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© 2012 Pearson Education, Inc. Lectures by Kathleen Fitzpatrick Simon Fraser University The Cell Cycle, DNA Replication, and Mitosis Chapter 19.

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Presentation on theme: "© 2012 Pearson Education, Inc. Lectures by Kathleen Fitzpatrick Simon Fraser University The Cell Cycle, DNA Replication, and Mitosis Chapter 19."— Presentation transcript:

1 © 2012 Pearson Education, Inc. Lectures by Kathleen Fitzpatrick Simon Fraser University The Cell Cycle, DNA Replication, and Mitosis Chapter 19

2 © 2012 Pearson Education, Inc. The Cell Cycle, DNA Replication, and Mitosis Cell growth is generally accompanied by cell division, whereby one cell gives rise to two new daughter cells All the genetic information in the nucleus must be accurately duplicated and carefully distributed to the daughter cells In doing this a cell passes through a series of stages known as the cell cycle

3 © 2012 Pearson Education, Inc. Overview of the Cell Cycle The cell cycle begins when two new cells are formed by division of a parent cell and ends when one of these cells divides again M phase is when the cells actually divide; the nucleus first, followed by the cytoplasm Nuclear division is mitosis and division of the cytoplasm is cytokinesis

4 © 2012 Pearson Education, Inc. Chromosomes in mitosis At the beginning of mitosis, chromatin folds and condenses to produce visible chromosomes DNA has replicated, so each chromosome is composed of two sister chromatids The microtubules of the mitotic spindle will distribute the chromatids to opposite ends of the cell

5 © 2012 Pearson Education, Inc. Figure 19-1A

6 © 2012 Pearson Education, Inc. Mitosis is a relatively short part of the cell cycle Cells spend very little time in M phase Most of the time is spent in interphase, which is composed of G1 phase, S phase (when DNA is replicated), and G2 phase The overall length of the cell cycle is called the generation time; in cultured mammalian cells this is about 18–24 hours

7 © 2012 Pearson Education, Inc. Figure 19-1B

8 © 2012 Pearson Education, Inc. The G phases G1 is quite variable depending on cell type; G2 is shorter and less variable During G1 a major decision is made, whether a cell will divide again; cells that arrest in G1, waiting for a signal to divide, are said to be in G0 Cells that exit the cell cycle are said to undergo terminal differentiation

9 © 2012 Pearson Education, Inc. DNA Replication DNA replication is a central event in the cell cycle The underlying mechanism depends on the double-helical structure of DNA One strand of every new DNA molecule is derived from the parent molecule and the other is new: semiconservative replication

10 © 2012 Pearson Education, Inc. Figure 19-2

11 © 2012 Pearson Education, Inc. DNA Replication Is Usually Bidirectional DNA replication is especially well understood in Escherichia coli Saccharomyces cerevisiae and the virus SV40 are used in studies of eukaryotic replication Replication is very similar in prokaryotes and eukaryotes

12 © 2012 Pearson Education, Inc. Early experiments in replication Cairns studied replication in E. coli; he grew cells in a medium containing 3 H-thymidine He visualized the circular chromosomes by autoradiography; he observed replication forks These are formed where replication begins and then proceeds in bidirectional fashion away from the origin

13 © 2012 Pearson Education, Inc. Figure 19-4A

14 © 2012 Pearson Education, Inc. Bacterial replication Replication forks move away from the origin, unwind the DNA, and copy both strands as they proceed This is called theta (  ) replication and is observed in replication of circular DNA molecules The two copies of the replicating chromosome bind to the plasma membrane at their origins; when replication is complete the cell divides by binary fission

15 © 2012 Pearson Education, Inc. Figure 19-4B

16 © 2012 Pearson Education, Inc. Figure 19-4C

17 © 2012 Pearson Education, Inc. Eukaryotic DNA Replication Involves Multiple Replicons In eukaryotes replication of linear chromosomes is initiated at multiple sites, creating replication units called replicons At the center of each replicon is a DNA sequence called an origin of replication, where synthesis is initiated by several groups of initiator proteins First, a multisubunit protein complex called the origin recognition complex (ORC) binds the replication origin

18 © 2012 Pearson Education, Inc. Figure 19-5A-E

19 © 2012 Pearson Education, Inc. Figure 19-5A

20 © 2012 Pearson Education, Inc. Figure 19-5B

21 © 2012 Pearson Education, Inc. Figure 19-5C

22 © 2012 Pearson Education, Inc. Figure 19-5D

23 © 2012 Pearson Education, Inc. Figure 19-5E

24 © 2012 Pearson Education, Inc. Eukaryotic replication (continued) Next, the minichromosome maintenance (MCM) proteins bind the origin The MCM proteins include several DNA helicases that unwind the double helix; a set of proteins called helicase loaders recruit the MCM proteins At this point all the DNA-bound proteins make up the pre-replication complex and the DNA is “licensed” for replication

25 © 2012 Pearson Education, Inc. Origins of replication DNA sequences that act as replication origins are greatly varied in eukaryotes Sequences that confer ability to replicate when introduced into DNA molecules are called autonomously replicating sequences or ARS After replication begins, two replication forks synthesize DNA in opposite directions, forming a replication bubble that grows as replication proceeds

26 © 2012 Pearson Education, Inc. Rates of replication S phase is very rapid in embryonic cells, where cell divisions occur in quick succession, but slower in adult cells, in which fewer and more widely spaced replicons are used During S phase in eukaryotes, not all replicons are activated at the same time; some are replicated early and others later Genes that are transcriptionally active are replicated earlier than inactive genes

27 © 2012 Pearson Education, Inc. Replication Licensing Ensures That DNA Molecules Are Duplicated Only Once Prior to Each Cell Division Licensing is provided by binding of MCM proteins to the origin, which requires both ORC and helicase loaders It ensures that after DNA is replicated at each origin, the DNA cannot be licensed for replication again until after mitosis After replication begins, the MCM proteins are removed from the origins and cannot bind again

28 © 2012 Pearson Education, Inc. Role of Cdk Cyclin-dependent kinase, cdk, has many roles in the cell cycle One form produced early in S phase activates DNA synthesis at licensed origins and prevents origins from being licensed again It catalyzes phosphorylation of ORC proteins and helicase loaders

29 © 2012 Pearson Education, Inc. Geminin Multicellular eukaryotes contain an additional inhibitor of relicensing called geminin It is made during S phase that blocks the binding of MCM proteins to DNA When the cell completes mitosis, geminin is degraded and Cdk activity falls so that licensing can occur for the next cycle

