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2. Cell Biology of Disease > Cytoskeleton - Microtubules > Flashcards

Cytoskeleton - Microtubules Flashcards

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1
Q

What is the cytoskeleton?

A

As eukaryotic cells are too large - sometimes diffusion isn’t possible so we need motors and tracks
Cytoskeletal systems are dynamic and adaptable and are made up of proteins only nm in size - which is helpful for disassembly/diffusion

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2
Q

What are the type of cytoskeletal filaments?

A

Three types of cytoskeletal filaments:
Actin filaments (microfilaments): 7nm in diameter
Polymers of actin, tracks for myosin (molecular motors)
This defines the shape of the cells surface, are needed for whole-cell movement and drives the pinching in telophase (forming a belt - contractile ring)
Found beneath the nuclear lamina

Microtubules: 25nm in diameter (largest)
Polymers of alpha-beta tubulin dimers
Tracks for kinesin and dynein (molecular motors)

Intermediate Filaments:
10nm in diameter
No motors associated with these filaments and non-polar = less dynamic
Strong rope like fibres - forms the nuclear lamina

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3
Q

What are accessory proteins?

A

All these filaments interact with hundreds of accessory proteins - these are essential for the controlled assembly of the cytoskeletal filaments

They regulate the length and stability of the cytoskeleton so they can form a variety of high-order structures - specifically regulating the spatial distribution and dynamic behaviour

They can: determine sites for new filaments, regulate partitions within a filament, alter filament kinetics, harness energy to generate force and link filaments to one another or other cell structures

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4
Q

What are motor proteins?

A

They bind to polarised cytoskeletal filament and use energy from ATP to move along it
They differ in direction, binding filament and cargo e.g. Membrane bound organelles like mitochondria
The direction of sliding depends on the polarity of the track

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5
Q

Describe the cytoskeleton in bacteria in most archaea?

A

FtsZ - homolog of tubulin, which polymerises into filaments - Z-ring (half-life of few mins)
The Z-ring generates a bending force to drive membrane invagination in telophase

MreB and Mbl - homologs of actin, found in rod or spiral shaped dynamic cells that move around the circumference of the cell
They serve as a scaffold to direct the synthesis of the peptidoglycan cell wall

Mutations cause extreme cell shape abnormalities
Caulobacter crescentus, harbours an intermediate homolog crescentin - that influences the crescent/sickle shape of this bacteria

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6
Q

Describe microtubules?

A

They are dynamic tubes growing from the centromere and then they collapse back
They are like highways along which motors carry membranes
They have moving vesicles along them, in both directions

Negative end is towards the centre and the positive end is towards the periphery of the cell (showing directionality)
○ Positive end = fast growing - B tubulin
○ Negative end = slow growing - a tubulin
They are the biggest type of filament in a cell - seen by immunostaining during interphase

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7
Q

What is the composition of microtubules?

A

They are made from tubulin
There are two types of tubulin: alpha and beta
There are around 6-7 genes for each alpha and beta tubulin (in mammals) = diverse types of tubulin
They are formed from the polymerisation of tubulin proteins - therefore the ‘building blocks’ are an alpha-beta tubulin complex (heterodimer)

The subunits of microtubules are asymmetrical and bind to one another head-to-tail = they all point in one direction

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8
Q

What is a protofilament?

A

Protofilament - a ‘stack’ of heterodimers forming a vertical line
Each protofilament is slightly offset creating a slightly helical structure
There are 13 protofilaments in a microtubule (diameter of 25 nm)

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9
Q

What is the centrosome?

A

The microtubules are nucleated at the centrosome in cells
It comprises of barrel shaped structures surrounded by ‘fuzzy material’ = pericentriolar matrix, containing various proteins that helps anchor the microtubules
Gamma tubulin rings - initiates microtubule polymerisation

The centrosome has a ‘mother’ (maternal) and ‘daughter’ centriole
A pair of centrioles each has ninefold symmetry
Only the maternal centriole has two sets of extra appendages - distal and subdistal
Subdistail seems to anchor microtubules

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10
Q

What is a property of microtubules?

A

They polymerise and depolymerise

GTP-tubulin subunits add to the fast growing plus end of microtubules
If the GTP tubulin ‘CAP’ is lost, the microtubule will start to depolymerise:
This is called ‘catastrophe’ (it happens rapidly)

Note both α and β tubulin bind GTP, but only β tubulin hydrolyses GTP
This subunit is exposed at the ‘plus’ (fast growing’ ends)

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11
Q

How can microtubules be post-translationally modified?

A

The C-terminal tail can be polyglutamylated, or polyglyclated, or the C-terminal tyrosine can be removed
C-terminal tails on the outside of the microtubule ‘tube’
This affects binding of MT proteins & their behaviour (& stability)

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12
Q

What are the main functions of microtubules?

