1. www.soran.edu.iq Cell and Molecular Biology Behrouz Mahmoudi The cytoskeleton-2 1

2. www.soran.edu.iq Intermediate filament and Microtubule 2

3. www.soran.edu.iq Intermediate filaments are composed of a variety of proteins that are expressed in different types of cells. More than 50 different intermediate filament proteins have been identified and classified into five groups based on similarities between their amino acid sequences 1. Acidic and basic keratins (types I and II) are typically expressed in epithelial cells. About 10 keratins are specific for “hard” epithelial tissues which give rise to nails, hair, and wool. cytokeratins, are more generally found in the epithelia that line internal body cavities. 2. Four proteins : vimentin, desmin, glial fibrillary acidic protein (GFAP), and peripherin — are classified as type III IF proteins. Vimentin expressed in leukocytes, blood vessel endothelial cells, keep the nucleus and other organelles. Desmin filaments in muscle cells are responsible for stabilizing sarcomeres. Glial fibrillary acidic protein forms filaments in the glial cells. Peripherin is found in neurons of the peripheral nervous system. 3. Neurofilaments (NFs). classified as type IV proteins. neurofilaments are responsible for the radial growth of an axon and thus determine axonal diameter. composed of three type : NF-L, NF-M, and NF-H 4. Lamins: are found exclusively in the nucleus that supports the nuclear membrane. 3

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5. Microtubules are responsible for various kinds of movements in all eukaryotic cells. Microtubules are involved in nucleic and cell division, organization of intracellular structure, and intracellular transport, as well as ciliary and flagellar motility. Building blocks" of microtubules - tubulins Alpha and beta tubulin spontaneously bind one another to form a functional subunit that we call a heterodimer. 5

6. www.soran.edu.iq Assembly of microtubules When intracellular conditions favor assembly, tubulin heterodimers assemble into linear protofilaments. Protofilaments in turn assemble into microtubules. Microtubules form a framework for structures such as the spindle apparatus that appears during cell division, or the whiplike organelles known as cilia and flagella. Cilia and flagella are the most well-studied models for microtubule structure and assembly, and are often used by textbooks to introduce microtubules. 6

7. www.soran.edu.iq Cilia and Flagella Cilia and flagella have the same basic structure. They are attached to structures known as basal bodies, which in turn are anchored to the cytoplasmic side of the plasma membrane. To form cilia or flagella, microtubules arrange themselves in a "9 + 2" array. Each of the two central microtubules consists of a single microtubule with 13 protofilaments arranged to form the wall of a circular tube 7

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9. Motor proteins are the driving force behind muscle contraction and are responsible for the active transport of most proteins and vesicles in the cytoplasm There are three superfamilies of cytoskeletal motor proteins: – Myosin: muscle contraction – Kinesin: transport cargo from the nucleus along microtubules – Dynein: transport carbon towards the cell nucleus along microtubules Motor proteins convert the chemical energy contained in adenosine-5’- triphosphate (ATP) into the mechanical energy of movement: Myosin, Kinesin, and Dynein ~ 7.3 kcal/mol 9

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11. www.soran.edu.iq Myosin motors act upon actin filaments to generate cell surface contractions and other morphological changes, as well as vesicle motility, cytoplasmic streaming and muscle cell contraction. Myosin is a large asymmetric molecule, it has a long tail and two globular heads the long tail portion forms the backbone of the thick filament Each head contains two light chains 11

12. www.soran.edu.iq Functions: 1.Myosin-actin binding: One of the biologically important properties of myosin is its ability to combine with actin. The complex formed is called actomyosin. The actin binding by myosin is highly specific; no other protein can substitute actin. Physiologically, when actin and myosin combine the muscle produces force. 12

13. www.soran.edu.iq The kinesin and dynein microtubule based motor superfamilies move vesicles and organelles within cells, cause the beating of flagella and cilia, and act within the mitotic and meiotic spindles to segregate replicated chromosomes. 2. ATPase activity of myosin: myosin is an enzyme that hydrolyzes ATP Actin is the physiological activator of myosin ATPase, the substrate is MgATP 2-. During muscle contraction the Ca 2+ concentration in the intracellular fluid is increased and the ATPase activity of myosin activated by actin 13

14. www.soran.edu.iq Kinesins are a large family of motor proteins, most of which walk along microtubules toward the plus end, away from the centrosome Mammalian cells have at least 40 different kinesin genes There are 2 copies each of a heavy chain and a light chain, Each heavy chain includes a globular ATP-binding motor domain at the N-terminus 14

15. www.soran.edu.iq Cargo proteins bound by kinesins are diverse: 1.Some organelle membranes contain transmembrane receptor proteins that bind kinesins. Kinectin is an endoplasmic reticulum membrane receptor for kinesin-I. 2.Scaffolding proteins, first identified as being involved in assembling signal protein complexes, mediate binding of kinesin light chains to some cargo proteins or receptors. 3.Some membrane-associated Rab GTPases, that provide specificity for vesicle transport and fusion, are known to bind particular kinesins BimC, a kinesin related protein involved in mitosis, has a tail domain that allows it to assemble into antiparallel dimers that can mediate sliding of microtubules relative to one another. This resembles the ability of myosin II to form bipolar filaments that mediate sliding of actin filaments 15

16. www.soran.edu.iq Some kinesins promote shortening of microtubules, perhaps by inducing curvature at ends of protofilaments 16

17. www.soran.edu.iq Dyneins are minus end-directed motor proteins. which is usually oriented towards the cell center Axonemal dynein causes sliding of microtubules in the axonemes of cilia and flagella and is found only in cells that have those structures Cytoplasmic dynein moves processively along the microtubule; that is, one or the other of its stalks is always attached to the microtubule so that the dynein can "walk" a considerable distance along a microtubule without detaching 17

18. www.soran.edu.iq Cytoplasmic dynein is a rather large multi-subunit protein complex composed of two identical heavy chains and about 6 intermediate chains. The heavy chains each contain four ATP-binding sites, including the ATP- hydrolytic site that provides the energy for its movement along microtubules, and a microtubule binding site intermediate chains are thought to bind the dynein to its cargo, whether that cargo is a membrane-bounded vesicle in a neuron, a golgi vesicle, a kinetochore or a mitotic spindle astral microtubule. The dynein then provides the force to move this cargo along a microtubule toward its minus end. 18

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