30 © 2012 Pearson Education, Inc. Figure 19-6

31 © 2012 Pearson Education, Inc. DNA Polymerases Catalyze the Elongation of DNA Chains DNA polymerase is an enzyme that can copy DNA molecules Incoming nucleotides are added to the 3 hydroxyl end of the growing DNA chain, so elongation occurs in the 5 to 3 direction Several other forms of DNA polymerase have been identified; the original is now called DNA polymerase I

32 © 2012 Pearson Education, Inc. Figure 19-7

33 © 2012 Pearson Education, Inc. Table 19-1

34 © 2012 Pearson Education, Inc. Temperature-sensitive mutations It is difficult to grow and study mutant strains that lack important functions such as DNA replication One approach involves using temperature- sensitive mutants, which produce proteins that function properly at 37 o C but lose their function when the temperature is raised to 42 o C

35 © 2012 Pearson Education, Inc. Observations on temperature- sensitive bacteria Bacterial strains have been identified in which DNA polymerase III functions normally at 37 o C but loses its ability to replicate when the temperature is raised to 42 o C The observations indicate that DNA polymerase III is central to bacterial DNA replication However, a variety of other proteins is needed for replication

36 © 2012 Pearson Education, Inc. Eukaryote DNA polymerases Eukaryotic cells contain several types of DNA polymerase; more than a dozen Of these, DNA polymerase , , and  are involved in nuclear DNA replication DNA polymerase  is used in mitochondrial DNA replication Those types remaining are involved in DNA repair or replication across regions of DNA damage

37 © 2012 Pearson Education, Inc. Biotechnology functions of DNA polymerases DNA polymerases have practical applications in biotechnology The polymerase chain reaction is a technique in which a DNA polymerase is used to amplify tiny samples of DNA

38 © 2012 Pearson Education, Inc. DNA Is Synthesized as Discontinuous Segments That Are Joined Together by DNA Ligase DNA is synthesized in the 5 to 3 direction, but the two strands of the double helix are oriented in opposite directions One strand (the lagging strand) is synthesized in discontinuous fragments called Okazaki fragments The other (the leading strand) is synthesized in a continuous chain

39 © 2012 Pearson Education, Inc. Okazaki’s experiments Okazaki isolated DNA from bacteria that were briefly exposed to a radioactive substrate incorporated into newly made DNA Much of the radioactivity was located in small fragments about 1000 nucleotides long With longer labeling the radioactivity became associated with longer molecules; this conversion did not take place in bacteria lacking DNA ligase

40 © 2012 Pearson Education, Inc. Figure 19-8

41 © 2012 Pearson Education, Inc. Figure 19-8A

42 © 2012 Pearson Education, Inc. Figure 19-8B

43 © 2012 Pearson Education, Inc. Okazaki’s observations illustrate how lagging strand synthesis occurs DNA synthesis from the lagging strand is synthesized in Okazaki fragments These are then joined by DNA ligase to form a continuous new 3 to 5 DNA strand Okazaki fragments are 1000–2000 nucleotides long in bacteria and viruses, but about one-tenth this length in eukaryotic cells

44 © 2012 Pearson Education, Inc. Figure 19-9

45 © 2012 Pearson Education, Inc. Proofreading Is Performed by the 3→ 5 Exonuclease Activity of DNA Polymerase About 1 of every 100,000 nucleotides incorporated during DNA replication is incorrect Such mistakes are usually fixed by a proofreading mechanism Almost all DNA polymerases have a 3 → 5 exonuclease activity

46 © 2012 Pearson Education, Inc. Proofreading Exonucleases degrade nucleic acids from the ends of the molecules Endonucleases make internal cuts in nucleic acid molecules The exonuclease activity of DNA polymerase allows it to remove incorrectly base-paired nucleotides and incorporate the correct base

47 © 2012 Pearson Education, Inc. Figure

48 © 2012 Pearson Education, Inc. Figure

49 © 2012 Pearson Education, Inc. RNA Primers Initiate DNA Replication DNA polymerase can only add nucleotides to the 3 end of an existing nucleotide chain Researchers implicated RNA in the initiation process based on several observations 1. Okazaki fragments usually have short stretches of RNA at their 5 ends

50 © 2012 Pearson Education, Inc. Observations about RNA involvement in DNA replication (continued) 2. DNA polymerase is able to add nucleotides to RNA chains as well as DNA chains 3. Cells contain an enzyme called primase that synthesizes short (~10 nt) chains of RNA using DNA as a template 4. Primase is able to initiate RNA strands without a pre-existing chain to add to

51 © 2012 Pearson Education, Inc. DNA synthesis requires RNA primers The observations led to the conclusion that DNA synthesis is initiated by the formation of short RNA primers These are synthesized by primase using a single DNA strand as the template In E. coli, primase is inactive unless accompanied by six other proteins, forming a complex called a primosome

52 © 2012 Pearson Education, Inc. Figure 19-11

53 © 2012 Pearson Education, Inc. The process of DNA synthesis Once the RNA primer is made, DNA polymerase III (or DNA polymerase , followed by  or , in eukaryotes) adds deoxynucleotides to the 3 end of the primer For the leading strand, just one primer is needed, but the lagging strand needs a series of primers to initiate each Okazaki fragment When the DNA chain reaches the next Okazaki fragment the RNA is degraded and replaced with DNA; adjacent fragments are joined together by DNA ligase

54 © 2012 Pearson Education, Inc. Unwinding the DNA Double Helix Requires DNA Helicases, Topoisomerases, and Single-Stranded DNA Binding Proteins During DNA replication the two strands of the double helix must unwind at each replication fork Three classes of proteins facilitate the unwinding: DNA helicases, topoisomerases, and single-stranded DNA binding proteins DNA helicases are responsible for unwinding the DNA, using energy from ATP hydrolysis

55 © 2012 Pearson Education, Inc. Helicases The DNA double helix is unwound ahead of the replication fork, the helicases breaking the hydrogen bonds as they go In E. coli, at least two different helicases are involved; one attaches to the lagging strand and moves 5 → 3, whereas the other attaches to the leading strand and moves 3 → 5 Both are part of the primosome

56 © 2012 Pearson Education, Inc. Topoisomerases The unwinding of the helix would create too much supercoiling if not for topoisomerases These enzymes create swivel points in the DNA molecule by making and then quickly sealing double-strand or single-stranded breaks Of the ~10 topoisomerases in E. coli, the key enzyme for DNA replication is gyrase

57 © 2012 Pearson Education, Inc. Single-stranded DNA binding protein Once strand separation has begun, molecules of SSB (single-stranded DNA binding protein) move in quickly and attach to the exposed single strands They keep the DNA unwound and accessible to the replication machinery When a segment of DNA has been replicated, the SSB molecules fall off and are recycled