A
  1. Trafficking of cargo (organelles, protein, RNA) in interphase cells
  2. Mitosis
  3. Cilium
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13
Q

What is used in trafficking within microtubules?

A

Kinesin and Dynein are motor proteins that use microtubules as ‘tracks’

The cell body of most motor neurons is in the spinal cord, and the synapses (neuromuscular junctions) can be over 1 metre away
Microtubule motors are important for this
Kinesin takes things out of the microtubule
Dynein will bring things in to the microtubule

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14
Q

Describe Kinesin?

A 
A dimer formed by two heavy chains
Motor domain - binds actin and nucleotide (hydrolyses Mg.ATP to generate movement)
Tail - Binds cargo
It has no lever
Mr - around 100 kDa
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15
Q

What is the function of kinesin?

A

Most kinesins walk to the ‘plus’ (fast growing) ends of microtubules at the cell periphery
Kinesin takes 8nm steps (same size as an αβ-subunit)
Walks straight along a single protofilament
Powered by ATP hydrolysis
The ATPase of kinesin is accelerated when the kinesin binds to microtubules
About 40 different types of kinesins, many specifically for mitosis

Mutations in kinesin-1 would interfere with trafficking organelles

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16
Q

What is the kinesin-1 ATPase cycle?

A
  1. Kinesin and ADP binds
    The microtubules accelerate ADP release x1000 fold
  2. ADP releases and the neck liner undocks
    There is a strong Apo binding state
  3. ATP binds to the kinesin and the neck linker docks
    During ATP state there is a strong microtubule binding
    Microtubules accelerate ATP hydrolysis x10 fold
  4. ATP hydrolysis results in an ADP.Pi state
    Here there is strong microtubule binding
  5. Pi is released - resulting in weak binding
  6. Kinesin detaches with the ADP
    ADP release is the rate limiting step
17
Q

What is significant about the two kinesin heads?

A

They are asynchronous (out of sync)
1. Rear head (ATP.Pi): neck linker docked, Front head (ADP) and neck linker undocked
2. ADP is lost by front head and Pi is lost by rear head
’Gating’ behaviour between the two heads for co-ordination
3. Rear head (ADP) detaches, neck linker will undock
4. Front head (was rear head) binds ATP, the Neck linker docks and helps to move rear (ADP) head forward

Powerstroke - this is the docking/undocking of the neck linker
It is the neck linker that is driven by the ATPase state of the motor

18
Q

Describe Dynein motors?

A

Minus-end directed microtubule motor
ATPase (4 nucleotide pockets per heavy chain)
Work coupled to release of products of ATP hydrolysis
A member of the AAA+ superfamily of mechano-enzymes

Composition:
Heavy chain(s) (>500 kDa each)
Accessory chains (I, LI, L- mainly cargo binding)
Heavy chain contains the motor domain (ATPase and microtubule-binding domains)
19
Q

What is the AAA+ superfamily of mechano-enzymes?

A

AAA+ proteins assemble into ring shaped oligomers: typically homo-hexamers
AAA+ mechanoenzymes perform diverse functions: unfolding/destabilising

Dynein’s contain 6 AAA+ domains in a single polypeptide chain
They convert energy from ATP hydrolysis into force/stepping along microtubules
ATPase kinetic scheme is similar to that of the actin-based motor myosin

20
Q

What is significant of the iosforms of dynein?

A

The family tree = all the isoforms that can be in one cell
Only two isoforms of cytoplasmic dynein heavy chains
Only one of these (cytoplasmic dynein 1) is the main trafficker
Multiple functions achieved through light chain/intermediate variation and additional regulatory proteins

21
Q

What is dynein’s structure?

A

This is the 9+2 organisation
Each of the 9 outer doublets has rows of dynein arms that reach out to the next doublet and cause it to slide
Driven by ATP hydrolysis
Both stalk and tail flexible
This means distance varies between cargo and MT-binding site
Important to allow Powerstroke of motor without simultaneous movement of cargo

22
Q

How does the powerstroke of dynein take place?

A

Tail connects to AAA1 ATPase domain in head via ‘linker’ domain
Linker changes its orientation relative to head and stalk
Linker movement could translate microtubule

We can see the linker’s change in conformation
When you hydrolyse ATP, The linker changes from straight to bent, causing it to come away form AAA4, this part of the molecule pulls on the stalk allowing the microtubule domain to bind = generation of the power stroke

23
Q

Describe the ATPase cycle for dynein?

A
  1. ATP hydrolysis and phosphate release, the linker is straight
  2. ADP is released, the linker docks and then ATP can bind
  3. ATP binding, causes the linker to bend
24
Q

What else does dynein require to work?

A

Dyactin activates the dynein
Dynactin is 1 mega Da
It binds to the tail of dynein
It makes sure the motor domains stick out correctly, in order to bind to the microtubule

25
Q

What are microtubule associated proteins (MAPs)?

