BIO 201 Cell Biology with Todd Hennessey Week Ten Notes
BIO 201 Cell Biology with Todd Hennessey Week Ten Notes BIO 201
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This 19 page Class Notes was uploaded by ChiWai Fan on Tuesday April 12, 2016. The Class Notes belongs to BIO 201 at University at Buffalo taught by TODD HENNESSEY in Spring2015. Since its upload, it has received 94 views. For similar materials see CELL BIOLOGY in Biology at University at Buffalo.
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Date Created: 04/12/16
Cell Bio on April 4, 6, 8, 2016 (All images taken from Professor Hennessey’s slide—edited by ChiWai Fan) Chromatin, chromatids, chromosome: we have 23 chromosomes Compact the chromatin and pair the chromatids (left) A Disperse chromatin. A thread-like single piece of double-stranded DNA (linear) with associated proteins, mostly histones (Seen in Interphase); disperse so you can compact it in Mitosis (right) Mitotic Chromosome (paired chromatids) there’s 46 of these in humans. (Seen in Mitosis) compacting DNA makes it easier to separate. Chromatid is COMPACTED Compacted and dispersed Chromatin Why have compacted chromatin in mitosis? It can’t be replicated or transcribed. So identical pieces of chromatin to be separated; so both daughters have 46 Why have disperse chromatin in interphase? So it can be transcribed and replicated (sisters are held together) 1. In M phase cell, the DNA and proteins in each Mitotic Chromosome form highly compact structure 2. In an interphase nucleus, chromatins are threadlike structures dispersed throughout the nucleus 3. During the interphase, DNA is replaced. Only a tiny portion of one piece of chromatin of many is shown 4. At the end of S the chromatids are held together at the centromere In what phases of the cell cycle can you see mitotic chromosomes? MITOSIS 2 chromatids (double stranded and genetically identical) paired together=1 mitotic chromosome Cell Cycle M-Phase: Nuclear division (mitosis) and Cell division (cytokinesis) YOU DON’T WANT TO DIVIDE CELL UNTIL YOU HAVE ALL Interphase: G1, S and G2. Transcription and translation happen mostly in G1 and G2. DNA is replicated in S phase only and very little transcription or translation in S G1: cell grows and carries out normal metabolism; organelles duplicate S: DNA replication and chromosome duplication G2: cell grows and prepares for mitosis by doubling amount of DNA Cell Cycle Highpoints Chromatin is dispersed in interphase for transcription and replication DNA replication only happens in S phase. Histones and centrioles are replicated in S-phase too in preparation for mitosis. (ie: We want four to go in mitosis so we can divide into 2 each normal ones) Chromatin condenses into chromatids and forms mitotic chromosomes in mitosis. Since it is condensed, transcription and replication stop. Little or no translation either in mitosis. Centrioles and centrosomes in the cell cycle Centrioles replicate in S-phase The mitotic spindle contains two centrosomes. Each centrosome has two centrioles Cell Cycle Exceptions (some): not all cells go through same type of cell cycle 1. No G1 or G2. Most or all of interphase is S-phase. Cells divide a lot but don’t grow much. Much mitotic activity. Examples: Embryonic cells, spermatogonia, etc. 2. Stuck in G1 or Go. No division at all. Terminally differentiated cells such as some neurons, some muscle cells, RBCs, etc. brian cells replicate like crazy! A cell not supposed to replicate like crazy starts to replicate like crazy= cancer 3. Inducible. Can progress from G1 to S if triggered to do so. For example, lymphocytes can be stimulated to proliferate by exposure to antigens. Liver cells regenerate after injury. Lizard tails can grow back after being cut off. Cancer cells start growing. Do soluble factors control the cell cycle? Assay: Fuse two cells together and see if the phase that one is in causes the other to enter that same phase Result: Fusion with a cell in S-phase stimulated the G1 cell to go into S-phase. How could you tell? 3 You’d see radioactive DNA (by autoradiography) if H radioactive thymidine was added. So did the cell become radioactive? How could you verify this?-- Microinjection 1. Take out a little of the cytoplasm of an S-phase cell 2. Microinject it into a G1 phase cell. Microinject water into a control G1 phase cell 3. Grow both cells in H thymidine, do autoradiography, see if they are radioactive. If the microinjected G1 cell is radioactive, it means it entered S phase (replication) Can G2 cells be stimulated to replicate DNA again? No change seen in either cell. Conclusion: G2 cells don’t respond to S-phase factor (whatever it is) Can G2 cells be affected by M-phase (mitosis) factor? How can you tell if the G2 cell starts mitosis? Result: The DNA in the G2 cell started to condense (compact), like they were starting mitosis. Also, G1 can be converted to M and S to M by “M-factor”. There’s a lot of factors floating around Conclusion: Both S-phase and mitosis are controlled by soluble factors Fusion of G1 phase cell with a cell in mitosis G1 chromatin becomes compacted due to exposure to the cytoplasm of a mitotic cell April 5, 2016 Fusion of a G2 phase cell with a mitotic cell If you fuse G2 cell and mitotic cell, you can tell which are compacted and which are dispersed A soluble factor from the mitotic cell causes the G2 chromatin to condense How can you tell when a G2 cell is starting to go into mitosis, the chromatin is condensed What are these soluble factors? 