7.2 Microfilaments I Flashcards
What are cytoskeletons required for?
Cell shape
Internal organization
Cell behavior
How are cytoskeleton destroyed and what are the effects of destroying it?
Drugs/chemical and Injury can destroy cytoskeleton like ischemia and when cytoskeleton is destroyed cells become spherical and they lose their attachments to each other
What is the form of actin when polymerized (assembled into filaments) ?
A single chain of monomers that has a shallow twist to it. It is called F-actin for filamentous actin.
What is the form of actin when not assembled into filaments? Where is it found?
Found on monomers in cells It is called G-actin for globular actin
What is the structure of actin?
Small globular protein (round), about 40kilodaltons in molecular weight. Small.
What happens when G-actin is bound to ATP? What about ADP?
G-actin bound to ATP can easily assemble into actin filaments
G-actin bound to ADP can’t assemble into filaments very well.
How do we know that prokaryotes also have cytoskeletal filaments?
Because prokaryotes contain proteins that may be evolutionary actin precursors and they resemble those found in eukaryotic cells. Mutation in these proteins affects the shape of bacteria
In a test tube, G-actin assembly is dependent on what?
salt concentration that is sufficient to promote assembly
What happens during the lag phase (nucleation)? AKA why don’t G-actin assemble into filaments immediately after adding sufficient salt concentration?
Lag phase period is a period in which no filaments are formed even though conditions for assembly are correct. Lag phase occurs during the period in which there are very few nucleation sites
What happens during a lag phase is that monomers interact w/each other, but for the most part, G-actin monomers form dimers. Dimers are unstable and fall apart.
When a dimer is able to add one more G-actin subunit, making a trimer, the resulting trimer is stable and can act as a nucleation site for additional growth and of the actin filament
What does the rapid growth phase (elongation) in G-actin assembly represent?
The addition of G-actin subunits to nucleation sites that have already been formed. (trimers formed in lag phase)
addition of g-actin subunits to trimers formed in lag phase
How can the role of nucleation be demonstrated?
By adding nucleation sites to a solution of G-actin at the start of the experiment. In this case, actin assembly begins as salt concentration is adjusted, and there is no lag phase.
Why is it good for a cell if actin assembly doesn’t occur even when all of the conditions for assembly are right?
b/c cells have control over where actin filaments assemble because of this, and they can provide nucleation sites only in specific areas of the cell where they want actin assembly to occur.
For example, leukocyte cells produces nucleation sites for actin assembly only at the front of the cell in the direction that it wants to move in
In G-actin assembly, why does assembly reach a steady state (equilibrium phase)?
This happens when the concentration of free G-actin falls to some critical value known as critical concentration (Cc). If the concentration of available G-actin is above the Cc, more growth occurs. If the concentration of G-actin falls below the Cc than existing filaments will shrink.
What is a critical concentration?
It’s the concentration of actin monomer at which a filament end exactly balances growth and de-polymerization. Cc is different value in different situations.
Aka where the rate of assembly is the same as the rate of hydrolysis for whatever end of the filament you’re looking at, and if the rate of addition is slower than the rate of ATP hydrolysis, the filament end will disassemble
In actin filament what is the pointed end? What is the name of the other end?
The direction that the myosin arrows are pointing (- end)
The barbed end of the filament (+ end)
Why is the barbed end of a filament called the plus end?
b/c it’s the side of the filament that grows the fastest
the rate of assembly of the plus and minus ends are both a function of what?
the concentrations of actin and the rates are different for each end.
What are the effects of ATP hydrolysis on the assembly of actin filaments plus and minus ends?
G-actin-ATP assembles readily but with ADP it does not assemble well, and can disassemble easily.
When an actin filament grows the growing tip of the filament has only ATP bound actin subunits attached to it.
After a short time the actin subunits hydrolyze the APT and so the subunit now has ADP bound to it
As long as the ADP subunits are held in the filament by their neighbors, they will remain in the filament.
If one of these subunits finds itself at the end of a filament it will disassemble out of the filament.
So when a filament is growing that means that the rate subunit addition at the end has to be greater than the rate of ATP hydrolysis by the assembled subunit.
What happens when the rate of addition of g-actin subunits is slower than the rate of ATP hydrolysis?
The filament end will disassemble
What happens when the rate of assembly at the plus end is greater than that of ATP hydrolysis but the rate of assembly is slower at the minus end?
Assembly is going to occur at the plus end, while at the same time, disassembly is going to occur at the minus end.
When does a filament exhibit treadmilling?
when one end of the filament grows while the other end of the filament shrinks
Subunits don’t change their position even though filaments appears to be moving from left to right. This is fundamental behavior of actin filaments
In filaments, fluorescent speckle microscopy allows tracking of what?
Allows you to track the position of individual filaments when they are grouped too closely together to otherwise see single filaments.
Which proteins interact with G-actin monomer and change how they assemble (regulate assembly directly)?
Profilin- promotes assembly
Thymosin- antagonistic to profilin
How does profilin interact with G-actin?
Binds to the end of G-actin monomer in a way that makes it easier for subunits to add to the filament.
It also binds to the end that would become the new plus end. it does not bind to the side of the monomer where it attaches to old filaments.
How does thymosin intreact with G-actin?
It binds to the part of the G-actin monomer where it would normally attach to a filament and so it prevents assembly of that monomer
Thymosin inactivates g-acting by binding to it (it’s a g-acting sequestering protein)
In addition to concentration of G-actin monomers, how else do cells regulate the balance of assembly and disassembly?
Proteins such as profilin and myosin
Apart from prolifin, what other proteins assist in actin assembly? What type of proteins are they?
Proteins that promote nucleation! Actin nucleation proteins are called actin related proteins (ARP) 2 and 3, aka ARP 2 and ARP 3
Structure of these proteins is very similar to that of G-actin and as a result when ARP 2 and 3 form complex in cells along w/a number of accessory proteins they act as a nucleation site for actin filament growth.
Apart from promoting actin assembly what else can Arp2 and Arp 3 do?
They can attach to the sides of an existing actin filament at an angle. When the attached ARP 2,3 complex nucleates a new filament it creates a y-shaped branching structure.
This gives actin filaments the ability to form 3-dimensional branching structure in cells and increases the complexity of shapes that a cell can create
How does gelsolin cause the disassembly of actin filaments?
It CAPS and CUTS actin filaments.
Gelsolin is activated by calcium and its activity cuts or breaks actin filaments. One of the cut ends is capped by gelsolin (can’t be used for further assembly unless gelsolin is disassociated). The two actions (cutting and capping) are how gelsolin causes actin filament networks to disassemble.
What does cofilin protein do?
It promotes actin disassembly
Cofilin assembles along the length of actin filaments and causes actin filaments to unwind slightly.
Actin filaments are twenty times less stable in the presence of cofilin
What is the difference between gelsolin and cofilin?
Cofilin doesn’t actually disassemble (it makes it much likely to disassemble on its own)
While gelsolin actually disassembles by cutting and capping.
What is capz?
Capz caps the plus end of the actin filament and prevents both assembly and disassembly at that end.
In the presence of capz, actin assembly occurs only at the minus end.
What does z-line do?
Forms an attachment point for stable actin filaments found in muscles cells and the protein capZ is a major component of that structure.
Which proteins promote assembly of actin filament
Profilin
ARP 2 and ARP 3
Which proteins are associated with disassembly or actually disassembly proteins?
Cofilin (promotes)
Gelsolin
Thymosin
Which protein stabilizes filament ends? Which side of the filament does it cap?
capZ and it caps the plus side. When it caps the plus side only the minus end will grow. When it’s not capped both minus and plus end grow
