Lecture 1- cell cycle

Major Changes in Mitosis

1) chromosome condensation: phos of condensins 2) formation of a mitotic spindle: microtubule instability 3) reorganization of the cytoskeleton 4) nuclear envelope breakdown: phos of lamins 5) organelle breakdown (fragmentation of Golgi and ER: phos of GM130) phosphorylation of substrates by MPF=cdc2/cyclin B (mitotic cyclin) is responsible for initiating many of the above changes.

Cdk/cyclin complexes

1. G1/S cyclins activate Cdks in late G1 and thereby help trigger progression through Start, resulting in a commitment to cell-cycle entry. Their levels fall in S phase. 2. S-cyclins bind Cdks soon after progression through Start and help stimulate chromosome duplication. S-cyclin levels remain elevated until mitosis, and these cyclins also contribute to the control of some early mitotic events. 3. M-cyclins activate Cdks that stimulate entry into mitosis at the G2/M transition. M-cyclin levels fall in mid-mitosis. 4. the G1-cyclins, helps govern the activities of the G1/S-cyclins, which control progression through Start in late G1.

26S proteasome structure

26S proteasome is composed of: 20S core (made up of 7 α and 7 β subunits x 2) Two 19S lids (made up of 12 RPN and 6 RPT ATPase subunits) ~30,000 proteasomes/mammalian cell 26S proteasome is in both nucleus & cytoplasm Ub is constantly recycled in this pathway designed for controlled proteolysis. Approximately 31 different subunits, which catalyzes protein degradation It contains a barrel-shaped proteolytic core complex (the 20S proteasome), capped at one or both ends by 19S regulatory complexes, which recognize ubiquitinated proteins. The regulatory complexes are also implicated in unfolding and translocation of ubiquitinated targets into the interior of the 20S complex, where they are degraded to oligopeptides.

formation of active MPF

Activation: MPF must be activated in order for the cell to transition from G2 to M phase. During G1 and S phase, the CDK1 subunit of MPF is inactive due to an inhibitory enzyme, Wee1. Wee1 phosphorylates the Thr-14 residues in yeast and Tyr-15 residues in humans of CDK1, rendering MPF inactive. During the transition of G2 to M phase, cdk1 is de-phosphorylated by CDC25. The CDK1 subunit is now free and can bind to cyclin B, activate MPF, and make the cell enter mitosis. There is also a positive feedback loop that inactivated wee1. In yeast, mutation of Tyr-15 to Phe-15 (cannot be phosphorylated) »» Mitotic Catastrophe (cells go into M-phase at all stages of cell cycle). In mammals both Thr-14 and Tyr-15 are inhibitory; both must be mutated for mitotic catastrophe.

FACS Analysis of Cell Cycle Phases

Analysis of DNA content with a flow cytometer: This graph shows typical results obtained for a proliferating cell population when the DNA content of its individual cells is determined in a flow cytometer. (A flow cytometer, also called a fluorescence-activated cell sorter, or FACS, can also be used to sort cells according to their fluorescence). The cells analyzed here were stained with a dye that becomes fluorescent when it binds to DNA, so that the amount of fluorescence is directly proportional to the amount of DNA in each cell. The cells fall into three categories: those that have an unreplicated complement of DNA and are therefore in G1, those that have a fully replicated complement of DNA (twice the G1 DNA content) and are in G2 or M phase, and those that have an intermediate amount of DNA and are in S phase. The distribution of cells indicates that there are greater numbers of cells in G1 than in G2 + M phase, showing that G1 is longer than G2 + M in this population. Histogram X axis= how much signal 1: certain amount of DNA 2: twice as much Normal distribution= bell curve. What causes this? Some have higher and lower signals. Just experimental variation due to inaccuracies. staining w DNA-binding fluorescent dyes: reveal the condensation of chromosomes in mitosis We can use flow cytometry to determine the lengths of G1, S, and G2 + M phases, by measuring DNA content in a synchronized cell population as it progresses through the cell cycle.

Nuclear Envelope Breakdown and Reformation

At the end of mitosis, (anaphase, telophase) there is a nuclear reassembly which is highly regulated in time, starting with the association of 'skeletal' proteins on the surface of the still partially condensed chromosomes, followed by nuclear envelope assembly. Novel nuclear pore complexes are formed through which nuclear lamins are actively imported by use of their NLS (nuclear localization signal). This typical hierarchy raises the question whether the nuclear lamina at this stage has a stabilizing role or some regulative function, for it is clear that it plays no essential part in the nuclear membrane assembly around chromatin. Lamin A that cannot be phosphorylated blocks nuclear lamina disassembly -Expression of lamin A mutant with 2 MPF phosphorylation sites changed from Ser to Ala (cannot be phosphorylated) - blocked nuclear envelope breakdown

Regulation of CDK/cyclin activity

Binding of Cdk inhibitor proteins (CKIs) inactivates cyclin-Cdk complexes. the three-dimensional structure of a cyclin-Cdk-CKI complex reveals that CKI binding stimulates a large rearrangement in the structure of the Cdk active site, rendering it inactive. the cyclin protein does not simply activate its Cdk partner but also directs it to specific target proteins. As a result, each cyclin-Cdk complex phosphorylates a different set of substrate proteins. There are different ways to effect it -association of CDK with cyclins -association with CKIs -inhibitory phosphorylation of threonine 14 and tyrosine 15 -activating phosphorylation of threonine around position 16 The ubiquitin-proteolytic pathway degrades Cyclins and CKIs ATP binding pocket has 2 inhibitory phos, that control G1 to mitosis.

Experimental approaches to study cell cycle regulation: Cell cycle mutants in yeast - genetic studies

Budding Yeast Cell Cycle: Genetic approach: creating mutations; take away function to discover things Use this approach to study cell cycle G1: round S: budding Mitosis: nucleus goes into other bud, spindle forms. Simple cell that divides by budding. Unusually short G2 phase. Saccharomyces cerevisiae (budding yeast) The most common mode of vegetative growth in yeast is asexual reproduction by budding.[37] Here, a small bud (also known as a bleb), or daughter cell, is formed on the parent cell. The nucleus of the parent cell splits into a daughter nucleus and migrates into the daughter cell. The bud continues to grow until it separates from the parent cell, forming a new cell.[38] The daughter cell produced during the budding process is generally smaller than the mother cell. under high-stress conditions such as nutrient starvation, haploid cells will die; under the same conditions, however, diploid cells can undergo sporulation, entering sexual reproduction (meiosis) and producing a variety of haploid spores, which can go on to mate (conjugate), reforming the diploid.[39] -The budding yeast Saccharomyces cerevisiae reproduces by mitosis as diploid cells when nutrients are abundant, but when starved, this yeast undergoes meiosis to form haploid spores.[42] Haploid cells may then reproduce asexually by mitosis.

