Lecture 17 Ch 9 (Cytoskeleton III, motor proteins)

If we make neck of myosin V longer, will we decrease or increase efficiency? Efficiency: For every ATP that is hydrolyze it will make it further down the microfilament.

Increase. For every ATP that is hydrolyzed, it will have a bigger step.

What is responsible for the outward movement of membranous organelles?

Kinesin

What determines step size?

Neck length

Define and characterize processivity of kinesin: explain why kinesin demonstrates high processivity by relating to the events of one motor cycle

Kinesin demonstrates high processivity because its molecule never ceases to have contact with the tubulin in the events of one motor cycle. CAN GO ON FOR LONG STRETCHES AND THE PROTEIN WILL NOT DIFFUSE AWAY - HIGH PROCESSIVITY: MOTOR PROTIEN CAN TRAVEL LONG DISTANCES WITHOUT LOSING CONTRACT WITH CYTOSKELETAL HIGHWAY)

Describe the molecular features of kinesin

Kinesin: ANTEROGRADE TRASNPORT. Kinesin only travel towards the (+) end. But how does it know? Kinesin is recognizing the shape of alpha-beta, alpha-beta in the microtubule. Its shape complementary only works this way! - Head domain: Force generating head that binds to the microtubule and catalyze ATP hydrolysis. - Neck domain: Neck is known as the mechanical element - Tail domain: Tail binds to the cargo being transported with specificity o Motor domain couples ATP hydrolysis to movement along microtubule. o Carbo binding domain binds vesicles and organelles

Very generally, relate the kinesin mechanism/structure to that of dynein and myosin motors.

Mechanism: (Kinesin, Dynein and Myosin) - Convert energy from ATP into mechanical energy - Move unidirectionally along their cytoskeletal track - Accomplish this via conformational changed powered by ATP hydrolysis - CARGO BINDING DOMAINS:bind vesicles and organelles - MOTOR DOMAINS: couples ATP hydrolysis to movement along cytoskeletal (kinesin and dynein: microtubule + myosin: microfilament) Motor proteins pass of cargo at cellular periphery Structure: Heads Neck in both kinesin and myosin, except dynein Tail Stalk in both dynein and kinesin except myosin While general structure of motor proteins, the exact code for domain may vary. Motor domains are more conserved, but tails are different because it depends on what cargo it bind to it. Neck length determines step size.

Location and function of motor domain, mechanical elements, and cargo binding domains of Myosin V: Identify which of these domains binds ATP, microtubules, microfilaments, vesicles or organelles

Motor domain: Couples ATP hydrolysis to movement along microtubule -Head: Binds to actin and catalyzes ATP hydrolysis -Neck:mechanical element Cargo binding domain: Binds vesicles and organelles - Tail: Binds to the cargo being transported in specificity

Location and function of motor domain, mechanical elements, and cargo binding domains of Kinesin: Identify which of these domains binds ATP, microtubules, microfilaments, vesicles or organelles

Motor domain: Couples ATP hydrolysis to movement along microtubule -Head: Binds to microtubule and catalyzes ATP hydrolysis -Neck: mechanical element Cargo binding domain: Binds vesicles and organelles - Tail: Binds to the cargo being transported in specificity

Location and function of motor domain, mechanical elements, and cargo binding domains of Dynein: Identify which of these domains binds ATP, microtubules, microfilaments, vesicles or organelles

Motor domain: Couples ATP hydrolysis to movement along microtubule -Stalk: Binds to microtubule and catalyzes ATP hydrolysis -Head: Binds to ATP (mechanical element) Cargo binding domain: Binds vesicles and organelles - Tail: Binds to the cargo being transported in specificity

Relate the helical structure of actin to the step size of myosin V

Myosin V has a really long neck, it has a step length that matches the distance required for the actin helix to repeat itself. It sees the same part actin helix, step by step - meeting the actin meeting the actin as it twists. (The actin helix repeats itself about 13 subunits - 36nm - which is just about the same step size of a myosin V molecule)

Explain why the myosin motor domain is highly conserved whereas significant variability exists in the cargo binding domains of various myosins

Myosin motor domains are highly conserved because specific genes code for them. However, they have significant variability existing in the cargo binding domains. This is because among myosin genes, motor domains are conserved but tail domain diverse because they can carry different types of cargo.

