Cell Bio Cytoskeleton Oct 22

microtubule-based molecular motors (bidirectional trafficking)

proteins that "walk" along microtubules, using the energy from ATP hydrolysis to transport cargo or generate movement

• kynesin & dynein

• energy conversion

• interaction with microtubules

• cargo transport

• bidirectional movement

kynesin

generally move cargo towards the plus-end of microtubules

dynein

generally move towards the minus-end of microtubules

bidirectional trafficking

a single cell can have both kinesin and dynein motors, leading to a "tug-of-war" or bidirectional transport of the same cargo

kinesin-1 motor organization

a heterotetramer composed of two identical kinesin heavy chains (KHCs) and two identical kinesin light chains (KLCs).

• 3 main domains

kinesin-1 main domains

• head

• stalk

• tail

dynein motor organization

• a multi-component structure built around a massive heavy chain that includes a six-ring AAA+ motor domain.

• This motor domain is attached to a tail domain that helps the motor complex bind to cargo and other subunits.

dynein domains

• motor domain

• tail domain

dynein motor domain

• AAA+ ring

• stalk

• buttress (or strut)

• linker

dynein tail domain

• cargo binding

• oligomerization

• subunit binding

kinesin-1 walking mechanism

a hand-over-hand mechanism, where its two heads alternate taking 8-nm steps along a microtubule

kinesin-1 walking mechanism steps

1. ATP binding

2. neck linker docking

3. diffusion and binding

4. binding and hydrolysis

5. head exchange

cargo adapters

Cargo adapters are proteins that act as a bridge between cellular "cargo" and motor proteins or vesicle coat proteins, facilitating the transport of specific cargo within a cell

cargo adapters functions

• sorting cargo into vesicles

• linking cargo to motor proteins

• regulating transport

• organelle-specific function

• expanding transport range

properties of intermediate filaments

stable, flexible, and extremely strong protein fibers that provide mechanical strength to cells

assembly of intermediate filaments steps

1. dimer formation

2. tetramer formation

3. protofilament and filament assembly

actin filaments (F-actin)

filamentous polymers of actin proteins that form the cytoskeleton and are crucial for cell structure, movement, and muscle contraction

kynesin function

Primarily responsible for moving cargo such as vesicles, mitochondria, and Golgi bodies

kinesin direction of movement

Many kinesins move cargo toward the plus-end of the microtubule, located at the cell periphery

kinesin structure

Consist of two heavy chains with two motor domains (heads) that bind to ATP, and two light chains

kinesin role in cell division

Crucial for the formation of the mitotic spindle, which separates chromosomes during cell division

dynein function

Transport cargo, position organelles, and generate the beating motion of cilia and flagella through axonemal dyneins.

dynein direction of movement

Move cargo toward the minus-end of the microtubule, located near the cell's center or nucleus

dynein structure

The motor domain is located in the C-terminal region and is composed of six ATPase-associated with diverse cellular activities (AAA) domains.

dynein role in cell division

Vital for positioning the mitotic spindle during mitosis

microtubule-based molecular motors energy conversion

The motor proteins convert the chemical energy from breaking down ATP into mechanical energy, which allows them to move along the microtubule.

microtubule-based molecular motors interaction with microtubules

• The microtubule-binding domain of the motor protein binds to the microtubule track

• cycle of ATP binding, hydrolysis, and release causes a change in the protein's shape, moving it along the filament.

microtubule-based molecular motors cargo transport

The tail domain of the motor protein is responsible for binding to cargo, such as vesicles or organelles, through adaptor proteins.

kinesin-1 head

• Located at the N-terminal (amino-terminal) end of each heavy chain.

• Contains the sites for binding to microtubules (the cytoskeletal tracks) and ATP.

• ATP hydrolysis provides the energy for the kinesin to "walk" along microtubules.

• The head's amino acid sequence is highly conserved across the kinesin family.

kinesin-1 stalk

• Comprises a long, central alpha-helical coiled-coil domain.

• Mediates the dimerization of the two heavy chains as they intertwine.

• Includes hinge regions that allow the stalk to bend, a crucial function for regulating the motor's activity

kinesin-1 tail

• A globular C-terminal (carboxy-terminal) domain of the heavy chains.

• Associates with the two kinesin light chains (KLCs).

