Cell Cycle, Mitosis, and Microtubules. LS1A Final Exam Review Friday 1/12/07. Processes occurring during cell cycle

Cell Cycle, Mitosis, and Microtubules LS1A Final Exam Review Friday 1/12/07 Processes occurring during cell cycle Replicate chromosomes Segregate chromosomes Cell divides Cell grows Cell Growth 1

The standard cell cycle G 0 Cell Cycle Checkpoints Trigger mitosis machinery to assemble the mitotic spindle Trigger anaphase and proceed to cytokinesis There is a control system comprised of sensors that determine whether the required events have occurred appropriately before releasing the cell into the next stage of the cycle. The places where these sensors function in the cell cycle are called checkpoints. If events have not occurred properly, the cell cycle will arrest at this stage and proceed NO FURTHER Trigger DNA synthesis and replicate DNA 2

Which stage of the cell cycle corresponds to which part of FACS profile? -stain cells with a dye that binds DNA (PI) and analyze fluorescence via FACS Number of Cells A = G0/G1 B = S C = G2/M A B C Relative amount of DNA per cell What would the FACS profile look like for a culture of serum-starved cells? Number of Cells Relative amount of DNA per cell 3

Prophase Prometaphase Metaphase Anaphase Telophase Cytokinesis Phases of Mitosis At which phase do the following events occur? Chromosomes are aligned midway between the spindle poles? Metaphase Two sets of daughter chromosomes arrive at poles of spindle and a new nuclear envelope assembles around each set forming 2 new nuclei? Telophase Replicated chromosomes condense and mitotic spindle assembles between the two centrosomes? Prophase Paired chromatids separate to form two daughter chromosomes and each is pulled toward the spindle pole it is attached to? Anaphase Breakdown of the nuclear envelope and chromosomes attach to spindle microtubules via kinetochores? Prometaphase 4

Question: In order to determine the function of a protein, A1, in cells you use a method called RNAi to knock down the levels of A1 in serum-starved cells. Following serum re-exposure, you observe the following results: Wild-type cells A1 knockdown cells Total DNA content in both cell types is the same What is a possible role of A1? -Chromosome segregation is faulty -A1 could affect kinetochore/centromere attachment, microtubule nucleation, or chromatid orientation The Cell Cycle Engine Cell cycle is driven by cyclin concentrations vary throughout cell cycle. Cyclins have no enzymatic activity must bind cyclin-dependent kinases (cdks) Cdk concentration remains constant 5

Different cyclins appear at different times in the standard cell cycle NORMAL Cancer cells lack G1 roadblocks Standard Cycle Cdk Inhibitors Lost Cyclin-dependent kinase inhibitors CANCER OR Too much G1 cyclin Cyclin-dependent kinase Xinhibitors CANCER Cyclin-dependent kinase inhibitors 6

Phosphorylation regulates protein activity Phosphorylation ACTIVATES OR Phosphorylation INACTIVATES Phosphorylation Regulates Inter-molecular binding Protein degradation Protein location Cyclin B-cdk1 complex and events of mitosis Cdk1-cyclin Phosphorylate nuclear lamins - allow breakdown of nuclear lamina. Phosphorylate condensins allow compaction of DNA/chromosomes. Make microtubules more dynamic. 7

Feedback of Cell Cycle Positive (Fast) Cyclin-cdk complex stimulates reactions that activate more cdk further accelerating reactions reaction gets faster and faster (amplified) Negative (Slow) Active cyclin-cdk complexes activate APC triggers destruction of cyclin. Occurs slowly. Cyclin Destruction Mediated by Anaphase Promoting Complex (APC). Mediates ubiquitination of specific lysine residues on cyclin. Ubiquitin is recognized by components of the proteasome. Proteasome cap recognizes ubiquitin residues and feeds the targeted protein through a central core lined with proteases proteolyze protein in an ATP dependent process. 8

What would happen if you? expressed a version of M-cyclin that could not be destroyed? Cell would stall in mitosis. expressed an M-cyclin that had all of its lysines mutated to alanines? Cyclin could not be degraded (ubiquitination occurs on lysine residues) and cell would stall in mitosis. injected active cyclin-cdk complex into a cell that was in interphase? Cell would enter mitosis. How could you tell if. Cyclin B must be ubiquitinated and targeted for destruction by APC how could you tell that a specific N-terminal 90 amino acid sequence is necessary for cyclin B degradation? Delete or mutate the sequence and see if cyclin B is still ubiquitinated and degraded by the proteasome How could you tell if this sequence was sufficient for ubiquitination by APC? Add the sequence to another protein that is not normally ubiquitinated/degraded and see if it is now degraded. 9

