Cytoskeleton and motility

Question 1

Microtubules overview

Answer 1

25nm in diameter (largest)
Hollow tubes, stiff, inextensible and resistant to bending.

Question 2

Microtubule molecule to structure sequence

Answer 2

α-tubulin + β-tubulin -> heterodimers -> protofilaments -> 13 protofilaments -> microtubules

Question 3

Microtubules are ______ (polar/nonpolar)

Answer 3

Polar! α = minus end binds ONLY GTP, β = plus end hydrolyses GTP to GDP

Question 4

Microtubule associated proteins function

Answer 4

Structural MAPs, increase microtubule stability and promote assembly (MAP1, MAP2, MAP4, tau)

Question 5

Tau MAP is necessary

Answer 5

If mutated, or hyperphosphorylated, microtubules are unstable and form tangled clumps associated with frontotemporal dementia.

Question 6

Dynamic MAPS

Answer 6

i.e. motor proteins, have been observed on squid giant axons with vesicles moving in both directions

Question 7

Kinesin

Answer 7

Anterograde movement towards the plus end (outside the cell), it is Kind and +positive, but has been Kicked out.

Question 8

Dynein

Answer 8

Retrograde movement towards the minus end (inside of cell), said “nein” to leaving and is DYiNg (-).

Question 9

Kinesin related proteins EXCEPTIONS

Answer 9

45 kinds in 14 families that all walk towards the plus end (anterograde) EXCEPT Kinesin-14 and Kinesin-13.

Question 10

Kinesin-14

Answer 10

moves towards the minus end

Question 11

Kinesin-13

Answer 11

doesnt move at all (unlucky!)

Question 12

Kinesin-1 structure

Answer 12

2 heavy chains composed of a globular region and a coiled α-helix, and a light chain. Globular region binds MT and hydrolyses ATP, 2 light chains in tail bind cargo.

Question 13

Kinesin movement mechanism

Answer 13

“Hand over hand” using ATP.
1. ATP binds leading head.
2. Power stroke of trailing head.
3. NEW leading head as it binds the MT.
4. ATP hydrolysis on the new trailing head, ADP release on the new leading head.

Question 14

Kinesin motility assay

Answer 14

Secure kinesin tails to coverslip (where a cargo would bind), add microtubules stained with a fluorochrome.
See the microtubules glide along the motor protein heads.

Question 15

Cytoplasmic dynein structure

Answer 15

HUGE, two head domains that hydrolyse ATP, an intermediate and light chain that binds cargo, and a region binding the MT near the heads.

Question 16

Dynactin

Answer 16

Complexes so that cargo can bind the dynein and MT.

Question 17

Microtubule organising centers (MTOCs), function and location

Answer 17

Organise MT-associated structures and organelles to orient transport, found perinuclear at the center of the cell. Polarity matters!!

Question 18

Nucleation

Answer 18

The initiation of growth of MTs

Question 19

Centrosomes

Answer 19

Two perpendicular centrioles (each made of nine triplet microtubules) and pericentriolar material.

Question 20

Pericentriolar material

Answer 20

A diffuse granular matrix surrounding the centrioles. Centrioles seem to recruit PCM (rich in gamma tubulin) to the centrosome to then nucleate the growth of microtubules.

Question 21

γ-TuRC

Answer 21

γ-tubulin ring complex.
Forms a base from which a microtubule can grow

Question 22

Where does microtubule growth occur.

Answer 22

Growth only occurs at the β-end.
Beta end is the Building end, adding (+) at the plus end.

Question 23

Dynamic instability model

Answer 23

Plus end start with a GTP cap where diners can join, growing the MT. With growth, GTP is hydrolysed to GDP (GDP cap). A β-tubulin bound to GDP is more likely to disassemble, leading to MT shrinkage (catastrophe).

Question 24

Flagellum axoneme structure

Answer 24

9+2 array - nine doublet MTs in a ring, with two normal, single MTs in the middle.

Question 25

Bending in cilia and Flagella depends on the _________ of the axoneme.

Answer 25

Crosslinks via sidearms; through ATP.

Question 26

Axoneme basal body

Answer 26

MTOC - structurally the exact same as the centrosome with nine triplet MTs.

Question 27

Intermediate filaments overview

Answer 27

10-12nm, unique to animals, 70 types with 6 classes. Rope-like but **not** hollow. Tough, flexible, extensible, elastic - tensile. No role in motility but very stable and provides mechanical support.

Question 28

Examples of intermediate filaments to know (3 classes)

Answer 28

Acidic keratins, basic or neutral keratins, nuclear lamins (A, B, C)

Question 29

IM structure sequence

Answer 29

Polar coiled-coil -> nonpolar tetrameric protofilament -> bundles of tetramers -> nonpolar intermediate filament.

Question 30

IM assembly mechanism relies on _____________

Answer 30

Phosphorylation!

Question 31

Phosphatases

Answer 31

Removal causes assembly

Question 32

Kinases

Answer 32

Addition causes disassembly

Question 33

Nuclear lamina

Answer 33

Thin, dense mesh work of fibers lining the inner surface of the inner nuclear membrane to support the nuclear envelope.