• The light chains and the tail of the heavy chains are responsible for binding to specific cellular cargo, such as vesicles, organelles, and adaptor proteins.

kinesin-1 regulation

regulation through conformational changes

• When not transporting cargo, kinesin-1 is in a folded, autoinhibited state to prevent wasted energy from futile ATP hydrolysis.

inactive (folded) kinesin-1 state

• The tail and light chains interact with the motor heads.

• The stalk bends at the hinge regions, bringing the cargo-binding tail into close proximity with the motor domains.

• This physically blocks the motor heads from binding to microtubules and inhibits their ATPase activity

active (extended) kinesin-1 state

• Cargo binding to the tail region triggers a conformational change that releases the motor heads from the autoinhibitory complex.

• The kinesin-1 molecule extends into an active, elongated form, allowing the motor heads to bind to a microtubule and begin processive movement.

kinesin-1 mechanochemical movement

Kinesin-1 walks along a single microtubule protofilament in a stepwise, "hand-over-hand" fashion, where the two motor heads take alternating steps. This movement is driven by a tightly coupled cycle of ATP binding, hydrolysis, and release that coordinates the heads.

dynein motor domain AAA+ ring

A ring of six AAA+ (ATPases Associated with diverse cellular Activities) domains forms the core of the motor, with AAA1 being the primary site for ATP hydrolysis to generate force.

dynein motor domain stalk

A stalk, extending from the AAA+ ring (specifically from the AAA4 domain), ends in a microtubule-binding domain that anchors the motor to the microtubule.

dynein motor domain buttress (or strut)

This structure, linked to the AAA5 domain, reinforces the base of the stalk and helps couple ATP hydrolysis to changes in microtubule affinity.

dynein motor domain linker

This element connects the AAA+ ring to the stalk and is a key component of the force-generating cycle. Its position changes significantly between ATP-bound and unbound states, which drives the movement of the motor along the microtubule.

dynein tail domain cargo binding

The tail domain extends from the motor and serves as an attachment point for the cargo to be transported.

dynein tail domain oligomerization

It can connect two or three dynein motors together, allowing for cooperative function.

dynein tail domain subunit binding

The tail domain is also a platform for associated subunits that are integral to the complex's overall function, stability, and regulation.

kinesin-1 walking mechanism step 1: ATP binding

The kinesin motor begins with one head bound to the microtubule, and the other head is either free or has just released ADP. ATP binds to the microtubule-bound head.

kinesin-1 walking mechanism step 2: neck linker docking

The binding of ATP causes the neck linker to dock with the motor domain, which swings the unbound "trailing" head forward by about 8 nm. The leading head remains bound to the microtubule.

kinesin-1 walking mechanism step 3: diffusion and binding

The newly forward head, now leading, begins a biased diffusional search for the next binding site on the microtubule. This step is ATP-dependent.

kinesin-1 walking mechanism step 4: binding and hydrolysis

The leading head binds to the next tubulin site. As this happens, it releases ADP and inorganic phosphate (𝑃𝑖), and the neck linker becomes untethered again. Meanwhile, the trailing head, now unbound, hydrolyzes its bound ATP and releases 𝑃𝑖

kinesin-1 walking mechanism step 5: head exchange

The head that just released 𝑃𝑖 now becomes the trailing head, and the other head has exchanged positions, ready to repeat the cycle. The molecule has now advanced by 8 nm along the microtubule.

kinesin-1 walking mechanism important features

• cooperative heads

• processive movement

• load-dependent stepping

kinesin-1 cooperative heads

Both heads work together to ensure that at least one head is always bound to the microtubule.

kinesin-1 processive movement

cooperative action allows kinesin-1 to move continuously for long distances, carrying cargo such as organelles and vesicles

kinesin-1 load-dependent stepping

The mechanism can be biased by external loads

• forward stepping is favored under low load

• high loads can lead to an increase in backward steps

cargo adapters: sorting cargo into vesicles

Adapters bind to transmembrane proteins or other cargo and help to package them into nascent vesicles for transport.

cargo adapters: linking cargo to motor proteins

They act as a bridge, linking a specific cargo to a motor protein (like myosin or dynein) that moves it along the cytoskeleton.

