The Cytoskeleton and Cell Motility

Cytoskeleton

A network of filamentous structures: microtubules, microfilaments and intermediate filaments. Each is a polymer of protein subunits held together by weak, non covalent interactions.
Each is highly dynamic, capable of rapid assembly and disassembly
Diverse roles in:
Cell shape, structural support and motility
Molecular, vesicle and organelle positioning and trafficking
Cell division
Signaling
Serves as a scaffold providing structural support and maintaining cell shape.
Serves as an internal framework to organize organelles within the cell.
Directs cellular locomotion and the movement of materials within the cell.
Provides a choring sites for mRNA

Live-Cell Fluorescence Imaging

Can be used to locate fluorescently-labeled target proteins
Molecular processes can be observed
Used to reveal the location of a protein present in very low concentrations

The Use of In Vitro Single-Molecule Assays

Can detect activity of an individual protein molecule in real time.
Can be supplemented with atomic force microscopy to measure the mechanical properties of cytoskeletal elements

Microtubules

Hollow, cylindrical structures. Each microtubule is a set of globular proteins arranged in longitudinal rows called protofilaments. Microtubules contain 13 protofilaments.

Protofilaments

The longitudinal rows of globular proteins that make up microtubules. Each protofilament is assembled from dimers of alpha- and beta-tubulin subunits assembled into tubules with plus and minus ends. The plus end is terminated by beta subunits. The minus end is terminated by alpha subunits.
This polarity plays an important role in the dynamic assembly/disassembly of microtubules.

Microtubule-Associated Proteins (MAPs)

A heterogeneous group of proteins that attach to microtubules to increase their stability and promote their assembly. MAPs are regulated by phosphorlyation of specific amino acid residues.

Microtubules as Structural Support and Organizers

Distribution of microtubules determines the shape of the cell.
Microtubules maintain the internal organization of cells.
Microtubules function in axonal transport.
Microtubules play a role in axonal growth during embryogenesis.

Microtubules as Agents of Intracellular Motility

Microtubules facilitate movement of vesicles between compartments.
Axonal transport: Movement of neurotransmitters along the length of the cell; movement away from the cell body (anterograde) and toward the cell body (retrograde); establish tracks for a variety of motor proteins

Motor Proteins

Motor proteins traverse the cytoskeleton. Molecular motors convert ATP into mechanical energy
Each motor moves unidirectionally along the cytoskeletal track in a stepwise manner.
Three kinds of molecular motors: Kinesin and dynein move along microtubule tracks; myosin moves along microfilament tracks.

Kinesins

Kinesin is a member of a superfamily called KLPs (kinesin-like proteins). Each is a tetramer of two identical heavy chains and two identical light chains. Each includes a pair of globular heads (motor domain) connected to a rod-like stalk. Kinesin is a plus-end-directed microtubule motor.
Kinesins move along a single protofilament of a microtubule at a velocity proportional to the ATP concentration.
Movement is processive = motor protein moves along an individual microtubule for a long distance without falling off.
KLPs move cargo toward the cell's plasma membrane (away from the cell body) - anterograde

Dynein

Retrograde movement (toward cell body; away from plasma membrane). Also responsible for the movement of cilia and flagella.

Cytoplasmic Dynein

Huge protein with a globular, force-generating head. It is a minus-end-directed microtubule motor. Requires an adaptor (dynactin) to interact with membrane-bound cargo.

Microtubule-Organizing Centers (MTOCs)

Specialized structures for the nucleation of microtubules. Control the number of microtubules, their polarity, the number of protofilaments, and the time and location of their assembly.

Centrosome

Animal-cell specific structure that initiates microtubules (MTOC). Contains two barrel-shaped centriole, surrounded by pericentriolar material (PCM). Centrioles are usually found in pairs.
Centrosomes are responsible for the initiation and organization of the micro tubular cytoskeleton. Microtubule minus-ends terminate in the PCM.

