The Cytoskeleton Flashcards
What is the definition of the cytoskeleton?
“The cytoskeleton is a dynamic structure made up of proteins which can self assemble into long polymers of repeating subunits”
What cellular processes/features is the cytoskeleton critical for?
- Cell Morphology
- Cell Migration
- Vesicle Transport
- Cell Division
- Cytokinesis
- Chromosome separation
What three networks of filaments comprise the cytoskeleton?
- Microtubules
- Microfilaments
- Intermediate Filaments
Rank the cytoskeletal filaments in order of size (smallest to largest)
- Microfilaments - smallest diameter at 7-9nm
- Intermediate Filaments - 10nm diameter
- Microtubules - 25nm - largest diameter.
What are fibroblasts?
Fibroblasts are a type of cell found in connective tissues. They secrete collagen and other components of the extra cellular matrix.
Describe the structure of microfilaments.
G-actin monomers self-assemble into linear polymers of F-actin. F-actin comprises or ~14 repeating G-actin subunits and is in a tight helix. F-actin polymers form the microfilament structure.
What does the ‘F’ in F-actin mean?
Filamentous
What does the ‘G’ in G-actin mean?
Globular
“G-actin is ubiquitously expressed in eukaryotes and has a highly conserved identity (~90%) across species.” True or False?
True
G-actin exists in multiple isoforms with slight sequence variations allowing for differences in function and tissue specificity. Give 3 examples.
Alpha-G-actin is expressed in vertebrate contractile muscles.
Beta-G-actin and Gamma-G-actin are expressed in non-muscle cells.
Describe the structure of G-actin with reference to its lobes and domains and state the functional importance of these structures in terms of actin polymerisation.
G-actin is structured in 2 lobes, with a total of 4 domains, I-IV. A hinge between domains I and III allows the two lobes to move relative each other, forming a nucleotide binding cleft.
This nucleotide binding domain, NBD, permits ATP binding. When ATP binds, the NBD folds, stabilising it’s structure. This allows self-assembly of G-actin into F-actin.
G-actin is able to self-assemble to F-actin freely, without the use of ATP. True or False?
False. G-actin is an ATPase and requires binding of ATP to induce a stabilising conformational change initiating actin polymerisation.
An actin trimer forms, serving as the centre for actin assembly, to which continuous addition of monomers occurs. What is the name of the process forming an actin trimer?
Nucleation - three monomers of G-actin forming an actin trimer nucleus.
The process of actin polymerisation is dependant on the concentration of G-actin. True or false?
True. Actin polymerisation will only occur if [G-actin] is at its threshold.
What is the meaning of “critical concentration” in actin assembly?
The critical concentration is the threshold concentration of G-actin at which actin polymerisation begins. Below the threshold, no actin polymerisation occurs.
The rate of filament assembly increases exponentially with increasing free subunit concentration. True or false?
False. Filament assembly rate increases linearly with [G-actin]
Actin polymers are polarised, with polymerisation occurring at different rates at either end.
Describe what is occurring at each end and explain the difference in rates.
- Affinity of ATP-actin to associate with other ATP-actin monomers is greater than affinity for ADP-actin.
- Higher rate of polymerisation of ATP actin at + end as associates more readily with neighbouring subunits.
- Lower rate of polymerisation of ADP actin at - end as dissociates more readily from neighbouring subunits.
What causes the difference in affinities in actin monomers at the + end versus actin monomers at the - end?
Actin at the + end is ATP-bound, which has a different confirmation from actin at the - end which is ADP-bound. The difference in conformation results in different binding affinities.
What is treadmilling?
The phenomenon where free G-actin/ATP-actin is being polymerised at the + end at a rate that is equal to the dissociation of ADP-actin at the - end.
One dissociated, ADP is exchanged for ATP which can then depolymerise start the + end and the cycle continues.
There is no net gain or loss in the length of the filament but a force is generated allowing movement.
G-actin is an ATPase with relatively high enzymatic activity. True or false?
False. It is a ATPase but has very low activity. The rate of ATP hydrolysis, and the release of the inorganic phosphate, is quite slow.
The longer it has been since a G-actin monomer was polymerised onto the filament, the more likely it is to have hydrolysed it’s ATP and have ADP bound
What are the critical concentration values for actin polymerisation?
[G-actin] > 1.0uM
polymerisation occurs at both ends but faster at + than -
0.1uM < [G-actin] < 1.0uM polymerisation only occurs at + end.
[G-actin] < 0.1uM
polymerisation does not occur.
Filament extension actively reduces the concentration of free G-actin
What is the leading edge?
Actin bundles form at the leading edge of the cell, and is in the direction the cell is moving in.
Where is the tail in relation to the leading edge?
The tail is present on the opposite side of the leading edge.
Stress fibres stretch from actin bundles at the leading edge to the tail. True or False?
True.
What are filopodia?
Fine cytoplasmic extensions/ruffles formed at the leading edge that act as an antennae for the cell to probe the environment.
They are typical of slower moving cells.
What are lamellipodia?
Broad membrane protrusions formed at the leading edge, typical of rapidly migrating cells.
What are ruffles?
Highly dynamic assemblies of micro-filamentous structures that do not form tight adhesions with the substrate.
Ruffles include filopodia and lamellipodia.
What is the substrate in terms of cell motion?
The surface/media that the cell is moving on/through.
What is the role of focal adhesions in cell migration?
Focal adhesions provide a mechanical link between the intracellular actin filaments and the extracellular substrate.