During mitosis, sister chromatids are held together by a complex of proteins that includes Scc1. Scc1 is cleaved by the protease separase to allow separation of the sister chromatids at anaphase. Securin is a protein that binds to separase and inhibits its activity. Separase securin Scc1 SeparaseScc1 Some cancer cells overexpress securin. These cells can undergo mitosis, but end up with abnormal numbers of chromosomes. 1. Explain how overexpression of securin could lead to abnormal chromosome number. Securin inhibits separase activity, and must be removed in order for separase to be active. Overexpression of the inhibitory securin keeps separase inactive, so sister chromatids are never separated, but cytokinesis still occurs. As a result, chromosomes either break or both sister chromatids go to one daughter cell, leading to chromosome duplication/loss in the daughter cells. During mitosis, sister chromatids are held together by a complex of proteins that includes Scc1. Scc1 is cleaved by the protease separase to allow separation of the sister chromatids at anaphase. Securin is a protein that binds to separase and inhibits its activity. Separase securin Scc1 SeparaseScc1 2. Ubiquitination by the anaphase promoting complex (APC) is necessary for sister chromatid separation. Is the likely target of the APC Scc1, separase or securin? Explain your answer and propose a role for the APC in sister chromatid separation. Securin is the likely target of the APC because it must be removed in order to activate separase. APC ubiquitinates securin, leading to its degradation. This releases active separase, which can then cleave Scc1 to allow sister chromatid separation. 10

Microtubule Structure Microtubules are polymers made of 2 tubulin proteins α and β. Polar : have + and end Assembled or disassembled by addition or loss at + end. Microtubule Assembly Dynamic instability involves GTP binding and hydrolysis by beta tubulin Beta tubulin in heterodimer can be bound to GTP or GDP Heterodimer is added to the plus end in GTP bound form. After addition, GTP is hydrolyzed to GDP with a slight delay. Therefore there is usually a cap of GTP bound heterodimers at the plus end. This is STABLE If heterodimers at the tip of the plus end have GDP bound but not GTP, microtubule rapidly depolymerizes. 12

Microtubule Function In interphase cells, microtubules radiate outward from centrosome near nucleus. - ends of microtubules located at centrosome + ends at edge of cell. Distribution suggests that microtubules might help organize the cytoplasm Microtubules are dramatically reorganized during mitosis. Mitotic cells have two centrosomes - Mitotic cells form mitotic spindle. This is important for separating. At late prophase the centrosome divides and the two daughters travel to opposite sides of the nucleus. Mitosis Interphase Increased nucleation Increased catastrophe Questions What would happen if you treated cells with a drug that binds tubulin and prevents its organization into mirotubules? -No movement or separation of the chromosomes, no cell division. Proteins have been identified that sever microtubules, how would these lead to an increase in the rate of microtubule dynamics? -If a microtubule gets severed in the middle, this exposes a (+) end that has GDP bound to it. This triggers catastrophe and creates 2 net (-) ends (1 on each fragment). Since depolymerization is favored at the (-) end, having more (-) ends = more depolymerization. 13

More Questions.. Dynamic Instability causes microtubules either to grow or shrink rapidly. Consider an individual microtubule that is in its shrinking phase. A) What would happen if you increased the tubulin concentration (assume sufficient concentration of GTP)? Why? the microtubule would stop shrinking and start elongation. Higher GTP-tubulin concentration (remember there is sufficient GTP), would cap the microtubule with GTP-tubulin which prevents shrinking. B) If no additional tubulin was added to the system, what would happen over time (i.e. after A, when you added a bunch of tubulin, you did not add more) and Why? Over time, switch to shrinking mode. The concentration of free microtubules will fall and the rate of GTP-tubulin addition will slow. At some point GTP hydrolysis will catch up with GTP-tubulin addition, and the GTP cap will be destroyed---therefore you start to switch. C) What would happen if the solution contained an analog of GTP that cannot be hydrolyzed? For how long would this happen? If a GTP analog were present, microtubules would grow until all the free tubulin subunits were used up. You have decided to characterize some microtubule associated proteins. To characterize the activity of the proteins that you identified you examine the effects of these proteins on microtubule dynamics in vitro. You add your purified proteins to an in vitro reaction that contains a MTOC, fluorescently labeled microtubules and GTP (note GTP is not limiting in this reaction). You let the reaction reach steady state and then examine the effects on individual microtubules under the microscope. This is what you observe: No protein A B C MT length MT length MT length MT length Time Time Time What are possible functions of proteins A, B, and C? Time Protein A: appears to be a (+) end capping protein. It prevents growth of the (+) end of the microtubule. Protein B: appears to promote microtubule depolymerization at the (+). Protein B might promote catastrophe by stimulating hydrolysis of GTP on ß-tubulin. Protein C: appears to promote microtubule polymerization at the (+) end. Protein C might promote microtubule growth by preventing hydrolysis of GTP on ß-tubulin. 14