BIOMG 1350 Lecture 12 - Cell Cycle

Cell Cycle

• The cell cycle involves cell growth and chromosome replication, chromosome segregation (and organelle secretion such as the mitochondria), and cell division. In other words it is the ordered series of growth and division events that a eukaryotic cell goes through to split into two genetically identical daughter cells

• The product of the cell cycle or mitosis is the production of two daughter cells.

• The most visible aspects of cell division are the segregation of the chromosomes at mitosis and division of the cytoplasm at cytokinesis. This part of the cell cycle is called M phase.

• The cell physically begins division during M-phases (mitosis and cytokinesis) The rest of the cycle is called ‘interphase’. During this part of the cycle, it grows by synthesizing proteins, lipids, etc. to double its mass and all the organelles

Differences in Cell Cycle Length Between Types of Cells is Due to…?

• The differences in cell cycle length are mostly due to differences in the time spent at the G1 checkpoint.

• Duration of the cell cycle varies greatly from one cell type to another.

  • Early fly embryo cells divide in 8 minutes whereas mammalian fibroblasts in culture divide every 20 hours and mammalian intestinal epithelial cells divide every 12 hours.

4 Stages of the Cell Cycle

• G1 or growth 1 phase. This includes cell growth and preparation for the S phase.

• S phase or the synthesis and DNA replication phase.

• G2 phase or growth 2 phase. This includes more cell growth and preparation for the M phase. 

• M phase which includes mitosis (nuclear division) and cytokinesis (cytoplasmic division).

• Note that the cell cycle can also be divided into 2 major phases, specifically interphase (G1, S, G2) and the M phase (where mitosis and cytokinesis occurs).

Cell Cycle Exit: Permanent vs Temporary (G0 Phase)

• Some cells also exit the cell cycle and never divide again after first being formed. They exit the cell cycle, such as nerve cells, and are referred to as "post mitotic" cells. Due to this fact neurons are as old as you are and are permanently in the G0 phase.

• Other cells temporarily stop dividing by exiting the G1 phase and entering the G0 phase.

  • Liver cells are usually in G0 but can reenter G1 and continue the cell cycle to make more liver cells.

Major Challenges Facing a Dividing Cell and Consequences of Proper Preparation for the Cell Cycle

What are the challenges facing a dividing cell?

• The cell must accurately duplicate all of its contents, then divide them equally between two daughter cells.

What would happen if a cell tried to divide before its DNA had replicated?

• It would be catastrophic as one or both of the daughter cells would lack part of its genetic material.

What would happen if a cell duplicated its DNA but then divided before all its other constituents had doubled?

• Daughter cells would lack some organelles and/or get smaller and smaller.

How do Cells Control the Order and Timing of Events in the Cell Cycle?

Cells have mechanisms to precisely coordinate cell growth, DNA replication, and segregation before undertaking cell division.

How do cells control the order and timing of events in the cell cycle?

• Cyclin dependent kinases (CDKs) where the activity of CDKs is regulated and changes throughout the cell cycle.

• Checkpoints which ensure one phase is properly completed, before the next phase begins. Checkpoints can be activated (cell cycle is stopped) or satisfied (cell cycle continues its normal progression).

Cyclin Dependent Kinases

• The order of events in the cell cycle is controlled by the CDKs.

• Cyclin Dependent Kinase can only phosphorylate other proteins, exchanging ATP for ADP and transfers a phosphate group onto a specific protein. CDKs can only phosphorylate when bound to cyclin which opens up the ATP within the CDK to be applied to proteins. Otherwise the ATP is lodged deep inside the CDKs.

• Cyclin binding to CDK pulls open the T look in the CDK, exposing the ATP and allowing the CDK to phosphorylate specific proteins, either activating or inactivating those proteins.

• CDKs associate with different cyclins to induce the different events of the cell cycle. One type of CDK can associate with different cyclins.

• Different cyclins will direct the CDK to phosphorylate different target proteins (substrates).

Regulation of CDK Activity by Cyclins

• Cyclin dependent kinases (CDKs) require cyclin proteins for their activity.

  • The concentration of cyclin varies during the cell cycle.

  • Kinases, specifically CDKs are present throughout the cell cycle, nearly at constant concentrations at any given time, but its activity is dependent on cyclins. However CDKs can only perform their “job” when bound to the correct cyclin and not inhibited.

