BIOL3000: Cell Biology

Exam 4 (Notes 31)

What is necessary for SUCCESSFUL cell division?

Successful cell division requires:

(1) DNA replication —> S phase (needs its full complement of DNA to replicate)

(2) Division of the DNA AND the cytoplasm —> M phase = Mitosis + Cytokinesis

What does one cycle consist of?

An M phase, an S phase, and part of the second M phase again (going into the second cycle)

Why is DNA replication important to cell division?

Two cells that result from cell division require the FULL complement of DNA for each cell, so each cell

How was the time period of each phase within a cycle determined?

• Howard and Pelc examined Vicia faba (fave beans) in the early 1950s — shined a lined on a lot of mammalian cell mechanisms

  • Saw the cells go through mitosis for 30 hours by looking at chromosome condensation and division (one M phase to the other)

  • Each mitosis phase is about 4 hours

Does this mean S phase takes 26 hours?

No

How did they find out how long it takes S phase to complete?

• Conducted an experiment

  • Radioactively labeled plant cells

  • Used microscopic autoradiography to track the events of S phase

• What did they find?

  • It took 2 hours for a cell to pick up the radioactive labeling

  • They counted the number of nuclei that showed the radioactive label

    • These nuclei were in the process of DNA synthesis

  • No matter how they did the experiment, 20% of the cells were labelled

  • 30 hour cell cycle x 20% (labeling after treatment) = 6 hours (the length of S phase)

What about the other 20 hours that are uncounted for?

• The 20 hours could be anywhere:

  • Between M and S

  • Between S and M

  • 10 hours between M and S and 10 hours between S and M

How did they determine what phases consisted of the 20 hours?

• Used microscopic autoradiography and counted the nuclei in M phase

• In this experiment, the Y-axis is the percent of labelled nuclei in division

  • The X-axis is time

• It takes 2 hours for the radioactive label to appear before there is a “trace” or dot of radioactive label

  • It takes 10 hours for an APPRECIABLE accumulation of radioactive label in the nuclei

  • There are 8 hours before it goes from incorporation to the radioactive label to actively seeing the radioactive label on the condensed chromosomes

What phase consists of the 8 hours?

• Period of time between S and M phase called the gap phase

• Unclear as to WHY this gap phase is called G2

What phase consists of the remaining 12 hours?

• This is called the Gap1 phase (G1)

• G1 is the time after M phase and before S phase

  • This is often the LONGEST period across different types of cells

What is interphase?

Everything but M phase (G1, S, and G2), the phase where the cell prepares for division and replicates its DNA.

Do cells ever rest?

• YES!

  • Ex. most neurons are non-dividing cells and spend their entire existence in a resting phase

  • A researcher noticed that if the cell cycle is inhibited before S phase, the cell would always take the same amount of time to re-enter the cell cycle NO MATTER what inhibitor is used

What is the fifth segment to the cell cycle?

• A G0 phase that’s a sidetrack

  • Somewhere after the M phase

  • G0 phase will end in the middle or later part of G1

• Rather than re-entering G1, a cell will enter G0 on the sidetrack and sometimes, not always, will return to G0

Quiescence

• Period of cycle

  • Ex. G0

• Doesn’t meant the cell is dead —> just means the cell isn’t moving through another round of cell division

Terminal G0

• In that experiment where cells in a culture dish have growth factors, the rate of culture will experience some growth over generations

  • It will plateau into constant growth after generations

  • Then, it will start to decrease over generations to 0

• Those cells that decrease to 0 have entered a terminal G0 phase — eventually, those cells will acquire properties or attributes that are signaling that the cells will eventually die

• This can go on for about 40-50 generations

Senescence

• Terminal G0

• Can never re-enter the cell cycle and the cells will eventually die

Is cell growth required for cell division?

• Cell growth is essential for cell division

• There are some examples in biology where increase in cell size doesn’t matter during division —> frogs

• There are others where increase in cell size does matter during division —> budding yeast

• However, GROWTH is important to cell division (division is a moot point)

  • Have to have a cell that can grow and divide to transmit genetic information

  • If a cell becomes smaller, the number of cells will run out

  • The important point is growth, not cell size

  • How do cells/organelles remain the same size at a constant rate? ??

Where does the cell cycle begin?

