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BIOMG 1350 Notes Week 4

by: genehan

BIOMG 1350 Notes Week 4 BIOMG 1350


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Week 4 notes
Introductory Biology: Cell and Developmental Biology
Garcia-Garcia, M; Huffaker, T
Class Notes
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This 5 page Class Notes was uploaded by genehan on Saturday September 3, 2016. The Class Notes belongs to BIOMG 1350 at Cornell University taught by Garcia-Garcia, M; Huffaker, T in Fall 2015. Since its upload, it has received 5 views. For similar materials see Introductory Biology: Cell and Developmental Biology in Molecular Biology and Genetics at Cornell University.

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Date Created: 09/03/16
BIOMG 1350 Professor Bretscher & Garcia­Garcia Spring 2016 Week 4: Lecture 1 of 1 Wednesday, Feb 17, 2016 Lecture Title: The Cytoskeleton and Molecular Motors  Lecture Keywords: GEF, GAP, Ras, cytoskeleton, microtubules, MTOC, kinesin, dynein, coiled­ coil dimerization, processive movement, actin filaments, myosin, sarcomere, Z­discs, thin and  thick filament I.  iClicker question – Which of the following statements is true? a. Enzymes increase reaction rates by eliminating the activation energy. (False – enzymes lower the activation energy.) b. Feedback inhibition involves the final product in a biochemical pathway inhibiting all the enzymes catalyzing the reactions in the pathway. (False – it inhibits the first reaction in the pathway.) c. Enzymatic catalysis can involve making covalent bonds with reactants. (True – it can make covalent bonds with intermediates) (from required reading) d. The tyrosine kinase activity of c-src is activated by phosphorylation of a C-terminal tyrosine. (False – it is inhibited by this.) e. If a protein has 2 cysteine residues it will form a disulfide bond to stabilize it. (False – it may only form a disulfide bond if the cysteines are appropriately positioned and only occurs on proteins in an oxidizing environment.) II. GTP binding proteins are time-controlled and location-controlled molecular switches. a. The “On” form is bound to GTP while the “Off” form is bound to GDP. b. GTP binding turns it on, which leads to downstream pathways, and GTP hydrolysis, by the GTP structure itself, turns it off. c. Proteins regulate this activity: Guanine nucleotide exchange factor GEF turns it on and GTPase activating protein GAP turns it off. d. The human genome has over 100 small GTPases encoded that regulate many downstream pathways and functions, such as membrane traffic, growth control, and the cytoskeleton, by a GEF and a GAP. III. A GTPase called Ras (rat sarcoma) regulates cell growth, and when mutated, can cause cancer/uncontrolled growth. a. When a growth factor signal sends a signal to GEF, it turns Ras on, as it is bound to GTP, and sends signals in a downstream pathway. b. When inactive, Ras is tightly bound to GDP through the facilitation of a RasGAP. c. Without GAP, you would have too much active Ras and defects in GAP cause Neurofibromatosis I. d. Another mutation is in Ras itself, so that it cannot hydrolyze GTP. This is known as Ras G12V mutation and causes cancer since it is always active. Figure from Slide #10 of “Lecture #6” Powerpoint IV. The cytoskeleton is a highly dynamic structure that can be assembled and disassembled that provides the cell with shape, organization, and motility. a. The cytoskeleton is made up of 3 filament types: microfilaments such as actin, microtubules made of alpha-beta tubulin dimers, and intermediate filaments that are rope-like structures. i. Actin filaments provide contractile machinery and network and provide the cell its shape. ii. Microtubules are involved in organization and long-range transport of organelles. V. Microtubules are hollow tubes of alpha-beta tubulin and organized by a microtubule organizing centers (MTOCs). a. During interphase, a centrosome is visible and in a dividing cell, spindle poles of 2 mitotic spindles (similar to centrosomes) organize the microtubules. b. The alpha and beta tubulin form a tubulin heterodimer to become a microtubule subunit and there are 13 that assemble to form the hollow tube circumference. i. The beta subunit is always on the plus end and the alpha subunit is always on the minus end, which means there is intrinsic polarity. ii. The plus ends are associated with the cell periphery while the minus ends are closer to the centrosomes. c. Microtubules transport cargo along a nerve cell axon. i. Nerve cells have one axon that can be very long and materials must be transported by microtubules. ii. Outward transport occurs to the plus ends and inward transport occurs to the minus end, associated with the cell body of the nerve cell. iii. The cytoplasm of a squid’s axon was “rolled out” to observe the motion of organelles. d. Kinesin transports specific cargo to the plus end of microtubules and dynein transports to the minus end. i. What do you think would happen to organelle transport if all the ATP was hydrolyzed to ADP and Pi in the cell extract? 1. The movement would stop and the cargo would fall off the microtubules. ii. The polarity of the microtubules is recognized by kinesin so that it knows to move towards the plus end. e. Different motor proteins transport different cargos that bind specifically to the tails of the proteins. f. In eukaryotic cells, microtubule motors arrange organelles. i. Kinesin will transport ER materials while dynein transports Golgi products. VI. The kinesin structure has N-terminus heads that bind ATP and C- terminus tails that bind cargo. Kinesins are held together by a coiled- coil dimerization. a. By coupling ATP hydrolysis, kinesin undergoes a conformational change. b. It is tightly bound when ATP is bound in the leading head, and the other head is weakly bound when ADP is bound. c. Binding ATP causes the neck linker to swing forward and the trailing head now becomes the leading head. d. The new leading head finds a binding site on the microtubule and it will release its ADP, which coordinates the new trailing head to hydrolyze its ATP and release phosphate and so the linker becomes undocked. Then, the cycle repeats itself down the microtubule. e. Kinesin moves processively, step-by-step, along the microtubule due to the coordination between the heads. Figure from Slide #25 of “Lecture #6” Powerpoint VII. Iclicker question – AMPPNP is an ATP analog that can bind to the kinesin active site but cannot be hydrolyzed. Given the mechanism of kinesin movement just described, what effect will AMPPNP have on kinesin? a. Kinesin will move along microtubules but more slowly. (No, kinesin will not move since it cannot by hydrolyzed.) b. Kinesin will not bind microtubules. (Kinesin remains tightly bound to microtubules in the ATP state) c. Kinesin will bind tightly to microtubules and release its cargo. (Kinesin will be tightly bound with its cargo d. Kinesin will move along microtubules but movement will not be unidirectional. (No, kinesin always travels towards the plus end.) e. None of the above will occur. (True – kinesin will be tightly bound to its cargo.) VIII. Microtubules are important for directed cell movement. The extract of the Autumn Crocus plant was used to treat gout, which King Henry suffered from. IX. Actin filaments define the shape of the cell. a. Actin filaments are thin, flexible protein threads in a helical structure. b. Like microtubules, actin filaments have a minus end favored for disassembling and a plus end favored for assembling. c. Actin monomers can be assembled to actin filaments, which then can form myosin, a motor protein. i. Myosin is most abundant in skeletal muscle. ii. Myofibrils are made up of repeating structures called sarcomeres. 1. A sarcomere is defined by 2 Z disks and is the contractile unit of muscle. iii. Actin is known as the thin filament and myosin is the thick filament. d. Myosin molecules associate to form bipolar myosin filaments held together by coiled-coil form. i. It has ATPase head domains that point in opposite directions to form a bare region in the middle. ii. The plus end associates with the Z-discs, and the minus end associated with the thick myosin filaments. iii. During contraction and relaxation, the thick filaments move closer to and then away from the Z-discs.


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