BMB508 - The cell cycle!

What is the cell cycle?

The regular sequence of growth and division that cells undergo

Which phases are involved in eukaryotic cell cycle?

G0, G1, S, G2 and M-phase

How are these phases divided up?

Interphase which includes the G0, G1, S and G2 phase

Metaphase which includes the M-phase

What happens in the G0 phase?

The cell begins functioning regularly. It's no longer dividing. The cell division has been put on a break this happens if:

- It doesn't receive any of the necessary signals to go from G1 to S-phase.

What happens in the G1 phase of interphase?

It prepares itself for cell division!

Cell growth, new organelles are formed and new proteins synthesized.

What happens in the S-phase of interphase?

DNA replication where it:

- Duplicates the chromosomes

- Duplicating the centrosomes

What happens in the G2 phase?

Many of the organelles and molecules required for cell division are produced (such as lipids and proteins)

- Reorganizing it's contents

- Preparation for mitosis

- Repairing eventually mistakes that happened under DNA-replication

What happens in the M phase?

Mitosis and cytokinesis occur!

DNA condensates and becomes closely packed together.

- Ensures that the chromosomes will be distributed correctly

- The m-phase ends with cytokinesis, where the cell will physically split into two daughter cells.

What are the steps in mitosis?

Prophase, prometaphase, metaphase, anaphase, telophase, cytokinesis

What happens in interphase?

G1, S, G2

What happens in prophase?

Chromosomes condense

Nuclear membrane disintegrates

Centrioles move to opposite poles

Spindle apparatus formed by microtubules

What happens in the prometaphase?

Chromosome attaches to the microtubule and starts to move

What happens in the metaphase?

Chromosomes line up in the middle of the cell

What happens in anaphase?

Sister chromatids split and move to either pole (Disjunction)

What happens in the Telophase?

Nuclear membrane reforms

Chromosomes decondense

What happens in the cytokines?

The cell membrane starts to pinch in the cell to separate in 2 new cells and the two new cells are back into interphase.

What checkpoints do we have in the cell cycle?

We have the G1, G2 and M-checkpoint!

What does the G1 checkpoint check for?

Cell size, nutrients, growth factors, DNA damage

What does the G2 checkpoint check for?

G2 checkpoint checks if the DNA has been successfully and correctly replicated and if the DNA doesn't have any damage!

- G2 stops the cell to proceed with the cell division if there is ANY DNA damage.

What does the M-checkpoint check for?

If the chromosomes are properly attached to spindle fibers

Why are unicellular yeast models used to study the cell cycle

Unicellular yeast models

- They are haploid organisms so they are easy to manipulate their genome

Fission yeast!

- They have a longer G1 phase, which makes it good for studying steps before DNA replication

- They have a longer G2 phase, which makes it good for studying steps before mitosis

- It is less ideal for biochemical analysis

Why are frog oocytes and early embryos a great model for biochemical characterization of the cell cycle machinery?

They have a higher eukaryotic model

- synchronized DNA replication and cell cycles

- Large number of eggs and large size

- NOT suitable for imaging

Why is it an ideal solution to under complex cell cycle regulation in mammalian cells by using a cell culture model such as U20S cells?

They are beneficial in nearly all categories such as:

- Genetics (siRNAs, shRNAs, CRISPR-Cas)

- Cell synchronization (Nocodazole- M, Thymidine- G1/S)

- Biochemistry (protein purification, immunoblotting, Mass spectrometry, inhibitors)

- Imaging (phase contrast, immunofluorescence)

What is cyclin-dependent kinase (Cdk)?

A protein kinase that is active only when attached to a particular cyclin.

- Cyclin dependent kinases (CDKs) must be bound to a cyclin to be partially active.

- The cyclin will furthermore direct the CDKs towards the right target protein.

- Example: G1-S phase cyclin send CDKs towards S phase target proteins which will promote DNA replication

Also known as the master regulators of the cell cycle!

What are the G1 cyclin-CDKs?

Cyclin D - CDK4/CDK6

Promote entry into the cell cycle

What are the G1/S-phase's cyclin-CDKs?

Cyclin E-CDK2

CDK2 => Functions as a G1/S phase CDK

What are the S-phase's cyclin-CDKs?