Cell cycle checkpoints

Checkpoints maintain the order of unrelated events by signaling if something goes wrong, e.g. Damage in G1 or G2, incomplete replication, incomplete establishment of mitotic apparatus. They are usually not essential however if anything goes wrong then checkpoint becomes essential to prevent continuation of the cell cycle. In checkpoint mutants, mechanical apparatus of division is intact, and the problem is regulatory. A checkpoint response consist of three broad components: something to generate the signal, something to transduce it, and something to receive it. The transducers are typically what we think of as "checkpoint proteins". • DNA damage checkpoint is effected by p53 (tumor suppression) • Unreplicated/Damaged DNA G2 checkpoint works by inactivation of Cdc25 phosphatase • Mitotic spindle checkpoint works by inactivation of APC

Redundant DNA Replication Licensing Controls

CDK phosphorylation induces most replication licensing events pre-RC formation: The replication origin is bound by the ORC (origin recognition complex) throughout the cell cycle. In early G1, Cdc6 associates with the ORC, and these proteins bind the DNA helicase, which contains six closely related subunits called Mcm proteins. The helicase also associates with a protein called Cdt1. Using energy provided by ATP hydrolysis, the ORC and Cdc6 proteins load two copies of the DNA helicase, in an inactive form, around the DNA next to the origin, thereby forming the prereplicative complex (preRC). prevention: At the onset of S phase, S-Cdk stimulates the assembly of several initiator proteins on each DNA helicase, while another protein kinase, DDK, phosphorylates subunits of the DNA helicase. As a result, the DNA helicases are activated and unwind the DNA. DNA polymerase and other replication proteins are recruited to the origin, and DNA replication begins. The ORC is displaced by the replication machinery and then rebinds. S-Cdk and other mechanisms also inactivate the preRC components ORC, Cdc6, and Cdt1, thereby preventing formation of new preRCs at the origins until the end of mitosis *A licensing factor is a protein or complex of proteins that allows an origin of replication to begin DNA replication at that site. involves the recruitment of the pre-replicative complex (pre-RC) to the replication origins; -replication licensing ensures that chromosomes are replicated only once per cell cycle. replication initiators: ORC, Cdc6, Cdt1, and the MCM complex proteins elevated CDK activity initiates replication at the origins and prevents rereplication by inhibiting origin re-licensing

The regulation of the G1 to S phase transition in yeast and mammals: CLNs

CLN genes: controls the Start transition in the budding yeast cell cycle is the point of most physiological regulation of cell cycle commitment Sic1 is the only non-redundant essential substrate of the CDK/CLN complexes (budding yeast, humans: cdc/CDK). *this CKI helps keep M-Cdk activity low after mitosis (M-Cdk and Sic1 inhibit each other) CLNs are required for cells to go from G1 to S phase (overlapping functions) If you overexpress any CLN, G1 gets shorter (cells get smaller) Therefore, CLN activity regulates entry into S phase CLN 1,2 and 3: G1 cyclin; regulatory subunit of CDK for cell cycle entry CLNs are functionally redundant: - remove any 1 CLN - G1 gets longer and cells get bigger - remove any 2 CLNs - G1 gets even longer and cells get even bigger - remove all 3 CLNs - G1 arrest (big cells) Therefore, the only essential function of the CLN's is to phosphorylate SIC1 so that it can be degraded and S phase can begin • if you get rid of SIC1 alone - early entry into S phase CLN level is regulated by degradation: CLNs have PEDST region in C-terminus PEDST = Proline (P), Glutamic Acid (E), Aspartic Acid (D), Serine (S), Threonine (T) PEDST region makes CLNs get rapidly degraded CLNs truncated without PEST region are stabilized and accumulate to higher level - G1 phase gets shorter - Cells get smaller

wee1 and cdc25 mutant phenotypes

Cdc25 deficit: elongated cells, increased G2 Wee 1 excess: elongated cells, increased G2 -In cdc25− cells, the inhibitory activity is unopposed and MPF activity is inhibited, blocking entry into mitosis and resulting in elongated cells. -overproduction of Wee1 inhibits MPF activity more than normal, delaying entry into mitosis and producing elongated cells. Cdc25 excess: small cells, decreased G2 Wee 1 deficit: small cells, decreased G2 -When Cdc25 is produced at higher-than-normal levels, it offsets the inhibitory effect of Wee1, so MPF activity rises faster than in wild-type cells, causing premature entry into mitosis, which results in small (wee) cells. -In wee1− mutants, the stimulatory effect of Cdc25 is unopposed, so MPF activity rises faster than normal, leading to premature entry into mitosis and small (wee) cells. Cdc25 protein stimulates the activity of MPF, whereas Wee1 protein inhibits MPF

Mitotic cyclin destruction box

Cyclin B is degraded at the metaphase-to-anaphase transition. Degradation of Cyclin B requires the presence of the mitotic destruction box sequence in Cyclin B. Removal of the destruction box stabilizes Cyclin B and transfer of the destruction box to other proteins causes them to be degraded in anaphase also - this indicates that the destruction box is both necessary and sufficient for anaphase degradation. We find that the APC/C binds to the D-box of cyclin B, whereas Cdc20 does not. Mutations in the D-box abolished this interaction. We show that this binding is regulated in the cell cycle, such that the APC/C in interphase does not bind to the D-box matrix. Mutants in substrates show that APC independently affects meta->ana transition and cyclin degradation/exit. Target sequence is called "destruction box". Cyclin B destruction box peptide blocks metaphase/anaphase transition -APC is required to go from metaphase to anaphase -Blocking APC with D box peptide, now have trouble going to metaphase from anaphase

Experimental approaches to study cell cycle regulation: Sea urchin oocytes - cell biology

