Lecture 4 - The Cytoskeleton: Microtubules Flashcards
Basic details of the 3 main types of cytoskeleton:
- What a protofilament is
- How they are nucleated
- Cellular roles for each type of cytoskeletal polymer
- Properties of MFs and MTs
- The existence of ABPs and MAPs
- Molecular motors
- How they work
- What they do
- Some examples
Functions of the cytoskeleton
• Bones and muscles of the cell
- cell shape/polarity
- cell motility
- cell plasticity
- segregation of chromosomes
• Railways of the cell
- endocytosis
- secretion
- segregation of organelles
- communication between organelles
- Many diseases associated with cytoskeletal abnormalities
- Route of entry for many bacterial and viral pathogens
Networks of intracellular filaments
Microtubules
- centrosome (MTOC)
Intermediate filaments - nuclear lamina, desmosome
Actin filaments (microfilaments) lamellipodium, stress fibre
Microtubules ILOs
o Function
o Structure
o Dynamic instability
o GTP cap
o Nucleation
o Regulators
o Links to disease
- Describe in some details how microtubules function
- Describe in some details how microtubules are nucleated
- Describe in some details how microtubules are regulated, notably by MAPs.
- Start to understand the implications of all of the above, for health and disease
- Interpret scientific data
Role of microtubules in interphase
- Structural support and shape
- Cell motility
- Positioning of organelles
- Movement of organelles and molecules
Role of microtubules in mitosis
- Chromosomes alignment and segregation
- Partitioning of organelles between daughter cells
- Definition of the site of cytokinesis
Microtubule structure and properties
Microtubules (MTs) are polarised hollow tubes made of tubulin heterodimers
alpha is always at the minus end
beta is always at the plus end
13 protofilaments make a MT
diameter of MT is approx 25nm
Dynamic instability of MTs
Parameters of dynamic instability:
‐ Rate of growth
‐ Rate of shrinkage
‐ Frequency of catastrophes
‐ Frequency of rescues
Data Handling Practice
Answer:
Microtubules are polarised
o The ‐ end is less dynamic, ie the 4 parameters are decreased.
o The 2 ends are not the same = polarised
o If grows faster at the + end and shrinks less at the – end, then, the
MT will necessary grow from its + end. Same for shrinkage!
Dynamic instability occurs at both ends, just faster at the plus end!
B‐tubulin is a GTPase
‘ase’ means it’s an enzyme
so b-tubulin is a GTP modifiying enzyme (hydrolyses GTP to GDP)
a-tubulin will bind GTP but does nothing to it
Dynamic instability is driven by GTP/GDP cycles
The GTP cap
The + end
= B-tubulin
= GTP/ GDP
= more dynamic
GTP cap protects the end of the microtubule, GDP is unstable -
GTP is added faster than GDP lost which is why you get growth and catastrophe - this is why you get the GTP CAP
How are microtubules formed (nucleated)?
Microtubule nucleation
Centrosome (spindle pole body in fungi):
‐ main MicroTubule Organising Centre (MTOC)
‐ Pair of centrioles + PeriCentriolar Material (PCM + ‐tubulin complex)
MTs are nucleated from the ‐ end
gamma tubulin makes the TUSC (G-tubulin Small Complex) and the TURC (G-tubulin Ring Complex)
centrosome is at the centre of the cell, all microbules come from it
Regulation of MTs dynamics
Data handling practice
A private company has developed a microfluidic chamber that changes the temperature very quickly on the microscope stage. Here they observed S. pombe cells expressing tubulin‐GFP. What conclusion(s) can you draw from this experiment?
A‐ MTs polymerise at low temperature
B‐ MTs are sensitive to the temperature
C‐ MTs polymerisation/ depolymerisation is reversible D‐ MT regrowth is favoured by higher temperatures
Answer: BCD
B‐ MTs are sensitive to the temperature
C‐ MTs polymerisation/ depolymerisation is reversible D‐ MT regrowth is favoured by higher temperatures
Some anti‐MTs drugs are used in the treatment of human diseases
Polymerisation inhibitor = destabilise
Depolymerisation Inhibitor = stabiliser
over stabilisation of MT is not good for cells either!
MAPs
Microtubule associated proteins
Stathmin
Stathmin regulates MTs dynamics by sequestering tubulin dimers
Data Handling Practice
After their death, the brains of 4 healthy patients (control) and 4 patients with Alzheimer’s disease (AD) were biopsied. Biologists prepared whole protein extracts from each sample and loaded them on a gel. They then used specific antibodies to detect Tau protein or Actin. What can they conclude from these experiments?
A‐ The anti‐Tau antibody does not work well in control patients
B‐ Tau binds actin in both control and AD patients
C‐ In AD patients, a form of Tau migrates at a high molecular weight D‐ Tau does not behave the same way in all AD patients
E‐ Actin was not a good loading control, tubulin would have been
Answer: C+D
Feedback:
This gel separates proteins on the basis of their size (KDa). Actin is used to check that the same amount of total protein was loaded. This is a goodloading control because actin is not expected to be affected in AD or by Tau. The Ab works because it detects Tau at 50 KDa in all samples. In AD, Tau shows an extra band at 150 KDa (aggregates? phosphorylation?). The amount changes between patients, maybe (?) due to the severity of the disease.
Tau and tauopathies
Tau is a MAP that normally binds MTs and stabilises them
In tauopathies (e.g. Alzheimer’s disease), Tau is hyperphosphorylated and detaches from MTs:
1) MTs become less stable and depolymerise;
2) Tau becomes insoluble and aggregates into filaments called Neurofibrillary tangles (NFT).
MTs motors use MTs as rails to transport organelles
- 2x coordinated heads “walk” by hydrolysing ATP
- Slow (kinesins= 0.02-2um/sec)
- Transport over long distances
- Do not detach easily from MTs
Cilia and flagella contain stable microtubules moved by special dyneins
- flagella long (~45u
- cilia short (~10um), many per cell (<200)m)
- primary cilia short (~10um), 1 per cell, non-motile
Examples of MTs use by the cell
Lecture 6: mitosis
Lecture 8 + practicals: cell movement
Lecture 7: cell polarity