58 © 2012 Pearson Education, Inc. Figure 19-12

59 © 2012 Pearson Education, Inc. Putting It All Together: DNA Replication in Summary Starting at the origin of replication, the machinery at the replication fork adds proteins required for synthesizing DNA These are DNA helicase, DNA gyrase, SSB, primase, DNA polymerase, and DNA ligase Several other proteins are used to improve the efficiency, e.g., a ring-shaped sliding clamp keeps DNA polymerase firmly attached to DNA

60 © 2012 Pearson Education, Inc. Figure 19-13, top

61 © 2012 Pearson Education, Inc. Figure 19-13, bottom

62 © 2012 Pearson Education, Inc. The replisome Proteins involved in DNA replication are all closely associated in one large complex called a replisome The activity and movement of the replisome is powered by nucleoside triphosphate hydrolysis As the replisome moves along the DNA it must accommodate the fact that DNA is being produced on both leading and lagging strands

63 © 2012 Pearson Education, Inc. Figure 19-14

64 © 2012 Pearson Education, Inc. Eukaryotic DNA replication Eukaryotes have much of the same replication machinery found in prokaryotes E.g. a DNA clamp protein acts along with DNA polymerase; one of these is called proliferating nuclear cell antigen (PCNA) PCNA is a clamp protein for DNA polymerase 

65 © 2012 Pearson Education, Inc. Replication factories and chromatin remodeling Studies addressing how many origins of replication can be coordinated suggest that the immobile structures called replication factories synthesize DNA as chromatin fibers are fed through them Unfolding chromatin fibers ahead of the replication fork is facilitated by chromatin remodeling proteins that loosen nucleosome packing

66 © 2012 Pearson Education, Inc. Telomeres Solve the DNA End- Replication Problem Linear DNA molecules have a problem in completing DNA replication on the lagging strand, because primers are required Each round of replication would end with the loss of some nucleotides from the ends of each linear molecule Eukaryotes solve this problems with telomeres, highly repeated sequences at the ends of chromosomes

67 © 2012 Pearson Education, Inc. Figure 19-15

68 © 2012 Pearson Education, Inc. Telomeres and telomerase Human telomeres have 100 to 1500 copies of TTAGGG at the ends of chromosomes These noncoding sequences ensure that the cell will not lose important genetic information if DNA molecules shorten during replication A polymerase called telomerase can catalyze the addition of repeats to chromosome ends

69 © 2012 Pearson Education, Inc. Telomerase function Telomerase is composed of protein and RNA In the protozoan Tetrahymena the RNA component of the telomerase (3-AACCCC-5) is complementary to the telomere repeat sequence (5-TTGGGG-3) This enzyme-bound RNA acts as a template for adding the DNA repeat sequence to the telomere ends

70 © 2012 Pearson Education, Inc. Protecting chromosome ends After telomeres are lengthened by telomerase, telomere capping proteins bind to the exposed 3 end to protect from degradation In many eukaryotes the 3 ends of the DNA also loop back and base-pair with the opposite strand to form a protective closed loop In multicellular organisms telomerase function is restricted to germ cells and a few other types of actively proliferating cells

71 © 2012 Pearson Education, Inc. Figure 19-16

72 © 2012 Pearson Education, Inc. Most cells have a limited life span Telomere shortening occurs with each cell division in most cells As a result, telomere length is a counting device for how many times a cell has divided; if a cell divides too many times, telomeres could be lost Cells at risk of loss of telomeres undergo apoptosis, programmed cell death

73 © 2012 Pearson Education, Inc. DNA Damage and Repair DNA must be accurately passed on to daughter cells In addition to ensuring that replication is faithful, this also means that DNA alterations must be repaired DNA alterations, or mutations, can arise spontaneously, or through exposure to environmental agents

74 © 2012 Pearson Education, Inc. DNA Damage Can Occur Spontaneously or in Response to Mutagens During DNA replication, some types of mutations occur through spontaneous hydrolysis reactions Depurination is loss of a purine base (A or G) Deamination is removal of a base’s amino group, changing its base-pairing properties Deamination may involve A, G, or, most often, C

75 © 2012 Pearson Education, Inc. Figure 19-17A

76 © 2012 Pearson Education, Inc. Figure 19-17B

77 © 2012 Pearson Education, Inc. DNA damage by mutagens DNA damage can be caused by mutation- causing agents, mutagens Environmental mutagens fall into two categories: chemicals and radiation Mutagenic chemicals alter DNA structure through a variety of mechanisms

78 © 2012 Pearson Education, Inc. DNA damage by chemical mutagens Base analogs resemble nitrogenous bases and are incorporated into DNA Base-modifying agents react chemically with DNA bases to alter their structures, forming DNA adducts Intercalating agents insert themselves between adjacent bases, distorting DNA structure

79 © 2012 Pearson Education, Inc. DNA damage by radiation Ultraviolet radiation alters DNA by triggering pyrimidine dimer formation – covalent bonds between adjacent pyrimidine bases X-rays and related types of radiation, called ionizing radiation, remove electrons from molecules, and generate highly reactive intermediates that damage DNA

80 © 2012 Pearson Education, Inc. Figure 19-17C

81 © 2012 Pearson Education, Inc. Translesion Synthesis and Excision Repair Correct Mutations Involving Abnormal Nucleotides A variety of mechanisms are used for DNA repair Some repair takes place during replication, with specialized DNA polymerases that carry out translesion synthesis This is synthesis across regions in which DNA is damaged (e.g., DNA polymerase η synthesizes new DNA across regions containing a thymine dimer)

82 © 2012 Pearson Education, Inc. Errors remaining after DNA replication Errors remaining after DNA replication are repaired by excision repair, in which abnormal nucleotides are removed and replaced E. coli has nearly 100 genes that code for proteins involved in this process Excision repair works by a basic three-step process

83 © 2012 Pearson Education, Inc. Excision repair Repair endonucleases are recruited to DNA by proteins that recognize damage 1. They cleave the backbone adjacent to the damage site; other enzymes remove the defective nucleotides 2. DNA polymerase (I in E. coli) replaces the missing nucleotides 3. DNA ligase seals the remaining nick in the repaired strand

84 © 2012 Pearson Education, Inc. Figure 19-18

85 © 2012 Pearson Education, Inc. Types of excision repair Excision repair pathways are classified into two types Base excision repair corrects single damaged bases E.g., deaminated bases are detected by DNA glycosylases, which recognize and remove the base by cleaving the bond between the base and the sugar

86 © 2012 Pearson Education, Inc. Base excision repair (continued) The sugar with the missing base is then recognized by a repair endonuclease that detects depurination It breaks the phosphodiester backbone to one side of the sugar and a second enzyme removes the sugar