Cyclin Synthesis and Degradation: Tim Hunt looked at S35-methionine labelled sea urchin embryos and observed that two proteins (cyclins A & B) were synthesized and degraded during each cell cycle. -cyclin: drives cell cycle; abruptly disappeared at the end of cell division and then gradually appeared again as eggs began the next round of division; family of proteins that control the progression of cells through the cell cycle by activating cyclin-dependent kinase (Cdk) enzymes, along w MPF cyclin and MPF rise during mitosis, fall during interphase Why use sea urchins/clams? if using human eggs, reaction blocks other sperm from entering after one has entered. Takes a long time. But these organisms fertilize very quickly. Took oocytes and preincubated them with S35-Met Cyclins going up and down, bound to CDK Whole thing was maturation promoting factor

cyclin B degradation

Degradation of cyclin B is by the APC APC = Anaphase Promoting Complex = ubiquitin ligase for degradation of proteins containing the mitotic destruction box motif APC in budding yeast = 12 components APC in Xenopus = 8 components identified Degradation of cyclin is essential to keep cell cycle moving forward. Making a cyclin mutant that cannot be degraded traps cells in M phase. -isolated components APC (anaphase promoting complex, or cyclosome) required for ubiquitination of substrates and targeting to proteasome. Two points of destruction: metaphase to anaphase transition, when chromosomes separate, and mitotic exit, when cyclin degraded. These were distinguished because expression of a non-degradeable cyclin did not prevent chromosome segregation -Activation of this APC/CDC20 requires mitotic CDK activity (makes sense since CDK promotes mitosis!)

Rb-E2F

E2F proteins encode proteins required for S-phase entry, including G1/S-cyclins, S-cyclins, and etc. In the absence of mitogenic stimulation, E2F is inhibited by retinoblastoma protein (Rb). When cells are stimulated to divide by mitogens, active CDK4/cyclin D phosphorylates Rb protein, reducing their binding to E2F. The liberated E2F proteins then activate expression of their target genes needed for S phase. CDK4/cyclin D is kept inactive by the CDK inhibitor (CKI) p16 until enough cyclin D is synthesized to form active complexes that don't have p16 bound. -If you overexpress p16 (CKD inhibitor), the whole complex will be shut down, because Rb cannot be phosphorylated and therefore does not release E2F. -if you overexpress p16 in Rb -/- cells » cells enter S phase

G0 phase

G0 = quiescence (resting stage) Terminal differentiation proceeds from G0 Metabolism is very low in G0 cells Entry and exit is mediated by availability of growth factors (ligands for RTKs) When they reach the restriction point, goes through the whole thing w/o interruption If extracellular conditions are unfavorable, cells delay progress through G1 and enter specialized resting state, G0 (can be days, weeks, years, or until death) If conditions become favorable and signals to grow/divide are present, they progress through commitment point at the end of G1 known as Start (in yeast) or restriction point (mammals). After passing, they are committed to DNA replication even when signals are removed.

cyclins in budding yeast

G1 phase cyclins = CLN1, 2, 3 S phase cyclin = CLB5, 6, 3, 4 M phase cyclin = CLB1, 2 All yeast cell cycle cyclins bind CDK1

Cyclin expression in humans

G1 phase cyclins = cyclins D1, 2, 3 (works with CDK4 & CDK6), cyclin E (works with CDK2) S phase cyclin = cyclin A (works with CDK1 and CDK2) M phase cyclin = cyclin B (works with CDK1)

Acetylation of histones facilitate transcription

HAT = histone acetyltransferase; acetylated histones; active chromatin HDAC = histone deacetyltransferase; deacytelated histones; inactive chromatin note: Histone tails have + charge, DNA - charge.

Major cyclins and CDKs

In yeast, only one CDK is responsible for cell cycle progression (budding yeast CDC28 and fission yeast cdc2; both aka CDK1) In higher eukaryotes there are multiple CDKs In both yeast & metazoa - different cyclins are synthesized at different times in the cell cycle. Availability of different cyclins determines which CDK/cyclin complexes form during each phase of the cell cycle.

Mitotic Checkpoint Complex (MCC)

MCC (Bub3, Mad2, Cdc20, BubR1) forms on the unattached kinetochore, and blocks APC/C activation (by binding and APC ubiquilates MCC), as a result, separate won't be released to cut cohesin & chromatids won't be separated MPF is also unable to exit to mitosis (?)

MPF creation and destruction

MPF is disassembled when anaphase-promoting complex (APC) polyubiquitinates cyclin B, marking it for degradation in a negative feedback loop. In intact cells, cyclin degradation begins shortly after the onset of anaphase (late anaphase), the period of mitosis when sister chromatids are separated and pulled toward opposite spindle poles. As the concentration of Cyclin B/CDK1 increases, the heterodimer promotes APC to polyubiquitinate Cyclin B/CDK1. Get out of mitosos by degrading cyclin B, signal is D box Addition of a peptide containing the Cyclin B destruction box was added to cycling extract to act as a competitive inhibitor of the degradation machinery. Adding more Cyclin B destruction box peptide blocked cell cycle progression in metaphase. As Cyclin B destruction is only required for progression through telophase, this indicated that another protein must be degraded to allow the metaphase-to-anaphase transition.

Experimental approaches to study cell cycle regulation: Xenopus oocyte maturation - biochemistry

Meiosis: In oocytes, polar bodies (each of the small cells that bud off from an oocyte at the two meiotic divisions and do not develop into ova; way easier to just get rid of DNA than to build up amount needed) arise from discarded 2n and 1n chromosomes from meiosis I and II Xenopus Oocyte Maturation: The oocyte grows without dividing for many months in the ovary of the mother frog and finally matures into an egg. Upon fertilization, the egg cleaves very rapidly—initially at a rate of one division cycle every 30 minutes—forming a multicellular tadpole within a day or two. The cells get progressively smaller with each division, and the embryo remains the same size. Growth starts only when the tadpole begins feeding. thus are capable of exceedingly rapid division in the absence of either growth or many of the control mechanisms that operate in more complex cell cycles. -is jam packed w Rna and maternal protein (all things needed to replicate) oocyte arrested in G2 phase of Meiosis I -->hormonal trigger-->M phase of Meiosis I -->polar body discarded--> oocyte arrested in M phase of Meiosis II -->fertilization/polar body discarded--> fertilized egg in interphase (diploid nucleus) -->cleavage & embryo

