Cytoskeleton and Cell Motility

Cytoskeleton and Cell Motility

• The cytoskeleton's functions, microtubules, microtubule-associated proteins, motor proteins (kinesins and dyneins), and microtubule dynamics are important components

Cytoskeleton

• A network of protein filaments extends throughout the cytoplasm
• Provides cell shape, support, and facilitates movement
• Marked animal cells in culture show major cytoskeletal systems
• Microtubules appear green, actin filaments are red
• Overlapping filaments appear yellow, DNA in the nucleus appears blue

Framework

• The cytoskeleton is made of three protein filaments
• Filaments forming the cytoskeleton differ in composition, mechanical properties, and roles

Cytoskeleton Functions

• Dynamic scaffold provides structural support
• Scaffold determines cell shape and resists deforming forces
• Internal framework positions organelles within the cell
• Network of tracks directs movement of materials and organelles
• Examples include mRNA delivery
• Force-generating apparatus enables cell movement
• Essential component of cell division machinery, separating chromosomes and splitting cells during mitosis and cytokinesis

Cell Structure functions

• Epithelial and neuron microtubules mainly support organelle transport
• Dividing cell microtubules form the mitotic spindle for chromosome segregation
• Intermediate filaments support epithelial cells and neurons
• Microfilaments support epithelial cell microvilli
• Microfilaments are integral to neuronal elongation and cell division

Microtubules

• Long, stiff, hollow protein tubes
• Microtubules organize eukaryotic cells
• They rapidly disassemble and reassemble
• Originate from the centrosome
• Create a cellular track system, which enables the transportation of vesicles, organelles, and other components

Microtubules and Organelles

• Most fluorescent green organelles (peroxisomes) are closely associated with red microtubules
• Microtubules serve as tracks for peroxisome transport
• Mammalian cells display green peroxisomes, aided by a peroxisomal protein fused with a fluorescent protein
• Microtubules are stained with a fluorescently labeled antibody
• Microtubules appear red

Hollow Tubes

• Microtubules are hollow tubes made of globular tubulin subunits
• Tubulin is a dimer of α-tubulin and β-tubulin protein
• Microtubules are found in the cytoskeleton, mitotic spindle, centrioles, and cilia and flagella
• They function in cell support and movement of materials between the cell body and axon terminals

Centrosome

• The centrosome is the major microtubule-organizing center
• Tubulin polymerizes from nucleation sites on the centrosome
• The centrosome, comprised of a pair of centrioles, is surrounded by a protein matrix
• The centrosome matrix includes ringshaped structures formed from γ-tubulin
• γ-tubulin ring complex serves as the starting point for microtubule growth
• The minus end of each microtubule is embedded in the centrosome
• The plus end extends into the cytoplasm

Microtubule Instability

• Each microtubule grows and shrinks independently
• The array of microtubules anchored in a centrosome is continually changing
• New microtubules grow (red arrows) and old microtubules shrink (blue arrows)
• Microtubules shrink partially, quickly start growing again, or disappear, replaced by those from from the γ-tubulin ring complex

Dynamic Instability

• Selective stabilization polarizes cells
• Newly formed microtubules persist only if both ends are protected from depolymerization
• Minus ends of microtubules are protected by organizing centers
• Plus ends are initially free but can be stabilized by binding to specific proteins
• A nonpolarized cell has microtubules growing randomly with some shrinking back
• Encounters with capping proteins in a cell region stabilize plus ends
• Stabilization orients the microtubule array and polarizes the cell

GTP Hydrolysis

• GTP hydrolysis controls the dynamic instability of microtubules
• Tubulin dimers carrying GTP bind more tightly than GDP-carrying dimers
• Rapidly growing plus ends of microtubules are capped by GTP-tubulin
• Plus ends tend to keep growing
• This occurs until GTP is hydrolyzed to GDP, then the microtubules are released causing shrinkage

Modified Dynamics by Drugs

• Colchicine binds tightly to free tubulin dimers and prevents polymerization into microtubules
• The mitotic spindle rapidly disappears and the cell stalls in mitosis
• Taxol has the opposite effect, it binds microtubules and prevents subunit loss
• New subunits can still be added, but the microtubules cannot compress
• Taxol has the same overall effect as colchicine arresting dividing cells in mitosis
• Such antimitotic drugs are used to treat human cancers

Cell Interior Organization

• Differentiated cells are polarized
• One end of the cell is structurally or functionally different from the other
• Microtubules transport organelles, vesicles, and macromolecules along a nerve cell axon
• Microtubules in the axon point with plus ends toward the axon terminal
• Oriented microtubules are tracks for directional transport of materials synthesized in the cell body
• Axonal transport from the spinal cord to a shoulder muscle takes about two days with outward and backward traffic using specific motor proteins
• The backward traffic includes worn-out mitochondria and materials ingested by the axon terminals
• Microtubule activity depends on accessory proteins