87 © 2012 Pearson Education, Inc. Nucleotide excision repair For removing pyrimidine dimers and other bulky lesions, a second type of excision repair is employed Nucleotide excision repair uses proteins that detect distortions in the DNA helix and recruit NER endonuclease (or exinuclease) that cuts the DNA backbone on either side of the lesion Helicase unwinds the DNA between the nicks, and frees it from the DNA; DNA polymerase and ligase complete the repair

88 © 2012 Pearson Education, Inc. The NER system is versatile The nucleotide excision repair system detects and corrects many types of DNA damage Sometimes it is recruited to regions where transcription is stalled because of DNA damage; this is called transcription-coupled repair People with xeroderma pigmentosum must stay out of the sun because of mutations that prevent them from carrying out NER

89 © 2012 Pearson Education, Inc. Mismatch Repair Corrects Mutations That Involve Noncomplementary Base Pairs Mismatch repair targets errors made during DNA replication that escape proofreading Mismatch repair is able to distinguish the original vs. the newly synthesized strand in order to correctly repair the mismatch E. coli uses a detection system based on methylation of A in the sequence GATC

90 © 2012 Pearson Education, Inc. Methylation in mismatch repair DNA methylation does not occur immediately after DNA replication Therefore, mismatch repair systems can distinguish the original DNA (methylated) from the newly made strand (non-methylated) The incorrect nucleotide in the newly made strand is excised and replaced

91 © 2012 Pearson Education, Inc. Damage Repair Helps Explain Why DNA Contains Thymine Instead of Uracil For some years it was not clear why DNA contained thymine instead of uracil But repair of deaminated nucleotides shows why DNA cannot contain uracil Deamination of cytosine converts it to uracil, which is detected and repaired; if DNA contained uracil normally, this type of repair could not be effected

92 © 2012 Pearson Education, Inc. Figure 19-19

93 © 2012 Pearson Education, Inc. Double-Strand DNA Breaks Are Repaired by Nonhomologous End- Joining or Homologous Recombination Double-strand breaks cleave DNA into two fragments It is difficult for the repair system to identify and rejoin the correct broken ends without loss of nucleotides Two pathways are used: nonhomologous end- joining and homologous recombination

94 © 2012 Pearson Education, Inc. Nonhomologous end-joining Nonhomologous end-joining uses a set of proteins that bind to ends of broken DNA fragments and join them together This is error-prone, because nucleotides can be lost from the broken ends, and there is no way to ensure the correct DNA fragments are joined

95 © 2012 Pearson Education, Inc. Homologous recombination Homologous recombination is a more precise method for fixing double-strand breaks If the DNA molecule from one chromosome is broken, the homologue is available as a template to guide accurate repair

96 © 2012 Pearson Education, Inc. Nuclear and Cell Division The two copies of each chromosome made during S phase are distributed into daughter cells during M phase M phase includes nuclear division (mitosis) and cytoplasmic division (cytokinesis)

97 © 2012 Pearson Education, Inc. Mitosis Is Subdivided into Prophase, Prometaphase, Metaphase, Anaphase, and Telophase Mitosis is divided into five stages based on changing appearance and behavior of chromosomes Events during each stage are directed toward the correct distribution of one copy of each chromosome into daughter nuclei

98 © 2012 Pearson Education, Inc. Prophase After DNA replication, cells exit S phase and enter G2 phase, where final preparations are made for entry into mitosis Toward the end of G2, chromosomes begin to condense into more compact, folded structures The G2 → prophase transition is not sharply defined but cells are in prophase when individual chromosomes become visible

99 © 2012 Pearson Education, Inc. Prophase In prophase, each chromosome has two chromatids In animal cells nucleoli disperse, but in plant cells nucleoli may still be visible The centrosomes near the nucleus, which function as microtubule organizing centers (MTOC), begin to migrate away from each other

100 © 2012 Pearson Education, Inc. Prophase (continued) As centrosomes move, they act as nucleation sites for microtubules (MTs), destined to form the mitotic spindle A dense starburst of MTs called an aster forms near each centrosome Within the centrosomes are microtubule- containing structures called centrioles

101 © 2012 Pearson Education, Inc. Figure 19-20A

102 © 2012 Pearson Education, Inc. Figure 19-21A

103 © 2012 Pearson Education, Inc. Prometaphase The onset of prometaphase is marked by the fragmentation of the membranes of the nuclear envelope Centrosomes complete their movement to opposite sides of the nucleus and the spindle MTs contact the condensed chromosomes MTs attach to chromosomes in the centromere region

104 © 2012 Pearson Education, Inc. Centromeres and kinetochores DNA in centromeres consists of simple, tandemly repeated CEN sequences, with considerable variation among species A common feature among species is the presence of a special histone H3 called CENP-A in humans CENP-A recruits additional proteins to the centromere to form the kinetochore, to which MTs attach

105 © 2012 Pearson Education, Inc. Kinetochores Kinetochore proteins begin to assemble on centromeres shortly after S phase During prometaphase spindle MTs bind the kinetochores associated with each chromatid Forces exerted by these kinetochore microtubules gradually move chromosomes toward the center of the cell; this is called congression

106 © 2012 Pearson Education, Inc. Figure 19-22A

107 © 2012 Pearson Education, Inc. Two other types of microtubules Polar microtubules interact with microtubules from the opposite pole of the cell Astral microtubules are shorter and form the asters at each pole Some of the astral microtubules interact with proteins lining the plasma membrane

108 © 2012 Pearson Education, Inc. Figure 19-20B

109 © 2012 Pearson Education, Inc. Figure 19-21B

110 © 2012 Pearson Education, Inc. Metaphase A cell is in metaphase when the fully condensed chromosomes are all aligned at the metaphase plate (a plane equidistant between the two poles of the spindle) Agents that interfere with spindle function (e.g., colchicine) are used to arrest cells at metaphase Examining metaphase cells allows chromosomes to be identified, generating a karyotype

111 © 2012 Pearson Education, Inc. Figure 19-20C

112 © 2012 Pearson Education, Inc. Figure 19-21C

113 © 2012 Pearson Education, Inc. Figure 19-22B

114 © 2012 Pearson Education, Inc. Figure 19-23

115 © 2012 Pearson Education, Inc. Anaphase Anaphase is the shortest phase of mitosis The two sister chromatids of each chromosome abruptly separate and move toward opposite poles In anaphase A, the chromosomes are pulled toward spindle poles as kinetochore MTs get shorter

116 © 2012 Pearson Education, Inc. Anaphase (continued) In anaphase B the spindle poles themselves move away from each other as polar MTs lengthen Depending on the cell type, anaphase A and B may take place at the same time, or anaphase B may follow anaphase A

117 © 2012 Pearson Education, Inc. Figure 19-20D

118 © 2012 Pearson Education, Inc. Figure 19-21D

119 © 2012 Pearson Education, Inc. Figure 19-24

120 © 2012 Pearson Education, Inc. Telophase At the beginning of telophase the daughter chromosomes arrive at the poles of the spindle Chromosomes uncoil into interphase chromatin Nucleoli reappear and nuclear envelopes reform During this period, cytokinesis also takes place

121 © 2012 Pearson Education, Inc. Figure 19-20E

122 © 2012 Pearson Education, Inc. Figure 19-21E

123 © 2012 Pearson Education, Inc. The Mitotic Spindle Is Responsible for Chromosome Movements During Mitosis The microtubule-containing apparatus responsible for separation of chromatids into daughter cells is the mitotic spindle

124 © 2012 Pearson Education, Inc. Spindle Assembly and Chromosome Attachment Microtubules have an inherent polarity (the two ends have different chemical properties) The end where MT assembly is initiated (the centrosome in the case of the spindle) is the minus end The end where most growth occurs is the plus end

125 © 2012 Pearson Education, Inc. Figure 19-25

126 © 2012 Pearson Education, Inc. Spindle assembly During late prophase, MT growth speeds up dramatically and initiation of new MTs at centrosomes increases When the nuclear envelope disintegrates, kinetochores and MTs can come into contact When the plus end of MTs and the kinetochore bind, the MT becomes a kinetochore MT

127 © 2012 Pearson Education, Inc. Kinetochores Each kinetochore is a three-layered structure made of proteins attached to CEN sequences at the centromere The two kinetochores are located at opposite sides of a chromosome and so they usually attach to MTs from opposite spindle poles The polar MRs make contact with MTs from the opposite pole at the same time

128 © 2012 Pearson Education, Inc. Figure 19-26

129 © 2012 Pearson Education, Inc. Cells without centrosomes Cells without centrosomes can still form spindles using a different mechanism, promoted by chromosomes This requires the involvement of Ran, the GTP- binding protein Ran binds to GTP due to a protein associated with mitotic chromosomes, and then binds importin and releases proteins that promote MT assembly

130 © 2012 Pearson Education, Inc. Chromosome Alignment and Separation Chromosomes migrate to the central region of the spindle through a series of movements generated by pulling and pushing forces from the microtubules Chromosomes reach the central region and stay there as a result of precisely balanced forces pulling them toward opposite poles Chromatid separation requires action of topoisomerase II and changes in adhesive proteins

131 © 2012 Pearson Education, Inc. Motor Proteins and Chromosome Movement Several motor proteins play active roles in mitosis They use energy from ATP to change shape and exert force that causes movement of attached structures Motor proteins play at least three distinct roles in movement of anaphase chromosomes

132 © 2012 Pearson Education, Inc. Roles of motor proteins in chromosome movement 1. Chromosomes are moved, kinetochores first, toward the spindle poles during anaphase A This is driven by kinesins associated with the kinetochore MTs One kinesin-like motor is at the plus end of the MT, embedded in the kinetochore, and moves the chromosome as it “chews up” the MT

133 © 2012 Pearson Education, Inc. Roles of motor proteins in chromosome movement (continued) The other kinesin-like motor is located at the minus end of the kinetochore MTs It is embedded in the spindle pole and induces depolymerization there, “reeling in” the microtubules and the attached chromosomes

134 © 2012 Pearson Education, Inc. Roles of motors in anaphase (continued) 2. Motor proteins play a role in the movement of the spindle poles away from each other during anaphase B Bipolar kinesin motors bind to overlapping polar MTs from opposite spindle poles, forcing the spindle poles away from each other

135 © 2012 Pearson Education, Inc. Roles of motors in anaphase (continued) 3. Cytoplasmic dynein is associated with astral microtubules that are connected to the cell cortex The cell cortex is a layer of actin microfilaments lining the inner surface of the plasma membrane The dynein moves toward the minus ends of the microtubules and appears to move the spindle toward the cortex

136 © 2012 Pearson Education, Inc. Figure 19-27A

137 © 2012 Pearson Education, Inc. Figure 19-27B

138 © 2012 Pearson Education, Inc. Figure 19-27C

139 © 2012 Pearson Education, Inc. Cytokinesis Divides the Cytoplasm After the chromosomes have separated, cytokinesis divides the cytoplasm in two It starts in late anaphase or early telophase, usually Certain cell types undergo many rounds of nuclear division without cytokinesis, forming a syncytium (multinucleate cell)

140 © 2012 Pearson Education, Inc. Cytokinesis in Animal Cells Cytoplasmic division is called cleavage; it begins with a slight puckering of the cell surface that deepens into a cleavage furrow The furrow continues to deepen until opposite surfaces make contact and split the cell in two The furrow deepens along a plane passing through the spindle equator

141 © 2012 Pearson Education, Inc. Figure 19-28

142 © 2012 Pearson Education, Inc. Cytokinesis Signals emanating from the central part of the spindle, the spindle midzone, are important for completing cytokinesis Cleavage depends on a beltlike bundle of actin microfilaments (the contractile ring) that form just below the plasma membrane in early anaphase As cleavage progresses, the ring tightens around the cytoplasm

143 © 2012 Pearson Education, Inc. Figure 19-29A

144 © 2012 Pearson Education, Inc. Myosin and cleavage Contraction of the ring is generated by interactions between actin and the motor protein, myosin Members of Rho-GTP binding proteins regulate assembly and activation of the contractile ring RhoA is recruited to the cleavage furrow to activate proteins needed for actin polymerization, and stimulate activation of myosin

145 © 2012 Pearson Education, Inc. Figure 19-29B

146 © 2012 Pearson Education, Inc. Cytokinesis in Plant Cells Plant cells cannot form a contractile ring because of the rigid cell wall They assemble a plasma membrane and cell wall between the two daughter nuclei In late anaphase or early prophase, a group of small vesicles from the Golgi complex align themselves across the equatorial region of the spindle

147 © 2012 Pearson Education, Inc. Cell Division Is Sometimes Asymmetric Cytokinesis is not always symmetric; sometimes the spindle forms in asymmetric fashion This can result in one large and one small cell These occur frequently during embryonic development; sometimes cells formed in this way have differing developmental potentials

148 © 2012 Pearson Education, Inc. Figure 19-31

149 © 2012 Pearson Education, Inc. Regulation of the Cell Cycle Variations are observed in –overall length of the cell cycle –relative length of time spent in various phases –how closely mitosis and cytokinesis are coupled The cell cycle is regulated to meet the needs of each cell type and organism