CDK/cyclin knockout

Microinjection studies indicate that cyclin D is required to pass the restriction point for entry into S phase from G1 in mammalian cells • knockout mice lacking all cyclin Ds (D1, D2, D3) die as embryos at day E16.5 However: -formation of most tissues occurs (defects in blood cells and heart) -mouse embryo fibroblasts (MEFs) from these mice can proliferate -Therefore, cyclin D is not essential for the cell cycle • knockout of CDK4 and CDK6 together produces similar phenotype to cyclin D triple knockout • knockout of CDK2 (binds cyclins E and A) produces mice that are viable adults with meiotic defects • double knockout of CDK2 + CDK4 produces mice that die at day E14.5 with more severe phenotypes than the single KO - still have embryonic proliferation, but organs are much smaller. CDK1 alone can drive embryonic cell divisions • knockout mice lacking G1 & S phase CDKs (CDK2-/-, CDK4-/-, CDK6-/-) are able to develop until day E12.5 - creating a large number of tissue types. • in CDK2-/-, CDK4-/-, CDK6-/- knock-out mice, CDK1 can bind to cyclin D2 and cyclin E. • CDK1/cyclin D and CDK1/cyclin E are able to phosphorylate Rb. • CDK1-/- knock-out embryos cannot develop at all. • Therefore, CDK1 - by itself - can replace the other cell cycle CDKs if they are missing - but the other CDKs cannot replace CDK1.

Nuclear lamina disassembly

Nuclear lamina composed of nuclear lamins A, B1, B2, C MPF phosphorylates all nuclear lamins the nuclear lamina regulates important cellular events such as DNA replication and cell division. Additionally, it participates in chromatin organization and it anchors the nuclear pore complexes embedded in the nuclear envelope. The nuclear lamina consists of two components, lamins and nuclear lamin-associated membrane proteins. The lamins are type V intermediate filaments which can be categorized as either A-type (lamin A, C) or B-type(lamin B1, B2) A phosphorylation event at the onset of mitosis leads to a conformational change which causes the disassembly of the nuclear lamina. At the onset of mitosis (prophase, prometaphase), the cellular machinery is engaged in the disassembly of various cellular components including structures such as the nuclear envelope, the nuclear lamina and the nuclear pore complexes. These different disassembly events are initiated by the cyclin B/Cdk1 protein kinase complex (MPF). Once this complex is activated, the cell is forced into mitosis, by the subsequent activation and regulation of other protein kinases or by direct phosphorylation of structural proteins involved in this cellular reorganisation. After phosphorylation by cyclin B/Cdk1, the nuclear lamina depolymerises and B-type lamins stay associated with the fragments of the nuclear envelope whereas A-type lamins remain completely soluble throughout the remainder of the mitotic phase.

Methods for determining cycle cycle stage: Mitosis is visible in microscope

One way is simply to look at living cells with a microscope. A glance at a population of mammalian cells proliferating in culture reveals that a fraction of the cells have rounded up and are in mitosis (Figure 17-5). Others can be observed in the process of cytokinesis. Similarly, looking at budding yeast cells under a microscope is very useful, because the size of the bud provides an indication of cell-cycle stage. From the proportion of cells in mitosis (the mitotic index), we can estimate the duration of M phase. Can see: - disappearance of nucleoli - nuclear envelope breakdown - mitotic spindle formation - cytokinesis (cell division)

Paul Nurse/Lee Hartwell

Paul Nurse found one mutant, cdc2, that arrested at both G1/S phase and G2/M phase cell cycle transitions Lee Hartwell found the ortholog CDC28, which also arrested at both G1/S and G2/M Paul Nurse cloned cdc2 by complementation (the repair of a mutational deficiency in an organism or cell culture by artificially introducing a gene that supplies the missing function) and found that it encoded a Ser/Thr kinase (Wee1) In 1987, Paul Nurse cloned the human cdc2 by complementation, expressing human cDNAs in yeast to rescue the cdc2 mutant. The finding that humans had counterparts to the yeast cell cycle machinery, started the current period of intense research that led to our current understanding of the cell cycle. Nurse set out to find a budding yeast homologue for fission yeast cdc2. Introducing plasmids with budding yeast genomic DNA into fission yeast mutants, he found one that restored mitotic control, cdc28, the same protein controlled both Start and mitosis. Nurse would then clone the human homologue of cdc2 (also known as CDK1) Found mutants all stopped at same point; One—cdc28—blocked all the early cell cycle events and identified a point in the cell cycle where cells are irrevocably committed to replicating their DNA. Hartwell called this crucial transition point Start. Why interested in two points? Not one point? If you mess up DNA replication, you will mess up S phase (lots of things to mess up DNA replication), but you have a lot of things that need to happen in mitosis. Its regulating the transition and determining if they go into S phase or mitosis. Decided to assume that human gene was same as yeast, put into complementation screen for cdc mutant. Sequences it and it looks just like CDC28. showed that these mutants are conserved even in humans. The 2001 Nobel prize was given to: Tim Hunt (identification of cyclins) Paul Nurse (fission yeast cdc mutants, cdc2 = CDK1) Lee Hartwell (budding yeast cdc mutants, CDC28 = CDK1)

Rb actively inhibits transcription by bringing histone deacetylase (HDAC) to E2F promoters

Rb can also repress transcription of endogenous cell cycle genes containing E2F sites through recruitment of histone deacetylase (HDAC), which deacetylates histones on the promoter, thereby promoting formation of nucleosomes that inhibit transcription.

