Mitosis & Cell Cycle - University BIO2133

• BIO2133 is a university-level course with university-level expectations
• Test 1 has been graded, with an average score of 60% and a normal grade distribution
• The MCQ average score is approximately 75%, while the problem section average is about 45%
• Less than half of the students attend classes, and 15% did not take Test 1
• UoO academic regulations define the workload for a 3-credit course as 135 hours per semester, roughly 10 hours per week
• The upcoming material is more molecular and conceptually demanding, it is highly recommended to attend class discussions

Mitosis as a Step in the Cell Cycle

• Mitosis ensures accurate distribution of genetic material to daughter cells
• Mitosis preserves chromosome integrity with the goal of maintaining genetic stability and cellular function
• Mitosis defects usually result in cell death, but sometimes lead to cancerous tumor formation

Contents

• Topics to be covered include:
• Mitosis as a step in the cell cycle
• G2 checkpoint
• Prophase
• Spindle assembly checkpoint
• (Pro)Metaphase
• Anaphase
• Telophase

Expected Learning Outcomes

• Keywords include:
• Prophase
• (Pro-)metaphase
• Anaphase
• Telophase
• DNA replication
• DNA condensation
• Mitotic spindle
• Centrosome
• Microtubule dynamics
• Kinetochore
• Chromosome
• Sister chromatids
• Nucleosome
• Condensin
• Histone protein
• Post-translational modification
• G2 and spindle assembly checkpoints
• Congression
• Polar ejection force
• Outline the initial and final specific steps of each mitotic phase
• Create an illustration depicting the key elements and role of the G2 checkpoint and provide a concise explanation of its primary purpose
• Make a drawing of a nucleosome, and identify its main components and discuss molecular alterations affecting its structural stability
• Make a drawing of a complex of two sister chromatids along with the binding molecular factors, and identify each of the components
• Make a drawing of the kinetochore complex and explain its main functions to mitosis
• Create an illustration depicting the key elements and role of the spindle assembly checkpoint and provide a concise explanation of its primary purpose
• Discuss the molecular forces leading to the proper alignment of sister chromatids at the cell equatorial plate
• Explain an experimental design used to study the dynamics of kinetochore microtubules leading to the proper alignment of sister chromatids at the cell's equatorial plate
• Make a drawing explaining the principle and physiological role of polar ejection forces
• Creation of a drawing illustrating the concurrent functions of the two mechanisms involved in chromosome pull during anaphase

Mitosis as a Step in the Cell Cycle

• All cellular growth and division cycles include "interphase," during which DNA replicates and other cellular constituents are made
• Mitosis is a type of cell division resulting in two daughter cells, each having the same number and kind of chromosomes as the parent nucleus
• During mitosis, cellular materials rearrange, so after cytokinesis each daughter cell can fully grow and divide again
• Budding yeasts make only one mistake in about 100,000 divisions

Checkpoint Entry Into Mitosis: G2 Checkpoint

• Checkpointing entry into mitosis is crucial for two main reasons:
• DNA Integrity: The G2 checkpoint ensures that all DNA has been accurately replicated and damage free before the cell enters mitosis
• Cell Size and Resources: The G2 checkpoint assess if the cell has sufficient size and resources to complete mitosis/support daughter cells

Checkpoint Entry Into Mitosis: G2 Checkpoint

• Mitosis is triggered by cyclin-dependent kinase Cdk1, and its activity is controlled by different factors:
• Activation of Cdk1 begins when it binds to its regulatory subunit cyclin B1, whose levels rise during G2 and peak in mitosis
• During interphase, Cdk1-B1 phosphorylates at inhibitory sites and is primarily cytoplasmic as its rate of nuclear export is greater than import
• During mitosis, multiple phosphorylations of B1 trigger a decrease in nuclear export and an increase in import
• Cdc25C is also regulated by phosphorylation and in interphase, phosphorylation-dependent binding of 14-3-3 inhibits import, resulting in cytoplasmic localization
• In mitosis, dephosphorylation and dissociation of 14-3-3 increase the rate of nuclear import
• Cdc25C on mitosis engagement exerts regulatory control

