week 8 - chromosome structure and organisation Flashcards
Definition of a chromosome in eukaryotes
chatgpt
DNA molecule + associated proteins (histones and non-histone proteins).
Levels of chromatin organization:
chatgpt
Nucleosome: DNA wrapped around histone octamers.
30 nm fiber: Coiling of nucleosomes.
Looped domains: Attached to a protein scaffold.
Higher-order folding: During mitosis into metaphase chromosomes.
Euchromatin vs. Heterochromatin:
chatgpt
Euchromatin: loosely packed, transcriptionally active.
Heterochromatin: tightly packed, mostly transcriptionally silent.
Role of Histone Modifications
chatgpt
Histones can be chemically modified on their N-terminal tails, which alters how tightly DNA is wound around them, influencing access to DNA by transcription machinery.
Levels of chromatin organization:
hierarchical structure:
chatgpt
nucleosome → 10nm fibre → 30nm fibre → chromatin loops → chromosome domains → territories.
Chromosome and Chromatin Organisation in Higher Eukaryotes
🔹 Functional Significance
chatgpt
Compaction: Enables 2 meters of DNA to fit in a 10 µm nucleus.
Accessibility: Dynamic chromatin remodelling allows transcription, replication, and repair.
Epigenetic Regulation:
Histone modifications: Acetylation (activating), methylation (can activate or silence).
DNA methylation: Typically silences gene expression.
Chromosome Segregation in Higher Eukaryotes
🔹 Meiosis vs. Mitosis
Unique Features
chatgpt
Mitosis
- One division, two identical diploid cells
Meiosis
- Two divisions, four non-identical haploid cells
Meiosis I
- Homologous chromosomes separate
Meiosis II
- Sister chromatids separate (like mitosis)
Chromosome Segregation in Higher Eukaryotes
🔹 Meiosis vs. Mitosis
Meiotic Recombination
chatgpt
Occurs in prophase I.
Involves formation of chiasmata.
Crucial for genetic diversity and correct segregation.
Key Factors for Accurate Chromosome Segregation
Component: Centromere
Anchor for kinetochore, site of spindle attachment
Key Factors for Accurate Chromosome Segregation
Component: Kinetochore
Links chromosome to spindle microtubules
Key Factors for Accurate Chromosome Segregation
Component: Cohesin Complex
Holds sister chromatids together
Key Factors for Accurate Chromosome Segregation
Component: Spindle Checkpoint
Delays anaphase until proper attachment
Key Factors for Accurate Chromosome Segregation
Component: Aurora B kinase
Ensures tension at kinetochores is correct
Key Factors for Accurate Chromosome Segregation
Component: APC/C (Anaphase-Promoting Complex)
Triggers cohesin cleavage, metaphase → anaphase
Key Factors for Accurate Chromosome Segregation
🔹 Tension Sensing
Only properly attached sister chromatids experience tension.
Aurora B kinase detects lack of tension → destabilizes incorrect attachments.
Consequences of Erroneous Chromosome Segregation
Examples
Aneuploidy
Abnormal chromosome number.
Down Syndrome: Trisomy 21.
Turner Syndrome: XO genotype.
Klinefelter Syndrome: XXY genotype.
Consequences of Erroneous Chromosome Segregation
Examples
Cancer
Aneuploidy is a hallmark of many tumors.
Consequences of Erroneous Chromosome Segregation
Examples
Cellular Consequences:
Apoptosis.
Senescence.
Mitotic catastrophe (leads to cell death).
Consequences of Erroneous Chromosome Segregation
🔹 Maternal Age Effect
Cohesin deterioration during the prolonged meiotic arrest in oocytes is a key contributor.
