Module 19 - Cytoskeleton Flashcards
List the diverse roles played by the cytoskeleton
- Mitosis
- Cell shape
- Cell migration and motility
- Intracellular trafficking
- Supports membranes
- Mechanically links adjacent cells
- Muscle contraction
Describe the common principles underlying the construction of all three major classes of cytoskeletal filaments
- Filaments are composed of repeating subunits.
- Intermediate filaments are formed from multiple protofilaments which bind laterally to each other.
- Actin and tubulin protofilaments assemble by head-to-tail binding of monomers.
- These cytoskeletal filaments are dynamic and adaptable
Describe the construction of intermediate filaments and appreciate how this construction gives mechanical strength to those filaments
Intermediate filaments are constructed from elongated protein subunits (protofilaments). These protofilaments are two antiparallel arrangements of dimers (tetramer) (extended α-helix); hence each end is identical (no polarity). Staggered arrangements of these protofilaments, held on by hydrophobic interactions, gives intermediate filaments rope-like properties and its strength.
Describe how actin filaments are constructed from actin subunits and why actin filaments are polar
Actin subunits (G-actins) assemble head to tail creating polar actin filaments (F-actin).
Actin protofilaments comprise two parallel filaments of actin monomers assembled end to end. Since each globular actin monomer has a positive and negative end, the protofilaments are polarized. Each monomer also has an ATP binding site.
Explain how actin filaments are assembled into complexes in cells and the assembly affects the functions of such complexes
There are three types of actin pattern formation:
- Contractile Bundle (Stress Fibres): contractile and exert tension
- Tight Bundle (Filopodia): allows extension
- Gel-like network (lamellipodia): supports plasma membrane and allows cell extension
F-actin self-assembles from actin subunits. Binding of actin monomers to the filament requires the hydrolysis of ATP to ADP in the ATP binding site. The rate of elongation in the positive end is much faster than the negative end; a conformational change is required for the monomer to bind to the negative end.
Polymerisation/Depolymerisation of these filaments may lead to extensions/contractions of the actin, which can be used to fo mechanical work in cells.
Explain how certain factors regulate actin polymerisation and depolymerisation
The concentration of free monomers is a factor that regulates polymerisation/depolymerisation of actin. Without sufficient G-actin monomers, the unstable Actin-GDP will dissociate from the filament and initiate depolymerisation. At a critical concentration, the rate of elongation will equal to shortening (treadmilling).
The concentration of local available G-actin monomers is regulated by Thymosin. Thymosin binds G-actin and prevents addition to either end of the F-actin. presence of Thymosin reduces local G-actin concentration.
Another regulatory factor is profilin, which binds to G-actins as well. In this case, the profilin-actin complex can bind to F-actin, but only to the plus-end, promoting rapid growth in that end.
Graphically show how nucleation is responsible for the lag phase in actin filament growth
Filament stability depends on the number of H-bonds between the G-actin subunits; small filament assemblies are unstable, while larger ones are more stable. Hence, filament formation is dependent on the unlikely/slow event of ‘nucleation’, where a sufficient number of monomers bind together to form a stable complex.
The progression of actin filament formation is shown in the graph below. The nucleation (lag phase) is shown as the rate-limiting step in F-actin formation.
What are ARP complexes? Describe their function.
ARP complex has a structure that is similar to actin. ARP2 and ARP3 (along with activating factors and other proteins) form a complex at which actin monomers can grow from the plus-end.
When the ARP2/3 complex binds to an existing actin filament, it will promote nucleation/branching of actin filaments at a 70-degree angle.
Describe the cycle of cellular events that underlie cell locomotion and the cytoskeletal rearrangements and cell-substrate adhesion during those events
- Protrusion of filopodia: by actin polymerisation (elongation of filament)
- Attachment: actin filaments connected (focal contacts that contains integrin) by TM proteins to the substratum
- Traction: contraction by myosin-actin interactions (rearranging actin cortex) move the rear of the cell forward.
- Focal contacts need to be assembled and disassembled
Describe the general structure of myosin proteins.
It consists of a coiled-coil of two alpha helices along with two heads of the N terminus, which are connected by linker protein chain.
Describe an example of how external molecule guide cells and axons by binding to cell surface receptors.
An example of this interaction is the interaction between neutrophil and bacteria. Bacteria produce chemoattractant, which binds to the leading edge’s surface G protein-coupled receptor.
This then activates 2 pathways:
- Rac –> which regulates actin polymerisation (lamellipodial extension)
- Rho –> regulates myosin activity (stress actin fibres)
These two pathways are mutually antagonistic.
Explain how microtubules are constructed from tubulin monomers and why microtubules are polar
Tubulin subunits assemble head to tail to create a polar protofilament. The subunit is a heterodimer made up of an α-tubulin (- end) and β-tubulin (+end). 13 protofilaments are required to constitute a microtubule, forming a tubular structure.
Explain the molecular basis for dynamic instability of microtubules and how this property can be regulated to make microtubules more or less unstable
Proteins that bind to microtubule ends can affect MT stability. Microtubule-associated proteins (MAPs) stabilize MT, while catastrophe factors (kinesin 13) destabilized MT.
Presence of GTP in β-subunit (GTP-capped end) promotes rapid growth of tubulin filament. However, accidental loss of GTP cap (through hydrolysis to GDP) makes MT more unstable and initiates depolymerisation (catastrophe). If the + end regain the GTP cap, polymerisation will be reinitiated (rescue)
Explain why nucleation is responsible for the lag phase in microtubule growth and how cells can eliminate that lag phase using preformed nuclei
Just like actin, initial polymerisation of microtubules is unstable unless there is a nucleation centre. MT nucleate at the γ-tubulin ring complex via their minus end (α), while allowing rapid polymerisation at their plus ends (β ).
What are centrosomes?
The centrosome is the major microtubule organising centre in animal cells. It contains as many as 50 γTuRCs. These complexes can be transported to various parts of the cell to initiate polymerisation at various sites.
Explain how different types of motor proteins mediate movement along microtubules
Kinesins and Dyneins are the microtubule motor proteins. These motor proteins attach themselves to MT through catalytic units (N-terminal), which is linked to organelles/vesicles at the other end of the coiled-coil chain.
Both motor proteins generate force by coupling ATP hydrolysis to conformational changes.
Remember: Kinesins drive (+/β) end-directed movement, while Dyneins drive (-/α) end-directed movement.
