lecture 10 cell bio: microfilaments

microfilament

One of the three fiber networks that compose the cytoskeleton, also called actin fibers and Involved in a wide range of cellular processes, including cell shape, movement, muscle contraction, cytokinesis, and cytoplasmic streaming.

microtubule

One of the three fiber networks that compose the cytoskeleton, play crucial roles in cell division, intracellular transport, and the formation of cilia and flagella.

intermediate filament

One of the three fiber networks that compose the cytoskeleton, Provide mechanical strength and support to cells and tissues, particularly in areas subject to mechanical stress.

G-actin

is the monomer form of actin, also called globular actin. Each actin protein can bind either ADP or ATP, and the negative charge of these molecules is countered by a Mg2+ ion

F-actin

is the polymerized form of actin, also called filamentous actin

pointed end

is the "-" end, which has an exposed ATP binding cleft.

barbed

is the "+" end, which has a protected ATP binding cleft, and is the site of more rapid monomer addition

nucleation

he first phase of actin fiber assembly, occurs when 3-4 actin monomers assemble into an unstable 'seed'

elongation

The second phase of actin fiber assembly, is the rapid addition of actin monomers to both ends of the fiber

catastrophe

The rapid depolymerization of a microfilament, often occurring when the filament loses its ATP:F-actin cap

critical concentration (Cc)

is the concentration of G-actin monomers at which filament growth or shrinkage occurs. There are two critical concentrations to consider for actin filaments: Cc+ and Cc-. At G-actin concentrations below Cc +, there is no filament growth. At G-actin concentrations between Cc + and Cc-, there is growth on the + end and loss at from the - end. At G-actin concentrations above Cc -, there is growth on both ends.

treadmiling

describes the dynamic behavior of actin filaments at steady state, where G-actin is added to the + end as fast as it is lost from the – end, resulting in a constant filament length

B-thymosin

Binds to G-actin monomers, preventing microfilament end addition. Sequesters G-actin in cytoplasm of rapid polymerization upon release.

profilin

binds to the + end of a G-actin molecule, preventing it from binding to the – end of a microfilament but allowing it to bind to the + end

gelsolin

breaks microfilaments and remains associated with the + end, preventing the addition or loss of actin monomers

CapZ

binds to the + end of a microfilament and prevents further elongation

Tropomodulin

binds to the - end of a microfilament, and its binding is enhanced by tropomyosin

Formin

is a nucleation factor that aligns two G-actin monomers and then recruits a third to create a nucleus for filament growth. It remains associated with the microfilament and promotes the addition of G-actin to the + end.

Arp2/3 complex

associates with an existing microfilament to nucleate a new microfilament that branches off at a 70 degree angle.

head

of a myosin protein possesses ATPase activity and is able to bind to actin microfilaments

neck

of a myosin protein is flexible and is bound by accessory proteins called light chains

tail

of a myosin protein interacts with other myosin tails and/or connects to a select cargo

ATPase

is an enzyme that catalyzes the hydrolysis of ATP to ADP and inorganic phosphate, releasing energy that can be used for cellular processes.

light chain

are accessory proteins that bind to the neck of a myosin protein and regulate its activity, specifically by influencing the conformation and flexibility of the neck region.

● The myosin neck acts as a lever arm that amplifies the small conformational changes in the head domain during the ATPase cycle into larger movements.

● The flexibility of the neck region is crucial for the power stroke, which is the force-generating step in myosin's movement along an actin filament.

● The myosin light chains can modulate the stiffness and length of the lever arm, thereby affecting the step size and force generated by the myosin motor.

processive

motor protein takes multiple steps along its track without detaching.

steady state

The third phase of actin fiber assembly, is reached when the actin monomer concentration decreases to the point that addition of monomers equals dissociation from fibers

Cofilin

binds to ADP F-actin, distorting the filament and causing it to break

Given Cc of the two different microfilament ends and the concentration of G-actin in solution, define which ends will elongate, catastrophe, or remain in steady state

● At G-actin concentrations below Cc+: There is no filament growth at either end.

● At G-actin concentrations between Cc+ and Cc-: There is growth on the + end and loss at the - end. This is because the concentration of G-actin is high enough to promote addition at the + end, but not high enough to overcome the inherent instability of the - end.