Methodologies to Study Chromosome Segregation
technique:
Giemsa Staining / G-banding
What It Shows
Chromosome structure
Pros
Simple, clinical use
Cons
Low resolution
Methodologies to Study Chromosome Segregation
technique:
FISH
What It Shows
Specific DNA sequence locations
Pros
Precise, colour-coded
Cons
Fixed cells only
Methodologies to Study Chromosome Segregation
technique:
Chromosome Painting
What It Shows
Whole-chromosome visualization
Pros
Detect translocations
Cons
Not live
Methodologies to Study Chromosome Segregation
technique:
Live-Cell Imaging with GFP
What It Shows
Real-time tracking of proteins/spindles
Pros
Dynamic insights
Cons
Requires transgenes
Methodologies to Study Chromosome Segregation
technique:
Immunofluorescence
What It Shows
Localize mitotic proteins
Pros
Specific
Cons
Requires fixation
Methodologies to Study Chromosome Segregation
technique:
CRISPR-tagging / RNAi knockdown
What It Shows
Functional studies
Pros
Genetic specificity
Cons
Time-intensive
Methodologies to Study Chromosome Segregation
technique:
Laser Ablation & Microscopy
What It Shows
Tension/signal response
Pros
Real-time biophysics
Cons
Advanced equipment needed
Human Genome Organisation
🔹 Composition
~3.2 billion base pairs.
~20,000–25,000 protein-coding genes.
Non-coding regions: introns, regulatory sequences, transposable elements.
Human Genome Organisation
🔹 Gene Distribution
Uneven: gene-rich regions (e.g. chromosome 19) vs gene-poor (e.g. Y chromosome).
GC-rich isochores associated with higher gene density.
Human Genome Organisation
🔹 Functional Elements
Exons/introns: Exons = coding, introns = spliced out.
Promoters/enhancers/silencers: Control expression levels and patterns.
Non-coding RNAs:
miRNA: Gene silencing.
lncRNA: Transcriptional/post-transcriptional regulation.
Human Genome Organisation
🔹 Genome Projects
Human Genome Project (2003): First full draft sequence.
ENCODE Project: Functional elements beyond protein-coding genes.
STRUCTURE AND ORGANISATION
Eukaryotic chromosomes during mitosis
- Chromosome structure is highly dynamic during the cell cycle
- E.g. onion roots under light microscopy
STRUCTURE AND ORGANISATION
Basic features of chromosomes
- Linear structures (one chromosome in one molecule)
- More than one; numbers vary over a wide range
- Generally larger than bacterial chromosomes, but vary dramatically in size
- Great variation in overall genome size
STRUCTURE AND ORGANISATION
Basic features of chromosomes
variation: number
- More than one; numbers vary over a wide range
o Crepis capillaris 3 pairs (DIPLOID)
o Budding yeast (Saccharomyces cerevisiae) 16 pairs
o Human 23 pairs
o Lily 12 pairs
o Crayish 200 pairs
o Some ferns ~1200 pairs
STRUCTURE AND ORGANISATION
Basic features of chromosomes
variation: genome size
e coli - 4 Mb - 1mm
yeast - 12.1 Mb - 3mm
(diploid)
human - 3,200 Mb - 2m
(diploid)
amoeba dubia -670,000 Mb -
STRUCTURE AND ORGANISATION
Basic features of chromosomes
naked DNA?