Basal Body

A structure where outer microtubules in a cilia and flagella are generated

Gamma-tubulin

The protein gamma-tubulin is found in all MTOCs and is critical for MT nucleation. It associates with a number of proteins forming a complex called the gamma-TuRC. It binds alpha-tubulin only, thereby nucleating microtubules, establishing their polarity. It also provides a protective cap, preventing gain or loss of additional subunits.

The Dynamic Properties of Microtubules

Newly-formed microtubules branch at an angle of pre-existing microtubules.
Changes in microtubule spatial organization is a combination of 2 mechanisms: rearrangement of existing microtubules and disassembly of existing microtubules/reassembly of new ones in different locations

Microtubule Assembly

Microtubules need GTP to assemble. alpha and beta subunits bind GTP. The Beta subunit is a GTPase, and hydrolyzes GTP once incorporated. This hydrolysis of GTP leads to a replacement of bound GDP by new GTP to "recharge" the tubulin dimer.
GTP-bound beta subunit promotes a stable cap, while the GDP-bound beta subunit makes the microtubule unstable.
During rapid assembly, the plus end (beta subunit) is stable. Once assembly stops and Beta subunits hydrolyze GTP to GDP, the plus end rapidly dissociates.

Dynamic Instability

Growing and shrinking microtubules can coexist in the same region of a cell.
A given microtubule can switch back and forth between growing and shortening phases; it is an inherent property of the plus end of the microtubule. Proteins called +TIPS regular the rate of growth and shrinkage

Cilia and Flagella

Hair-like motile organelles with similar structure but different motility. Cilia tend to occur in large numbers on a cell's surface. Flagella exhibit a variety of different beating patterns (waveforms) depending on cell type.
Degree of asymmetry in the pattern of the beat in cells regulated by the internal calcium ion concentration.
Cilia are important in developmental processes, and mutations lead to a range of abnormalities.

Structure of Cilia and Flagella

Central core = axoneme. Consists of microtubules in a 9+2 arrangement. The axoneme includes a central sheath, connected to the tubules of peripheral doublets by radial spokes. Doublets are interconnected to one another by an inter doublet bridge.
Cilia and flagella emerge from basal bodies (gamma tubulin). The growth of an axoneme occurs at the plus ends of microtubules

Intraflagellar Transport (IFT)

The process responsible for assembling and maintaining flagella. Depends on the activity of both plus-end and minus-end directed microtubules (kinesin and dynein, respectively). Kinesin transports IFT particles toward the plus end, while dynein recycles kinesin back to the cell body. These processes also play important roles in cilia-mediated signaling events.

Ciliary (axonemal) dynein

Required for ATP hydrolysis, which supplies energy for locomotion. Axonemal dynein vary in the number of heavy chains; depends on the organism and cell type. Like cytoplasmic dynein, each heavy chain is composed of a globular head and a stalk which binds betatubulin. The stem binds the adjacent tubulin doublet. Rotation of the head serves as the basic driving force for ciliary/flagellar motion.

The Mechanism of Ciliary and Flagellar Locomotion

"Swinging cross-bridges" generate forces for ciliary/flagellar movement. Dynein arm on an A tubule binds to a B tubule. Undergoes conformational change, sliding tubules past each other. Dynein releases B tubule and arms slide back into original positions.

Situs Inversus

A syndrome in which the left-right body symmetry is reversed. One cause of situs inverses is mutations in the gene encoding ciliary proteins. Patients with situs inverses suffer from respiratory infections and male infertility.

Polycystic Kidney Disease

Caused by mutations in primary cilia

Intermediate Filaments (IFs)

Heterogeneous group of proteins, divided into five major classes. IF classes I-IV are used in the construction of filaments; type V (lamina) are present in the inner lining of the nucleus. Ifs radiate through the cytoplasm of a wide variety of animal cells and are often interconnected to other cytoskeletal filaments by thin, wispy cross-bridges.

Assembly of Intermediate Filaments

Basic building block is a rod-like tetramer formed by two antiparallel dimers. Both the tetramer and the IF lack polarity. Ifs are less sensitive to chemical agents than other types of cytoskeletal elements. Assembly and disassembly of Ifs are controlled by phosphorylation and dephosphorylation.