cargo adapters: regulating transport

Adapters can "switch on" motor proteins and control the timing and location of cargo transport. They can also play a role in the assembly and disassembly of transport structures, such as vesicle coats.

cargo adapters: organelle-specific function

Different types of adapters are used at different organelles to ensure the correct cargo is sorted for each destination.

cargo adapters: expanding transport range

In some cases, such as intraflagellar transport (IFT), adapters are necessary to carry a diverse set of proteins and protein complexes that cannot be transported directly by the core transport machinery.

intermediate filaments mechanical properties

• tensile strength and elasticity

• strain hardening

• flexibility

• stability

intermediate filaments structural properties

• cell-type specific composition

• hierarchical assembly

• lack of polarity

• network integration

intermediate filaments dynamic behavior

• dynamic in vivo

• subunit exchange and movement

• bidirectional movement

intermediate filaments tensile strength and elasticity

Intermediate filaments are remarkably strong and can be stretched to multiple times their resting length without breaking.

intermediate filaments strain hardening

The more they are stretched, the more resistant they become, allowing them to absorb mechanical energy and prevent catastrophic fracture.

intermediate filaments flexibility

Despite their strength, they are the most flexible of the cytoskeletal filaments and have a low persistence length, meaning they can bend easily.

intermediate filaments stability

They are more stable than actin filaments and microtubules, and can persist even after the cell dies.

intermediate filaments cell-type specific composition

They are made of a diverse family of proteins that vary depending on the cell type, such as keratins in epithelial cells or vimentin in connective tissue cells.

intermediate filaments hierarchical assembly

They assemble hierarchically, unlike actin and microtubules which polymerize from monomers or dimers. This structure allows for their unique mechanical properties.

intermediate filaments lack of polarity

Unlike actin filaments and microtubules, intermediate filaments are not polar, meaning subunits can be exchanged along their entire length

intermediate filaments network integration

They form an extensive network that integrates other cytoskeletal components and extends from the nucleus to the plasma membrane. They can connect to other structures like desmosomes to link cells together and to the extracellular matrix

intermediate filaments dynamic in vivo

Despite being very stable in vitro, they are highly dynamic in live cells, with subunits and entire filaments constantly being remodeled.

intermediate filaments subunit exchange and movement

Dynamic processes allow for subunits to be exchanged and for the filaments to move and assemble into new networks in response to cellular needs.

intermediate filaments bidirectional movement

Particles of intermediate filament proteins can move bidirectionally along microtubules, facilitated by motors like kinesin and dynein.

assembly of intermediate filaments step 1: dimer formation

• Individual intermediate filament (IF) proteins have a central 𝛼-helical rod domain.

• Two of these proteins wrap around each other in a coiled-coil structure, forming a dimer.

• The N- and C-termini of the proteins are at the ends of this dimer.

assembly of intermediate filaments step 2: tetramer formation

• Two dimers associate in an antiparallel and staggered orientation to form a tetramer.

• This step can be accomplished solely by the central rod domains.

• The resulting tetramer is symmetrical

assembly of intermediate filaments step 3: protofilament and filament assembly

• Tetramers align end-to-end to form protofilaments.

• These protofilaments then associate laterally and longitudinally.

• Approximately eight protofilaments twist together in a ropelike fashion to form the mature intermediate filament.

• This final filament has a diameter of about 11 nm.

actin filaments (F-actin) formation

F-actin filaments are formed from individual globular actin subunits (G-actin).

actin filaments (F-actin) structure

The two long helical strands are stabilized by interactions between actin subunits.

actin filaments (F-actin) role in cytoskeleton

They are a major component of the cytoskeleton, forming networks and contractile bundles, particularly concentrated at the cell's periphery.

actin filaments (F-actin) force generation

F-actin filaments are key to force generation, acting as a track for motor proteins like myosin for muscle contraction and other movements.

actin filaments (F-actin) are essential for which cellular processes?

essential for processes such as cell division, cell movement, and maintaining cell shape.

actin filaments (F-actin) regulation

• F-actin's polymerization and depolymerization are tightly regulated by a large number of actin-binding proteins (ABPs).

• These proteins can control filament assembly, localization, and mechanical properties, allowing cells to fine-tune force production for specific tasks.

• Signaling molecules and post-translational modifications also play a role in regulating actin dynamics.