Lecture 16: Cell Cycle - Part 1

Stages of Interphase

• G1 (Gap1)

  • Growth

• S (Synthesis)

  • Genome and centrosome duplication

• G2 (Gap2)

  • More growth

How DNA Content Changes During The Cell Cycle

• Terminology

  • 2N: 2 copies of each chromosome

    • Meaning that there will be 46 individual chromosomes (or 23 pairs)

    • Visually, each single chromosome is a single strand with the centromere in the middle

  • 4N: Each chromosome has duplicated (has sister chromatids)

    • meaning that each chromosome will now have two identical strands that will be connected by that original centromere

• G1 phase is 2N

• S phase is 2N to 4N

• G2 phase is 4N

• Mitosis remains 4N until the very end

Flow Cytometry

• A technique used to measure DNA content of cells

• Process

  • Label cells with a fluorescent DNA dye

  • Flow cells through a machine that measures the fluorescent signal

  • After DNA replication, the cell has twice as much DNA, so it twice the fluorescent signal

  • Therefore, this technique tells you how much DNA is in the cell

• Reading/Interpreting Graph

  • The graph shows a population of cells that are all at different stages of the cell cycle

  • A higher peak indicates that the phase corresponding to the peak takes longer because more cells are associated with that peak

  • Cell in between the two peaks are in S phase

Cell Type Duration Takeaway

• Cell cycle duration varies between cell types

• Usually, the G1 stage is the longest in many cell types, but there are exceptions

Properties of the Cell Cycle

• Recursive: Cells must remain the same size so cell growth must be tightly coupled to cell division

• Ordered: DNA replication always has to precede chromosome segregation

• Linked cycles: The chromosome cycle and growth cycle have to be linked

Control System

• A control system drives the events of the cell cycle and makes sure that events occur in order

Cyclin-Dependent Kinases (Cdks)

• Cdks phosphorylate proteins to drive key cell cycle events

• These kinases need to be bound to a protein called cyclin to be active

• Cdks switch the machinery required at different cell cycle stages on and off at the correct times

  • Cdks are present throughout the cell cycle but they are only activated at specific times

  • This is mediated by the concentration of cyclins, which unlike Cdks, varies in a cyclic fashion during the cell cycle

  • There are different cyclin-Cdk complexes that act at different times in the cell cycle

Cyclin-Cdk Complex That Drives Entry Into Mitosis

• M-Cdk: M-phase Cdk

• M-Cyclin: M-phase Cyclin

Role of M-Cdk

• M-Cdk is a kinase

• Phosphorylation of nuclear lamins

  • The nuclear envelope breaks down upon entry into M-phase, allowing for the spindle to capture chromosomes

  • Nuclear lamins must diassemble in order for the nuclear envelope to break down

  • Lamins and nuclear pore proteins are phosphorylated by M-Cdk at the start of mitosis, which leads to nuclear envelope disassembly

• M-Cdk inactivation

  • Leads to nuclear envelope reformation

  • When M-Cdk is inactivated at the metaphase-to-anaphase transition, lamins and nuclear pore proteins need to be dephosphorylated for the nuclear envelope to re-form in telophase

  • Vesicles form around chromosomes and fuse together to form the nuclear envelope

Cyclin Changes in Cyclin Concentration Drives M-Cdk Activation

• M-cyclin needs to be synthesized before M-Cdk can drive entry into mitosis

  • M-Cdk is inactive without cyclic

• When M-cyclin levels drop, this inactivates M-Cdk and drives entry into interphase

Additional Levels of M-Cdk Regulation

• M-cyclin concentration increases gradually during interphase, BUT M-Cdk activation is much faster, so additional levels of regulation are necessary

• The activation of M-Cdk is regulated by a kinase, Wee1, and a phosphatase, Cdc25, which antagonize each other

• Wee1 is a kinase that phosphortylates M-Cdk and inhibits M-Cdk activity

• Cdc25 is a phosphatase must remove those phosphates to activate M-Cdk activity (dephosphorylation)

Two Things Have to Happen for M-Cdk Activation

• Binding of M-Cdk to M-cyclin (these complexes accumulate gradually during interphase)

• Removal of inhibitory phosphates by Cdc25 (Only happens at the end of G2)

  • Leads to fast M-Cdk activation

Positive Feedback Accelerates M-Cdk Activation

• Why Positive Feedback

  • Positive feedback loops allow systems to be activated quickly and to become locked in the “activated” state

  • Ensures that the cell cycle only goes forward and does not slip backwards

• Process

  • Once some M-Cdk is activated, it phosphorylates Cdc25

  • This phosphorylation activates Cdc25

  • More active Cdc25 means that additional molecules of M-Cdk get their inhibitory phosphates removed

  • This “positive feedback” leads to rapid M-Cdk activation

  • Active M-Cdk also inactivates Wee1 (through phosphorylation also), which further accelerates M-Cdk activation

Inactivating M-Cdk

• M-cyclin levels fall rapidly at the end of M-phase

• When cyclin levels drop, this inactivates M-Cdk and drives entry into interphase

Cyclin Destruction: Modification by Polyubiquitination

• Cell targets cyclin for degradation

• Ubiquitin (a small protein) is covalently coupled to cyclin

• Multiple ubiquitins are added in a chain (polyubiquitination)

• This polyubiquitin chain targets the protein for degradation

Proteasome

• Multisubunit complex

• A region at the top of the complex recognizes proteins tagged with ubiquitin chains

• Also has a central pore where proteolysis occurs (contains proteases)

• Degrades proteins in cells

  • There is a poly-ubiquitin binding site on one end of the proteasome

  • After binding, the targeted proteins are unfolded and woven through the pore, where they are degraded

  • The ubiquitins are recycled so they can be re-used on other proteins

APC

• A ubiquitin ligase that facilitates M-cyclin destruction

  • The APC is a protein complex that attaches ubiquitin to M-cyclin

• Note: There are many ubiquitin ligases in the cell (different ubiquitin ligases attach ubiquitin to different target proteins)

Switch-like Cell Cycle Transitions

• M-Cdk is rapidly activated through psitive feedback loops and rapidly inactivated via cyclin degradation

• These rapid transitions act as switches between cell cycle states and ensures they only go in one direction