5: Cytoskeleton Flashcards
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
Q
Lamins
A
- examples of IF proteins
- form nuclear lamina just below the nuclear envelope
26
Q
Keratins
A
- example of IF proteins
- found in epithelial tissues and in structure that grow from skin in animals
27
Q
Role of keratin IFs in cell attachment
A
- desmosomes
- cell to cell attachment structure
- hemidesmosomes
- cell to ECM attachment structure
28
Q
Desmosomes
A
- 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
Q
Hemidesmosomes
A
- cell to extra cellular matrix attachment
- keratin filaments extending outward into the cytoplasm
- bind to membrane spanning integrins
30
Q
Keratin mutations
A
- can lead to cell adherence disorders
- eg. Epidermolysis bullosa simplex
- skin shedding off easily
31
Q
Microtubule associated proteins
A
- 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
Q
Squid giant axon
A
- large axon (0.5-1mm diameter)
- observed that vesicles were moving in both directions along the axon
- vesicles were moving along microtubules
33
Q
Kinesin related proteins
A
- 45 different kinesins
- all walk toward + end of microtubule
- EXCEPTIONS:
- Kinesin 1 moves toward - end
- Kinesin 5 goes either direction
- Kinesin 13 doesnt move at all
34
Q
Kinesin 1
A
-composed of:
- heads:
- binds MT and hydrolyze ATP
- do the walking
- Heavy chain
- the stalk
- holds everything together
- Tail
- binds cargo
- attach to vesicles
35
Q
Kinesin movement
A
-hand over hand mechanism
- ATP binds to the leading head
- must have ATP to continue
- This causes a power stroke and the lagging head swings to the front
- The new leading head binds to the microtubule
- Leading head releases its old ADP and binds new ATP
- Lagging head hydrolyzes its ATP
36
Q
Kinesin motility assays
A
- Secure kinesin tails to a coverslip
- Add microtubules stained with fluorochrome
- See microtubules glide along the motor protein heads
37
Q
Cytoplasmic dynein
A
- 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
Q
Internal structure of cilia and flagella
A
- internal structure called axoneme
- arranged in a 9+2 pattern
- 9 doublet microtubules
- 2 normal microtubules in centre
39
Q
Other axonemal proteins
A
- 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
Q
Microtubule organizing centre for axoneme
A
- called basal body
- composed of 9 triplets instead of doublets
-each cilia has own basal body
41
Q
Myosin
A
- molecular motor of actin
- plus end directed motors (moves toward barbed end)
- head domain hydrolyzes ATP
- conventional myosin
- unconventional myosin
42
Q
Conventional myosin
A
- 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
Q
Unconventional myosin
A
- myosin I or V
- one or two heads
- no filament formation
- tail binds vesicles and membrane
- more similar to dynein and kinesins
44
Q
Cell locomotion
A
- 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
- Signal activates WASP
- WASP activates Arp2/3 to promote branching
- Nucleation on sides of filaments
- Barbed ends elongate
- membrane pushed forward in direction it wants to go
- Capping protein terminates elongation (CapZ) on MFs in the back so that it doesnt grow in wrong direction
45
Q
Cell locomotion steps
A
- cell protrusion
- extension
- adhesion (via focal adhesions)
- translocation of cell body (leftover of cell gets pulled forward)
- myosin
46
Q
Focal adhesions
A
- 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