- Chromosomes do not simply consist of naked DNA, but have a complex structure and organisation
- They consist of DNA associated with histones (and other proteins) to form a nucleoprotein complex called chromatin
- They are organised into higher order structures with differentiated regions (e.g. centromere/telomeres)
STRUCTURE AND ORGANISATION
Euchromatin and heterochromatin
- Chromatin exists in different forms
STRUCTURE AND ORGANISATION
Euchromatin
o Decondensed
o Associated with gene coding sequences
o Accessible to transcription machinery
o GC rich
STRUCTURE AND ORGANISATION
- Heterochromatin
o Is highly condensed
o Low density of genes
o AT-rich
o Contains long tandem arrays of repetitive sequences
o So-called constitutive heterochromatin is often found in centromeric regions
STRUCTURE AND ORGANISATION
Euchromatin and heterochromatin
staining
- Heterochromatin/euchromatin differentiated by specific stain techniques applied to chromsomes
o E.g. Giemsa stain
Heterochromatin +ve and give dark band
Eucharomatin -ve and give light band
STRUCTURE AND ORGANISATION
Visualising chromatin
- G-banding
- G-banding reveals heterochromatin and euchromatin
o Can be used for defining chromosomes and analysing regions on chromosomes - G-banding is used in clinical cytogenetics to identify chromosomal abnormalities
o For example: Prader-Willi syndrome
This leads to developmental and neurological disorders associated with a deletion on chromosome 15
STRUCTURE AND ORGANISATION
Visualising chromatin
- Fluorescence imaging techniques
- Fluorescence imaging techniques are now frequently used and are based on hybridization of fluorescent DNA probes
STRUCTURE AND ORGANISATION
Visualising chromatin
- FISH (fluorescence in situ hybridization)
- FISH (fluorescence in situ hybridization) is used to identify specific region of the chromosomes
o Different fluorescence can see different colours
o E.g. centromere or individual genes
STRUCTURE AND ORGANISATION
Visualising chromatin
Chromosome painting
Chromosome painting can be used to detect chromosomal aberrations
- Bloom’s syndrome
o Rare autosomal disease leading to a predisposition to cancer
o Defect in DNA helicase
Problem repairing DNA DSB
This leads to chromosomal fragmentation
STRUCTURE AND ORGANISATION
Visualising chromatin
- Chromosome specific probes
- Chromosome specific probes are also available – variant FISH on referred to as chromosome painting
STRUCTURE AND ORGANISATION
Global chromosome organization
- Organisation of the chromosomes in the nucleus is now known to be…
non-random and dynamic
STRUCTURE AND ORGANISATION
Global chromosome organization
- They organised into chromosome territories
- Segregation of chromosomes is important
- Colour intact
- Chromosomes stay in a specific region of the nucleus
STRUCTURE AND ORGANISATION
Global chromosome organization
significance
- The significance/rules of this is not fully understood but ageing, senescent, apoptotic and cancer cells show differences compared to healthy cells
STRUCTURE AND ORGANISATION
Chromosome territories (CTs)
Chromosome territories (CTs) have a complex organisation
- Summary model based on experimental evidence
o Actively transcribed DNA can from loops at the CT surface
- Chromosome arms form territories distinct from centromeric DNA (heterochromatin rich)
- Actively transcribed genes are located away from the centromeric region
- Whereas silent genes are in close proximity
STRUCTURE AND ORGANISATION
Chromosome territories (CTs)
- chromosomes form…
higher order chromatin structures that places actively transcribed genes at the surface of the CT whereas silent genes are internal
STRUCTURE AND ORGANISATION
Dynamic changes in chromosome organization
- The signal comes from the territory
STRUCTURE AND ORGANISATION
Nuclear organisation contributes to the…
the efficient spatial and temporal organization of processes such as transcription, replication and DNA repair
STRUCTURE AND ORGANISATION
- The organisation of DNA repair “factories” on damaged DNA…
– similar replication/transcription factories are observed in cells
o Localized DNA damaged induced by radiation. Immunolocalization of Mre11 (a repair protein – green) (DNA in red)
o Colocalization of an active gene (red) with a transcription “factory” gene (FI-U) using FISH
STRUCTURE AND ORGANISATION
Eukaryotic chromosome organisation
- Two important considerations:
o DNA packaging
o DNA accessibility
STRUCTURE AND ORGANISATION
Eukaryotic chromosome organisation
