicking the shape of actin subunits so G-actin will add on
- Arp2/3
22
Q
Intermediate filaments
A
- 8-12nm
- unique to animal cells
- rope-like, not hollow
- mechanical support
- very stable, resist tension
- no role in motility
- chemically heterogenous (made of many proteins)
23
Q
Intermediate filament assembly
A
- two monomers coil together to form a dimer
- they coil parallel to each other so that both Amino ends are together and both carboxy ends are together
- dimers assemble to form a tetrameric protofilament
- assemble ANTIPARALLEL to eachother so they are NONPOLAR
- protofilaments assemble end to end and laterally
- form a NONPOLAR IF
- neither ATP nor GTP involved in IF assemble
- phosphatases remove PO4 (assembly)
- kinases add PO4 (disassembly)
24
Q
Plectin
A
- intermediate filament associated protein
- forms bridges with other IF or MT
25
Lamins
- examples of IF proteins
| - form nuclear lamina just below the nuclear envelope
26
Keratins
- example of IF proteins
| - found in epithelial tissues and in structure that grow from skin in animals
27
Role of keratin IFs in cell attachment
- desmosomes
- cell to cell attachment structure
- hemidesmosomes
- cell to ECM attachment structure
28
Desmosomes
- cell to cell attachment
- network of IF provides tensile strength to entire sheet of cells
- found in tissues subjected to mechanical stress
- cardiac muscle
- epithelial layers
- uterine cervix
29
Hemidesmosomes
- cell to extra cellular matrix attachment
- keratin filaments extending outward into the cytoplasm
- bind to membrane spanning integrins
30
Keratin mutations
- can lead to cell adherence disorders
- eg. Epidermolysis bullosa simplex
- skin shedding off easily
31
Microtubule associated proteins
- dynamic MAPs (motor proteins)
- kinesin (move towards outside of cell + end)
- dynein (move toward inside of cell - end)
- kinesin and dynein mediate transport of organelles and vesicles moving through endomembrane system
- polarity very important
32
Squid giant axon
- large axon (0.5-1mm diameter)
- observed that vesicles were moving in both directions along the axon
- vesicles were moving along microtubules
33
Kinesin related proteins
- 45 different kinesins
- all walk toward + end of microtubule
- EXCEPTIONS:
1. Kinesin 1 moves toward - end
2. Kinesin 5 goes either direction
3. Kinesin 13 doesnt move at all
34
Kinesin 1
-composed of:
1. heads:
- binds MT and hydrolyze ATP
- do the walking
2. Heavy chain
- the stalk
- holds everything together
3. Tail
- binds cargo
- attach to vesicles
35
Kinesin movement
-hand over hand mechanism
1. ATP binds to the leading head
- must have ATP to continue
2. This causes a power stroke and the lagging head swings to the front
3. The new leading head binds to the microtubule
4. Leading head releases its old ADP and binds new ATP
5. Lagging head hydrolyzes its ATP
36
Kinesin motility assays
1. Secure kinesin tails to a coverslip
2. Add microtubules stained with fluorochrome
3. See microtubules glide along the motor protein heads
37
Cytoplasmic dynein
- moves toward - end of MT
- bigger than kinesins, but functions in similar way
- head domain hydrolyzes ATP
- intermediate and light chains bind cargo
- DYNACTIN required for dynein to bind cargo
- complex allows vesicle to bind to dynein
38
Internal structure of cilia and flagella
- internal structure called axoneme
- arranged in a 9+2 pattern
- 9 doublet microtubules
- 2 normal microtubules in centre
39
Other axonemal proteins
- radial spokes
- extend from the doublets to the single microtubules
- nexin link complex
- connects doublets
- stretchy protein
- axonemal dynein
- extends from an alpha tubule toward neighbouring doublet
- walks along neighbouring doublet to force a bend
- results in flagella moving back and forth causing propelling motion
40
Microtubule organizing centre for axoneme
- called basal body
- composed of 9 triplets instead of doublets
-each cilia has own basal body
41
Myosin
- molecular motor of actin
- plus end directed motors (moves toward barbed end)
- head domain hydrolyzes ATP
- conventional myosin
- unconventional myosin
42
Conventional myosin
- myosin II
- In muscles for contraction
- ATP dependent
- first ones discovered
- two head and long tail
- no cargo
- they twist to form bipolar filaments
43
Unconventional myosin
- myosin I or V
- one or two heads
- no filament formation
- tail binds vesicles and membrane
- more similar to dynein and kinesins
44
Cell locomotion
- microfilament motility in non-muscle cells
- filpodium=thin pointed protrusions
- lamellipodium=leading edges
- cell protrusion occurs due to actin polymerization beneath membrane
- Arp2/3, CapZ, Profilin all involved
1. Signal activates WASP
- WASP activates Arp2/3 to promote branching
2. Nucleation on sides of filaments
3. Barbed ends elongate
- membrane pushed forward in direction it wants to go
4. Capping protein terminates elongation (CapZ) on MFs in the back so that it doesnt grow in wrong direction
45
Cell locomotion steps
- cell protrusion
- extension
- adhesion (via focal adhesions)
- translocation of cell body (leftover of cell gets pulled forward)
- myosin
46
Focal adhesions
- temporary attachment sites between cell and substrate below
- how cells attach to dishes in culture
- hemidesmosomes = IF attachment
- strong/permanent
- focal adhesions = actin attachment
- temporary/strong
BIOL 2020
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