Methods for determining cycle cycle stage: Labeling of S phase cells

S phase can be observed by pulse labeling with 3H-thymidine/autoradiography - or - bromodeoxyuridine (BrdU) and then anti-BrdU antibody staining If cells in S phase, it will be replicating DNA. Put nucleotide in DNA that's labeled radioactively, take cells and add an overlay of silver (when light hits, grains will condense and the rest is washed away). Get little silver particles on top and ones that didn't bind are washed away Book: antibodies that recognize specific cell components such as the microtubules (revealing the mitotic spindle). S-phase cells can be identified in the microscope by supplying them with visualizable molecules that are incorporated into newly synthesized DNA, such as the artificial thymidine analog bromodeoxyuridine (BrdU); cell nuclei that have incorporated BrdU are then revealed by staining with anti-BrdU antibodies. From the proportion of cells in such a population that are labeled, we can estimate the duration of S phase as a fraction of the whole cell-cycle duration. -red: cells -green: antibodies

cell cycle stages

S phase: chromosome duplication, 10-12 hrs, occupies half the cell cycle time. M phase: chromosome separation and divison, <1 hour, 2 major events: mitosis (nuclear division; copied chromosomes are distributed into pair of daughter nuclei) and cytokinesis (cytoplasmic division; cell divides into two) End of S phase, DNA in each pair of duplicated chromosomes are intertwined and held together by protein linkages. Prophase (early mitosis): chromatin condenses into chromosomes. Nucleolus disappears. Mitotic spindle forms. Prometaphase: nuclear envelope breaks down. microtubules begin to capture chromosomes and align them. Metaphase: all chromosomes are aligned at metaphase plate and captured by spindle, attached by kinetochores of each chromosome. Spindle checkpoint. Anaphase: destruction of sister chromatid cohesion at the start which separates the sister chromatids and they are pulled to opposite sides of the spindle. Spindle is disassembled Telophase: segregated chromosomes are packaged into separate nuclei. chromosomes start to decondense. nucleolus reappears. Cytokinesis cleaves the cell into two so each daughter cell inherits one of the two nuclei. interphase: S, G1, and G2

budding yeast re-replication control

Saccharomyces cerevisiae cells prevent rereplication by directly regulating pre-RC assembly through the CDK-mediated phosphorylation of the pre-RC components Cdc6, MCM2-7, and the ORC subunits.[4] The phosphorylation of these components is initiated at the onset of S phase and is maintained throughout the rest of the cell cycle as CDK activity remains high. Phosphorylated Cdc6 is bound by the ubiquitin-protein ligase SCF which leads to its proteolytic degradation. CDK-dependent phosphorylation of the MCM2-7 proteins results in the complex's export from the nucleus. (Cdt1 which associates with the MCM complex is similarly exported from the nucleus). Phosphorylation of the ORC subunits presumably disrupts the ORC's ability to bind other pre-RC components.[4] Thus, multiple mechanisms ensure that the pre-RC cannot be reassembled on postreplicative origins.

Fission yeast cell cycle

Schizosaccharomyces pombe *remember: P goes w fission -reproduce by fission instead of budding,[37] thereby creating two identically sized daughter cells. -The haploid fission yeast Schizosaccharomyces pombe is a facultative sexual microorganism that can undergo mating when nutrients are limiting. -Major cell cycle regulation in G2 phase

Anaphase Entry

Securin has a mitotic destruction box motif Expression of Securin that has its destruction box removed leads to the stabilization of Securin and to a metaphase arrest Therefore, degradation of Securin is required for entry into Anaphase APC mutants » no sister chromatid separation APC mutants + Securin mutant » has sister chromatid separation Therefore, Securin is the only substrate that must be degraded at the Metaphase-Anaphase transition Securin mutants - normal timing of chromatid separation - not premature Therefore, There are other mechanisms to ensure timing besides Pds1 (securin gene)

Sister chromatid separation in yeast

Separase is a protease that is kept inactive by inhibitory binding by Securin. At the metaphase-to-anaphase transition, APC is activated and it targets the degradation of Securin (which has destruction box motif). Active Separase is released, which degrades the cohesin proteins that hold the two sister chromatids together. Mitotic progression is dependent upon S phase: Cohesion between sister chromatids is established during S phase Absence of cohesion leads to premature chromosome separation and segregation defects Normal cohesion is required to establish tension between the chromosomes on the mitotic spindle, and also to orient the chromosomes properly for the mitotic divisions. Cohesin connections are dissolved by ESP1 (separin) when PDS1 (securin) is destroyed by APC • Separase cleaves Scc1 (encodes a component of the cohesin complex in budding yeast) during mitosis to allow sister chromatid separation

Preparation of Xenopus cycling extract

Take oocytes blocked before their final division (like M phase), ask is it possible to induce them to enter M phase by injecting them with cytoplasm from post-M phase eggs. Answer: Yes. Purify and find two proteins comprising an activity called MPF. G2 arrested frog egg-->impose electric voltage, remove buffer-egg fall to bottom w oil->centrifuge-->all eggs crush and separates components roughly. Collect cycline/undiluted cytoplasm extract

Methods for determining cycle cycle stage: Fluorescence Activated Cell Sorter (FACS)

Taking cells in suspension (fixed=dead), add sheath fluid, goes through thin nozzle, sorts DNA if you want cells with a certain receptor, signal can be interpreted as it moves thru nozzle. It will add negative charge as droplet is being made. Ones that have - charge is one with signal, passes thru magnets and shows up in compartment. Can sort the cells you are interested in! are live cells. How do they add neg charge? Electricity We can gain additional clues about cell-cycle position by staining cells with DNA-binding fluorescent dyes (which reveal the condensation of chromosomes in mitosis) or with antibodies that recognize specific cell components such as the microtubules (revealing the mitotic spindle). Another way to assess the stage that a cell has reached in the cell cycle is by measuring its DNA content, which doubles during S phase. is approach is greatly facilitated by the use of fluorescent DNA-binding dyes and a flow cytometer, which allows the rapid and automatic analysis of large numbers of cells (Figure 17-8). We can use ow cytometry to determine the lengths of G1, S, and G2 + M phases, by measuring DNA content in a synchronized cell population as it progresses through the cell cycle. adv:High-throughput, can be combined with different fluorescent labels dis: indirect measurement of size, does not track individual cells measures: volume and shape; protein content

Regulation of cell cycle entry at START

The cell cycle commitment point in G1 is called START -yeast cells check for availability of food/nutrients before START, correct size of yeast, and the presence of mating pheromones/factors -if no food, or not large enough, or in the presence of mating factor then don't go through START (once past START yeast have to continue with cell cycle) Mammals also have a START that is regulated by availability of growth factors Start point: are conditions good? Chooses between mating and cloning. G1: growth phase Cell-cycle control system: -the switches are generally binary (on/off) and launch events in a complete, irreversible fashion. -remarkably robust and reliable, partly because backup mechanisms and other features allow the system to operate effectively under a variety of conditions and even if some components fail -highly adaptable and can be modified to suit specific cell types or to respond to specific intracellular or extracellular signals. 3 major regulatory transitions: first is Start (or the restriction point) in late G1, where the cell commits to cell-cycle entry and chromosome duplication. Enter cell cycle and proceed to S phase if env is favorable. the second is the G2/M transition, where the control system triggers the early mitotic events that lead to chromosome alignment on the mitotic spindle in metaphase. Enter mitosis if all DNA is replicated and env is favorable. third is the metaphase-to-anaphase transition, where the control system stimulates sister-chromatid separation, leading to the completion of mitosis and cytokinesis. Triggers anaphase and proceeds to cytokinesis if all chroms are attached to the spindle. the control system blocks progression through each of these transitions if it detects problems inside or outside the cell.