  • Example: "M cyclin" binding to CDK creates the M-CDK kinase complex and this is specifically present during mitosis to prepare the cell for the process involved in the M phase. The M cyclin concentration increases and then peaks during the M cyclin activity portion of the cell.

2 Major Cyclin and CDK Types Present in the Cell Cycle

• There are 2 major cyclin and CDK types.

  • The first is the S cyclin synthesis which peaks at the end of G1 and the transition into the S phase. The S cyclin activates its respective CDK and then the S phase substrates, usually involved in DNA replication, are activated.

  • The second is the M cyclin synthesis which peaks at the end of G2 and the transition into the M phase. The M cyclin activates its respective CDK and then the M phase substrates, involved in mitosis, are activated.

• Some other cyclin/CDK types present in the cell are G1-CDK and G1/S-CDK which are discussed further in a different flashcard.

4 Types of CDKs and Their Function

Cyclin–CDK complex	Main function
G1-CDK	Promotes progression through G1
G1/S-CDK	Triggers the transition from G1 into S phase
S-CDK	Initiates DNA replication during S phase
M-CDK	Triggers entry into mitosis (M phase)

Note: G1-CDK and G1/S-CDK functionality and ihibition at checkpoints or near checkpoints.

Cell Cycle Checkpoints

• The timing of the events in the cell cycle are controlled by checkpoints.

  • A checkpoint is a molecular mechanism that pauses the cell cycle to ensure that one step is completed before the next is started.

• There are 3 main cell cycle checkpoints G1/S, G2/M, and M checkpoint.

• Checkpoints ensure that the conditions conductive to proper cell division are met before moving on.

  • A checkpoint is activated if conditions are not met and the cell stops/pauses at that stage of the cell cycle.

  • A checkpoint is satisfied only when conditions are met. This causes the removal or inactivation of the negative regulation of the checkpoint allowing the cycle to proceed (double negative).

  • Active checkpoints mean conditions for proceeding with cell division are not met. A checkpoint is satisfied when the conditions are met.

3 Major Cell Cycle Checkpoints and their Purpose

• There are 3 major cell checkpoints.

  • One is the G1/S checkpoint which ensures that the cell has enough nutrients, that the DNA is not damaged, and that proper growth signals were received so that synthesis of the DNA can occur.

  • The second is the G2/M checkpoint which checks to make sure if the DNA is all replicated properly and that any present DNA damage is properly repaired so that mitosis can start.

  • The last cell checkpoint is the mitosis checkpoint (M checkpoint) or the spindle assembly checkpoint whose main purpose is the ensure all the chromosomes are properly attached to the mitotic spindle so that mitosis can finish and the chromosomes can be pulled apart.

3 Major Ways in Which CDK activity is Regulated

There are multiple mechanisms that regulate CDK activity. Each checkpoint involves distinct mechanisms for regulation of CDK activity:

• CDK Inhibitor Proteins: In the G1/S checkpoint, CDK inhibitors block entry into S phase if the checkpoint is activated and conditions are not met.

• Phosphorylation: In the G2/M checkpoint, activating phosphatase (Cdc25) is inhibited which blocks the entry into mitosis as the checkpoint is activated and conditions are not met.

• Protein Degradation: In the M or spindle assembly checkpoint APC/C activation is inhibited which delays the exit from mitosis as the checkpoint is activated and conditions not met.

G1/S Checkpoint

• The G1/S checkpoint is the point at which cells decide whether to enter the cell cycle based on environmental signals.

• Growth factors (like the epidermal growth factor or EGF) signal animal cells to proceed through the G1/S checkpoint and enter the S phase.

• This checkpoint greatly influences the cell cycle duration. CDK inhibitors are the main factors in blocking the entry to the S phase. If the cell chooses it can withdraw into G0 during the G1 phase in the cell cycle.

• During G1, S-CDK complexes are formed but bound to p27 inhibitor proteins. To satisfy the G1/S checkpoint, cells reduce the level of CDK inhibitor proteins and can only do this if the conditions are favorable for continuing the cell cycle.

  • P27 is the specific CDK inhibitor protein that inhibits the S-CDK complex and then cells reduce its levels to satisfy the G1/S checkpoint.

