Understanding Cell Cycle Regulators: The Role of Cyclins and CDKs
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The Hong Kong University of Science and Technology**We aren't endorsed by this school
Course
LIFS 2010
Subject
Medicine
Date
Dec 12, 2024
Pages
46
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Identification of Regulators of the Cell CycleNobel Prize in Physiology or Medicine 2001Sir Paul M. NurseLeland H. HartwellTim Huntgenetics(budding yeast)genetics(fission yeast)biochemistry(sea urchin)
The Cell Cycle as you Probably Know It
All cells need to undergo cell division to growWe now understand that the events and much of the machinery that controls cell division are evolutionarily conserved in eukaryotes
The process of cell division is observable by microscopy
Mammalian tissue culture cells in various stages of growth
What does cell division and growth look likein a developing organism?
Stages of growth in budding yeast cells
Cell division (at the molecular level) is a very complex process subject to a high degree of regulationHow do we “know” how it works?Are there some “fundamental / basic principles”that apply to this process?
Tim HuntAt Woods Hole in the Summer of 1982 using the sea urchin (Arbacia punctulata) egg as his model organism, he discovered the cyclin proteins.He was studying the process of translation and choose the sea urchin. Upon fertilization sea urchin embryogenesis startsand with that the synthesis of new proteins commences Discovery of “cyclins”
Eggs and sperm are readily produced by these creatures in the laboratory (only a little filtered sea water is needed)The embryos are transparent and well-suited for microscopy.The remarkable synchrony of egg development made biochemical and molecular characterization of the phases of cell division straightforward.
In Tim Hunt's classic experiment, fertilized eggs from the sea urchin Arbacia were labeled with radioactive methionine, and extracts were resolved on SDS-polyacrylamide gels. The autoradiograph of the protein gel shows the clear result that one protein, called cyclin, accumulates and is precipitously destroyed in mitosis (Evans et al., 1983).
Hunt found that cyclins begin to be synthesized after the eggs are fertilized and increase in levels during interphase, until they drop very quickly in the middle of mitosis in each cell division. ribonucleotidereductase
“ It is difficult to believe that the behavior of the cyclins isnot connected with processes involved in cell division, butat this stage we have no direct evidence that it is. Thesynthesis and periodic destruction of proteins have longbeen proposed to account for various features of the cellcycle(Mitchison, 1971; John et al., 1981). Mitotic spindlescome and go; chromosomes condense and extend; thenuclear envelope dissolves and reforms (Mazia, 1961). Toinduce the de novo synthesis of an enzyme and subsequentlydestroy it is one way of turning its activity on andoff. “Tim Hunt, 1983
The cyclin mechanism of cell division is fundamental to all living organisms (excluding bacteria)and thus the study of the process in simple organisms helps shed light on the growth of tumours in humansHe and others subsequently showed that cyclins bind to and activatea family of protein kinases, now called the cyclin-dependent kinases, one of which had been identified as a crucial cell cycle regulator by Paul Nurse.Tim Hunt
The Cyclin / Cyclin-Dependent Kinase (CDK) Paradigm
CDKs are a family of multifunctional enzymes that can modify various protein substrates involved in cell cycle progression.CDKs phosphorylate their substrates by transferring phosphate groups from ATP to specific stretches of amino acids in the substrates. Yeast have only a single CDK, whereas vertebrates have four different ones. What Are Cyclin-Dependent Kinases?
CDKs require the presence of cyclins to become active. Cyclins have no enzymatic activity of their own but activate CDKs by binding to them.
How Do CDKs Control the Cell Cycle?When cyclin levels decrease, the corresponding CDKs become inactive. All CDKs exist in similar amounts throughout the entire cell cycle. In contrast, cyclin manufacture and breakdown varies by stage — with cell cycle progression dependent on the synthesis of new cyclin molecules.