Temperature Sensitive Cell Division Cycle (CDC) mutants

The genes affected by these mutations are known as cell-division-cycle genes, or cdc genes. Many of these mutations cause cells to arrest at a specific point in the cell cycle, suggesting that the normal gene product is required to get the cell past this point. Screen for temp sensitive mutants Temp sensitive mutant: hypersensitive to temperature variation; unstable structure, usually missense mutations; why would they need a temp sensitive mutant? Its inducible, cant use a knockout mutation cause it will die, must be able to go thru cycle Grow at low temp, shift to high temp, look how its stopped at different points (arrest) For both yeasts: key insight was the idea that it might be possible to identify genes required to regulate cell division by looking for mutants. Key to this: haploid cells. expect genes for cell division to be essential. Look for mutants that are conditional: that is mutant only in one condition, not another. Use temperature. TS (temperature sensitive) mutants are unable to do their job at high temperature, presumably due to altered protein structure. At low temps, proteins stay intact and can grow cells. Isolate cells with mutations, ask what happens at high temp? cells will get "stuck" at a particular cell cycle stage. picture: (A) At the permissive (low) temperature, the cells divide normally and are found in all phases of the cycle (the phase of the cell is indicated by its color). (B) On warming to the restrictive (high) temperature, at which the mutant gene product functions abnormally, the mutant cells continue to progress through the cycle until they come to the specific step that they are unable to complete (initiation of S phase, in this example). Because the cdc mutants still continue to grow, they become abnormally large. By contrast, non-cdc mutants, if deficient in a process that is necessary throughout the cycle for biosynthesis and growth (such as ATP production), halt haphazardly at any stage of the cycle—depending on when their biochemical reserves run out In budding yeasts, a uniform cell-cycle arrest of this type can be detected by just looking at the cells: the presence or absence of a bud, and bud size, indicate the point in the cycle at which the mutant is arrested

Maturation (M-phase) Promoting Factor (MPF) Assay

Yoshio Masui showed that a cytoplasmic substance from frog oocytes (egg precursor cells) induced egg maturation and called the substance maturation promoting factor (MPF). -makes oocyte mature into M phase -MPF proved to consist of two protein subunits, one cyclin B and the other cdc2. So both were necessary to generate a "cyclin-dependent kinase" driving the cell cycle, in both developing and mature cells. -induces the condensation of chromosomes

condensins

activation of protein kinases of MPF phos & activates condensin complex and induces chromatin condensation. contain two SMC proteins SMC = structural maintenance of chromosomes condensin complex (I and II): SMC2 & SMC4 (form condensins) + Cap proteins = condense chromosome cohesin complex: SMC1 & SMC3 (form cohesins) + Cap proteins =bind sister chromatids condensin I and condensin II, each of which is composed of five subunits.[3][4] Condensins I and II share the same pair of core subunits, SMC2 and SMC4, both belonging to a large family of chromosomal ATPases, known as SMC proteins (SMC stands for Structural Maintenance of Chromosomes). -play a central role in chromosome assembly and segregation during mitosis and meiosis. Cohesin is a protein complex that regulates the separation of sister chromatids during cell division, either mitosis or meiosis. Cohesins hold sister chromatids together after DNA replication until anaphase when removal of cohesin leads to separation of sister chromatids. -SMC 1 and 3

cdc2 mutant phenotypes

aka CDK1 controls entry into mitosis in fission yeast, conserved in all including humans Wild-type cell (cdc2+) is depicted just before cytokinesis with two normal-size daughter cells. A recessive cdc2− mutant cannot enter mitosis at the nonpermissive temperature and appears as an elongated cell with a single nucleus, which contains duplicated chromosomes. A dominant cdc2D mutant enters mitosis prematurely before reaching normal size in G2; thus, the two daughter cells resulting from cytokinesis are smaller than normal, that is, they have the wee phenotype.

Early Embryonic Cell Cycle

alternates between S and M phases without intervening G1 and G2 phases. independent of cell cycle control

cyclin

cdc2 = cyclin-dependent kinase 1 (CDK1) CDKs generally required binding to a cyclin to become active CDK/cyclins = cell cycle machinery the most important of these Cdk regulators are proteins known as cyclins. Cdks, as their name implies, are dependent on cyclins for their activity: unless they are bound tightly to a cyclin, they have no protein kinase activity the levels of the Cdk proteins, by contrast, are constant. Cyclical changes in cyclin protein levels result in the cyclic assembly and activation of cyclin-Cdk complexes at specific stages of the cell cycle. The original name of Cdk1 was Cdc2 in both vertebrates and fission yeast, and Cdc28 in budding yeast.

isolation of CDC28 gene by complementation

cdc28, one of several genes required for cell division in budding yeast, S. cerevisiae, has been isolated on recombinant plasmids Recombinant plasmids capable of complementing cdc28 mutations were isolated by transformation of a cdc28ts strain and selection for clones capable of growth at the restrictive temperature. Plasmids responsible for complementing the cdc28ts phenotype were shown to recombine specifically with the chromosomal cdc28 locus, confirming the identity of the cloned sequences. In addition, one of the recombinant plasmids was capable of complementing a mutation in tyr1, a gene genetically linked to cdc28. This method of gene isolation and identification should be applicable to all yeast genes for which there are readily scorable mutants. cdc28 TS cells grown at 25 C-->transformed with WT S. cerevisiae DNA--> transformed cdc28 TS cells grown at 35 C-->those w cdc28 gene will be at various cell cycle stages and are isolated--> results in CDC28 Mutant cells grown at the permissive temperature are transformed with a gene "library" and selected for repair of function at the restrictive temperature.