• If you are a mammalian cell —> restriction point or R-point that exists in late G1

  • Cell is sensing whether there are growth factors present

  • Regardless of what we’re sensing, the cell cycle in regular cells starts around late G1

• If you are a fungal cell —> START

  • Cell is sensing nutrients and growth to support the entire process of DNA synthesis and cell division

How do we know the restriction point exists?

• The general rule after starting the R point, there is going to be cell growth, and the cell cycle is going to start

• They used an experiment — using an asynchronous culture, which means all of the cells in the culture are at different points (not all at G1, not all at S)

• Ex.

  • The researchers changed the medium, which lacked serum (which is part of the blood that has growth factors) —> 1-hour serum starvation

  • If a cell has just exited M phase and you starve the cell, there is a delay in the cell entering the next round of the cell cycle at G1, even if you return fresh medium that returns growth factors

• However, in other cells:

  • Cells starved in G1 enters the cell cycle with no delay

  • Cells starved in S, and G2, enter the cell cycle with no delay

• Once the cell has PASSED the restriction point, you’re committed to the cell cycle regardless of what happens

Is there only ONE route through the cell cycle?

• Rao and Johnson developed an experimental technique where they could fuse cells together

• Because the cells were two different cells, this was the process of heterokaryon formation

  • Two different nuclei (S and G1, S and G2, and G1 and G2)

• Rao and Johnson watched how the cells responded to each other when they are in the presence of each other

G1 and G2 Cell Fusion Experiment

• G2 nucleus sits there and doesn’t change

• Over time, the G1 nucleus would become an S phase nucleus where DNA replication was occurring

• Phases of the cell cycle CAN’T be skipped (can’t go from G1 into G2, has to go through S phase)

S and G2 Cell Fusion Experiment

• G2 nucleus sits there and doesn’t change

• S phase nucleus sits there and also doesn’t change —> eventually becomes G2 but not at this particular time point

  • G2 nucleus did not return to become an S phase

  • DNA replication occurred already in the nucleus and is just sitting there

• S phase is in the process of DNA replication and will eventually become G2

• Cells have blocks that prevent themselves from returning to a previous cell cycle state (can’t go backwards in the cell cycle)

  • DNA is licensed to replicate only once per cell cycle

• S also stays in S in the presence of G2 without going to M - because there’s DNA that hasn’t finished replicating, there’s no entry into M phase

  • The nucleus in S phase must finish DNA replication before entering M phase - suggests cell is checking whether the cell cycle is complete or not before moving into M (until both are in G2, then both nuclei will move into M phase)

S and G1 Cell Fusion Experiment

• G1 acquires characteristics of S phase —> DNA replication begins

• This is not a “skip ahead” of the cell cycle - which is a rule violation

• The cell cycle can be accelerated because this G1 nucleus is in the process of an S phase nucleus

  • The G1 transitions into S phase more rapidly than it normally would

• Rao and Johnson suggests there is a diffusible activating signal - an S phase promoting factor (SPF) - exists at or past the R-point

Hartwell’s experiment identified genes that regulated the cell cycle

• He used yeast because he could spread them out on a plate

  • Every yeast colony is derived from a SINGLE cell

  • On the left plate, there is an asynchronous culture where every cell is in a different phase of a cell cycle

    • Large budded cell is M phase

    • Large/medium cell is G2 phase

    • Medium cell is G1 phase

    • Small budded cell is S phase

• Spread the cells on a plate at 25 degrees C and raised the temperature to 37 degrees C

  • Found one cell that did not divide at 37 degrees C

  • Mutagenized the cells using double-mutant analysis to identify the order of events of cell division

  • Got the cells to grow by raising the temperature

• Called these cell division cycle mutants —> the 28th mutant in his collection, when temperature shifts, those cells DON’T enter the cell cycle and stay in G1

  • If they were in S or G2, they would divide

  • The 28th mutant defined start in G1 - before start and lose important protein activity, you don’t pass start

  • cdc28ts encoded an enzyme

How did they find the enzyme cdc28ts coded for?

• Each DNA molecule in the molecular library represents part of the yeast genome

• If you put gene X into these cells through transformation and raise the temp, the cells don’t grow

  • Whatever the gene that cdc28ts encodes for is defected

  • The piece of DNA contains part of the yeast genome that contains a gene that is rescuing the defect in these cells

  • This gene is then called cdc28, which encodes a kinase —> what they discovered is the MASTER KINASE that regulates the cell cycle