150 © 2012 Pearson Education, Inc. The Length of the Cell Cycle Varies Among Different Cell Types At one extreme are cells that divide continuously to replace cells that are constantly lost or destroyed –E.g., cells involved in sperm formation, and stem cells At the other extreme are extremely slow growing tissues or even some (mature nerve or muscle cells) that do not divide at all Some cells do not divide unless stimulated

151 © 2012 Pearson Education, Inc. Variations in generation time Most of the variations in generation time are based on differences in the length of G1, though S and G2 can also vary Cells that divide very slowly may spend days, months, or years in the offshoot of G1 called G0 Cells that divide very rapidly have a short G1 or may skip it entirely

152 © 2012 Pearson Education, Inc. Cell growth and the cell cycle Although in some cases cell growth is not essential to the cell cycle, the two are generally linked A protein kinase called TOR (target of rapamycin) plays a role in the signaling network that controls cell size and coordinates it with cell cycle progression The network activates TOR in the presence of nutrients and growth factors

153 © 2012 Pearson Education, Inc. Cell growth and the cell cycle (continued) Activated TOR stimulates molecules that control the rate of protein synthesis, leading to increased cell mass Some of the molecules also facilitate entry into S phase TOR is therefore an important regulator of both cell size and cell cycle progression

154 © 2012 Pearson Education, Inc. Progression Through the Cell Cycle Is Controlled at Several Key Transition Points Control of the cell cycle must –1. Ensure that events of each phase are carried out in the correct order and at the appropriate time –2. Ensure that each phase is completed before the next one begins –3. Respond to external conditions

155 © 2012 Pearson Education, Inc. Key transition points At key transition points in the cell cycle, conditions in the cell determine whether or not it will proceed The first control point occurs in late G1; the G1→ S progression is called Start in yeast In animal cells, the comparable control point is called the restriction point; the ability to pass it is influenced by the presence of extracellular growth factors

156 © 2012 Pearson Education, Inc. The restriction point Cells that successfully pass the restriction point are committed to S phase Those that do not, enter into G0 and reside there until a signal allows them to reenter G1 and pass through the restriction points

157 © 2012 Pearson Education, Inc. A second transition point Another transition point occurs at the G2  M boundary, where the commitment is made to enter mitosis In some cell types, the cell can be arrested in G2 indefinitely and the cell enters a state analogous to G0 In most cells the G1 arrest is the more prevalent type of control, but in some, such as frog eggs, the G2 arrest is more important

158 © 2012 Pearson Education, Inc. A third transition point A third important transition point is during M phase, between metaphase and anaphase Here the commitment is made to move the two sets of chromosomes into the new cells Before cells can begin anaphase, all the chromosomes must be properly attached to the spindle

159 © 2012 Pearson Education, Inc. Figure 19-32

160 © 2012 Pearson Education, Inc. Studies Involving Cell Fusion and Cell Cycle Mutants Led to the Identification of Molecules That Control the Cell Cycle Cell fusion experiments in the 1970s gave hints about the identity of molecules that drive the cell cycle Two cultured mammalian cells were fused to form a cell with two nuclei—a heterokaryon If one cell is in S phase and the other in G1, the G1 nucleus quickly begins DNA replication, suggesting that S phase cells contain molecules that trigger the G1 → S progression

161 © 2012 Pearson Education, Inc. Figure 19-33A

162 © 2012 Pearson Education, Inc. Other observations Fusion of cells in M phase with cells in G1, S or G2 causes the interphase nuclei to immediately begin mitosis These experiments suggest that molecules in the cytoplasm drive cells from G1 to S or G2 to M Yeast cells were used to try to identify the molecules

163 © 2012 Pearson Education, Inc. Figure 19-33B

164 © 2012 Pearson Education, Inc. Temperature-sensitive cell cycle mutants in budding yeast Yeasts carrying a temperature-sensitive mutation affecting the cell cycle can be grown at a lower (permissive) temperature Their cell cycles will be blocked at high temperatures Hartwell and colleagues identified many genes involved in cell cycle regulation using this type of mutation

165 © 2012 Pearson Education, Inc. Progression Through the Cell Cycle Is Controlled by Cyclin-Dependent Kinases (Cdks) Phosphorylation of target proteins by protein kinases and dephosphorylation by protein phosphatases is a common mechanism for controlling the cell cycle Cell cycle progression is driven by protein kinases that are active only when bound to a cyclin These kinases are cyclin-dependent kinases (Cdks)

166 © 2012 Pearson Education, Inc. Cyclins The concentration of cyclins varies with phases of the cell cycle –Mitotic cyclins are required for the G2  M transition and bind mitotic Cdks –G1 cyclins are required for passage through the G1 restriction point (or Start) and the Cdks to which they bind are called G1 Cdks –S cyclins are required for DNA replication

167 © 2012 Pearson Education, Inc. Mitotic Cdk-Cyclin Drives Progression Through the G2-M Transition by Phosphorylation Key Proteins Involved in the Early Stages of Mitosis Evidence of a control molecule triggering mitosis came from experiments involving frog eggs (Masui) Mature eggs develop from oocytes through meiosis; the oocyte arrests shortly after meiosis begins until a hormone signal is received Injecting the cytoplasm of a mature egg into an immature oocyte causes it to immediately proceed through meiosis

168 © 2012 Pearson Education, Inc. Figure 19-34

169 © 2012 Pearson Education, Inc. Maturation-promoting factor Masui hypothesized that a cytoplasmic chemical that he named maturation-promoting factor (MPF) induced oocyte maturation Subsequent experiments showed that MPF triggered mitosis when injected into fertilized frog eggs Comparable molecules were soon detected in a broad range of organisms

170 © 2012 Pearson Education, Inc. MPF is a mitotic Cdk-cyclin complex MPF was shown to be composed of a cyclin and a Cdk (a Cdk-cyclin complex) The mitotic Cdk portion of the complex is almost identical to the protein product of the yeast cdc2 gene Yeast cells with a defective cdc2 gene can function perfectly well if the human equivalent is provided to them

171 © 2012 Pearson Education, Inc. Control of Cdk-cyclin complexes Mitotic Cdk is found consistently throughout the cell cycle It is active only when bound to mitotic cyclin and the concentration of mitotic cyclin gradually increases through G1, S, G2 until it reaches a crucial threshold at the end of G2 Halfway through mitosis, it is abruptly degraded

172 © 2012 Pearson Education, Inc. Figure 19-35

173 © 2012 Pearson Education, Inc. Activation of mitotic Cdk Activation of mitotic Cdk involves phosphorylation and dephosphorylation The binding of mitotic cyclin to mitotic Cdk forms a cyclin-Cdk complex that is initially inactive (1) Inhibiting kinases phosphorylate two sites on the Cdk, blocking the active site (2)

