Cell and Molecular Biology Behrouz Mahmoudi Cell cycle 1

This cell has been triple stained for f-actin (red), microtubules (green), and DNA (blue) 5 minutes after anaphase-onset 2

Cell Cycle – The basics The cell cycle can be divided into 3 phases: Interphase is the longest phase where the cell will grow if no division occurs. Mitosis is shorter and refers to the segregation of the DNA (karyokinesis). Cytokinesis is the shortest phase and refers to the division of the cytoplasm. 3

Cell Cycle – Key Elements 4

Saccharomyces cerevisiae (diploid budding yeast) divides through budding 5

Expression patterns of about 800 yeast genes whose level of transcription varies systematically through the cell cycle revealed through microarray analysis. The two cell populations are: synchronized dividing cells in culture (cDNA is labeled with a red fluorochrome) non-dividing cells (cDNA is labeled with a green fluorochrome) 6

Cell Cycle – Genetic Analysis of the Cell Cycle Determining the time of function of a cell-cycle protein by shift of a temperature-sensitive mutant to the restrictive temperature allows for precise molecular analysis. Recall that conditional mutations are expressed in restrictive, but not permissive conditions. Growth of temperature-sensitive mutants can be analyzed at restrictive and permissive temperatures. cdc mutants (cell division cycle) mutants are used. Cells arrest at stage when defective protein is needed. 7

Cell-cycle arrest in temperature-sensitive cdc13 mutants. 8

Cell Cycle – Progression Through the Cell Cycle Two types of molecule control the progression through the cell cycle: Cyclins Cyclin-Dependent-Protein Kinase (CDK) Cyclin-CDK complexes Yeast have various cyclins, but only one CDK (Cdc28 in budding yeast, Cdc2 in fission yeast). Higher eukaryotes have 4 CDKs, 7 cyclins. Each CDK is only active as a kinase to phosphorylate target proteins when it is bound to specific cyclin(s). 9

Fluctuations of cyclin levels during the cell cycle. Expression of cyclins E, A, and B (mitotic cyclins) are periodic, whereas cyclin D is expressed throughout the cell cycle in response to mitosis- stimulating drugs (mitogens). 10

Targets of cyclin-CDK complexes are proteins. Protein kinases add phosphates to –OH group of ser, thr, tyr residues. Cyclin binds specific target and brings the CDK there; after phosphorylation, complex dissociates. Phosphorylation may activate or inhibit proteins, enzymes. Phosphatases take off phosphates from target proteins; reset system. Activities of cyclin-CDK are controlled by phosphorylation: Cyclin D-CDK complexes are controlled by inhibitor p16 protein and by dephosphorylation. 11

Cells at rest at the G1 restriction point (start). Retinoblastoma protein (RB) hold the cells at the G1 restriction point by binding to the transcription factor E2F. Pre-replication complexes formed of the Origin Recognition Complex (ORC) and CDC6 proteins initiate DNA replication. Cells have two main transition phases: G1/S and G2/M transitions: G1/S transition: G2/M transition: Cells move from G2 to M through the Maturation- Promoting Factor (MPF) composed of cyclinB-Cdc2 complexes. CyclinB-Cdc2 complexes move to the nucleus (phosphorylation). Target proteins are involved in the duplication of the spindle and breakdown of nuclear envelope. 12

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Protein degradation regulates cell cycle: Activated Anaphase-Promoting Complex (APC/C) controls metaphase to anaphase transition and marks proteins for degradation by proteasome. 14

Cell Cycle – Checkpoints Checkpoints permit pause to repair damage and failure to stop at checkpoints causes aneuploidy, polyploidy or mutations. In addition, unregulated cell division is characteristic of cancer: 1.DNA damage checkpoint (G1/S or G2/M) 2.Centrosome duplication checkpoint (G2/M) 3.Spindle checkpoint (metaphase/anapha se) 15

The tumor suppressor protein p53 is essential in regulating the cell cycle at DNA damage checkpoints, thus preventing genome mutations that can lead to cancer. Mutated p53 leads to cancer 16

Downstream events triggered by p53 include transcriptional activation of the genes for p21, GADD45, σ, Bax, maspin, Apaf1, and miRNA34a and b/c. 17

Transcription factors Bax and Bcl2 are involved in apoptosis (programmed cell death). p53 activates Bax Bcl-2 protect cells from apoptosis Balance between the two factors is necessary for normal cell cycle regulation 18