- Biological processes that must take place
o Transcription
o Replication
o DNA repair
o Mitosis
o Meiosis
Must package chromosomes to enable them
STRUCTURE AND ORGANISATION
Chromosome organisation – packaging problem
- Interphase chromosomes must be sufficiently compact to fit in the nucleus
- Also they must undergo further compaction during cell division
o
STRUCTURE AND ORGANISATION
Chromosome organisation – packaging problem
for example:
For example:
Human genome 2m DNA must fit into interphase nucleus, ~10um diameter
At mitosis the smallest human chromosome with a length of 1.4cm is condensed to only 2um at metaphase (~7000:1 packing ratio)
This packaging is an important and essential part of chromosome organisation and involves the organisation of DNA into chromatin fibre
STRUCTURE AND ORGANISATION
Chromosome organisation – packaging problem
chromatin
- Chromatin fibres = basic units of organisation
o 10nm fibre – interpreted as a sub-unit of higher order 30nm fibre
STRUCTURE AND ORGANISATION
Chromosome organisation – packaging problem
chromatin - Composition (relative proportions)
o DNA – 100
o Histones – 114
o DNA binding protein – 33
o RNA species – 7
o + minor components (metabolites e.g. zinc, magnesium)
STRUCTURE AND ORGANISATION
Chromosome organisation – packaging problem
Nucleosome
- DNA + 2x(H2aA, H2B, H3, H4)
- Nucleosome is the first step of packaging
- Nucleosome is very dynamic
- Histones
- Highlights how important flexibility in packaging is
STRUCTURE AND ORGANISATION
Chromosome organisation – packaging problem
The 10nm nucleosome fibre:
- Nucleosome core
o 146 bp DNA + histone octomer
Histones – positively charged DNA bind to negatively charged DNA
Overall packing ratio 7:1
STRUCTURE AND ORGANISATION
Chromosome organisation – packaging problem
The 10nm nucleosome fibre:
electrogram
- Electron microscope of an isolates chromatin fibre showing the. Nucleosomes organized in a “beads of string” arrangement along the DNA
STRUCTURE AND ORGANISATION
Higher-order chromatin fibre
- In the nucleus the chromatin is organised into a more compact structure than “beads on the string”
- There is an on-going debate as to the exact organisation
STRUCTURE AND ORGANISATION
Classic view of the 30nm fibre structure
- Nucleosomes are coiled to form a solenoid producing the 30nm fibre structure
o 6 nucleosomes per turn - The 30nm fibre is further organised into chromatin loops to give a higher order organisation
STRUCTURE AND ORGANISATION
Classic view of the 30nm fibre structure
incorrect
Recent studies suggest that the “classical view” of the 30nm fibre structure is incomplete/incorrect
- It is now clear that the structures that have been determined in vitro for the 30nm fibre are influenced by the extraction conditions
o Divalent metal ions - It seems that the structure is highly dynamic reflecting the fact that in vivo the chromatin must be capable of dynamics changes in organisation
o E.g. during transcription, DNA repair
STRUCTURE AND ORGANISATION
Higher order organisation
- Packaging based on nucleosome is ~36 fold
- Too little to account for the ~10,000 fold maximal condensation observed at mitosis metaphase I
- Lammli proposed that additional scaffold proteins fold the 30 nm fibre to achieve this
STRUCTURE AND ORGANISATION
Higher order organisation
chromatin loops
- Chromatin loops are organised to a protein scaffold by scaffold attachment regions (SARs)
o 1-2kb regions that are very AT rich - Scaffold protein = topoisomerase II and SCII (condensing)
- The 30nm fibre loops of chromatin that are associated with a protein scaffold
STRUCTURE AND ORGANISATION
Schematic view of the many levels of DNA folding in the cell:
duplex DNA
7nm length
nucleosome core (147bp wrapped DNA/histone core)
60nm length
chromatin (extended beads on a string)
200nm length
compact chromatin (stabilized by cis- and trans- nucleosome interactions via histone tails) - hypothetical structure
metaphase chromosome
2cm length
STRUCTURE AND ORGANISATION
The organized dynamic packaging of the genetic information
- Compaction increases but DNA is still highly accessible
STRUCTURE AND ORGANISATION
The organized dynamic packaging of the genetic information
- Chromatin is a…
a dynamic structure
o Global changes during mitosis and meiosis
o But also undergoing constant remodelling during interphase
E.g. remodelling is very important for gene transcription
STRUCTURE AND ORGANISATION
The organized dynamic packaging of the genetic information
