Part 4 (final exam)Molecular Motors and Cell Behavior- Myosins- motor proteins associated with F-actino Myosins are a huge family of motor proteins that bind to microfilamentso Family can be divided into “conventional” type II myosins and 14 types of“un-conventional” myosins - Myosin II structure :o Heteromer with 6 polypeptide chains- one pair of heavy and two pairs of light chains Each heavy chain contains:- An “S1” head with ATP-ase activity (on N-terminus)- A “neck” region- A coiled-coil tailo Dimerization occurs when the 2 alpha helices of the heavy chains wrap around each other to form a coiled-coil, driven by the association of regularly spaced hydrophobic amino acidso The coiled-coil arrangement makes an extended rod in solution tail)o The heavy chain can be split into isolated, soluble, S1 heads and coiled-coil tails by mild proteolytic enzymeso Isolated S1 heads bind to actin filaments and if the heads are attached to a coverslip and provided w/ ATP, they move filaments across the coverslip Therefore, they contain all the necessary motile machineryo Myosin II molecules can associate into filamentso These filaments are highly stable in muscle cells, where bipolar myosin filaments form a basic structural unit of the contractile machineryo In non-muscle cells, myosin II filaments are only formed transiently, as needed, to move elements of the cytoplasm aroundoo Myosin II can move actin filaments by attaching to actin filaments, moving (“power stroke”) and then detaching- Cellular motility mediated by actin and myosin (examples):o Skeletal muscle contraction in animalso Cytoplasmic streaming in many types of cellso Movement of cargo along actin filamentso Amoeboid movement of cell or “crawling”- Cellular locomotion- non-muscle motilityo Muscle contraction o Multinucleated cells form by fusion of many muscle cell precursors- myoblastso Activation of skeletal muscle contraction: Action potentials are triggered by the nervous system within the muscle fiber. The propagate along the muscle fibers plasma membrane and penetrate to the center of the fiber by following invaginations of the plasma membrane called “t-tubules”- T-tubules come into close contact with a specialized form of smooth ER called the sarcoplasmic reticulum- a calcium ion storeo When the action potential reaches the junctions between t-tubules and sarcoplasmic reticulum, it triggers Ca@+ release from the sarcoplasmic reticulum into the muscle cell’s cytosol around the myofibrilso Released Ca2+ enter the muscle fiber cytosol and bind to troponin on the actin filaments of the myofibrilso Tropinin changes conformation and pushes tropomyosin out of a groove in the actin filament, allowing myosin head to bind to actin and move it, producing muscle contractiono- Myosin I- only has a single “head” (unconventional myosin)o It can bind to and push against actin filaments, but single molecules of Myosin I find it difficult to “walk” along actin filaments since, with only one head, it must “hop” and stand a chance of detaching during the hops- Cytoplasmic streamingo Some unconventional myosins are attached to the plasma membrane and can be used to drag microfilaments and attached cytoplasm across the inner surface of the membrane- Cargo Movemento Some unconventional myosins can bind to various types of “cargo” and can move them along microfilament “tracks”o Most move cargo towards the (+) end of microvilli E.g. localization of mRNA to growing tips of budding yeast cells-- Amoeboid movement of Fibroblast and keratocytes- crawlingo Actin and myosin play a leading role in many forms of non-muscle motility, such as the “crawling” movement of some cells across surfaceso Motile cells like human fibroblastsor fish keratinocytes (pigmented skin cells), as well as axons developing neurons, have a specialized “leading edge” called the lamellipodium (or nerve growth cone in neurons)o These cells/axons are capable of moving/growing towards a chemical signal The leading edge of these cells is filled with a network rich in actinmicrofilamentso Fibroblast locomotion: Reception of a chemical signal molecule by a receptor protein in the plasma membrane of the leading edge of the cell triggers the assembly of an actin cytoskeleton via a protein complex called the Arp2/3 The assembly of the actin network at the leading edge of the lamellipodium pushes it against the plasma membrane and extends it forward (protrusion) Contraction at the rear of the cell propels the cell forward to relax some of the tension (traction). New focal contacts are made at the front (onto the underlying surface in the new areas over which the plasma membrane extends) The actin network at the trailing edge of the lamellipodium is disassembled by an enzyme, cofilin, retracting the cytoplasm at thetrailing edge, allowing the lamellipodium to move forward- a form of actin filament “treadmilling” The lamellipodium drags the rest of the cell after it, enabled by detachment of the cell from substrate at the trailing edge and flow of cytoplasm from the trailing edge towards the lamellipodium- This flow of cytoplasm- “cytoplasmic streaming” and is mediated by myosin motor proteins (this is common on many other types of cells-- moves cytoplasm around even non-motile cells) - Microtubule-based motility and motor proteinso Vesicular transport- the example of fast axonal transport in neurons Proteins, peptides are synthesized in the cell bodies of nerve cells, close to the nucleus but are requires throughout the nerve cell Most of these materials are compartmentalized into vesicles in the ER and Golgi complex of the cell body which are then transported down the axon along microtubule “tracks” Other vesicles are moving in the opposite direction, carrying wastematerials and regulatory factors back from nerve terminals to the cell body The “fast axonal transport” can proceed at rates up to 5 microns per second (400 mm per day) This transport is mediated by motor proteins that move along the microtubules- it requires ATPo A sensory nerve cello Kinesins- microtubule-associated motor protein The 1st kinesin was isolated as a motor protein responsible for moving vesicles and organelles along nerve axons from the cell body to the synaptic terminals Composed of 2 light chains and 2 heavy chains Heavy chains entwined to create a
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