Microfilaments

What is cytokinesis and how does it occur?

The division of the cytoplasm after mitosis has occurred; it occurs via the purse-string model in which pulling forces pull the outside of the cell towards the center; these pulling forces are due to myosin II sliding actin microfilaments past each other, which contracts the contractile ring

Is cytokinesis also symmetric?

No, it can also be asymmetric

What are microfilaments composed of?

Actin

Do microfilaments have polarity?

Yes, there is a plus (barbed) end and a minus (pointed) end

How did researchers figure out the polarity of microfilaments?

They “decorated” the actin microfilament with myosin heads; they took myosin and cut it twice with trypsin until only the heads were left; they mixed these heads with the microfilament, and the heads binded to the microfilament; on and EM, this showed a clear pattern along the microfilament where on end is “pointed” and the other is “barbed” like an arrowhead

What is the structure of myosin?

It has two heavy chains wrapped around each other and they bunch up at the end to form two heads that bind to actin

Which end of the microfilament is barbed?

The plus end

Which end of the microfilament is pointed?

The minus end

What is F-actin vs. G-actin

F-actin is actin in the polymer (microfilament) and G-actin is the free actin monomer

How does actin polymerize into microfilaments?

G-actin subunits must reach Critical concentration for polymer assembly to begin; during assembly, ATP-bound subunits get added on to the barbed end of the microfilament; after these subunits get added, this ATP gets hydrolyzed into ADP and Pi, so the pointed end of the microfilament generally has ADP bound subunits

Once you reach critical concentration, what happens to the amount of monomer in the solution?

It plateaus because any new monomer produced is being added into the polymer

At critical concentration, what is true of the rates of assembly and disassembly of subunits?

The rate of assembly is equal to the rate of disassembly

Above critical concentration, what is true of the rates of assembly and disassembly of subunits?

the rate of assembly is greater than the rate of disassembly

Below critical concentration, what is true of the rates of assembly and disassembly of subunits?

the rate of assembly is lower than the rate of disassembly

Does actin bind to any nucleotide triphosphates?

Yes, actin monomers bind ATP in the center and act as ATPases, so they can hydrolyze this ATP into ADP

What type of molecule will eliminate the lag phase of polymerization without changing Cc?

A nucleator

When microtubules or microfilaments are at steady state, is their mass changing?

No, the overall mass of microtubules or microfilaments is constant at steady state, even though microtubules (and sometimes microfilaments) and shrinking and growing

Do microfilaments undergo dynamic instability?

No

Does assembly occur at the pointed or barbed end of a microfilament?

It can occur at either end because the Cc’s of either end are very close to each other, but it occurs more quickly at the barbed end because the Cc is lower at the barbed end than it is at the pointed end

How does Kd related to the assembly of microfilaments?

the Kd = free ATP-bound subunit / ATP-bound subunit in microfilament; at the barbed end, the Kd is lower, meaning there is more ATP-bound subunit in the microfilaments; at the pointed end, the Kd is higher, meaning there is more free ATP-bound subunit; remember that low Kd is high affinity and high Kd is low affinity → ATP-bound subunits have higher affinity for barbed end than for pointed end; so Kd can approximate Cc for the barbed and pointed ends

How does treadmilling occur in microfilaments?

When concentration of actin monomers is above Cc of barbed end but below Cc of pointed end, there is no net growth because even though subunits are being added onto the barbed end, subunits are also being lost at the pointed end, so the rate of assmebly = rate of disassembly and the length of the microfilament remains constant

What concentration of actin subunits is necessary for a microfilament to grow in length?

The concentration of actin subunits must be greater than the Cc of both the pointed and barbed ends of the microfilament

Do microfilaments typically treadmill?

Yes, microfilaments in cells are often treadmilling because concentration of actin is typically in between Cc of barbed end and Cc of pointed end

What are some examples of what Actin associated proteins can do to regulate microfilaments?

• They can stabilize microfilaments → bundling, cross-linking

• They can destabilize microfilaments → severing (cofilin), disassembly

• They can sequester monomers → thymosin

• They can link microfilaments to other structures

• They can act as nucleators → formin

What is the motor protein for actin microfilaments?

Myosin

What are some characteristics of Myosin?

There are 24 superfamilies, all bind actin, but can have different functions; they all have barbed-end (plus-end) directed movement (except for one class); myosin is processive, meaning they can remain attached to microfilaments for a long time

How does myosin “walk”?

The Cross-Bridge Cycle; a myosin head is attached to the microfilament; this association is strong when there is no ATP bound to the myosin (nucleotide free); when ATP bind to myosin, the head detaches from the microfilament and undergoes a conformational change; the ATP is then hydrolyzed and Pi is released and the ADP bound myosin reattaches to the microfilament; the ADP is then released and the myosin goes back to its original conformation, this is called the power stroke and it moves the microfilament

How does myosin II work?

It tends to fold up on itself, but if myosin light-chain kinase (MLCK) is present, it will phosphorylate the light chains of the myosin II at the base of the heads, and this results in the myosin II assuming an elongated structure; the tail domains of this elongated myosin II can interact with other tail domains of other myosin II in an antiparallel fashion, forming bipolar filaments of myosin, called “thick filaments”

How do myosin II thick filaments interact with actin thin filaments during cytokinesis?

The actin filaments are attached to the plasma membrane; the myosin II filament sits between the thin filaments and each head walks towards the barbed end of opposite filaments, sliding the filaments past each other and decreasing the diameter of the contractile ring

How do myosin II thick filaments interact with actin thin filaments in sarcomeres during muscle contraction?

There is a central thick filament surrounded by many thin filaments, forming an array; these thin filaments are held in place by a Z disc (made of Z proteins) that associates with the barbed ends of the microfilaments; the myosin II heads want to walk towards the barbed ends, bringing the Z discs together in a contractile motion

What is the minimum structure of a sarcomere?

At least 2 thin filaments and 1 thick filament

What is the order of structure in muscle cells?

many sarcomeres bundle together to form a Myofibril → many myofibrils bundle together to form an Individual Muscle Cell → many muscle cells bundle together to form a bundle of muscle fibers → many muscle fibers bundle together to form a muscle

How is muscle contraction coordinated and regulated?

On actin, the binding site for myosin is blocked by a protein called tropomyosin; the position of tropomyosin is regulated by the troponin complex; when a muscle contraction occurs, an intracellular signaling process occurs that released Ca2+; this Ca2+ binds to the troponin complex, causing tropomyosin to move away from the myosin binding site on the actin, allowing the myosin II to bind to the actin

In the presence of Ca2+, is the myosin binding site of actin free or blocked?

It is free in the presence of Ca2+

How does the Ca2+ get released during muscle contraction?

It is released from the endoplasmic reticulum of the muscle cells (sarcoplasmic reticulum) when the sarcoplasmic reticulum senses voltage changes in the plasma membrane of the muscle cell; it can do this because the plasma membrane of muscle cells dives deep into the cell such that it is sitting right next to the sarcoplasmic reticulum

What are some functions of microfilaments?

• Organelle Transport → the microfilaments act as side streets, whereas microtubules are the superhighways

• Cytokinesis → purse-string model

• Maintain cell structure → makes up microvilli and stereocilia (sensing of sound waves in ear)

• Cell motility → contraction of rear end of cell (stress fibers) and branched actin network at front of cell

• Morphogenesis