The Role of p53 as the Guardian of the Genome in the G1/S Checkpoint

When DNA damage is present, protein kinases phosphorylate p53 already present in the cell which stabilizes and activates it.

This activated p53 protein bind to the regulatory region of the p21 gene. Then the p21 gene is expressed via this p53 protein binding to the DNA which causes the production of the p21 mRNA and p21 protein. This p21 protein then can inhibit the currently active S-CDK and G1/S-CDK preventing further cell replication.

• P53 is mutated in about 50% of cancers. Loss of p53 function results in more rapid accumulation of mutations as damaged DNA is not repaired. Conversion of a normal cell to a cancer involves the accumulation of mutations in specific genes.

• P53 is synthesized during G1, but is rapidly degraded by the proteasome unless DNA damage occurs. This is an important regulator of the G1 checkpoint.

G2/M Checkpoint

• The G2/M checkpoint checks two important aspects of DNA replication. First, it checks to ensure that all DNA is replicated. Second, it checks to make sure there is no DNA damage.

• M-cyclin is constantly accumulating during S and G2, so why is M-CDK activity triggered so abruptly? (We see a graph demonstrating the steady increase of M-Cyclin but then bursts of M-CDK activity in mitosis)

• The Wee1 kinase enforces the G2/M checkpoint and Cdc25 phosphatases activity satisfies the G2/M checkpoint or allows the checkpoint to be passed.

• Activating the M-CDK kinase will satisfy the G2/M checkpoint, meaning that checkpoint is no longer active.

• Inhibition of activating phosphatase (Cdc25) blocks entry to mitosis as nothing reactivates the deactivated M-CDK from the Wee1.

How Wee1 and Cdc25 Function in the G2/M Checkpoint to Activate and Satisfy

• Wee1 kinase and checkpoint enforcement process

  • The Wee1 inhibitory kinase attaches 2 inhibitory phosphates onto the M-CDK complex inactivating it. The inactive M-CDK prevents the cell from progressing along its cell cycle.

  • Then the activating phosphatase (Cdc25) removes the phosphate groups on the inactive M-CDK switching it to the active M-CDK form.

  • Cdc25 Phosphatase is activated right during the transition from interphase to mitosis allowing the cell to progress through its cycle.

M-CDK and Cdc25 Positive Feedback Loop

• Cdc25 phosphatase and M CDK participate in a positive feedback loop. Active M-CDK activates Cdc25 phosphatase which in turn activates more M-CDKs through dephosphorylation.

• M-CDK phosphorylates Wee1 and this inactivates Wee1 so that Wee1 no longer phosphorylates CDK as well.

• Thus, once M-CDK levels reach a critical threshold concentration, small amounts of active M-CDK lead to the activation of the entire pool of M-CDK.

Spindle Assembly Checkpoint (in Mitosis)

• The spindle assembly checkpoint ensures that all chromosomes are properly attached to spindle microtubules through their kinetochores before the cell proceeds from metaphase to anaphase.

• If any chromosome is not properly attached, the checkpoint delays anaphase by inhibiting the anaphase-promoting complex (APC/C). Once all chromosomes are correctly attached, the checkpoint is satisfied, allowing sister chromatids to separate.

Function of the Spindle Assembly Checkpoint or how M-CDK is Inactivated

• If Cdc25 positive feedback maintains M-CDK in the active state, how is M-CDK inactivated in cells?

• M-cyclin protein levels drop abruptly at the end of mitosis, or in other words, satisfying this spindle assembly checkpoint involves the loss of M-CDK activity. But the question is how does this occur.

• M-CDK inactivation at the spindle assembly checkpoint is triggered by M-cyclin degradation.

  • APC/C is a protein complex that attaches ubiquitin (ubiquitin chains) to cyclin. This ubiquitin marks the protein for destruction by the proteasome. Destruction of the M-Cyclin in the M-CDK/Cyclin complex inactivates it.

  • Ubiquitin is a small protein and several ubiquitin molecules can be covalently linked to proteins as a chain called poly ubiquitylation.

Protein Degridation via Ubiquitin Proteasome System

• Protein degradation via the ubiquitin proteasome system is performed as follows.