Ubiquitin-Proteasome Proteolytic Pathway

degrades Cyclins and CKIs Most cytoplasmic proteins are be degraded by the ubiquitin-mediated proteolytic pathway. The ubiquitin-activating enzyme (E1) uses one ATP molecule to link a 76 aa ubiquitin protein to itself in a high energy thiol-linkage. The E1 transfers the activated ubiquitin to a ubiquitin-conjugating enzyme (E2). The E2 can transfer the ubiquitin directly to substrates, or more usually, does so with the assistance of a ubiquitin protein ligase (E3) which binds to the substrate. the E2 binds to E3 and then transfers the ubiquitin to the substrate. Multiple ubiquitin are tandemly added to substrate proteins. The ubiquitinated substrates are recognized by a 26S proteasome that degrades the substrate to peptides and recycles the ubiquitin proteins.

SCF Complex

not on ppt ?? a multi-protein E3 ubiquitin ligase complex catalyzing the ubiquitination of proteins destined for proteasomal degradation. It has important roles in the ubiquitination of proteins involved in the cell cycle and also marks various other cellular proteins for destruction -3 core components: F-box, Skp1, and Cullin (Cul1) -also Rbx1 SCF activity depends on substrate-binding subunits called F-box proteins. activity is constant during cell cycle its major role in the cell cycle is to ubiquitylate certain CKI proteins in late G1, thereby helping to control the activation of S-Cdks and DNA replication. SCF is also responsible for the destruction of G1/S-cyclins in early S phase. Ubiquitylation by SCF is controlled instead by changes in the phosphorylation state of its target proteins

p27

p27 is a CKI that is important for ensuring cells enter G0 by inhibiting CDK In mammals, the CDK-inhibitor p27Kip1 appears to function as a check to S phase entry expression of growth factors » decrease p27 no growth factors » turn on p27; G1 phase arrest no growth factors + antisense p27 » cycling cells Knockout p27 in mice » get large mice

humans re-replication control

pre-RC assembly is regulated by the anaphase-promoting complex (APC) in addition to CDKs. APC, an E3 enzyme, ubiquitinates the protein geminin and targets it for degradation.[4] Geminin normally prevents origin licensing by binding to and inhibiting Cdt1 (DNA replication factor). In G1, APC activity is adequate to suppress the accumulation of geminin, thereby indirectly promoting pre-RC assembly. At the end of G1, APC is inactivated and geminin can accumulate and prevent origin re-licensing.

Unattached spindle checkpoint

spindle checkpoint: separate mechanism monitors whether cells assemble their chromosomes and spindles correctly. This works by affecting APC activity, in all systems. Don't want to divide in absence of aligned chromosomes, or might lose one. In yeasts this pathway is not essential for life, but in other organisms, it is required. This may indicate that more complex genomes are more prone to problems. -all kinetochores need tension and must be attached Mad 2 and Bub 3: checkpoint protein for attachment 3F3/2 Phosphoepitope: checks for tension and alignment; part of a cell cycle checkpoint by which the onset of anaphase is inhibited until complete metaphase alignment is achieved *absence of both required to continue. Once all kinetochores attached and spindle is ready, MAD/BUB release and mitosis continues. **purpose of detecting tension and microtubule attachment: to ensure APC/C only functions when its attached to the kinetochore. Why would cell care If there is tension at kinetochore: prevent nondisjunction

APC/C

wasn't on ppt? the key regulator of the metaphase-to-anaphase transition is the anaphase- promoting complex, or cyclosome (APC/C), a member of the ubiquitin ligase family of enzymes. they polyubiquitylate specific target proteins, resulting in their destruction in proteasomes. -is an E3 ubiquitin ligase that marks target cell cycle proteins for degradation by the 26S proteasome. the APC/C catalyzes the ubiquitylation and destruction of two major types of proteins. -first is securin, which protects the protein linkages that hold sister-chromatid pairs together in early mitosis and binds to separase/separase, making it nonfunctional. Destruction of securin in metaphase activates a protease that separates the sisters and unleashes anaphase - activation of the APC/C by Cdh1: This continued activation prevents the accumulation of cyclin that would trigger another round of mitosis and instead drives exit from mitosis. the S- and M-cyclins are the second major targets of the APC/C. Destroying these cyclins inactivates most Cdks in the cell. As M-Cdk gets degraded later in mitosis, Cdc20 gets released and Cdh1 can bind to APC/C, keeping it activated through the M/G1 transition-->APC/C^Cdh1 then continues working in G1 to tag S and M cyclins for destruction. However, G1/S cyclins are not substrates of APC/C^Cdh1 and therefore accumulate throughout this phase and phosphorylate Cdh1-->By late G1, enough of the G1/S cyclins have accumulated and phosphorylated Cdh1 to inactivate the APC/C until the next metaphase. As metaphase begins, the spindle checkpoint inhibits the APC/C until all sister-kinetochores are attached to opposite poles of the mitotic spindle--> the spindle checkpoint is silenced and the APC/C can become active-->M-Cdks phosphorylate subunits on the APC/C that promote binding to Cdc20-->Securin and M Cyclins (cyclin A and cyclin B) are then targeted by APC/C^Cdc20 for degradation-->Once degraded, free separin is released which degrades cohesin (what keeps chromatids together) and sister chromatids are prepared to move to their respective poles for anaphase. *separin/separase: protease that degrades protein linkages and cohesin *securin: binds to separin and makes it nonfunctional

DNA damage checkpoint

works through Cdc25 Chk1 is a kinase that becomes active upon DNA damage and phosphorylates Cdc25 to inhibit it from activating CDK/cyclin b. Once CDK/cyclin b is active, it will go into mitosis Chk1 is the damage-response kinase. It is activated by DNA damage, irradiation, or S phase progression (during which cells generate chromosomal structures characteristic of DNA damage). It phosphorylates Cdc25 protein which is inactivated and also exported from the nucleus. Chk1 mutants are viable, but sensitive to irradiation and other forms of damage. Kinase checkpoint is activated with damage