nucleosome phasing
- The beads on a string arrangement is referred to as nucleosome phasing
o This phasing is dynamic and subject to chromatin remodelling
o Whereby the nucleosome organization in a region may be transiently modified
Particularly important during gene expression to allow access of the transcription machinery to gene promoter regions
STRUCTURE AND ORGANISATION
chromatin remodelling is…
is energy-dependent
STRUCTURE AND ORGANISATION
chromatin remodelling: energy dependent
- ATP-dependent chromatin remodelling
e.g. SWI/SNF
STRUCTURE AND ORGANISATION
chromatin remodelling: energy dependent
SWI/SNF
- SWI/SNF works in conjunction with histone-modifying proteins such as histone acetyl transferase
o Acetylated chromatin has more “open structure”
o Histone H3/H4 are targets or acetylation
STRUCTURE AND ORGANISATION
chromatin remodelling: energy dependent
SWI/SNF
mutations
o Mutations in Histone acetyl transferases HAT are commonly reported in colorectal, pancreatic, breast and gastric carcinomas
STRUCTURE AND ORGANISATION
chromatin remodelling:
Histone acetylation in chromatin remodelling is…
reversible
STRUCTURE AND ORGANISATION
SUMMARY
- DNA Sequence Level (bottom left):
The double helix includes single-copy genes, introns, spacer DNA, gene families, simple-sequence DNA, and mobile elements.
This is the linear, uncondensed form of DNA.
- Nucleosome Formation:
DNA wraps around histone proteins forming nucleosomes — the first level of compaction.
This structure looks like “beads on a string”.
- 30-nm Fibre:
Nucleosomes further coil to form a 30-nm chromatin fibre, a more compact and organized structure.
This step provides ~7x compaction.
- Loop Domains and Chromosome Scaffold:
The 30-nm fibre forms loops anchored to a chromosome scaffold (via scaffold attachment regions or SARs).
These loops increase DNA compaction and help organize functional domains.
- Higher-Order Chromatin Folding:
These loops undergo additional folding into even more compact structures during mitosis and chromosome condensation.
- Chromosome Territories (Nucleus):
In the interphase nucleus, chromosomes occupy distinct, non-overlapping chromosome territories.
This spatial organisation helps regulate gene expression, replication, and repair.
CHROMOSOME SEGREGATION
Regulating chromosome partition, segregation and disjunction
- The key players:
Centromere - And associated proteins (kinetochore)
Cohesins - The cohesin complex
Microtubules - The microtubule spindle
CHROMOSOME SEGREGATION
mitotic cell cycle
Interphase
G1: Cell grows
S: DNA replicates
G2: Prepares for mitosis
Mitosis
Prophase: Chromosomes condense, spindle forms
Metaphase: Chromosomes align
Anaphase: Sister chromatids separate
Telophase: New nuclei form
Cytokinesis
Cell splits into two identical daughter cells
CHROMOSOME SEGREGATION
chromosome terminology
2 pairs:
homologous chromosome: homologues
(Chromosome 1 for mum and dad are homologues )
1 pair:
chromosome
1/2 of pair:
chromatid
diploid species: 2n - 6 chromosomes
CHROMOSOME SEGREGATION
meiosis
Meiosis I (reductional division)
Prophase I: Chromosomes condense, homologous pairs form, crossing over occurs
Metaphase I: Homologous pairs align
Anaphase I: Homologous chromosomes separate
Telophase I: Two haploid cells form
Meiosis II (similar to mitosis)
Prophase II: Chromosomes condense
Metaphase II: Chromosomes align
Anaphase II: Sister chromatids separate
Telophase II: Four haploid cells form
outcomes:
4 non-identical haploid cells
Increases genetic diversity (via crossing over & independent assortment)
CHROMOSOME SEGREGATION
The centromere
- Functional role in accurate chromosome transmission
- Site of interaction with microtubules of the mitotic/meiotic spindle apparatus
CHROMOSOME SEGREGATION
The centromere
how many per chromosomes
- Usually one per chromosome (monocentric chromosome)
Metacentric chromosome - Centromere in central position
Acrocentric chromosome - Centromere in of centre position
Telocentric chromosome - Centromere at one end
CHROMOSOME SEGREGATION
The centromere
in a few cases…
- In an few cases (e.e. C. elegans) no single centromere defines
o Instead chromosomes have multiple sites where microtubules attach
Holocentric chromosome
CHROMOSOME SEGREGATION
Centromere structure
- The centromeric DNA contains tandem arrays of repeat sequences
o E.g. satellite repeats in humans are several hundred kb in length
CHROMOSOME SEGREGATION
Centromere structure:
divergences between species?
