BSCI421: Cytoskeleton: made of microtubules and microfilaments and intermediate filaments.

MT and MF are dynamic, needed for polarity and stability of cells. MT and MF have vectorial movement, polymers of individual subunits, form filamentous networks which interact with each other or other proteins (ex: motor proteins). Different proteins on each end (polar) MT: long cylindrical structures, 3 start helix with 13 columns, - end is closer to centriole, + end grows out. Brains have lots of tubulin. When cooled, tubulin breaks down. MT formation: nucleation and elongation. Exhibit dynamic instability (constantly grow/shrink). If not, cells dont grow as quickly and they move slower. Tubulin subunit has alpha and beta subunit with GTP bonded to each. + end is beta end. When beta GTP is exposed=MT is stable. When GTP is hydrolyzed MT collapses. Gamma subunit is the template that the MT grows off of. At low Ca MT form, at high Ca MT fall apart. Colchicine degrades MT. Taxol stabilizes MT, freezes spindle and halts mitosis (cancer treatment). Motor proteins: either + or directed. Myosin moves only in one direction. MAPS: MT associated proteins-can stabilize, break up or cap MT. Katenin cuts MT, without it, the cell slows down. It is needed to keep everything under control. Kinesin 1 moves to + end and hydrolyzes GTP, degrading MT. Dynein goes to end. MF: hold cell together, important in cell structure. If MF are degraded, the cell will pop. Centrosome: has centriole. Pericentriolar matrix is important for MT formation. Head groups bind ATP, bending occurs at hinge region. Go through cycles of conformation with ATP with hydrolysis and release. Vesicles are bound to both kinesin and dynein, depends what signals they have to know where to go. Dynactin binds vesicle to dynein, AP1 binds vesicle to kinesin. Actin: pear shaped, has Mg and ATP binding sites, can form a rod of parallel double helix strands. G-actin binds ATP f-actin which forms filaments. Some proteins change actin growth patterns. Reynolds number: affects diffusion. At cellular level, there is no motion without energy (i.e. cant just float across).

How does muscle movement occur? Muscle has a-band and i-band and h-band and z-

band They slide and dont squish. If you get rid of myosin only z bands are left, showing myosin is made of a-bands. If you add ATP you see sliding when you graph mass=absorbance vs. distance. (z-bands on the outside, a-bands in the middle) Myosin thick filament: highly ordered. 2 alpha helix coil tail. If you cut myosin with trypsin, it will separate by mass, light chains polymerize and make filamentous chains, heavy chains bind actin and in presence of ATP will detach from actin. Cleft, actin binding site and ATP binding site on head. Cycle of movement: ATP binds, head group releases actin. ATP hydrolyzes, causes conformational shift which binds back to actin. ADP falls off power stroke Optical trap: take actin filament with fluorescent bead at the end, glue myosin to the actin, add ATP and myosin will start moving down actin use laser to push the bead so you can see how much force is needed to make the bead move. TIRF microscopy: can see single labeled molecule FIONA: fluorescence and absorbance (increases when myosin is flat against the actin) show that to move the myosin heads alternate/walk Actin 6 walks the same way but with uneven steps Myosin only moves in one direction. We know because in the sarcomere all the myosin faces the same direction. Skeletal muscle only contracts in presence of calcium, because otherwise actin is blocked. Troponin binds to actin and binds to tropomyosin which blocks the actin. in presence of ca, tropomyosin is moved and allows other things to bind (myosin) Scallops dont need calcium to contract Fluorescence speckle microscopy: fluorescence is uneven against the spindle and they all move rapidly to the middle Cilia and flagella: axoneme, moves by power stroke. Cilia (singular): power stroke is through the whole axoneme, flagella: moves by standing wave

The axoneme can beat in either manner depending on conditions. Axoneme is extension

of cytoplasm. To show movement is by sliding: Ian gibbons and keith summers. When the filaments bend, will the longer one be longer (a) if the inside one is longer slides if the outside one is longer bends Basal body at the base, acts like a centromere. Trypsin cuts nexin links between a-fibers. Beat frequency Take axoneme, strip membrane add salt to kill dynein arms. The longer its in the salt the lower the beat frequency, but it will go down to 30, after that its the inner arms. If you add arms and ATP, it will regain beat movement (even if arms are from a different species) Plus ends of the MT are at the tips of the axoneme. How do you get stuff to the tip to make it grow? Intraflagellar transport mechanism: add latex beads to a paralyzed cilium, the bead will move up and down the outside of the cilium. Integral membrane proteins move with a protein raft up and down the cilium. Mitosis: Prophase: replicated chromosomes condense within the nuclear envelope. Mitotic spindle begins to grow, nucleus compresses and disperses Prometaphase: chromosomes start moving to center, kinetochores bind to + MT and are pushed to the center by + end motors, nuclear membrane begins to break down Metaphase: biconical spindle with focus poles, chromosomes align in center Anaphase: Chromosomes pop apart. MT attach to the kinetochores and shorten, then the poles start to move apart Telophase: chromosomes de-condense, nuclear membrane reforms and spindle falls apart http://www.life.umd.edu/cbmg/faculty/wolniak/wolniakmitosis.html Spindle exists in dynamic equilibrium, flux polar wind: fluorescence shows that tubulin is going to the middle=spindle formation Chromosomes move by MT shortening in anaphase A. do they shorten from the chromosomal or spindle end? Add fluorescent tubulin, kill fluorochrome in one band of the spindle during anaphase, spindle stays, centromere pulls itself in to the spindle. Anaphase B: spindles slide against each other by + end motor kinesins Sister chromosomes separate by a complicated cycle

