BIOL 200 7.1 Cell Cycle & Checkpoints

Introduction to the Cell Cycle & Checkpoints

Cell Cycle

-divided into 4 separate stages

Gap 1 (G1) Phase

-gap between end of cytokinesis and start of DNA synthesis

Synthesis (S) Phase

-initiation and termination of DNA synthesis

Gap 2 (G2) Phase

-second gap phase lasts from end of DNA synthesis to the onset of mitosis

Mitosis (M) Phase

-division occurs

-there must be provision for cells to get out of the cell cycle and into other developmental mathways (mating, meiosis, differentiation) or to arrest

-necessary for cells to get from these alternate fates back into cell cycle

-controls are placed on the progression of cell cycle where the cell is required to meet certain criteria before it’s allowed to proceed

  • each checkpoint is controlled by at least one gatekeeper proteins that respond to cellular conditions → only allows cell to move forward into next phase of cell cycle if certain conditions are met

  • checkpoints help ell to ensure that certain conditions are met before cell cycle is allowed to continue

G1/S Checkpoint

-transition from G1 to S phase

-restrict cells from beginning DNA synthesis before cell is ready

-conditions that need to be met

  • nutrients (carbon source, energy source, Pi, S, N, vitamins) must be present at specific concentrations

  • chromatid separation (from previous mitosis) must be complete

  • no detectable DNA damage

  • cells must have reached a critical threshold size

  • external factors must be appropriate

    • ex: in yeast, if mating factor is present → cells can’t proceed to S-phase and are switched to alternative pathways (sexual pathway — in mammalian cells, appropriate growth factors must be present to allow cells to press Start)

G0 Phase

  • sometimes cells are stalled for extended periods of time

    • removed themselves from cell cycle

  • long term deprivation of nutrients or other resources required for cell division

  • normal part of development of certain cell types

    • ex: stem cells for specific tissues enter G0 for shorter periods of time until replacement cells are required → tissues are able to grow to a certain size and then stop growing and maintain a relatively stable size and distribution

  • some cells undergo terminal differentiation

    • the form cells take at maturity is the functional form that is maintained

    • cells don’t undergo mitosis

    • ex: muscle cells → fuse together at maturity to produce multinucleated fibers

    • ex: neurons → long axons

    • ex: osteocytes → bone cells that are intricately embedded in calcified matrix of bone

G2/M Checkpoint

-stops cell from entering mitosis before it’s ready

-commitment to division → cell can’t stop the process of mitosis once it has started

  • checkpoint controls transition from G2 to M phase

-conditions that need to be met

  • DNA replication must be complete

  • DNA must be undamaged

  • cell must have reached a certain minimum size

-cells can only move beyond checkpoint and into next stage of cell cycle when they have met required conditions

-ex: biotin missing from growth medium → yeast cells won’t start even if all other conditions are perfect

  • low nutrient levels reduce the growth rate, which if severe enough can make it so that conditions required for passing checkpoint can’t be met at all

Studying the Cell Cycle — Experimental Techniques

Chemically Synchronizing Cells in a Population

-scientist applies a block that stalls the cell cycle at a known location

-cell cycle blocks can be placed at other check points too

-when these blocks are applied → cells continue to process through cell cycle until they hit the block where they can go no further

Fluorescence-Activated Cell Sorting (FACS)

-using fluorescently labeled cells

  • use its fluorescence to differentiate between cells

-machine (flow cytometer) measures the fluorescence using a laser and separates a mixed population of cells

-result of this technique is two-fold

  1. synchronized population of cells without using chemical inhibitors or temperature-sensitive mutations → can be used for further experiments

    • cells in G1 will have 1 full set of their DNA while cells in G2 and M-phase have duplicated their DNA

    • cells in S-phase will have more than 1 set but less than 2 sets as replication has started but isn’t complete

  2. flow cytometer produces a graphical readout that summarizes how many cells were found with each amount of fluorescent material (DNA)

Interpreting FACS Readouts

-frequency histogram

-Y-axis represents number of cells counted at each point on the X-axis

-X-axis measures amount of DNA

-since amount of DNA correlates to phase of cell cycle → determine how many cells are in each stage of cell cycle at the moment at which they were measured

  • second peak is replicated DNA

    • 2 phases that both have replicated DNA (G2 and M)

    • FACS can’t differentiate these so they are lumped together in histogram

  • part in between the peaks is important