  • Proteins are marked by chains of a small protein called ubiquitin (ubiquitination) for degradation. Proteasome then binds to ubiquitinated proteins and actively unfolds them (through the energy of ATP) and then transfers the unfolded peptide chain into the proteolytic chamber to be degraded/hydrolyzed.

  • Proteasome is composed of the polyubiquitin binding site in the regulatory particle, and the proteolytic chamber

Summary of Checkpoint Mechanisms

• G1/S Checkpoint

  • S-CDK inhibitor proteins maintain the checkpoint and the checkpoint is satisfied when inhibitor levels are reduced.

• G2/M Checkpoint

  • Phosphorylation of M-CDK keeps it inactive and checkpoint is satisfied when M-CDK is dephosphorylated.

• Spindle Assembly Checkpoint

  • M-CDK remains active at the checkpoint and the checkpoint is satisfied when the M cyclin is degraded and M-CDK activity is lost.

Cyclin Concentration Fluctuations Throughout the Cell Cycle

• S cyclin increases near the end of G1 and decreases near the beginning of the M phase. M cyclin increases near the end of G2 and decreases near the middle of the M phase.

• It emphasizes that cyclin concentrations rise and fall throughout the cell cycle, while CDK concentrations remain relatively constant. These fluctuations regulate CDK activity and help control the timing of different cell cycle events.

S Cyclin

A regulatory protein that binds to and activates CDKs to form S-CDK complexes, which initiate DNA replication during S phase.

S cyclin levels increase near the G1/S transition and remain elevated through S and G2 phases before being degraded during mitosis.

M Cyclin

A regulatory protein that binds to and activates CDK1 to form the M-CDK complex, which triggers entry into mitosis.

M cyclin concentrations gradually increase during interphase, peak during early mitosis, and rapidly decrease during the metaphase-to-anaphase transition as M cyclin is degraded, allowing the cell to eventually exit mitosis.

S-CDK

• A cyclin–CDK complex formed when S cyclin binds to and activates a CDK (typically CDK2). S-CDK promotes entry into S phase and initiates DNA replication by activating proteins involved in DNA synthesis.

• It also helps prevent DNA from being replicated more than once during the same cell cycle.

• S-CDK is involved with progressing the cell past the G1/S checkpoint

M-CDK

• A cyclin–CDK complex formed when M cyclin binds to and activates CDK1. M-CDK triggers entry into mitosis by phosphorylating proteins involved in chromosome condensation, nuclear envelope breakdown, and mitotic spindle formation.

• M-CDK activity decreases when M cyclin is degraded during mitosis, allowing the cell to exit mitosis.

• M-CDK is involved with progressing the cell past the G2/M checkpoint.

Cdc25

A protein phosphatase that activates M-CDK by removing the 2 inhibitory phosphate groups from M-CDK. This activation allows the cell to transition from G2 into M phase and begin mitosis.

Cdc25 also participates in a positive feedback loop in which active M-CDK further activates Cdc25, causing rapid M-CDK activation.

Wee1

A protein kinase that inhibits M-CDK by adding an 2 inhibitory phosphate groups to M-CDK, preventing premature entry into mitosis.

Wee1 helps keep M-CDK inactive during G2 until the cell is ready to enter M phase.

When M-CDK becomes active, it inhibits Wee1 through a negative feedback mechanism, promoting rapid M-CDK activation.

APC/C (Anaphase-Promoting Complex/Cyclosome)

A protein complex that functions as an E3 ubiquitin ligase, tagging specific proteins for degradation by the proteasome during mitosis.

APC/C promotes the transition from metaphase to anaphase by triggering securin degradation, allowing sister chromatids to separate.

It also promotes M cyclin degradation, reducing M-CDK activity and allowing the cell to exit mitosis.

p27

A cyclin-dependent kinase inhibitor (CKI) that binds to and inhibits certain cyclin–CDK complexes, particularly G1/S-CDK and S-CDK, preventing progression from G1 into S phase.

p27 helps regulate cell cycle arrest in response to signals such as the absence of growth factors, preventing cells from dividing when conditions are unfavorable.

p21

A cyclin-dependent kinase inhibitor (CKI) that binds to and inhibits cyclin–CDK complexes, particularly G1/S-CDK and S-CDK, preventing progression into S phase.

In response to DNA damage, p53 stimulates p21 production, which inhibits CDK activity and arrests the cell cycle, allowing time for DNA repair.