1. A soluble factor that causes progression from G1 to S 2. Another (different?) soluble factor that causes progression from G2 to mitosis MPF and Cyclin Yeast=eukaryotic Yeast is doubling in mitosis; MPF: Maturation-promoting factor. This stimulates entry into mitosis, as seen by compaction of DNA. MPF activity is a protein kinase activity. There are many protein kinase, they phosphorylate specific protein depending on cell function Kinases are enzymes that phosphorylate specific proteins Cyclin: Part of the MPF complex which regulates its activity; Cyclin levels goes up or down to signalize Need both MPF kinase and cyclin regulator to go into mitosis The MPF is not active until cyclin binds to it Cdk is a kinase; Cdk Cyclin complex is starter Activated MPF phosphorylates a protein It is the phosphorylated protein that stimulates progression to the next cycle Cdk protein is always present but its active site is not exposed (until it gets conformational change by binding with Cyclin) Cyclin protein is made only at a certain point in the cell cycle 1. Cyclin binding changes Cdk, exposing its active site 2. A protein substrate and ATP bind to Cdk. The protein substrate is phosphorylated 3. The phosphorylated protein regulates the cell cycle. Each Cdk has specific protein targets Two cyclins in yeast, a model eukaryotic cell One cdc kinase but two different cyclins that can bind to it (the Cdc/Cdk Kinase is always there) G1 cyclin stimulates progression from G1 to S-phase Mitotic cyclin stimulates progression from G2 to mitosis Kinase has to be stimulated to do things Cell cycle regulation Note: The Cdk is always there but it is not active until the cyclin appears This cyclin gene gets transcribed in G1; genes are there but some genes are not expressed until signaled Why is turned on and then turned off? It needs stimulant Is this a G1 cyclin or a mitotic cyclin? It is a G1 cyclin Cdk is present in Mitosis but without cyclin it is not active Cyclin synthesis begins during G1 and Cyclin bines to Cdk, which becomes active. Cyclin breaks down then Cdk is inactive in DNA synthesis (S phase) Cell Cycle Checkpoints: Cell cycle arrest (a factory) 1. “Sensors” detect DNA damage or cellular abnormalities; like some mutation 2. The cell cycle is arrested, stalling progression to the next step; all along the way 3. During this delay, DNA damage is repaired or the cell defect is corrected CdK inhibitor proteins can act as “molecular brakes” to stop the cell cycle from progressing to the next step; until you fix the problem. What happens if the checkpoint isn’t working well? Mutation, not the right amount of DNA. It may progress into a dead cell, a defective cell or cancer Specialized Inspectors (proteins/ complexes) can check the checkpoint, if a problem exists, you stop the whole thing, all in a guy to try to fix it then proceed Inspectors everywhere An example of a checkpoint protein p27 is a CdK inhibitor, it binds to cyclin complex as cell cycle arrest. It must be removed before the cell can move into S phase. What would happen if a cell was missing p27? You can get a spontaneous mutation (random at any time) and lose p27. A lot of p27 activation is false alarm Effects of a mutation in a checkpoint protein The brown mouse is a p27 knockout mouse. It has no p27. Therefore, there is no cell cycle arrest protein to pause progression into S phase. No p27 = More S-phase cells, more cell division, more cells, bigger mouse Higher cancer risk? Yes. If cell damage, there’s no p27 to fix it If defective or removal of p27, higher chance of cancer risk Mitosis First a little bit about eukaryotic DNA Telomeres and Centromeres Telomeric and centromeric regions of DNA are replicated but not transcribed; Not all DNA is Transcribed; How DNA is Packed into a Mitotic Chromosome DNA wraps around histones to form nucleosomes; organizing and shorten DNA so you can split it in half easily This compacts, loops and forms mitotic chromosomes with a packing ratio of about 1:10,000 The core Particle is an octamer of 8 histones Two domains (parts) to each histone in the core: 1. Hydrophobic: Responsible for histone assembly to make the core by hydrophobic aggregation 2. Hydrophilic: Positively charged, hydrophilic, faces the water. (Remember, DNA is negative at pH=7.0). DNA wraps around the outside of the core (chromatin) April 8, 2016 In general, eukaryotic DNA is either compacted (ex.: mitotic chromosome) or dispersed. Why? Dispersed chromatin is easier to replicate and transcribe. However, it is difficult to separate in mitosis Two pieces of compacted chromatin (mitotic chromosome) are easier to separate. However, it is more difficult to replicate and transcribe Condensin helps