174 © 2012 Pearson Education, Inc. Figure 19-36

175 © 2012 Pearson Education, Inc. Activation of mitotic Cdk (continued) An activating phosphate is then added by an activating kinase (3) The final step is the removal of the inhibiting phosphates by a specific phosphatase enzyme (4) Once the phosphatase begins removing the phosphates, activated Cdk stimulates the phosphatase, amplifying the activation

176 © 2012 Pearson Education, Inc. Onset of mitosis Once activated the Cdk-cyclin phosphorylates lamin proteins of the nuclear lamina This causes lamina breakdown and destabilization of the nuclear envelope It also phosphorylates condensin, which is involved in chromosome condensation, and microtubule-associated proteins to facilitate assembly of the mitotic spindle

177 © 2012 Pearson Education, Inc. Figure 19-37

178 © 2012 Pearson Education, Inc. The Anaphase-Promoting Complex Coordinates Key Mitotic Events by Targeting Specific Proteins for Destruction Mitotic Cdk-cyclin phosphorylates and contributes to activation of the anaphase-promoting complex The anaphase-promoting complex functions as a ubiquitin ligase, which adds ubiquitin to proteins to target them for destruction One target is securin, an inhibitor of sister chromatid separation

179 © 2012 Pearson Education, Inc. Function of securin Prior to anaphase, sister chromatids are held together by adhesive proteins called cohesins Securin maintains this attachment by inhibiting the separase protein that would otherwise degrade the cohesins When the anaphase-promoting complex triggers destruction of securin, separase cleaves cohesin, freeing the sister chromatids

180 © 2012 Pearson Education, Inc. Figure 19-38A

181 © 2012 Pearson Education, Inc. Other activities of the anaphase- promoting complex The anaphase-promoting complex targets mitotic cyclin for destruction, causing the kinase activity of mitotic Cdk to fall Many changes associated with exit from mitosis— cytokinesis, chromosome decondensation, nuclear envelope reassembly—depend on degradation of mitotic cyclin

182 © 2012 Pearson Education, Inc. G1 Cdk-Cyclin Regulates Progression Through the Restriction Point by Phosphorylating the Rb Protein G1 Cdk-cyclin phosphorylates the key target Rb protein Nonphosphorylated Rb binds the E2F transcription factor Unbound E2F activates transcription of genes coding for proteins that initiate DNA replication

183 © 2012 Pearson Education, Inc. Rb and E2F While Rb remains bound to E2F, the E2F molecule is inactive and the cell cannot enter S phase But when cells are stimulated to divide by growth factors G1 Cdk-cyclin is activated, and phosphorylates Rb Rb releases E2F, which initiates transcription of genes needed for entry of S phase

184 © 2012 Pearson Education, Inc. Figure 19-39

185 © 2012 Pearson Education, Inc. Checkpoint Pathways Monitor Chromosome- to-Spindle Attachments, Completion of DNA Replication, and DNA Damage If cells proceeded from one phase of the cell cycle to the next without completing each step, daughter cells might be abnormal –E.g., aneuploidy (incorrect number of chromosomes) could result Cells use a series of checkpoints that ensure each phase is completed properly before the next one begins

186 © 2012 Pearson Education, Inc. The mitotic spindle checkpoint The mitotic spindle checkpoint prevents anaphase from beginning before the chromosomes are all attached to the spindle Kinetochores that remain unattached to microtubules produce a “wait” signal that inhibits the anaphase-promoting complex Members of the Mad and Bub protein families are involved

187 © 2012 Pearson Education, Inc. Mad and Bub One model suggests that Mad and Bub proteins accumulate at unattached kinetochores They are converted into a multiprotein complex that inhibits the anaphase-promoting complex by blocking the action of Cdc20 protein Once all the chromosomes are attached, Mad and Bud are no longer converted and the inhibition is lifted

188 © 2012 Pearson Education, Inc. Figure 19-38B

189 © 2012 Pearson Education, Inc. The DNA replication checkpoint The DNA replication checkpoint ensures that DNA synthesis is complete before the cell exits G2 and begins mitosis Cells that are prevented from completing DNA replication fail to undergo the final dephosphorylation step in the activation of mitotic Cdk-cyclin

190 © 2012 Pearson Education, Inc. The DNA damage checkpoint A multiple series of DNA damage checkpoints monitor DNA for damage and halt the cell cycle at various points (late G1, S, and late G2) by inhibiting different Cdk-cyclin complexes p53 protein, the “guardian of the genome,” plays a central role in these checkpoint pathways

191 © 2012 Pearson Education, Inc. Response to double-stranded DNA breaks Breaks in DNA trigger activation of an enzyme called the ATM (ataxia telangiectasia mutated) protein kinase ATM phosphorylates checkpoint kinases, which then phosphorylate p53 (and other targets) Phosphorylated p53 is unable to bind to Mdm2; Mdm2 marks p53 for destruction

192 © 2012 Pearson Education, Inc. Response to double-stranded DNA breaks (continued) Phosphorylated p53 is protected from degradation and activates two types of events: cell cycle arrest and cell death p53 activates the gene coding for p21, a protein that halts progression of the cell cycle by inhibiting activity of different Cdk-cyclins ATR (ATM-related) acts similarly in response to single-stranded DNA breaks

193 © 2012 Pearson Education, Inc. Figure 19-40

194 © 2012 Pearson Education, Inc. Cell death p53 stimulates production of enzymes involved in DNA repair But if the damage cannot be repaired, p53 activates genes needed to trigger cell death by apoptosis A key protein in this pathway is called Puma (p53 upregulated modulator of apoptosis), which inactivates Bcl-2, an apoptosis inhibitor

195 © 2012 Pearson Education, Inc. Putting It All Together: The Cell Cycle Regulation Machine The “machine” that regulates the eukaryotic cell cycle involves two interacting mechanisms –1. An autonomous clock goes through a fixed cycle over and over again via the synthesis and degradation of cyclins –2. The clock is adjusted as needed

196 © 2012 Pearson Education, Inc. Figure 19-41

197 © 2012 Pearson Education, Inc. Growth Factors and Cell Proliferation In simple unicellular organisms presence of nutrients in the environment is the primary factor determining whether cells grow and divide In multicellular organisms extracellular signaling proteins, growth factors, control the rate of cell proliferation Growth factors are called mitogens

198 © 2012 Pearson Education, Inc. Stimulatory Growth Factors Activate the Ras Pathway Mammalian cells with plenty of nutrients but no growth factors arrest in G1 Growth and division can be triggered by adding blood serum, which contains several stimulatory growth factors, such as PDGF (platelet-derived growth factor) Another is epidermal growth factor (EGF)