A

They are important for microtubule function e.g. TAU MAPs:

Mainly in axons
Stabilises microtubules
May regulate trafficking
Some tau in dendrites – function unclear
Some tau in nucleus – for genomic integrity
Protein is mostly unfolded
Tau affinity for MTs decreases when hyper-phosphorylated

26
Q

Describe another MAP?

A

Spastin
Spastin severs ‘stable’ regions of microtubules (M87 - higher activity than M1)
Recognises C-terminal tails and it can pull/cut microtubules
Polyglutamylation of C-terminal tubulin tail by TTLL6 (tubulin polyglutamylase) stimulates severing

27
Q

How do microtubules function in mitosis?

A

They are highly dynamic
Dynein and kinesins also involved (specific mitotic kinesins)
The chromosomes are attached to microtubules, via the kinetochore (MAP)
Depolymerisation of the MTs as well as molecular motors (kinesin and dynein) are responsible for separation of the chromosomes in anaphase

28
Q

How do microtubules function in Cilia?

A

They function as motile whips or sensory devices on the surface of the cell
Dynein makes the flagellum/cilium bend in motile cilia
Kinesin and cytoplasmic dynein-2 traffic intra-flagellar (IFT) cargoes: important for building/maintaining all cilia

29
Q

What can disease affect within the microtubule network?

A

Microtubules
Kinesins
Microtubule associated proteins
Proteins in the dynein complex

30
Q

What are some diseases that affect microtubules?

A

α-tubulin (TUBA1A)
Polymicrogyria, multiple small gyri (folds) - not formed correctly

β-tubulin (TUBB2A, TUBB2B,TUBB3, TUBB4A, TUBB)
Diffuse pachygyria, very few gyri – smooth brain

ɣ-tubulin (TUBG1) (found at minus ends of MTs)
Associated with “Tubulinopathies” range of brain malformations

31
Q

Describe the microtubulin b-tubulin disease: lissencephaly?

A

Lissencephaly - smooth brain
Absence of normal folds (gyri) in the cortex
Patients have an abnormally small head (microcephaly)
Unusual facial appearance, difficulty swallowing, failure to thrive, muscle spasms, seizures, and severe psychomotor retardation
Most children die before the age of 2

32
Q

What do mutations within kinesin cause?

A

Mutations in kinesins cause hereditary spastic paraplegia ‘transportopathy’

A failure in transport towards synapse = synapse deterioration and fail to connect with their target organ
Reduced trafficking (particularly mitochondria) to synapses: results in their gradual degeneration
33
Q

What is a disease associated with the MAP: Tau?

A 
Implicated in:
Alzheimer’s disease
Progressive supranuclear palsy
Cortico- basal degeneration 
Frontotemporal dementia
Parkinsonism

Majority are missense (but can change splicing)
Others are splicing mutations
Most are in the microtubule binding domain
Reduced affinity for microtubules = reduced trafficking
Increased tendency to aggregate

34
Q

What happens if Tau gets into the wrong place?

A

If it gets into the dendrites

Spastin in recruited to microtubules when tau is there
It polyglutamylates microtubules, and severs them
This will then cut the microtubules where it shouldn’t be cut

35
Q

What is a disease associated with the MAP: Spastin?

A

Mutations in Spastin cause >40% of cases of Hereditary spastic paraplegia
Autosomal dominant
Most mutations reduce activity of Spastin

Progressive lower limb spasticity
Degeneration of sensory and spinal cord neurones

Loss of Spastin – fewer dynamic MTs (shorter microtubules move more rapidly)

36
Q

What is a disease within the dynein compelx?

A

Mutations in dynein heavy chain (DNAH5) cause primary cilia dyskinesia (PCD)
Currently >220 mutations identified (most are missense)

This leads to missing dynein arms (one or both)
Likely to affect ATPase/motor activity or it may not be present at all

37
Q

What are some chemical inhibitors of microtubules?

A

Taxol
Monastrol

As microtubules help within mitosis - this is a good target for cancer drugs

38
Q

Describe Taxol?

A

A microtubule stabilizing drug: used in cancer treatments
Derived from Yew Trees
Used to treat metastatic breast cancer, non-small lung cell cancer, ovarian, gastric and prostate cancers

Taxol stabilizes microtubules
Induces mitotic arrest
Increases number of abnormal spindles
Cell death results from chromosome mis-segregation on multipolar spindles

39
Q

Describe Monastrol?

A

As microtubules in the spindle are highly dynamic, then stabilising the microtubules interferes with normal cell division
Cells that don’t divide properly are more likely to apoptosis
This chemical interferes with one of the kinesins involved in cell division - Eg5, which helps microtubules to slide past each other to build the spindle properly

2. Cell Biology of Disease (17 decks)

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