to compact and supercoil DNA in mitosis In which part of the cell cycle would you expect to see condensin first appear? M phase Condensin helps to compact DNA Cohesin helps to keep the two pieces of chromatin together at the centromere after S-phase, through G2 and into the beginning of prophase Phosphorylation of cohesin in early prophase causes a loss of cohesin (after S phase) between homologous chromatids except near the centromere How could phosphorylation regulate this? Conformational change and change in charges by adding negative charge causing repel In which part of the cell cycle would you first see cohesin? S phase The kinetochore is a protein complex that is attached to the centromere o DNA is blue o Kinetochores are green; does not wrap all around the centromere; o Cohesin is red Cohesin holds the sister chromatids together at the centromere; The kinetochore is not always at the center of mitotic chromosomes Regulation of gene expression by histone modifications (FYI) Closed chromatin DNA inaccessible: transcription repressed Open chromatin DNA accessible: transcription activated Human karyotype: Not all centromeres are at the center Spectral karyotyping (SKY) Spectral karyotyping is a molecular cytogenetic technique used to simultaneously visualize all the pairs of mitotic chromosomes in an organism in different colors Karyotypes of some eukaryotes Mitotic Chromosomes are different in different cells. Different number of mitotic chromosomes, different sizes, different shapes DNA content and Ploidy Haploid: Single set of genetic information. Single genome 23-sex cells (1x) Diploid:Two full sets of genetic information. Two copies of the genome 46 (2x) Polyploid: Many sets of genetic information. Many genomic copies (up to 800 in cotton) Ploidy (n): Number of sets of genetic information. Number of copies of the genome. x: Mass of DNA or amount of DNA c: Number of base pairs (size of DNA) Human cells are mostly 2n2x2c in G1 In this course, the x and c numbers will always be the same Changes in human DNA content during the cell cycle In G1: 22 autosomes and 1 sex chromosome (XX female or XY male) That’s 23 kinds of disperse chromatin in G1. But there are two sets of each because we are diploid (2n) so: 46 pieces of disperse chromatin in G1. They are all linear, double-stranded DNA. In S: Each piece doubles so: 92 pieces of disperse chromatin in S-phase. The nucleus is still diploid (2n). Same thing in G2. In mitosis: The 92 pieces of disperse G2 chromatin condense in mitosis to form 92 chromatids. The 92 chromatids are paired to form 46 mitotic chromosomes. Still diploid (2n) DNA changes during the cell cycle For example, here’s a G1 NUCLEUS (human) right after fertilization S-phase The disperse chromatin doubles in S-phase There are 92 pieces of disperse chromatin in S-phase How many mitotic chromosomes in human S-phase cells? None; only mitotic chromosomes in M phase DNA ploidy and content during the cell cycle Goal of mitosis: Make two copies of cells with the same ploidy and DNA content as the mother. This is: Equational Division—no change in Ploidy (n) Mitosis Mitosis is preparation for cytokinesis Phases of Mitosis Would you expect to see any cohesin connections in anaphase? No. Condensin? Yes 1. Condensin goes to work in prophase 2. Cohesin holds together the sister chromatids at their centromeres 3. Cohesin lets go in anaphase 4. Condensin starts to disappear in telophase and the chromatin becomes disperse Phases of Mitosis Prophase Chromatin cohesion in S-phase and compaction (condensing) in prophase of mitosis How can you tell you’re in prophase? 1. The chromatin is compacted. 2. Mitotic chromosomes are seen but they aren’t clustered near the middle 3. MTs from the mitotic spindle have not made contact with any mitotic chromosomes Prophase: Spindle formation 1. Pericentriolar material surrounding centrioles serves as the nucleation site (MTOC) for cytoplasmic microtubules 2. The initial spindle pole forms outside of the nucleus 3. Breakdown of the nuclear envelope, ER and golgi happens in prophase Not charges, they’re poles for plus end etc Prometaphase Chromosomal microtubules from the mitotic spindle begin to make contact with kinetochores Mitotic chromosomes begin to arrange in Amphitelic orientation Amphitelic means that each sister chromatid faces opposite poles Prometaphase 1. A protein complex on the centromere is called the kinetochore. 2. The plus ends of chromosomal MTs start to “capture” mitotic chromosomes at their kinetochores 3. Mitotic chromosomes begin to move (oscillate) and end up near the spindle equator at the end of Prometaphase 4. Sister chromatids end up in amphitelic orientation. Each faces opposite poles. In Prometaphase Three types of cytoplasmic MTs in the mitotic spindle: Not all cytoplasmic MT are the same 1. Astral MTs: Eminate from centrosome into cytoplasm to anchor and position the aster (mitotic spindle) 2. Chromosomal MTs: Connect from pericentriolar material of centrosome to kinetochores 3. Polar MTs: Extend from centrosome to equator but interact with other polar MTs instead of chromosomes