Checkpoint Entry Into Mitosis: G2 Checkpoint

• Molecular mechanisms control the activation of Cdc25C and Cdk1-B1 at the onset of mitosis -Cdc25C Activation:
  • Cdc25C is normally inhibited by the binding of the 14-3-3 protein to its phosphorylated serine 216
  • Dephosphorylation at serine 216 leads to the dissociation of the 14-3-3 protein
  • Free Cdc25C is further activated by extensive phosphorylation of its amino terminus, which is carried out by Polo-like kinase (Plk) and by Cdk1 itself (positive feedback)
  • Activated Cdc25C further interacts and activates Cdk1-B1 by dephosphorylating Cdk1 and phosphorylating B1
  • Phosphorylation of cyclin B1 then promotes its translocation into the nucleus in late prophase, where it initiates events such as nuclear envelope breakdown
• DNA damage has a dual inhibitory effect on Cdc25C activation
• DNA damage controls this mechanism to ensure checkpoint regulation, specifically preventing mitosis initiation

Checkpoint Entry Into Mitosis: G2 Checkpoint

• The presence of unreplicated DNA or DNA damage blocks the activation of cyclin-B1-Cdk1 complexes, preventing mitotic events until replication is complete or DNA damage is repaired
• Expression of Cdc25C-S216A, a mutant protein that cannot be phosphorylated at Ser216, stimulates premature mitosis in cells arrested in G2 by DNA damage
• The ability of cyclin B1 to stimulate nuclear envelope breakdown is abolished by mutations in its cytoplasmic retention sequence

Mitosis as a Step in the Cell Cycle

• Interphase covers G1, S, and G2 phases and account for most of a cell's life
• Mitosis stages include:
  • Prophase:
    • DNA condenses
    • Disappearance of nucleolus
  • Prometaphase:
    • Appearance of microtubules (mitotic spindle)
    • Disappearance of the nuclear membrane
  • Metaphase:
    • Alignment of chromosomes at the center of the cell
  • Anaphase: Chromosomes move apart
  • Telophase: Formation of nuclear membrane surrounding newly formed nuclei
• In cytokinesis, the cell splits into two daughter cells

Mitosis Sequential Steps

• Prophase (start of mitosis)
  • Chromosomes condense
  • Chromosomes become visible
• Pro-metaphase
  • The nuclear membrane begins to disintegrate
  • Microtubules extend from the two polar, cytosolic centrosomes and attach to kinetochores
• Metaphase
• Chromosomes align at the cell's equatorial (metaphase) plate
• Anaphase
• Sister chromatids (the two halves of each chromosome) pulling apart to opposite poles
• Telophase (end of mitosis)
• Microtubule disintegration
• Chromatids reach the poles as nuclear membranes form around each chromosome set, which begin to de-condense
• Cytokinesis (following mitosis)
• Cell cytoplasm divides resulting in two separate daughter cells

Prophase: Cells Get Ready for Division

• The onset of chromosome condensation marks beginning of prophase
• Human linear DNA sums to nearly 2m (0.34 nm per nucleotide), but this is shortened by nearly 100,000 times during condensation (from 2m down to 20-40 um)
• Compaction acts on strands of chromatin so that DNA forms as it wraps around histone cores forming nucleosomes
• As compaction occurs, transcription shuts down

Prophase: Regulatory Control of DNA Condensation

• Post-translational modifications of histone proteins occur,
• Histone proteins are highly conserved, positively charged amino acids such as lysine, histidine, and arginine enable ionic bonding with the ribose-phosphate backbone of DNA tightening the nucleosome structure
• Acylation of these positive charges loosens nucleosomes, promoting decondensation
• Nucleosomes stabilize through H1 histone -Unlike the core histones (H2A, H2B, H3, and H4) forming the nucleosome "bead," H1 binds to the linker DNA between nucleosomes -This binding stabilizes the higher-order structure of chromatin, facilitating the compaction of DNA into a more condensed form