- It is very remarkable that these repeat sequences are divergent between even closely related species
CHROMOSOME SEGREGATION
Centromere structure:
histone variant
The histone variant CEMP-A (CENH3) replaces conventional H3 at the centromere
CHROMOSOME SEGREGATION
Kinetochore
- The kinetochore (a large protein complex) forms at the centromere o f each sister chromatid during meiotic prophase and meiotic prophase I
- These are the sites o attachment for microtubules that form the mitotic and meiotic spindle
CHROMOSOME SEGREGATION
Kinetochore: general organisation
- Kinetochores are proteinaceous structures that provide the major contact between spindle microtubules (MTs) and chromosomes
CHROMOSOME SEGREGATION
Kinetochore: general organisation
regions
- Kinetochores comprise 3 region
inner plate: associated with CENP - a nucleosome
outerplate
fibrous corona
o With ~80 proteins
- E.g. in budding yeast the kinetochore is small, binding a single MT
CHROMOSOME SEGREGATION
Kinetochore: general organisation
- Most organisms kinetochores
o 100-500nmm diamenter
o Binds 3-40 microtubules
highly conserved
CHROMOSOME SEGREGATION
Kinetochore: functions
- Site of attachment of MTs and associated motor proteins
- Monitors correct MT attachment (senses tension) and removes incorrectly attached MTs
- Provides a link to cell cycle as checkpoint control to ensure all chromosomes are accurately aligned at metaphase before anaphase
CHROMOSOME SEGREGATION
The cohesin complex
- Protein complex that maintain the close alignment of the sister chromatids established during/immediately after replication
- The cohesin complex encircle the sister chromatids along their entire length
CHROMOSOME SEGREGATION
The cohesin complex
tension
- It is essential to provide tension to allow organization and correct alignment of the chromosome on the equator of the mitotic/meiotic spindle prior to chromosome disjunction
CHROMOSOME SEGREGATION
The cohesin complex
metaphase/anaphase transition
- It is proteolytically cleaved at the metaphase/anaphase transition
o Allowing the sister chromatids to separate
CHROMOSOME SEGREGATION
The cohesin complex: components
The cohesin complex forms a ring-like structure that encircles the sister chromatids:
SMC1 (pink)
Structural Maintenance of Chromosomes protein – one half of the core ring.
SMC3 (purple)
The second SMC protein – forms the other half of the ring with SMC1.
SCC1 (also known as RAD21) (green)
Connects SMC1 and SMC3 to complete the ring and lock the chromatids inside.
SCC3 (yellow/orange)
Regulatory subunit that stabilizes the complex and helps with loading/unloading.
CHROMOSOME SEGREGATION
The cohesin complex
function
The cohesin ring physically encircles the sister chromatids, maintaining cohesion until anaphase.
At anaphase, an enzyme called separase cleaves SCC1, allowing chromatids to separate.