Kinesin: type of cytoskeletal element along which they move; does it only move in one direction or in both directions along cytoskeletal element

Such plus end directed motor protein accomplished movements along microtubules o Kinesins move pigment containing vesicles in anterograde direction along microtubules

Relate the pattern in neck size of motor proteins to the anticipated step size/rate of movement; predict the impact of lengthening or shortening neck size on step size/rate of movement

The length of the neck determines step size. Example: Myosin II, V, VI. If we make the neck of Myosin II longer, will we increase their efficiency of the speed of transport, or will we decrease? We will increase, because for every ATP that tis hydrolyzed the further it will travel. EFFICINECY MOVING FORAWARD, THE LONGER THE NECK THE LONGER THE STEP SIZE

Retrograde transport:

Toward minus end

Describe the directionality by which kinesin, dynein and myosin V traverse cytoskeletal highway

Unidirectional Microtubules Kinesin: Anterograde, towards the positive end. Dynein: Retrograde towards the negative end. Microfilaments Myosin V: They typically travel towards the positive end of actin filaments. However, overall, there are several different types of myosin that move to opposite directions of each other, so they can move to either positive or negative end.

Predict the impact of inactivating dynein mutations on spindle formation and chromosomal movement during mitosis or movement of vesicle components from the plasma membrane to the Golgi

inactivating dynein mutations would cause phenomena to transport in a retrograde fashion. On the other hand, activating dyne mutations: endocytotic vesicles as well as organelles and chromosome movement would be disrupted

Explain the findings presented in Figure 9.10

members of the kinesin superfamily tend to move vesicles and organelles (e.g., peroxisomes and mitochondria) in an outward direction toward the cell's plasma membrane. This is illustrated in the micrographs in FIGURE 9.10. The pair of micrographs labeled a and c shows a cell that was isolated from a normal 9.5‐day mouse embryo and stained to reveal the location of its microtubules (green) and mitochondria (orange). The pair of micrographs labeled b andd shows a cell that was isolated from a 9.5‐day mouse embryo lacking both copies of the gene that encodes the KIF5B kinesin heavy chain (a kinesin‐1 family member). The mitochondria of the KIF5B‐deficient cell are absent from the peripheral regions of the cell, as would be expected if this plus end‐directed kinesin is responsible for the outward movement of membranous organelles

Why are motor proteins considered enzymes?

-Motor proteins are considered to be enzyme because they power the movement along the cytoskeletal elements.

Knocking out which gene would result in mitochondria being accumulated around the nucleus?

Mitochondria of the kinesin cell are absent from the cell's peripheral regions. Why? Because kinesin is responsible for moving mitochondria to the periphery of the cell.

Order the steps in the chemical cycle that power one motor protein 'step' of kinesin

Steps: - ATP bind to motor domain #1 - Hydrolysis of ATP - Release of Pi - Release of ADP

Dynein: type of cytoskeletal element along which they move; does it only move in one direction or in both directions along cytoskeletal element

Such minus ended directed motor proteins accomplished movement along microtubules (retrograde)

Predict the impact of inactivating kinesin mutations on subcellular localization of chromosomes, exocytic vesicles, and mitochondria in cells

inactivating kinesin mutations will lead to peripheral regions of the cell to contain chromosomes, exocytic vesicles and mitochondria?

Distinguish between anterograde and retrograde transport between the Golgi and, for example, the plasma membrane; compare and contrast the involved machinery and the orientation of the molecular "highway" in anterograde and retrograde transport

(Idea) - Kinesin traveling in microtubules travel in a anterograde transport, to the plasma membrane (+ end) - Dynein traveling in microtubules travel in a retrograde transport, back to the cell body (- end) Cytoplasmic dynein 2 drives minus-end-directed intraflagellar transport Cytoplasmic dynein 1 drives minus-end-directed cargo transport (transports membranes, mRNA, proteins, and viruses) Cytoplasmic kinesin drives plus-end-directed intraflagellar transport Golgi to the plasma membrane. (Kinesin) Vesicle coming back to the Golgi (Dynein)

Motor proteins

+Use cytoskeletal structures to accomplish movement of cell, cell protrusions, and material within the cell. - Transport materials from one cell compartment to another. o Ex: Neurotransmitters transported down the axon; RNA being moved to site of cellular outgrowth and microvilli; movement of microtubules of cilia and flagella to accomplish "beating"; sliding of actin filaments to accomplish cytokinesis, muscle contraction, plasma membrane morphology change, spindle formation.

Explain what is meant by "motor proteins convert chemical energy to kinetic energy"; Describe the dependence of motor protein activity on [ATP]

- Convert ATP to mechanical energy --> kinetic energy (movement) - In order to traverse the cytoskeleton dragging an entire mitochondrion behind it, motor protein will have to convert chemical energy form ATP into mechanical energy (kinetic energy).

