Nucleating proteins: nucleation is the rate limiting step1) Formins- used to form long filaments.Domains:1) FH2: formin homology, does the nucleating function for the protein. Every formin has 2 subunits of FH2. Form a C shape and fit an actin monomer in the middle. The idea is that together the C shape adds a new actin monomer to the positive end.2) FH1: proline rich, acts as a landing or docking site to increase the local amount of ATP-actin3) RBD: rho binding domain, regulatory domain, Rho is a monomeric GTPase that is very similar to RAS. RAS was involved with the growth factor pathways. Rho has a single tail anchored in the plasma membrane. It is a GTP binding protein.Arp2/3 complex- branched chains of actin, done/formed by Arp2/3.Nucleation promoting factor (NPF): has a W domain that actually binds to actin. C is just a connector. The A domain is what will bind to the Arp2/3. Arp2/3 does the actual nucleation. The complex binds to the actin chain. In order to elongate, the NPF is done to bring over or bind to new actin monomers to be added to the growing chain. When the complex binds to it, it will assemble a positive end. When it nucleates out, it has a very distinct angle, 70.Regulation of Arp2/3 complex: requires monomeric GTPase called Cdc42. Related to RAS in that it activated by growth factors. In the normal state of the cell, this NPF is in folded over confirmation. Has a Rho binding domain. However, instead of binding to Rho, it will bind to Cdc42. Regulation is done by G proteins.Organization of Actin Based Structures: Shapes and structures are maintained by actin binding proteins. Their function is to cross them into actin filaments with each other or cross link actin with membrane or extracellular matrix. Maintain the parallel shape of the actin or skeletal muscles. Longer tails provide a lot more flexibility. In muscle cells, dystrophies all arise as a diseases due to the mutations in the dystrophin. Carried on the X chromosome, therefore it is more prevalent in males. The mutation will cause weakening of dystrophin. Dystrophin will start to break and weaken as the muscle contracts.Know types of actin, functions.Myosins: Actin Based Motor ProteinsMyosins- motor proteins that can move along actin filaments powered by ATP hydrolysis. The actin filaments act as tracts. Proteins that move along the tracts are called myosins. Humans have 40 different genes divided into many classes. Myosin is involved in skeletal muscle contractions. All cases of myosin have a head domain (mouth-like) where the myosin protein binds to the actin (actin binding functions), ATP hydrolysis occurs. All head domains also have a neck domain, the neck domain has light chains (separate molecules), each type has different numbers and functions. The light chains act to stiffen the neck so it isn’t flopping around and to regulate when the myosin molecules move along the actin tract (regulatory function). In myosin I, the tail interacts with the plasma membrane This is used for endocytosis. Myosin V has two heads, necks, and tails. The head is used to bind to vesicles. Myosins move to the positive ends of actin, no matter what.Myosin II-involved in skeletal contraction.2 light chains: 1) essential for stiffening, 2) regulatory: can be phosphorylatedHydrolysis of 1 ATP needed for each step taken by myosin II. Myosin uses actin as a track. Takes 1 step for each ATP hydrolyzed. When no ATP< myosin head is bound to an actin filament. ADP + Phosphate remain bound to head. Longer the neck, the longer it can stretch. Phosphate gets released, then power stroke. Moves the length of the neck domain.Rigor mortis: After death, ATP goes away. When there is no ATP around, the myosin is bound tightly to the actin filaments. This causes stiffness after death.Myosin Powered MovementsMuscles cells are long and skinny. 1-40 cm in legth, multiple nucleiWithin a muscle cell, there are bundles of actin and myosin that are bundled together as myofibrils.Sarcomere: unit of contractionThick filaments are the myosin II bundlesThin filaments are actin.Myosin movement along the actin tracts cause muscle contraction. Z disks on the left and right delineate the sarcomere. Embedded in Z disk are parallel structures of actin. Attaches to Z disk at positive end. For a muscle contraction, lots of ATP has to be used.Other proteins in the sarcomere: Do not want tread milling.CapZ- works on positive end, embedded in Z disk.Tropomodulin- negative end.Nebulin- black, extends from Z disk on one end to the Tropomodulin and back around to Z disk. Maintains integrity or length of the actin fiber.Titin- orange, from Z disk through the myosin II bundle.Sarcoplasmic reticulum (SR) and Ca 2+Normally Ca2+ levels are low in cytoplasm, kept in ER, mitochondria, or outside the cell.SR is a specialized ER, exclusive for skeletal muscle cells.The SR runs through the Z discs. The Z disks might have transverse tubules. These transverse tubules are a conduit for nerves (have nerves running through them).Nerves send an impulse through the transverse tubule. In the SR, there are voltage gated Ca channels. The never impulse stimulates the gated calcium channels to open up. Calcium floods out into the rest of the sarcomere. Calcium is floods out into the rest of the sarcomere. Calcium is a regulatory molecule, it regulates myosin’s ability to bind to actin. It works because there are two other proteins in the sarcomere called tropomyosin and troponin. Tropomyosin forms a rope and binds to seven actin monomers. Essentially, there is a continuous rope as long as the actin fiber is. Tropomyosin exists bound to actin monomers (normally) but it covers myosin binding sites. Otherwise, with ATP myosin would start walking and contract muscles. Troponin is a regulatory protein, it binds to tropomyosin and has a series of EF hands that bind to calcium. When troponin EF hands bind to calcium, the conformation change essentially moves tropomyosin out of the way.Regulation in a smooth muscle or nonmuscle cells.Smooth muscles involve the light chains. It remains inactive. The head is tucked back in near the tail.Calcium binds to calmogulen.17.7Cell Migration: Signaling and Chemotaxis1) Membrane extensionLeading edge = lamallepodiaArp 23 complex will nucleate actin into branched networks. Works for Lamallepodia and Filopodia (finger)Proflin provides ready supply of ATP for monomers and removes ADP). Coflin breaks off actin monomers.2) Cell substrate adhesions: