Cell and Molecular Biology Cell cycle Behrouz Mahmoudi

Cell and Molecular Biology Cell cycle Behrouz Mahmoudi 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 (c. DNA is labeled with a red fluorochrome) • non-dividing cells (c. DNA 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. 7

Cell-cycle arrest in temperature-sensitive cdc 13 mutants. 8

Cell Cycle – Progression Through the Cell Cycle Two types of molecule control the progression through the cell cycle: Cyclin-CDK • Cyclins complexes • Cyclin-Dependent-Protein Kinase (CDK) • Yeast have various cyclins, but only one CDK (Cdc 28 in budding yeast, Cdc 2 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 mitosisstimulating 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 p 16 protein and by dephosphorylation. 11

Cells have two main transition phases: G 1/S and G 2/M transitions: G 1/S transition: • Cells at rest at the G 1 restriction point (start). • Retinoblastoma protein (RB) hold the cells at the G 1 restriction point by binding to the transcription factor E 2 F. • Pre-replication complexes formed of the Origin Recognition Complex (ORC) and CDC 6 proteins initiate DNA replication. G 2/M transition: • Cells move from G 2 to M through the Maturation. Promoting Factor (MPF) composed of cyclin. B-Cdc 2 complexes. • Cyclin. B-Cdc 2 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 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 (G 1/S or G 2/M) 2. Centrosome duplication checkpoint (G 2/M) 3. Spindle checkpoint (metaphase/anapha se) 15

The tumor suppressor protein p 53 is essential in regulating the cell cycle at DNA damage checkpoints, thus preventing genome mutations that can lead to cancer. Mutated p 53 leads to cancer 16

Downstream events triggered by p 53 include transcriptional activation of the genes for p 21, GADD 45, 14 -3 -3σ, Bax, maspin, Apaf 1, and mi. RNA 34 a and b/c. 17

Transcription factors Bax and Bcl 2 are involved in apoptosis (programmed cell death). • p 53 activates Bax • Bcl-2 protect cells from apoptosis • Balance between the two factors is necessary for normal cell cycle regulation 18