199 © 2012 Pearson Education, Inc. Action of growth factors Growth factors such as PDGF and EGF act by binding receptors on the plasma membrane This activates the tyrosine kinase activity of the receptors, which triggers a complex cascade of events that ends with the cell passing the restriction point The Ras pathway plays a central role in these events

200 © 2012 Pearson Education, Inc. The Ras pathway in the cell cycle Binding of a growth factor to its receptor leads to Ras activation (1) Activated Ras leads to phosphorylation and activation of a protein kinase called Raf, which starts a phosphorylation cascade (2) Raf phosphorylates a protein kinase called MEK, which phosphorylates a group of MAP kinases (mitogen-activated protein kinases)

201 © 2012 Pearson Education, Inc. The Ras pathway in the cell cycle (continued) Activated MAPKs enter the nucleus and phosphorylate specific genes, including Jun and members of the Ets family of transcription factors (3) These transcription factors turn on transcription of “early genes” The early genes code for production of other transcription factors including Myc, Fos, and Jun

202 © 2012 Pearson Education, Inc. The Ras pathway in the cell cycle (continued) Genes such as Myc, Fos, and Jun activate transcription of a family of “delayed genes” One of these encodes the E2F transcription factor, covered earlier The delayed genes include several genes coding for Cdk or cyclin molecules that form Cdk-cyclin complexes that phosphorylate Rb and trigger G1  S transition

203 © 2012 Pearson Education, Inc. Figure 19-42

204 © 2012 Pearson Education, Inc. Stimulatory Growth Factors Can Also Activate the P13K-Akt Pathway Activated growth factor receptors may trigger other pathways besides Ras One is the PI3-kinase-Akt pathway, that begins with receptor-induced activation of phosphatidyl- inositol 3-kinase which catalyzes formation of PIP 3 (phosphatidylinositol-3,4,5-trisphosphate) It leads ultimately to Akt phosphorylation and activation and suppression of apoptosis by Akt

205 © 2012 Pearson Education, Inc. Figure 19-43

206 © 2012 Pearson Education, Inc. Inhibition of cell cycle arrest Akt inhibits cell cycle arrest through activation of a monomeric G protein called Rheb This leads to activation of TOR, a key regulator of cell growth The net effect of the PI3-kinase-Akt signaling pathway is to promote cell survival and proliferation

207 © 2012 Pearson Education, Inc. Inhibitory Growth Factors Act Through Cdk Inhibitors Some growth factors inhibit cell proliferation, e.g., transforming growth factor  (TGF  ) TGF  binding to its receptor phosphorylates Smad proteins that move into the nucleus and activate expression of genes coding for proteins that inhibit proliferation Two Cdk inibitors that block cell cycle progression are p15 and p21

208 © 2012 Pearson Education, Inc. Apoptosis Damaged or diseased cells need to be eliminated In such cases, the process must not damage surrounding cells Multicellular organisms accomplish this through a programmed cell death—apoptosis

209 © 2012 Pearson Education, Inc. Apoptosis and necrosis Cell death called necrosis sometimes follows tissue injury Necrosis involves swelling and rupture of injured cells, whereas apoptosis involves a specific series of events that lead to dismantling of the cell contents

210 © 2012 Pearson Education, Inc. Steps of apoptosis The cell’s DNA segregates near the periphery of the nucleus and the cytoplasm decreases (1) The cell produces small cytoplasmic extensions and the nucleus begins to fragment (2) DNA is cleaved by an apoptosis-specific endonuclease and the cell is dismantled into small pieces called apoptotic bodies

211 © 2012 Pearson Education, Inc. Steps of apoptosis (continued) Inactivation of a phospholipid translocator (flippase) causes accumulation of phosphatidylserine in the outer leaflet of the plasma membrane This serves as a signal for the remnants of the affected cell to be engulfed by nearby cells via phagocytosis (3)

212 © 2012 Pearson Education, Inc. Figure 19-44A

213 © 2012 Pearson Education, Inc. Figure 19-44B,C,D

214 © 2012 Pearson Education, Inc. Caspases Apoptosis proceeds through the activation of a series of enzymes called caspases They are produced as inactive precursors called procaspases and are cleaved to create active enzymes

215 © 2012 Pearson Education, Inc. Apoptosis Is Triggered by Death Signals or Withdrawal of Survival Factors There are two main routes by which cells can activate caspases and enter apoptosis Activation can occur directly, e.g., when human cells are infected by viruses, cytotoxic T lymphocytes are activated and induce apoptosis This is triggered when cells receive cell death signals

216 © 2012 Pearson Education, Inc. Apoptosis in cell infected by viruses Two death signals are tumor necrosis factor and CD95/Fas CD95 is a protein on the surface of infected cells; lymphocytes have a protein on their surfaces that binds CD95, causing it to aggregate Adaptor proteins attach to the CD95, which recruits procaspase-8 to the sites of clustering

217 © 2012 Pearson Education, Inc. Initiator and executioner caspases When the procaspase is activated it acts as an initiator caspase, initiating the cascade Initiator caspases also activate an executioner caspase, caspase-3, which is important for activating many steps in apoptosis

218 © 2012 Pearson Education, Inc. The second type of apoptosis One of the best-studied cases of the second type of apoptosis involves survival factors When survival factors are withdrawn, a cell may enter apoptosis The site of action is the mitochondrion Healthy cells have several anti-apoptotic proteins in the outer mitochondrial membrane

219 © 2012 Pearson Education, Inc. The second type of apoptosis (continued) The proteins are structurally related to a protein called Bcl-2 which, together with other proteins, counteracts proteins that promote apoptosis (pro- apoptotic proteins) When cellular signals shift in balance toward pro- apoptotic proteins, the cell is more likely to undergo apoptosis One pro-apoptotic protein is called Bad (Bcl-2- associated death promoter)

220 © 2012 Pearson Education, Inc. Mitochondria trigger apoptosis Mitochondria trigger apoptosis by releasing cytochrome c into the cytosol after accumulation of pro-apoptotic proteins lead to formation of channels in the outer mitochondrial membrane Cytochrome c stimulates calcium release from mitochondria and ER, where it binds IP 3 receptors It also activates an initiator procaspase, procaspase-9, which then activates caspase-3

221 © 2012 Pearson Education, Inc. Damaged cells can trigger their own apoptosis If a cell suffers such damage that it can’t repair itself, it may trigger its own demise It can enter apoptosis through the activity of p53, which acts through the protein Puma, which binds and inhibits Bcl-2

222 © 2012 Pearson Education, Inc. Figure 19-45


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