Yeast Cell Shape Indicates Cell Cycle Position

yeast control size primarily by regulating division in response to growth. This requires detection of the nutrient concentrations that determine growth rate, growth rate itself, or cell size. S. pombe (fission) cells enter G2 at different sizes following S-phase. They grow in a bilinear fashion and enter mitosis upon reaching a threshold size, so that smaller cells spend more time in G2 than larger cells, as indicated. S. cerevisiae daughter cells are born at different sizes and grow exponentially. Smaller cells spend more time in G1 prior to Start than larger cells (as indicated), which partially compensates for initial size variation. ***Size control is a function of nutrient conditions and growth rate and is exerted at G2-M transition in S. pombe and within G1-S transition (start) in S. cerevisiae. In S. pombe, evidence for a size requirement for division came from experiments that tracked the growth and division of cells following synchronization by S-phase arrest -not a steady curve In S. cerevisiae, early single-cell studies showed that small cells spend a longer time in G1, which allows them to grow more than initially larger cells comparison of the two cell cycles (A) The fission yeast has a typical eucaryotic cell cycle with G1, S, G2, and M phases. In contrast with what happens in higher eucaryotic cells, however, the nuclear envelope of the yeast cell does not break down during M phase. The microtubules of the mitotic spindle (light green) form inside the nucleus and are attached to spindle pole bodies (dark green) at its periphery. The cell divides by forming a partition (known as the cell plate) and splitting in two. The condensed mitotic chromosomes (red) are readily visible in fission yeast, but are less easily seen in budding yeasts. (B) The budding yeast has normal G1 and S phases but does not have a normal G2 phase. Instead, a microtubule-based spindle begins to form inside the nucleus early in the cycle, during S phase. In contrast with a fission yeast cell, the cell divides by budding. As in fission yeasts, but in contrast with higher eucaryotic cells, the nuclear envelope remains intact during mitosis, and the spindle forms within the nucleus.

Rb tumor suppressor protein

• Rb = tumor suppressor protein (loss produces retinoblastomas). • Alfred Knudson proposed the two-hit hypothesis to explain familial retinoblastomas (multiple cancers in both eyes) from sporadic retinoblastoma (one cancer in one eye)

Ubiquitin Degradation Regulates S phase entry

• SCF complex = ubiquitin ligase Sic1 is phosphorylated in 7 places by CDK/CLN complex at G1/S-->SCF complex binds to Sic1 and degrades it-->enters S phase with DNA replication • If SCF components are inactivated by mutation, then cells arrest at G1/S boundary - because Sic1 cannot be degraded • The trigger for SIC1 degradation at G1/S is phosphorylation by CDK/CLN complex • The G1 cyclins CLNs are also phosphorylated by CDK/CLN and targeted for degradation by SCF complexes • the SCF complex only binds to phosphorylated substrates

Activating Phosphorylation of the CDK

• The T-loop blocks access to the ATP-binding cleft (active site) • Phosphorylation of the T-loop makes it move out of the way, resulting in partial activation of the Cdk2 • Full activation of the cyclin-Cdk complex then occurs when a separate kinase, the Cdk-activating kinase (CAK), phosphorylates an amino acid near the entrance of the Cdk active site. T-loop phosphorylation is by a CDK-activating kinase (CAK). This causes a conformational change that further increases the activity of CDK, allowing the kinase to phosphorylate its target proteins effectively and thereby induce specific cell-cycle events -Phosphorylation of Cdk2 (by CAK) at a threonine residue in the T-loop further activates the enzyme by changing the shape of the T-loop, improving the ability of the enzyme to bind its protein substrates

Inhibitory Phosphorylation of the CDK

• Tyrosine 15 and Threonine 14 are in the ATP-binding pocket. • Phosphorylation of Y15 and T14 block the transfer of the gamma-phosphate to the substrate. • The inhibitory kinase is Wee1 or Myt1. The active cyclin-Cdk complex is turned off when the kinase Wee1 phosphorylates two closely spaced sites above the active site. Removal of these phosphates by the phosphatase Cdc25 activates the cyclin-Cdk complex. Also Binding of Cdk inhibitor proteins (CKIs) inactivates cyclin-Cdk complexes. Cells use CKIs primarily to help govern the activities of G1/S- and S-Cdks early in the cell cycle. -in humans: The p27 (a CKI) binds to both the cyclin and Cdk in the complex, distorting the active site of the Cdk. It also inserts into the ATP-binding site, further inhibiting the enzyme activity.

Cytokinesis regulation

• cytokinesis is accomplished by a contractile ring of actin and myosin II. • myosin II light chain is phosphorylated by MPF keeping myosin II inactive. • When MPF is destroyed at metaphase/anaphase transition, myosin light chain II can be dephosphorylated and become active. Myosin II contracts actin filaments during cytokinesis Only occurs later bc cyclin B must be degraded. Regulated because of myosin sliding actin filaments, where they get smaller to cause a constriction.

p53-mediated cell cycle arrest

• p53 is a tumor suppressor protein that is activated by DNA damage In a normal cell, p53 is inactivated by its negative regulator, mdm2. Upon DNA damage or other stresses, various pathways will lead to the dissociation of the p53 and mdm2 complex. Once activated, p53 will induce a cell cycle arrest (by CKI p21) to allow either repair and survival of the cell or apoptosis to discard the damaged cell. How p53 makes this choice is currently unknown. • in the absence of DNA damage, p53 is constantly degraded by the MDM2 (an E3 ubiquitin-protein ligase) • upon DNA damage, MDM2 is inactivated, and p53 protein levels accumulate • p53 is a transcription factor that works as a homo-tetramer. Only the higher level of p53 protein allows the active homo-tetramer to form. • p53 induces the transcription of genes that promote apoptosis and cell cycle arrest (cell cycle arrest genes include the CKI p21 that inhibits CDK/cyclin complexes) *p21: Suppresses G1/S-Cdk and S-Cdk activities following DNA damage • p21 does not inhibit PCNA (clamp that goes around DNA and binds on end of DNA polymerase, cant come off DNA) in cells, instead p21 binding to chromatin-bound PCNA causes p21 to get degraded thereby ending the checkpoint after DNA is repaired (which chromatin-bound PCNA).