● At G-actin concentrations above Cc-: There is growth on both ends. This occurs because the concentration of G-actin is sufficiently high to drive addition at both the + end, which has a higher affinity for G-actin, and the - end.

Explain how myosins select their cargo.

● Tail Domain Interactions: . Different myosin isoforms have distinct tail domains that may contain specific binding sites for particular cargo proteins or lipids. Ex: myosin V, which transports organelles, is known to interact with specific receptor proteins on the surface of organelles.

● Adaptor Proteins: These proteins bind to both the myosin tail domain and the cargo molecule, bridging the two components.

● Regulation by Light Chains: different myosin light chains can modulate the conformation and flexibility of the myosin neck, which may affect its ability to bind and transport specific cargo molecules.

● Post-Translational Modifications: Post-translational modifications, such as phosphorylation, can alter the binding affinity of myosin for its cargo.

Define the direction microfilaments are oriented in a cell and the direction most myosins travel on the microfilaments.

Myosin motor proteins, with the exception of myosin VI, move directionally along microfilaments, always traveling from the minus (-) end towards the plus (+) end.

● Muscle Contraction: myosin II forms thick filaments that interact with actin thin filaments. The sliding of these filaments past each other, powered by myosin II's movement towards the plus end of actin, leads to muscle contraction.

● Cytokinesis: During cell division, a contractile ring composed of actin and myosin II forms at the cleavage furrow. The contraction of this ring, driven by myosin II's movement towards the plus ends of actin filaments, pinches the cell in two, completing cytokinesis.

● Organelle Transport: Myosin V is a processive motor protein that transports organelles along actin filaments. By moving towards the plus end of actin filaments, myosin V can carry cargo to specific destinations within the cell.

● Cell Migration: Actin polymerization at the leading edge of a migrating cell pushes the plasma membrane forward, while myosin motors are thought to pull the cell body forward by interacting with actin filaments. The precise roles of different myosin isoforms in cell migration are still being investigated.

defines 5 steps in the myosin power stroke

● Step 1: Myosin is attached to actin in a "rigor" state. In the absence of ATP, myosin binds tightly to actin. This is the state of muscle after death, called rigor mortis.

● Step 2: ATP binding causes myosin to detach from actin. Binding of ATP to the myosin head group reduces its affinity for actin, causing the myosin head to release from the actin filament.

● Step 3: ATP hydrolysis cocks the myosin head. The hydrolysis of ATP into ADP and inorganic phosphate (Pi) remains bound to the myosin head. This hydrolysis event triggers a conformational change in the myosin head, "cocking" it into a high-energy state.

● Step 4: The cocked myosin head reattaches to actin. The cocked myosin head, with bound ADP and Pi, now has a higher affinity for actin and reattaches to the actin filament at a new site, further along the filament.

● Step 5: The power stroke occurs. The release of Pi from the myosin head initiates the power stroke, a force-generating conformational change in the myosin neck region that pulls the actin filament towards the minus end

Actin Polymerization Pressing on a Membrane

Cells can extend membrane protrusions by assembling actin filaments at the leading edge. The force generated by the polymerization of actin monomers into filaments pushes against the cell membrane, causing it to extend outward. This mechanism is similar to the one used by the bacteria Listeria to propel itself through the cytoplasm of infected cells.

Myosin Hauling Cargo

Myosin motor proteins, such as myosin V, can transport cargo, including organelles and vesicles, along actin filaments. These myosin proteins bind to their cargo through their tail domains and "walk" along actin filaments towards the plus (+) end, using energy from ATP hydrolysis. This directed movement of myosin effectively hauls the attached cargo to specific locations within the cell.

Contractile Rings

During cell division, a contractile ring composed of actin filaments and myosin II assembles at the cleavage furrow. The myosin II motor proteins slide along the actin filaments towards their plus (+) ends, causing the ring to contract and pinch the cell in two. This process is essential for the completion of cytokinesis, the final stage of cell division.

cytoplasmic streaming

In some algae, myosin V motor proteins drive cytoplasmic streaming by continuously pulling membrane vesicles in a circular path along actin filaments. This coordinated movement of vesicles creates a bulk flow of cytoplasm within the cell. Cytoplasmic streaming facilitates the rapid distribution of materials, such as nutrients and organelles, throughout the cell.