Cyclin A-CDK2

CDK2 => Functions as a S phase CDK

What are the M-phase's cyclin-CDKs?

Cyclin A-CDK1

Cyclin B-CDK1

CDK1 = Is a mitotic CDK

What determines the activity of CDKs?

Cyclins!

Cyclins bind to and activate CDKs. The activity and substrate specificity of any given CDK is primarily defined by the particular bound cyclin.

- Cyclins are present only during the cell cycle stage that they trigger and are absent from other cell cycle stages.

- In addition to triggering a particular cell cycle stage, cyclins also set in motion a series of events in preparation for the next cell cycle stage. In this way, they propel the cell cycle forward

What can inhibit cyclin-dependent kinase (Cdk)?

Multiple biochemical mechanisms ensure that the CDKs are active only in the stages of the cell cycle they trigger. These molecular mechanisms are organized into feedback loops that amplify the process of CDK activation and inhibition

- Kinase inhibitors (CDK-inhibitors

- Phosphatases

- Kinases

- Protein degradation by the ubiquitin-proteasome system (SCF and APC/C)

- Cell cycle checkpoints

How can kinases regulate protein activity?

Switch protein activity on/off

Increase/decrease protein activity

Modulate protein-protein interactions

Assembly/disassembly protein complexes

What are Cdk inhibitors (CKIs)?

Intracellular molecules with low molecular weight specifically inhibiting the activities of cyclin-dependent kinases during cell cycle progression

What are phosphatases?

Enzymes that remove phosphate groups from proteins

What does the CAK kinase?

Activates CDKs in all organisms

What does the Wee1 kinase?

Inhibits CDKs in all organisms

Normally phosphorylates CDK1 on Y and T residues => inhibition.

What does cdc25 phosphatase do?

Activates CDKs in fission yeast

Removes the phosphate added by wee1 kinase, reactivating the Cdk.

Dephosphorylates CDK1 => active

What does the Cdc25B phosphatase?

Makes sure the CDK-cyclin complex becomes active right before the entry into mitosis.

- Cdc25B starter phospatase & Cdc25C is the primary activation of the M-CDK complexes.

- These phosphatases remove inhibitory phosphate groups from CDK1.

What does Cdc14 phosphatase do?

Activates vertebrate S phase CDKs

Removes phosphates!

What does Sic1 do?

Binds and inhibits S phase CDKs in budding yeast

What does Rb do?

Binds to E2F, which is necessary for progression of the cell cycle.

This prevents transcription of multiple cell cycle genes in animal cells

What is the SCF ubiquitin ligase?

G1/S transition by targeting G1 and G1/S cyclins

G2/M transition by targeting inhibitory proteins.

The specificity is defined by the appearance of F-box proteins, which are responsible for substrate recognition through specific sequence interaction that often involves phosphorylation.

These sequence specific motifs are often referred to as degradation motifs. Due to this it is the substrates that are phosphorylated to target them for degradation.

What is E3 ligase?

It is a E3-ligase that transfer ubiquitin from an E2 ubiquitin-conjugated enzyme to one or more lysine residues on the E3-ligase's substrate proteins, making long polyubiquitin chains that mark these proteins for degradation by proteasomes.

What is the APC/C ubiquitin ligase?

Active in the onset of mitosis and throughout the next G1-phase.

It targets S- and M-cyclins, and anaphase inhibitory proteins (securin).

The specificity is deterred by of substrate-targeting module (cdc20 or cdh1). Binding of cdc20 initiates anaphase and the degradation of M-cyclin (hence exit of mitosis) and the binding of cdh1 causes the degradation/inhibition of S and M cyclins.

Activation of the complex is dependent on phosphorylation from mCDKs (the complex is phosphorylated).

APC/C scaffold have two modes:

Cdh1: Degradation of cyclin B

Cdc20: Degradation of securin.

What is the definition of cyclins?

A family of proteins that rise and fall in concentration in step with the eukaryotic cell cycle, thereby regulating the activity of the crucial protein kinases that control progression through the cell cycle.

- Cyclin proteins binds one or more specific protein kinases ( CDKs) to increase their activity

- CDKs are present throughout the cell cycle, but their activity is regulated by association with cyclins.