The use of “genetics” to identify genes requiredfor the cell division cycleLeland H. HartwellWas pioneered by Leland Hartwell
Leland H. HartwellUsed budding yeast to identify mutants with defects in cell cycle progressionThese were termed “CDC” for cell division cycleHe (also) identified mutants with defects in initiation of the CDC – a process that he called “START”
Hartwell had the crucial insight that mutants with defects in growth (cell division) should stop growing at particular stages of growthLife cycle of budding yeast
He postulated (based on his genetic studies) that there must be “checkpoints” in the CDC process reasoning that……“The occurrence of specific cell-cycle events requires the prior completion of other, earlier events; for instance, assembly of the mitotic spindle is dependent upon completion of DNA synthesis.”
How did Hartwell identify CDC mutants?
While studying at Caltech, Hartwell was influenced by thework of Bob Edgar who worked on the bacterial virus calledphage T4.Edgar pioneered the use of conditional mutants for elucidating the organization of essential cellular pathways.These conditional mutants were temperature-sensitive for growth (Ts)……. and were used to decipher the process of phagereplication in bacterial cells
Temperature sensitive (Ts) mutants grow at some temperatures but not at others. Yeast strains growing on the surface of nutrient agar plates
Why was the choice of conditional mutants important?
Ts mutations are typically missense mutations, which retain the function of a specific essential geneat a standard (permissive) low temperature, and lack that function at a defined high (non-permissive) temperature.Essential genes, by definition, encode products with critical cellular functions that are not “buffered” by redundant functions or pathways
Ts mutants make possible the analysis of physiologic changes that follow controlled inactivation of a gene or gene product by shifting cells to a non-permissive temperature, offering a powerful approach to the analysis of gene function.
To begin his study Hartwell generated a library (or collection) of Ts yeast mutants.How could you do this?What does this mean?Why do this at all?
Hartwell’s Ts library of yeast mutants contained ~ 400 individual mutantsThis mutant library was “screened” visually using light microscopes - for cell division cycle (CDC) phenotypesHartwell isolated 35 CDC mutants from this library whichwere named CDC1 - CDC35
A “cdc” mutant phenotype in budding yeast Wildtype“cdc”
Once isolated (CDC mutants) how could the correspondinggene defective in the CDC mutant be identified?Why is this important?….because this might reveal the protein’s function
Sir Paul M. Nurseborn January 25, 19491970 B.Sc., University of Birmingham1973 Ph.D., University of East AngliaDirector of ICRF / Cancer UKPresident of Rockefeller UniversityPresident of the Royal SocietyDirector and Chief Executive of the UK Centre for Medical Research and InnovationChief Executive The Francis Crick Institute
Nurse’s work on the cell cycle was inspired by that of Lee Hartwell – although he chose a different yeast species
S. pombe cells and asci.(A) A haploid cell containing a fission plate.(B)Diploid cells.(C)Zygotic asci formed by the mating of two haploid cells.(D)Azygotic asci formed when diploid cells go through meiosis.Unlike the budding yeasts S. pombe divides by binary fission (like our cells)
Nonetheless fission yeast could also be used to screen visually for cell cycle mutants
Nurse has said good scientists must have passion'to know the answer to the questions' that interest them, along with good technical ability, and a set of attitudes including mental honesty, self-criticism, open-mindedness and scepticism
Because I failed at so many things so often, because I was in a mess at school ... it gave me a sort of internal discipline—you take less note of what other people think of you, what other people say, because you don’t get off on being praised about things. I had to be resilient inside.… I was constantly comparing me to me when I did well, and not with other people. I realise it’s very odd, but it’s really useful, because when I … failed examinations, I couldn’t get into university, I couldn’t get a job, when you put all that together, it was a constant low to medium level [of] failure about things. So when I got to difficult problems and I failed, I didn’t go into depression or anything. And when you get into research, it’s constant failure all the time, and I was perfectly trained for it.Nurse’s thoughts on failure…..
How Paul Nurse (finally) got into University
Nobel Laureate Sir Paul Nurse - The Beauty of Science