Lecture 5 - The Cytoskeleton: IF and Actin Flashcards
Networks of intracellular filaments
Intermediate Filaments - nuclear lamina, desmosome Actin Filaments - lamellipodium, stress fibre
ILOs
Actin
o Structure and polarity
o Treadmilling
o Nucleation (Arp2/3 or Formins)
o Regulators
o Collaboration with MTs
‐ IF
o Structure
o The example of nuclear lamins
- Describe in some details how IF and actin are nucleated
- Describe in some details how actin functions
- Describe in some details how actin is regulated, notably by ABPs.
- Start to understand the implications of all of the above, for health and disease
- Compare and contrast with MTs
- Interpret scientific data
What does actin do ?
- Cytokinesis (contractile ring)
- Cell adhesion (wound healing); migration
- Endocytosis
- Transport of cargos (trafficking)
- Polarity
- Infection (e.g. parasites use actin)
F‐ actin structure and properties
Only one type - G actin (globular actin) makes F actin (filamentous actin)
Actin Filaments (F-actin = Microfilaments = MFs) are polarised threads of G actin
- they have a plus end (barbed) and a minus end (pointed)
- barbed end is where actin monomers are added
- Actin uses ATP, it is an ATPase. ATP added at plus end, ADP comes off at the minus end

Actin is an ATPase
Rate of addition at barbed end = rate of loss at pointed end
Length is constant but the polymer moves
Action is called Treadmilling
(very different from dynamic instability of MTs)

Treadmilling
Rate of addition at barbed end = rate of loss at pointed end Length is constant but the polymer moves

How is actin nucleated?

2 modes of nucleation for 2 types of actin organisation
- Straight (bundled) filaments by formins (e.g. stres fibres)
- Branched filaments nucleated by ARP2/3 complex (e.g. lamellipodia)

Nucleation by formins
‐ Works as a dimer; each monomer adds 1 actin monomer - homodimer (MT is heterodimer)
‐ Each monomer has 2 domains: 1 for actin, 1 for regulators
‐ Nucleates from the + end
‐ Nucleates straight filaments

Nucleation by Arp2/3

- Complex of proteins, incl Arp2 and Arp3
‐ Activated by a Nucleation Promoting Factor (NPF)
‐ Initiates polymerisation because it mimics G‐actin
‐ Nucleates from the – end (similar to MTs but different from formins)
‐ More efficient when nucleates from an existing filament - ie forms a branched network

Regulation of actin
Data handling practice
Based on these 2 figures:
A‐ cytochalasin D prevents actin polymerisation B‐ cytochalasin D prevents actin depolymerisation C‐ cytochalasin D affects both actin and MTs
D‐ Jasplakinolide induces actin polymerisation
E‐ Jasplakinolide induces actin depolymerisation

Answer: A, D
The ration F/G tells you which of the actin Filament or G actin subunits prevails.
High F/G = more F or less G, ie the drug induces polymerisation of G actin into Filaments.
Low F/G= more G or less F, ie the drug prevents G actin polymerisation into Filaments. Other figure confirms cytochalasin depolymersises F actin. It has however no effect on MTs (less clear, in fairness!).
ABPs
Actin binding proteins

Thymosin and profilin compete to bind to G‐actin

Thymosin sequesters actin monomers - anti‐polymerisation
Profilin favours ATP exchange on G‐actin - pro‐polymerisation
They bind to the same site on actin
Most myosins walk towards the plus (barbed) end of the filament
Molecular motors are also actin binding proteins

- Each head “walks” by hydrolysing ATP, independently of other head
- Kinesins swing on 2 feet, but by contrast myosins detach, tilt and attach again. Myosins are faster than kinesins
- Fast (0.2- 60 mm/sec)
- Transport over short distances
- Just a few steps on actin before let go
MTs for long range transport and actin for local delivery
- Kinesin from cell centre to periphery (long range transport, e.g. along axons), dynein from periphery to centre, long range;
- Myosin, local distribution

Data handling practice
In this experiment, pre‐labelled G‐actin is polymersised in vitro on a microscope slide and imaged by TIRF microscopy in the presence of Cip4, Dia or both. Which are true:
A‐ Cip4, Dia and actin all form polymers
B‐ Cip4 inhibits actin polymerisation
C‐ Dia promotes actin polymerisation
D‐ Cip4 inhibits actin polymerisation by Dia

Answer: C,D
ABPs can themselves be regulated
We are looking at F- actin, not Cip4 or Dia! We see many more actin filaments when Dia is added alone. However, when added together with Cip4, we see less F-actin. This is interesting because on its own, Cip4 has no effect on actin. Hence, the ability of the formin Dia to nucleate F- actin is negatively regulated by Cip4.
Note: Dia is a formin; Cip4 is a membrane-associated protein.
More examples of actin use by the cell
- Cytokinesis
- Cell movement
- Trafficking
Intermediate filaments (IF) are strong and flexible
- Dimers (//), tetramer (anti- //). In the final IF, each monomer points in a different directionno polarity. No motor!
- No GTP or ATP required for assembly
- Strength because of overlapping lateral interactions. Flexibility because of helical structure.
- IFs are NOT POLARISED
- this is why they have no molecular motors (as they wouldnt know which way to go)

4 classes of IF straighten specialised cells against mechanical stress

- Every tissue has a distinctive set of IFs (e.g. keratins in the cornea, guts or nails are all different)
- An IF can be made of several types of proteins of the same class (e.g., different types of keratin)
4 types of tissues:
muscles (vimentin), neural (neurofilaments), connective, epithelial (keratin filaments)
Data handling practice
Progeria is a rare genetic disorder that causes premature aging. In 2003, scientists discovered it was due to mutations in Lamin A. Based on this figure, how do these mutations affects patient cells?
A‐ cells are misshaped
B‐ cells are glued to each other
C‐ cells have an abnormal nuclear envelope
D‐ Lamin A is delocalised
E‐ cells have less chromosomes
F‐ chromosomes are improperly distributed

Answer: C,F

We are looking at the nucleus (it encloses the DNA!), not the whole cell. Lamin A labels the nuclear envelope, which is lobed in progeria patient cells. The resolution of the microscope is not high enough to tell if Lamin is inserted or not in the PM (see next slide). The number of chromosomes (centromeres) was not counted here. The DNA and centromeres aggregate at the centre of the nucleus
Examples of IFs use by the cell
- Cell‐ECM interaction
- Cell‐cell junctions