- Cyclin concentrations flucturate during the cell cycle

- Thus, driving the activation of specific cyclin-CDK complexes that regulate cell cycle progression

- The coordinated action of cyclin-CDK complexes ensure proper timing and control of cell cycle events

What transitions do we have in the cell cycle?

G1 to S-phase commitment, preparation for DNA replication

S-Phase: DNA replication

G2 to M phase: Preparation and Mitosis (M-phase)

How is the E2F transcription factor activated before entry into the S-phase?

(regarding the G1/S-phase transition)

E2F is bound to Rb => inactive in G1

Active G1 CDKs (cyclin D-CDK4/6) phosphorylates Rb => dissociation of Rb, leaving E2F "free" to start transcription

E2F => activation of genes encoding proteins involved in DNA replication

E2F stimulates transcription of genes encoding G1/S phase cyclin E and S phase cyclin A

- Cyclin A forms complex with CDK2

- Leading to more phosphorylation of Rb

- positive feedback loop

- boosting S phase entry

What needs to be done for the transition of the S-phase to happen?

It is necessary to degrade Sic1 since it is an S-phase inhibitor that inhibits the S-phase and M-phase CDK complexes

- For DNA replication to happen, the Sic1 is degraded by SCF ubiquitin-protein ligase

- This degradation of the Sic1 is induced by phosphorylation caused by the G1/S phase CDKs.

- It has to be phosphorylated at no fewer than six sites.

How is DNA replication at each origin initiated once and only once during the cell cycle?

1. Preparation: DNA replication origins are prepared during early G1 phase.

2. Complex Formation: Complexes are assembled at replication origins, including the loading of the MCM helicase complex.

3. Complex Activation: Activation of replication complexes occurs in S phase, allowing DNA replication to begin.

4. DNA Replication: The actual copying of DNA takes place.

5. Dephosphorylation: Regulatory proteins are dephosphorylated to reset the system for the next round of replication.

Look at figure 19.19

What happens in the preparation phase:

After mitosis during early G1 phase when CDK activity is low

The origin-recognition complex (ORC) bind to all DNA replication origins, marking them for replication in the upcoming cell cycle

The MCM helicase is then loaded onto the DNA with help from Cdc6 and Cdt1, preparing the DNA for replication initiation

These proteins can only bind when unphosphorylated!!!

What happens in the complex formation?

During S-phase when CDK activity is high

DDK and CDK enzymes phosphorylate proteins involved in DNA replication, facilitating the assembly of complexes necessary for DNA unwinding and replication

Specifically, they target the MCM helicase, Sld2, and Sld3 to facilitate the loading of MCM helicase activators, such as Cdc45 and GINS complexes.

S phase CDKs prevent re-assembly of replication complexes to ensure each DNA segment is replicated only once.

They achieve this by phosphorylating Cdc6 and Cdt1, promoting their release from the replication origins.

This leads to their nuclear export or degradation by ubiquitin ligases.

What happens after DNA replication?

After DNA replication, S phase CDKs trigger the disassembly and export of replication machinery from the nucleus, marking completion of replication

Phosphorylation of MCM helicase by S phase CDKs leads to export from the nucleus when the helicases disengage from the DNA when replication is complete

What happens at the end of mitosis?

Dephosphorylation at the end of mitosis prevents re-initiation of DNA replication from occurring, ensuring that each origin initiates replication only once per cell cycle.

Also:

By the end of mitosis APC/C-cdh1 is active which leads to degredation of Geminin.

Since Geminin is no longer binding and inhibiting Cdt1 it is free to participate in the loading of MCM helicase

How are the sister chromatids held together along their length until they separate at mitosis?

After replication sister chromatids are hold together along their length by the cohesin protein complex.

Cohesin protein complex dissolves during mitosis => separation of the sister chromatids

What events in the M-phase are stimulated by the M-CDK complex?

Nuclear envelope breakdown

Assembly of the mitotic spindle

Bipolar attachment of sister chromatids - Mitotic spindle formation

chromosome condensation

Separation of the copied sister à initiation of spindle formation

Rearrangering of cytoskeleton og Golgi à microtubuli reaches from centrosomes into the nuclear area.

How many kinetochores are present in a normal human cell in the early M-phase?

92 kinetochores

46 chromosomes in humans

Each have 2 sister chromatids = 92