Types and Functions of IFs

IFs containing keratin form the protective barrier of the skin, and epithelial cells of liver and pancreas. Ifs include neurofilaments, which are the major component of the network supporting neurons

Microfilaments

Composed of actin monomers. Play key roles in cell motility, muscle contraction and intracellular movement. Actin polymerization is ATP dependent (as opposed to GTP for tubulin). Each end of the actin filament has different structural characteristics and dynamic properties. All actin subuinits point in the same direction; therefore the filament is polarized. Polymer composed of two intertwined helical strands with a pointed (minus) and barbed end (plus).

Myosin

A family of motor proteins that are plus-end directed with one exception.
Myosins share a characteristic motor head for binding actin and hydrolyzing ATP.
Numberous light chains are either essential to function or regulate activity of the motor domain.
Myosins are divided into two groups: Conventional (type II) and unconventional (Types I, III-XVIII)
Non-muscle activities of myosin include: cell division, tensile strength of focal adhesions, cell migration and the direction of axonal growth cone movement.

Actin Assembly and Disassembly

Depends on concentration of ATP-actin monomers. The plus end has a higher affinity for actin monomers. At low ATP-actin concentration, polymerization is favored at the plus end. Eventually, the minus end loses monomers faster than it adds.

Treadmilling

At low ATP-actin concentrations (normal physiology), filaments reach a steady state where addition to the plus end equals loss from the minus end.

Drug Disruption

Actin polymerization can be a force-generating mechanism in cells. Controlling rate of polymerization/depolymerization promotes changes in cell shape and motility. Dynamics of polymerization can be altered pharmacologically

Cytochalasin D

Drug that blocks plus ends of actin filaments

Phalloidin

Drug that binds to intact filaments and prevents turnover

Latrunculin

Drug that binds free monomers and prevents incorporation

Type II Myosins

Generate force in muscles and some non-muscle cells.
Each myosin II is composed of two heavy chains, two light chains and two globular heads.
All machinery required for motor activity is contained in a single globular head (called the S1 subunit). The tail portion plays a structural role, allowing the protein to form bipolar filaments.

Unconventional Myosins

Have a single head and do not assemble into filaments. Several are associated with cytoplasmic vesicles and organelles, and participate in intracellular transport. Function cooperatively with microtubule motors to deliver targets to correct locations. Some function as dimers, while others are monomeric. As with dynein and kinesin, various adaptors link myosin to its cargo.
Myosin motors, such as myosin Va, can transport their cargo over microfilaments, including those present in the peripheral regions of the cell. Microtubule and microfilament motors may cooperate. Xx pigment granules are transferred from microtubules to actin filaments.

Stereocilia

Hair-like structures that project from the apical surface of the cell into the fluid-filled cavity of the inner ear. Displacement of the stereo cilia by mechanical stimuli leads to the generation of nerve impulses that we perceive as sound. Each stereo cilium is supported by a bundle of parallel actin filaments.

Muscle Fibers

Multinucleate cells resulting from fusion of myoblasts in the embryo. Each muscle fiber contains hundreds of cylindrical strands called myofibrils.

Myofibrils

Cylindrical strands that make up muscle fibers. Made up of a repeating array of sarcomeres.

Sarcomeres

Muscle contractile units composed of actin and myosin II fibers. Each sarcomere has a stereotypical banding pattern that gives muscle fibers a striated appearance. Banding pattern due to overlap of two distinct types of filaments: thin and thick filaments.

Z line (sarcomeres)

Where actin is bound. Border of the sarcomere.

I band (sarcomeres)

Thin filament: actin filaments only.

A band (sarcomeres)

Thick filaments: myosin filament superimposed over actin

H band (sarcomeres)

Myosin only

M band (sarcomeres)

Central cross connecting disc in the center of the sarcomere H band

The Sliding Filament Model of Muscle Contraction

Skeletal muscle works by sliding fibers. Myosin is plus-end directed. A bands remain constant in length. H ad I bands decrease in width. Z lines on both ends of sarcomere move inward.