Prophase: Attachment of the Two Sister Chromatids

• During prophase, two sister chromatids attach to each other at a region called the centromere
• The sister chromatid attachment gets facilitated by the protein complex known as cohesins -Cohesins connect by forming a ring-like structure around the sister chromatids, ensuring they remain Connected -An ATPase tightens the cohesin complex to bring the two sister chromatids closer together -Cohesins are essential in segmenting chromosomes during cell division

Prophase: Changes in the Cytosol

• Messenger RNAs experience Increased stability, which may compensate for lack of transcription till end of mitosis due to chromosome condensation
• Interphase microtubules form a loose 3D network (A), at the start of prophase, centrosomes initiate new microtubules/mitotic spindle (B)
• Prophase ends when the nuclear envelope disperses

Prometaphase: Chromosomes Engage with the Spindle and Become Organized

• Centrosome-initiated microtubules grow into the nuclear space as soon as the nuclear envelope disperses
• Chromosomes attach to the microtubule spindle at kinetochores
• The sister chromatids can be pulled apart at anaphase
• The chromosomes become connected on both
• Kinetochores are built from approximately 100 different polypeptides
• After activation and translocation into the nucleus, CDK1-B1 exerts two roles:
• Promotes assembly of the outer kinetochore on a constitutive kinetochore platform
• Controls the attachment of sister chromatids

Stepback Recapitulative Strategy: A Color-Coded Overview of our Discussion So Far

Stages:

• Prophase: Chromosomes (blue) condense inside a nucleus while microtubules (green) organize in the cytoplasm
• Prometaphase: Microtubule spindle (red) accesses the chromosomes (blue) attaching to the kinetochores (yellow)
• Metaphase: Chromosomes align on the spindle midplane
• Anaphase: Chromosomes segregate, move toward spindle poles, and the spindle elongates

Prometaphase: The Spindle Assembly Checkpoint

• During mitosis (and meiosis), the spindle assembly checkpoint (SAC) maintains genome stability by delaying cell division until accurate chromosome segregation can be guaranteed
• Accuracy requires correct chromosome attachment to the microtubule spindle
• If chromosomes are incorrectly attached, kinetochores activate the M CC network, blocking cell cycle progression by inhibiting the APC-C
• When all kinetochores become stably attached to the spindle, the checkpoint inactivates, cell cycle block alleviates and, chromosome segregation/cell division proceeds (anaphase engagement)

Metaphase: Formation of the Cell's Metaphase Plate

• After spindle attachment, chromosomes need to align at the spindle equator/forming metaphase plate (congression)
• The metaphase plate is not universal, but is still common, as its value minimizes chances of a single chromosome being left behind

Metaphase: Congression Mechanism: Kinetochore Microtubule Dynamics

• Microtubules are polar structures with distinct plus (+) and minus (-) ends
• The + end exposure of beta-tubulin subunits is the site where microtubule growth (polymerization) occurs rapidly
• The - end has alpha-tubulin subunits exposed and grows more slowly compared to the + end
• Microtubule polarity is crucial for the transport of cellular components and the proper mitotic spindle function

Metaphase: Congression Mechanism: Kinetochore Microtubule Dynamics Visualization

• Experimental design of:
• Biotin-labeled tubulin (biotin is easy to visualize with fluorescence microscopy) is microinjected in cell
• The process is stopped with a fixative to prevent the biotin incorporation for electron microscope observation
• Incorporation of new (red) and old (green) tubulin can distinguish
• Kinetochore is the site for microtubule assemblance -In sister kinetochore, microtubules assemble but disassemble at the other

Metaphase: