MCB 2210 1st EditionExam # 3 Study Guide Actin: Structure and Biochemistry- Actin Overview:o Subunit: actino Organization: bundles, networkso Smallest of the 4 cytoskeletal polymerso Location: cytoplasm, plasma membrane, cell junctionso Functions: locomotion, shape change, cytokinesis, adhesion- Actin is one of the most abundant proteins in eukaryotic cells. o It comprises from 1-20% of the total protein in cells. o Cytoskeletal subunit proteins span the cell and are generally abundant. o The sequence of actin has been highly conserved through evolution (homology)o Actins contain ~375 amino acids and have a molecular weight of ~42 kDa. o Actins from amoeba and humans are >80% identical to each other in sequence. o Simple eukaryotes like yeast have one actin gene. o Mammals have several genes that produce multiple types of actin:  α-actin in muscle (sarcomeres) β- and γ- actin in non-muscle cells o (Do bacteria have a cytoskeleton?) - Actin filaments are helical polymers composed of globular actin monomers that bind ATPo Monomers= bound to ATPo Polymers (filaments)= can hydrolyze ATP to ADP- Structure:o Actin monomer is separated into 2 lobes by a deep cleft that binds ATP or ADP complexed with Mg2+o Actin exists in two forms in the cell: G-actin (globular actin): a monomer F-actin (filamentous actin): filamentous polymer- Helical polymer of G-actin subunits held together by noncovalent (hydrophobic and ionic) interactions- A polar polymero All of the subunits within an actin filament have the same polarity  Oriented in the same directiono The 2 ends of a filament are different—have different dynamic propertiesThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute. One end (-)- ATP cleft exposed Other end (+)- Contacts a neighboring subunit  When the monomers form filaments, + ends bind to – ends - Polarity can be determined by decorating (labeling) the filaments with the actin-binding “head” domain of the myosin II molecule (an actin motor protein that will interact with actin) (called subfragment-1 or S1)o Isolate actin filaments o When S1 is mixed with actin filaments, it attaches to the sides at an angle, creating an arrowhead-like pattern One end of the filament is called the pointed end (minus (-) end) and the other is called the barbed end (plus (+) end)- How do we study actin assembly?o Actin can be isolated and then we can get pure actin (G-actin monomers) Actin polymerization (assembly) can be initiated by adding salts (Mg2+, K+) to a solution of pure G-actin (in-vivo) - This process is reversible—if salt is removed, actin will depolymerizeo However, because the ionic composition of the cytoplasm is constant, actin polymerization can’t be regulated by this mechanism in cells (in-vitro) The assembly and disassembly properties of pure actin are different from those for actin in the cello Actin polymerization in-vitro can be monitored by: Measuring the scattering of light - Polymer scatters more light than monomer Attaching a fluorescent tag (pyrene) to actin -Fluoresces more brightly when it’s incorporated into F-actin than in G-actin—associated with the change in conformation from a monomer to being part of a polymer - Pyrene-actin assembly can be measured using a spectrofluorometer  Visualizing filaments by fluorescence or electron microscopy (physically see it, depending on the limitations of the type of microscopy) Conducting sedimentation analysis-Because F-actin is larger than G-actin, F-actin sediments more readily by centrifugation - Actin polymerization proceeds in 3 steps in vitro:o Nucleation: formation of a stable seed (“nucleus”) consisting of 3 actin monomers, which can elongate to form a filament If 3 actin monomers come together, a nucleus is formed—more stable arrangement than dimers because there are stronger interactions)  This lag phase is slow because actin dimers are very unstable The lag can be eliminated by adding nucleating factors (or in actin filaments themselves “seeds”)o Elongation: growth of filament Subunits are added to the nuclei- Monomers are added to both ends to increase the length of the filament  Fast phaseo Steady state: no net increase or decrease in the amount of polymerized actin—same size  Elongation monomer addition is balanced by shrinkage due to subunit loss- Monomers are added, but some also fall off—so they balance each other out - As actin filaments elongate, the concentration of free subunits (G-actin) decreases until the system reaches a steady state o The concentration of free actin at a steady state is called the critical concentration (Cc) At Cc, the rate of subunit addition (kon[C]) = rate of subunit loss (koff) Cc=kof/kon- Cc for actin polymerization under typical conditions in vitro is about 2.0 μM (33:00min of audio) If the free subunit concentration is above Cc, subunits will add onto the ends of filaments If the free subunit concentration is below Cc, subunits will be lost from the ends of filamentso At Cc the concentration of G-actin in equilibrium with F-actin  At monomer concentrations below the Cc, polymerization does not take place When polymerization is induced at monomer concentrations above the Cc, filaments assemble until steady state is reached and the monomer concentration falls to the Cc- The + and – ends of actin filaments are physically and kinetically differento The polymerization rate is different at the 2 ends of an actin filamentBecause actin is a polar polymer, the 2 ends have different propertieso The barbed + end elongates up to 10x faster than the pointed – end Growth at + end is much faster than – end o This can be demonstrated experimentally by mixing G-actin with F-actin seeds that have been decorated with myosin S1 fragment to mark the filament polarity The newly assembled filaments are much longer at the end- Critical Concentration:o Most G-actin monomers are bound to ATP Shortly after binding to another to form a filament, ATP is hydrolyzed so that ADP is bound to the actin and Pi is released from ATP o ATP hydrolysis accompanies polymerization. Each actin carries an ATP that is hydrolyzed to ADP soon after its assembly into polymer.  ATP hydrolysis (ADP) causes a conformational change that destabilizes interactions within the