A. The cytoskeleton is a dynamic structure with many roles.
1. It serves as a scaffold, providing structural support and maintaining cell shape.
2. It serves as an internal framework, organizing organelles within the cell.
3. It assists in movement of materials within the cell and cellular locomotion.
4. It provides anchoring sites for MRNA.
5. It serves as a signal transducer.
B. The cytoskeleton is a network of three filamentous structures: microtubules, microfilaments, and intermediate filaments.
A. Fluorescent microscopy of live cells locates fluorescently-labeled proteins.
B. Video microscopy and in vitro motility assays enable observation of intracellular movements.
C. Molecular biology and genetic mutants identify genes for essential cytoskeletal proteins.
D. The electron microscope can be used to visualize the cytoskeleton of a treated cell.
A. Molecular motors convert chemical energy of ATP into mechanical energy.
B. Molecular motors move by stepwise, conformational changes corresponding to a mechanical cycle.
C. Three types of molecular motors have been described.
1. Kinesins and dyneins move along tracks of microtubules.
2. Myosins move along microfilament tracks.
A. Structure and composition:
1. Microtubules are hollow cylindrical structures found in the cytoskeleton, n-dtotic spindle, cilia, and flagella.
2. The microtubule is a polymer made up of globular tubulin subunits arranged in longitudinal rows called protofilaments.
3. Most microtubules contain 13 protofilaments.
4. Heterodimers of (a- and B-tubulin subunits are assembled into tubules with plus and minus ends.
B. Microtubule-associated proteins (MAPs):
1. A MAP contains a globular "head" that attaches to a microtubule and a filamentous "tail" that extends away from the microtubule's surface.
2. MAPs not only form crossbridges, organizing microtubules into bundles, they also stabilize, alter the rigidity of, or influence the assembly of microtubules.
3. MAPs are regulated by phosphorylation of specific amino acid residues.
C. Microtubules as structural supports:
1. The distribution of microtubules corresponds to the shape of the cell.
2. Nerve axons have microtubules oriented parallel to the long axis of the axon.
a. In mature axons, microtubules function in axonal transport of vesicles.
b. During embryonic development, microtubules play a key role in axon growth.
3. Microtubules in plant cells organize cellulose-synthesizing enzymes that produce the cell wall.
4. Microtubules play a role in the location of organelles.
D. Microtubules as agents of intracellular motility:
1. Microtubules facilitate the movement of vesicles traveling between compartments.
2. Axonal transport:
a. Neurotransmitters are synthesized in the cell body and released from synapses at the ends of axons.
b. Both anterograde transport (away from the cell body) and retrograde transport (toward the cell body) involve microtubules.
E. Kinesins:
1. Kinesin is a large protein with a pair of force-generating globular heads and a fan-shaped, cargo-binding tail.
a. The motor domains of kinesin and kinesin-like proteins are similar.
b. Tails sequences differ reflecting the variety of cargo these proteins can haul.
2. Kinesin is a plus end-directed microtubular motor responsible for anterograde transport.
3. Kinesin functions via an ATP-dependent cross-bridge cycle.
4. Kinesinlike proteins are found in all eukaryotic cells.
F. Cytoplasmic Dyneins:
1. Dyneins are associated with cilia and flagella but are also found in the cytoplasm.
2. Cytoplasmic dyneins are large proteins with globular, force-generating heads.
3. Dyneins move toward the minus ends of microtubules and are involved in chromosomal movement during mitosis, minus end-directed movement of vesicles, and retrograde axonal transport.
G. Microtubule organizing centers (MTOCs):
1. Centrosomes have two centrioles surrounded by pericentriolar material.
a. Centrioles have nine fibrils, each with three microtubules.
b. Centrioles are found in pairs oriented at right angles to each other.
c. Microtubules grow from the pericentriolar material of a centrosome.
d. Centrosomes form the poles from which the mitotic spindle emerges.
2. Basal bodies and other MTOCS:
a. Basal bodies are the MTOCs for cilia and flagella.
b. Spindle pole bodies serve as MTOCs in fungi.
c. The protein 7-tubulin occurs in all MTOCs and is essential for microtubule assembly.
H. The dynamic properties of microtubules:
1. Microtubules assemble and disassemble readily.
2. Tubulin dimers are added or removed at the plus end, away from the MTOC.
I. Factors that influence assembly and disassembly:
1. GTP on the B-subunit of the tubulin dimer is hydrolyzed during polymerization.
a. Dimers bound to GTP have a higher affinity for one another than those bound to GDP.
b. GTP hydrolysis provides a mechanism for regulation of assembly and disassembly.
2. Temperature, Ca2+ concentration, and MAPs influence microtubule stability.
J. Cilia and flagella:
1 Cilia and flagella have similar structures but different motions.
2. Cilia are relatively short and occur in large numbers on cell surfaces.
3. Flagella, longer and fewer in number, are used primarily for motility.
K. The structure of cilia and flagella:
1. The central core (axoneme) of both cilia and flagella contain microtubules in a 9 + 2 arrangement.
a. The peripheral doublets contain both an A and a B tubule.
b. Radial spokes connect the A tubules to the central tubules.
2. Cilia and flagella emerge from basal bodies.
3. Basal bodies contain nine triplet microtubules and no central microtubules.
L. The dynein arms:
1. The machinery for ciliary and flagellar function resides in the axoneme.
2. Ciliary dynein is needed for ATP hydrolysis.
M. The mechanism of ciliary and flagellar locomotion:
1. Swinging cross-bridges generate the forces for ciliary or flagellar movement.
2. The dynein arm of an A tubule binds to a neighboring B tubule and forces a conformational change that slides the tubules past each other.
3. Sliding alternates from one side of the axoneme to the other.
4. Radial spokes and interdoublet links provide resistance.
N. Ciliary and flagellar locomotion is regulated by Ca 2+ and CAMP.
A. Intermediate filaments (IFs) are a heterogeneous group of proteins, each with a central helix flanked by globular domains.
B. IFs assemble by several mechanisms that do not include nucleotide hydrolysis.
1. The basic unit of assembly is a tetramer formed by two antiparallel dimers.
2. Both the tetramer and the IF lack polarity.
C. IFs resist tensile forces and IFs containing keratin form the protective barrier of skin.
D. Assembly and disassembly of IFs are controlled by phosphorylation and dephosphorylation.
E. IFs include neurofilaments, the major component of the structural framework supporting neurons.
F. IFs may provide specialized mechanical support in certain cell types.
1. Defective IFs may lead to extreme fragility.
2. Overexpression of IFs in neurons can lead to degenerative diseases.
A. Microfilaments are made of actin and are involved in cell motility.
B. Using ATP, globular actin (G actin) polymerizes to form actin filaments (F actin).
C. Actin is a major contractile protein in muscle but is present in all cell types.
D. G actin is added at the plus end and removed from the minus end, causing treadmilling.
E. Most motile processes involving actin require myosin.
F. Myosin II-class proteins generate forces in muscle and some non-muscle processes.
1. Myosin II has a tail composed of two heavy chains and two globular heads.
2. Nonhelical sections of the heavy chain form "hinge" regions.
3. Each head region has actin- and ATP-binding sites on the heavy chain.
4. Myosin forms bipolar filaments with tails all oriented toward the center.
G. Myosin I is found near cell surfaces and has only a single head.
A. Skeletal muscle fibers are large, multinucleate cells packed with myofibrils that contain repeating arrays of sarcomeres.
B. Thick and thin filaments in the sarcomere overlap.
1. Sarcomeres extend between Z lines.
2. 1 bands contain only thin filaments, the H zone contains only thick filaments, and the A band contains both thick and thin filaments.
C. Skeletal muscle functions by shortening fiber length.
D. Three classes of filaments are found in muscle cells: actin, myosin, and titin.
1. Titin filaments provide tension in resting muscles and support myosin filaments.
2. Tropomyosin molecules occupy the groove between the two actin molecules.
3. Heterotrimeric troponin molecules are spaced evenly along thin filaments.
4. Myosin head project laterally from the thick filaments.
E. During contraction, the myosin heads form cross-bridges with the actin filaments.
1. The myosin heads bend, sliding the thin actin filaments over the thick filament.
2. Full contraction of a sarcomere requires 50 to 100 cycles of cross-bridging.
F. Energy is provided by ATPase activity in the myosin head.
1. ATP hydrolysis provides energy for "cocking" the myosin head.
2. The binding of the head to the thin filament initiates the power stroke.
3. The binding of a new ATP releases the head from the thin filament.
4. The absence of ATP prevents dissociation of the thick and thin filaments and is the basis for rigor mortis, the condition following death.