Question 34

Plectins

Answer 34

Intermediate filament associated protein that will form bridges with IFs, MTs, and MFs.

Question 35

Keratins

Answer 35

Tethered to the nuclear envelope and outer edge of the cell, desmosomes and hemidesmosomes.

Question 36

Role of keratin IFs in epithelial cell attachments

Answer 36

Hemidesmosomes and desmosomes

Question 37

Desmosomes

Answer 37

Cell to cell attachment structures along the basolateral region. Provides tensile strength to a sheet of cells

Question 38

Hemidesmosomes

Answer 38

Cell to epithelial-extracellular matrix attachment structures, attaching the basal lamina of the basement membrane.

Question 39

Epidermolysis bullosa simplex disorder cause

Answer 39

Issues with cell adherence due the keratin mutations.

Question 40

Microfilaments

Answer 40

Smallest fibers (6-8nm) made of actin protein. Flexible, inextensible helical filament. Contractile and can generate tension important for movement within a cell and of the cell itself.

Question 41

G-actin structure

Answer 41

Monomer that has lobes, each with two domains, binding ATP in the between cleft.

Question 42

F-actin structure

Answer 42

Polymer that, in mature filaments, wraps around each other to become helical.

Question 43

Actin assembly provides MF with polarity

Answer 43

At the minus end, the ATP-binding pocket is exposed and the plus end is buries it in the filament.

Question 44

S1 decoration experiment

Answer 44

Mixing purified S1 fragments of myosin and actin microfilaments results in an arrowhead formation demonstrating that actin MFs are polar.

Question 45

Microfilament functions (5)

Answer 45

Cell shape Migration Transport of vesicles and organelles Cytokinesis Muscle contraction

Question 46

Microfilament assembly occurs at the ____nend

Answer 46

Plus!!

Question 47

Molecular motor of actin

Answer 47

Plus end directed. Conventional (II) and unconventional (I and V)

Question 48

Myosin II

Answer 48

Conventional. Two globular domain heads and a long two heavy chain tail with no cargo. Twist with each other to form bipolar filaments.

Question 49

Myosin I or Myosin V (smaller)

Answer 49

Unconventional I - single head (discovered first) V - two heads No filament formation as tail binds vesicles and membranes.

Question 50

Thick filaments of skeletal muscle structure

Answer 50

Hundreds of myosin II.

Question 51

Cyclic process of muscle contraction involving actin and myosin filaments (CCPPAA)

Answer 51

Cocked (ATP has been hydrolysed), crossbridge, Pi released, power stroke, ADP released, ATP rebinds (myosin detaches).

Question 52

Actin bundles and networks

Answer 52

Bundles - parallel fibers found in filopodia. Networks - 2D or 3D.

Question 53

Microfilament organisation is mediated by _______-______ in __ ways.

Answer 53

Actin-binding; 8.

Question 54

Actin binding proteins function

Answer 54

regulate polymerisation and length of filaments

Question 55

Arp2/3

Answer 55

Nucleating protein. Forms a nucleating center, mimicking the shape of actin so that G-actins add on.

Question 56

Thymosin β4

Answer 56

Monomer sequestering. Controls the amount of G-actin available for polymerisation.

Question 57

CapZ and Tropomodulin

Answer 57

End blocking. - CapZ caps the plus end (shrinkage). - Tropomodulin caps the minus end (growth).

Question 58

Profilin

Answer 58

Monomer polymerising. Increases actin filament growth rates by promoting G-actin addition to the filament plus ends. Competes with thymosin for G-actin monomers.

Question 59

Cofilin

Answer 59

Depolymerising proteins. Binds at the minus end and causes depolymerisation.

Question 60

Filamin and Villin

Answer 60

Filamin - Crosslinking, holds filaments at right angles. Villin - Bundling, holds filaments in parallel.

Question 61

Gelsolin

Answer 61

Filament severing protein. Breaks up the MF network causing the actin gel to soften and liquefy **AND** caps the newly-exposed plus ends to prevent further depolymerisation.

Question 62

Dystrophin and Vinculin

Answer 62

Membrane binding. Secures the microfilament to the membrane so that the membrane follows actin movements.

Question 63

Steps of cell locomotion

Answer 63

1. extension 2. adhesion 3. translocation of cell body 4. de-adhesion (all four steps involve actin)

Question 64

lamellipodium

Answer 64

Leading edge

Question 65

Filopodia

Answer 65

Thin, pointed protrusions

Question 66

Protrusion of the lamellipodium

Answer 66

1. WASP complex activates Arp2/3 2. Nucleation of the side filaments 3. Barbed ends elongated and push the membrane forwards 4. Capping proteins terminate elongation 5. Cofilin depolymerises the pointed ends on older filaments. Monomers exchange ADP for ATP and profilin adds them to the barbed ends of existing filaments.

Question 67

Extension

Answer 67

LEading edge extends via polymerisation of actin filaments at its tip in a branched formation.

Question 68

Focal adhesions

Answer 68

Temporary attachment sites between cell and substrate made of actin and integrins.