Recognize cellular events in which each of the three types of motor proteins play critical roles

- Convert energy from ATP into mechanical energy - Move unidirectionally along their cytoskeletal track - Accomplish unidirectional via conformational changes powered by ATP hydrolysis Dynein and kinesin help move neurotransmitters down the axon until they are dropped off at microfilaments. In general microtubules are used for longer distance movement while microfilaments are used in the periphery. Myosin is most commonly known for its function in muscle contraction. All three are also used for general movement of raw material in the cell and throughout axons. Finally movement of microtubules is what causes the swinging in microtubules and flagella. Actin filaments play a role in cytokinesis and microtubules in metaphase and anaphase. Microtubules are also pivotal to chromosomal separation during anaphase.

Using pigment producing cells as an example, contrast the roles of microtubules/kinesin versus microfilaments/myosin in cargo transport

- Kinesin move pigment containing vesicle in anterograde direction along microtubules. - Actin monomers continually bind to the barbed end of each filament, treadmill through the body of the filament, and dissociate from the pointed end. This process is captured in the fluorescence micrograph of Figure 9.46c, which shows the incorporation of GFP‐labeled actin subunits at the barbed end of each filament. Microtubules are for the larger part of the cell while microfilaments are used for the minute movements in the periphery.

Unconventional myosin : type of cytoskeletal element along which they move; does it only move in one direction or in both directions along cytoskeletal element

1. Accomplishes movement along microfilaments o Myosin V moves toward (+) end in a hand over hand fashion o Other myosins move in different ways but + end directionality is common

Order the molecular events that define the stepwise movement of kinesin and the manner in which it is driven by ATP hydrolysis

1. We begin in the first panel, the end of the last cycle. We are left with domain #1 (dark green), bound to tubulin but it doesn't have any nucleotide associated with it (nucleotide binding domain empty). Behind it is domain #2 (light green); bound to ADP and is dangling free. 2. ATP enters the picture and binds to the empty domain #1 that is bound to tubulin; the binding of the ATP causes the neck region to swing domain #2 light green up ahead domain #1. (ATP caused a conformational change to kick the back-leg forwards). Mechanism: motor domain #1 conformation change elicits lever-like motion in neck, or mechanical element of motor domain #2 (motor domain swing forward) but it doesn't get associated with tubulin until ADP dissociates, and leaves. 3. ADP bound to motor domain #2 dissociate and leaves, thus allowing domain #2 to be associated with tubulin (microtubule). Now the light green domain is empty and can bind to tubulin. We have ATP bound domain #1, and empty domain #2. Momentarily both bound to the tubulin. 4. But now domain #1 hydrolysis ATP to ADP + Pi. After phosphate group dissociates, what was domain #1 is able to lift, and become domain #2. (process repeats)

Predict the impact of altering the amino acid composition of the stalk, head and/or tail of dynein, unconventional myosin, or kinesin on: type of cytoskeletal element used as a highway, efficiency/speed of transport, or the type of cargo carried by the motor protein.

Altering the head would alter the highway that it is bound to in kinesin and myosin, altering the head in dynein would altering the neck/ stalk in myosin and kinesin would alter its speed/efficiency while it would change the highway in dynein. Altering the tail in any of the three would lead to the vesicle or cargo that the motor protein carries, to change or not be able to bind.

Describe the molecular features of myosin

Conventional myosin's: Aid in muscle contractions Unconventional Myosin They traverse microfilaments. They are a lot of unconventional myosin, some are + directed some are - directed. Because there is a collection of such proteins, then movement can be accomplished as needed in either direction. - Head domain: Force generating head that binds to the microtubule and catalyze ATP hydrolysis. - Neck domain: Neck is known as the mechanical element - Tail domain: Tail binds to the cargo being transported with specificity o Motor domain couples ATP hydrolysis to movement along microfilament o Cargo binding domain binds vesicles and organelles

List functions associated with conventional myosin versus unconventional myosin

Conventional myosin: - Tissue distribution - Bipolar: it has the motor domains on both ends, and t can pull actin from each part of the muscle cell: muscle contraction!*** Unconventional myosin: - Movement along microfilaments (microfilament rearrangements) - Microfilament rearrangement - Not bipolar Myosin's in general: - Muscle contraction - Cytokinesis - Tension on membrane - Cargo transport at periphery - Tethering of organelles

- Which motor proteins travels from the plus end to the negative end of the microtubule?

Dynein

Describe the molecular features of dynein

Dynein: RETROGRADE TRASNPORT. Dynein only travels towards the (-) end. It recognizes the shape beta-alpha, beta-alpha in the microtubule. Its shape complementarity only works if it goes in a retrograde fashion. - Head: where ATP is hydrolyzed and where force is generated - Tail: where we get specificity with cargo - Stalk: binds microtubules o Motor domain couples ATP hydrolysis movement along microtubule o Cargo binding domain binds vesicles and organelles