G. Muscle fibers innervated by branches of a single motor neuron form motor units.
1. The contact between nerve and muscle occurs at the neuromuscular junction.
2. Linking the nerve impulse to the shortening of the sarcomere is called excitation-contraction coupling.
3. Action potentials are carried into the cell interior by transverse (T) tubules.
4. T tubules terminate near the sarcoplasmic reticulum (SR), which sequesters Ca2+.
5. In the relaxed state, cytoplasmic Ca2+ levels are low. Action potentials open calcium channels in the SR, releasing Ca2+.
6. The binding of Ca2+ to troponin C causes a conformational change, shifting the tropomyosin and exposing the myosin binding site.
7. Ca2+ is removed from the cytoplasm by the Ca2+-ATPase activity in the SR, causing tropomyosin to hide myosin-binding sites.
A. Actin-binding proteins organize actin filaments into functional assemblies.
1. Monomer-sequestering proteins bind to G actin and prevent polymerization.
2. End-capping proteins regulate the length of actin filaments.
3. Cross-linking proteins link together two or more separate actin filaments.
a. Rod-shaped cross-linking proteins promote the formation of networks that have the properties of elastic gels.
b. Globular actin-binding proteins bundle actin filaments into tight parallel arrays which are found in microvilli and stereocilia.
4. Filament-severing proteins shorten filaments, decreasing cytoplasn-dc viscosity.
5. Membrane-binding proteins link contractile proteins to the plasma membrane.
B. Actin filaments and myosin motors are responsible for cytokinesis, phagocytosis, cytoplasmic streaming, vesicle trafficking, movement of integral proteins in membranes, cell locomotion and more.
C. Stress fibers attach to the substrate at focal contacts and contain bundles of actin filaments.
1. Stress fibers contain other proteins found in muscle cells.
2. Stress fibers contract isometrically, creating tension between cell and substrate.
D. Cells lacking cilia or flagella move by "crawling" over the substrate.
1. Cultured fibroblasts crawl by forming lamellipodia.
2. The protrusion of lamellipodia involves the organization of actin filaments.
3. Force generation in lamellipodia occurs by the addition of actin monomers to cortical filaments and/or the movement of myosin motors along cortical filaments.
4. Cell movement requires both the formation and breakdown of microfilaments and adhesions, occuring simultaneously in different regions of the cells.
E. Contact inhibition of movement occ,-irs when motile cells in culture make contact.
F. Axonal outgrowth occurs in embryonic nerve cells.
1. Highly motile growth cones contain several structures filled with actin.
2. Growth cones follow defined markers during embryonic development.
G. The cytoskeleton is responsible for cell shape changes that occur during development.
A. A techniques called video-enhanced contrast, differential interference microscopy, or AVEC-DIC, allowed high-resolution images of living cells, facilitating the study of axonal transport.
B. Allen showed that vesicular movements along linear elements are both anterograde and retrograde.
C. In 1985, Vale, Schnapp, Reese and Sheetz showed that vesicles of different sizes move along linear elements at equal rates, indicating that a single motor drives fast axonal transport.
1. Organelles attached to single filaments move in both directions, even passing one another, suggesting that each filament has more than one track.
2. The filaments were the diameter of microtubules and bound immunofluorescent antibodies to tubulin.
D. Vale's group reconstituted the axonal transport system from separate components.
1. The addition of a supernatant fraction (S2) to the mixture of tubulin and organelles promoted transport in only the anterograde direction.
a. The motor protein kinesin was isolated from S2.
b. Kinesin was a identified as a plus end-directed motor.
2. A retrograde motor was identified using affinity chromatography to remove kinesin from the extracts.
3. Drug sensitivity suggests that the retrograde motor may be a dyneinlike protein.
E. A kinesin receptor molecule on transported vesicles, called kinectin,
is part of a larger family of vesicle-transport proteins.