Spindle can detect anaphase onset even without chromosomes. Spindle will change on one side in response to a change on the other side. Chromatin and MT can form spindle Cell cycle: Gap 1, synthesis, gap 2, mitosis. Gaps are for cells to grow In embryonic cell, only M and S phases. Checkpoints throughout the cycle, when something goes and kills its precursor, so the cell must continue on through the cycle, it cant go back. Can synchronize populations in their cell cycle to get different cells in different stages. If you fuse S and M cells, the S chromosomes will act like they are going through mitosis: something in M is more powerful. Fuse G1 and S, waits in S for G1 to catch up. Except for mitosis, the one that is further ahead will wait for the earlier chromosomes to catch up. Restriction point in G1 can use dye to see if DNA replicated X. laebus: frog female with fully grown oocyte, add progesterone cell will begin to split and wait in metaphase of meiosis 2. Take cytoplasm from cell in meiosis 1 and add it to a new oocyte that wasnt exposed to progesterone (in pre-meiosis phase), the cell will immediately go into meiosis 1. there is a maturation promoting factor (MPF) that is transferable. MPF is released by the cell. What is MPF? Add cycloheximide (blocks translation) MPF was disabled, so there is some connection with proteins. Add progesterone to a bunch of oocytes, there is an increase in MPF. MPF levels go up and down in the cell, up in Meiosis 1 prophase through metaphase, down through anaphase and telophase, goes up in meiosis 2 until metaphase. When sperm is added, MPF goes down and there is a burst of it during mitosis. MPF is the harbinger of mitosis. Urchin eggs treated with radiolabels. Fertilize them and isolate proteins every 10 minutes, make tons of radioactive proteins because the cell is growing. But cyclin fluctuates, related to MPF, matches MPF pattern. (Cyclin doesnt cause maturation like MPF does) cyclin is proteolytically degraded as it goes down, and if cyclin doesnt get degraded the cell arrests. What is MPF? Cyclin + CDK (cyclin dependent kinase). MPF phosphorylates lots of things, starts a huge cascade which starts meiosis/mitosis. Temperature sensitive yeast mutants: used to discover cell cycle checkpoints. The gene that they knocked out is for a protein that binds to cyclin. Wee-1 mutant divides before the cells are fully grown. When wee1 is there cells are regular sized, when it is not there, the cells are small and not fully grown. CDC25 mutant arrests late in G2. when you make a double mutant, the cells progress normally. Wee1 encodes a kinase (adds phosphoryl group which slows the cell cycle down), CDC encodes a phosphatase (dephosphorylates which speeds the cell cycle up) and they both work on the same thing. So when they are both mutated, the cell can proceed normally through the cycle. Isolated cDNA in human genes and put it in the CDC25, it worked, showing that the sequence is highly conserved. Add frog eggs in centrifuge, bottom layer is debris, middle is cytoplasm and top is lipid. Take cytoplasm and add sperm cells, they will begin to swell and divide because there is so much MPF. What is cyclin doing? Add cycloheximide to the same experiment: nuclei

of sperm will swell, but no division. Add RNase to the same experiment: nuclei swell, no division. Degrade RNase, add mRNA of cyclin division. So cyclin is integral to mitosis. It doesnt matter if the cyclin is from frogs or not, it is highly conserved. Cyclin is necessary to start mitosis, but needs to be degraded for the cell to go all the way through mitosis Domain in the middle of cyclin which binds to Ubiquitin and gets destroyed to allow the cell to proceed through the cycle. If you add something that inhibits CDK to cells in metaphase, the cells will go into anaphase, but not normally. Remove the inhibitor the cells will go back to metaphase.

Checkpoint before S is called start. Checkpoints throughout cycle that check and repair DNA and other things. Cells wait and grow while waiting at start. P53 gene manages checkpoints at G2, DNA is sensitive to UV light Retinoblastoma: affects checkpoint during S. These both can cause cancer.