Motor Proteins

• Motor proteins move along cytoplasmic microtubules
• Two families exist: kinesins and dyneins
• Kinesins move toward the plus end
• Dyneins move toward the minus end, toward the cell body
• Kinesins and cytoplasmic dyneins are dimers with two globular ATP-binding heads and a single tail, while ciliary dyneins have a different structure
• Globular heads move along microtubules
• Kinesins move toward the plus end, dyneins to the minus end
• Each dimer has two globular heads to bind and hydrolyze ATP
• Heads interact with microtubules with a tail that interacts with cargo

Motor Proteins Drive Transport

• Kinesin and dynein globular heads are enzymes that have ATP-hydrolyzing (ATPase) activity
• Kinesin "walking" includes hand-over-hand movement along a filament
• Two heads use ATP to move
• ATP hydrolysis and phosphate release loosens the rear motor head from the microtubule
• ADP release and ATP binding, a conformational change causes a rear head flip to the front
• This completes a single step for directional movement

Cargo Transport

• Kinesin and dynein heads interact with microtubules stereospecifically
• Motor proteins attach to microtubules in one direction
• The tail binds to a cell component, such as a vesicle or organelle, which decides the cargo
• Motor proteins convert ATP chemical energy into mechanical energy for movement
• cargo moves, and muscle cells contract using movement caused by these proteins
• Cargo towards plus ends is transported by kinesin motors, each for specific vesicles, organelles, or molecules
• The kinesin tail binds directly to the cargo
• Different adaptor proteins enable the same type of kinesin to carry different cargos
• Transport toward the minus end is mediated by cytoplasmic dynein, which uses adaptor proteins
• Selected cargo is transported with cytoplasmic dynein

Kinesins

• Kinesins move along MT filaments powered by ATP hydrolysis and perform cell functions
• These functions include mitosis, meiosis, axonal transport, and moving cargo
• Kinesin-1 consists of two heavy chains that wrap into a common stalk form with two light chains at the globular ends of heavy chains
• The human genome encodes three heavy chains and four light chains for kinesin-1
• Force-generating heads bind to the microtubule and the tail binds to the cargo being transported
• Kinesin-1 transports mitochondria, endoplasmic reticulum, and Golgi-derived vesicles to carry messenger RNAs

Cytoplasmic Dynein

• Discovered in 1963 for moving cilia and flagella
• Only two cytoplasmic dyneins enable management unlike kinesins that have adapted functions
• Dynein positions the spindle and moving chromosomes during mitosis, acting as a force-generating agent
• As a microtubular motor, dynein positions the centrosome and Golgi complex
• Dynein moves organelles, vesicles, and particles through the cytoplasm

Cytoplasmic Dynein transport

• Contains two dynein heavy chains and smaller intermediate and light chains at the base
• Each dynein heavy chain contains a force-generating globular head
• A protruding stalk contains a binding site for the microtubule, and a stem for movement
• Two vesicles move bidirectionally, kinesin moves it toward the plus end
• Cytoplasmic dynein moves it toward the minus end along the same microtubule
• Each vesicle contains inactivated kinesin or dynein
• Integral and peripheral membrane protein, and a soluble protein complex called dynactin allows attachment for vesicles

Organelles Positioning

• Microtubules and motor proteins position organelles in eukaryotic cells
• Endoplasmic reticulum (ER) tubules are located near the edge of the cell, and the Golgi apparatus is located near the centrosome
• The ER expands along microtubules
• Kinesins pull the ER outward, and dyneins pull the Golgi apparatus inward
• Regional differences are vital for cell functions

Movememnt from Dynein

• Hairlike cilia project from the respiratory tract
• A cilium beats using repetitive movements of a power stroke and a recovery stroke
• Flagella use wavelike motion

Cilia and Flagella

• Microtubules in a cilium or flagellum are arranged in a “9 + 2” array with nine doublets and two central microtubules
• Unicellular alga cross sections are examined
• The nine outer microtubules (paired) carry two dynein rows
• Dynein heads arms reach toward the adjacent doublet microtubule
• Dynein heads connect and move along it, producing ciliary beating

Dynein and Bending

• Dynein slides microtubules and enables movement
• In sperm flagella, doublets slide with ATP present
• In intact flagella, there is bending rather than sliding with flexible protein links
• Ciliary dynein generates the bending motion

Dynein Defects

• Dynein defects cause Kartagener’s syndrome in humans
• Syndrome causes defects, dyskinesia, and situs inversus totalis
• Symptoms include respiratory infections, sinus infections, ear infections, and nasal congestion
• Men with a sperm disorder are infertile
• Increase susceptibility to bronchial infections because paralyzed cilia cannot clear lungs
• Mutations in many genes can cause the syndrome and lead to cells being either immotile or dysmotile

Motor Proteins superfamilies

• Motor proteins are grouped into kinesins, dyneins, and myosins
• Motor proteins convert ATP, chemical energy into mechanical energy
• Motor proteins are used to create force, contract muscle cells, and move cellular cargo
• Kinesins and dyneins move along microtubules
• Myosins move along actin filaments
• Motor proteins move unidirectionally along the cytoskeletal track
• The protein undergoes conformational changes during each mechanical cycle that provide energy