Titin

A skeletal muscle protein that extends from the Z line to the M band. Titin filaments are elastic and prevent the sarcomere from being pulled apart during muscle stretching. Titin and nebulin molecules act as "molecular rulers" by regulating the number of actin monomers that assemble into a thin filament.

The Composition and Organization of Thick and Thin Filaments

Actin thin filaments also contain tropomyosin and troponin. Tropomyosin occupies the gap between two actin molecules. Troponin molecules are in contact with both actin and tropomyosin.

The Molecular Basis of Contraction

Each myosin II molecule is non-processive: it remains attached only a fraction of a second and moves the actin filament about 10 nm. The neck acts as a level arm to amplify the small cane in head conformation into the 10 nm shift. Hundreds of myosin's firing out of sync contract the sarcomere several hundred nm in milliseconds.

Actinomyosin Contractile Cycle

Two cycles, mechanical (interaction of myosin with actin) and chemical (hydrolysis of ATP) begin:
Binding of ATP causes detachment of the head from the actin filament. Hydrolysis of ATP to Pi and ADP promotes weak binding to the actin filament. Release of Pi enhances attachment and promotes the power stroke that moves the thin filament toward the center of the sarcomere. Release of ADP sets the stage for another cycle. Absence of ATP prevents dissociation of cross-bridges, causing rigor mortis .

Sarcoplasmic Reticulum

Stores calcium and releases it into the cytosol.

Excitation-contraction coupling

Linking of the nerve impulse to the contraction of sarcomeres.

Neuromuscular junction

Contact between nerve and muscle

Transverse (T) tubules

Part of the plasma membrane that is connected to the SR. Propagates action potential in muscles into the cell interior

Actin-binding proteins

Affect localized assembly/disassembly of actin filaments

Nucleating proteins

Provide a template for adding actin monomers (ex. Arp2 and Arp3)

Monomer-sequestering proteins

Bind to actin-ATP monomers and prevent them from polymerizing

End-blocking (capping) proteins

regulate the length of actin filaments

Monomer-polymerizing proteins

promote the growth of actin filaments

Actin filament depolymerizing proteins

Bind actin-ADP subunits for rapid turnover of actin filaments

Cross-linking proteins

Alter the three-dimensional organization of actin filaments

Filament-severing proteins

Shorten filaments and decrease cytoplasmic viscosity

Membrane-binding proteins

Link contractile proteins to plasma membrane

Microvilli

Present on the apical surface of the epithelia. Function in absorption of solutes (such as the lining of the intestine). Each microvillus contains about 25 actin filaments maintained in an ordered arrangement by the bundling proteins villain and fimbrin.

Arp2/3

Recruited by ActA protein on one side of bacteria for actin polymerization

Cell locomotion

Step 1. Protrusion of leading edge (lamellipodium).
Step 2. Adhesion of lower surface of lamellipodium to substratum (mediated by integrins).
Step 3. Movement of the bulk of the cell foreword over the site of attachment (stationary). Accomplished by contractile force exerted against the substratum.
Step 4. Cell after the attachments with the substratum have been severed and the rear of the cell has been pulled forward.

Lamellipodium

Leading edge of a cell, protrudes and guides cell movement along substratum. Force generation in lamellipodia occurs by adding actin monomers to filaments.

WASP

Protein that mediates Arp2/3 complex activation.

Wiskott-Aldrich syndrome

Disorder with crippled immune system because white blood cells lack a functional WASP protein and fail to respond to chemotactic signals.

Growth Cone

tip of axon, highly motile, shows several types of locomotor protrusions

Microspikes

a locomotor protrusion that points outward to the edge of lamellipodium, found on the growth cone of axons

Filopodia

elongations that extend and retract during motile activity. responds to physical, chemical stimuli

Neural plate

Formed by elongated ectodermal cells as microtubules become oriented parallel to the cell's axis during embryonic development.