CHROMOSOME SEGREGATION
The cohesin complex:
cohesins crucial role
Cohesins plays crucial role in meiosis and mitosis
homologues linked by chiamsa and cohesin during meiosis
in meiosis centromeric cohesin is protected at the first division
remaining centromeric cohesin cleaved at the second meiotic division
CHROMOSOME SEGREGATION
The cohesin complex:
release of cohesin
Release of cohesin at metaphase/anaphase transition
- Correct MT attachment and tension in monitored by the chromosome passenger complex (part of the kinetochore)
- When orientation/tension is established APC (anaphase promoting complex) is activated and cohesion is cleaved
CHROMOSOME SEGREGATION
The anaphase promoting complex:
- Prometaphase
o Spindle-assembly-checkpoint proteins (Mad2 and BubR1) are activated at kinetochores that are not fully attached with microtubules
o Activated Mad2 and BubR1 inhibit the capability of the anaphase-promoting complex (APC) to target (ubiquitination) the degradation of securing and cyclin B
This prevents anaphase andm mitotic exit
CHROMOSOME SEGREGATION
The anaphase promoting complex:
- Metaphase:
o When all kinetochores are attaches to microtubules APC targets the degradation of securing and cyclin B
o Thereby activating the protease separatase and inactivates the cyclin-depending kinase 1 (CDK1)
o Separase cleaves cohesion complexes to iniate sister chromatid separation
o CDK1 inactivation leads to the dephosphorylation of CDK1 substrates by protein pohoshatases
Thereby enabling exit from mitosis
CHROMOSOME SEGREGATION
The anaphase promoting complex:
The APC/C is a large E3 ubiquitin ligase complex that regulates progression through mitosis by tagging specific proteins for degradation via the proteasome.
CHROMOSOME SEGREGATION
The anaphase promoting complex:
Key Functions
Triggers anaphase by:
Ubiquitinating securin, which normally inhibits separase.
Degradation of securin → separase is activated → cleaves cohesin → sister chromatids separate.
Promotes mitotic exit by:
Ubiquitinating cyclin B, leading to the inactivation of CDK1 (cyclin-dependent kinase 1).
Allows the cell to exit mitosis and enter G1.
CHROMOSOME SEGREGATION
The anaphase promoting complex:
regulation
Activated by Cdc20 during metaphase → initiates anaphase.
Later activated by Cdh1 for mitotic exit and G1 maintenance.
Inhibited by spindle assembly checkpoint proteins (e.g. Mad2, BubR1) if chromosomes aren’t properly attached to spindle → prevents premature anaphase.
CHROMOSOME SEGREGATION
Regulating chromosome partition segregation, and disjunction
by what:
- Mitotic chromosomal segregation is regulated by kinases and phosphatases
CHROMOSOME SEGREGATION
The chromosome Passenger complex (CPC)
- The CPC is required for the formation of a bipolar spindle and its stability rom prophase/prometaphase to anaphase
- Important for different steps/stages in mitotic division
CHROMOSOME SEGREGATION
The chromosome Passenger complex
1. Prophase:
o On chromosome arms
o Role: phosphorylates histone H3
CHROMOSOME SEGREGATION
The chromosome Passenger complex
2. Prometaphase
o At centromeres
o Role: maturation of kinetochores
CHROMOSOME SEGREGATION
The chromosome Passenger complex
3. Metaphase
o At centromeres
o Role: in centromeric cohesion and the regulation of kinetochore-microtubule attachments
CHROMOSOME SEGREGATION
The chromosome Passenger complex
4. Anaphase
o At the spindle midzone (at the cortex)
o Role: involved in the formation of the central spindle
CHROMOSOME SEGREGATION
The chromosome Passenger complex
5. Telophase
o At the cleavage furrow and at the midbody
o Role: required for completion of cytokinesis
CHROMOSOME SEGREGATION
The chromosome Passenger complex:
Aurora B Gradient
The CPC produces spatial gradients of Aurora B activity (phosphorylation)
- Tension changes the physical space
CPC creates a spatial phosphorylation gradient.
High Aurora B activity at centromeres → weak attachments are destabilized.
As tension increases (correct attachments), substrates move away → attachments stabilize.
CHROMOSOME SEGREGATION
The chromosome Passenger complex:
microtubules attached…
to both sides
tension pulls them apart
proteins can access
CHROMOSOME SEGREGATION
The chromosome Passenger complex:
what
The CPC is a multi-protein complex that regulates chromosome alignment, segregation, and cytokinesis during cell division.
CHROMOSOME SEGREGATION
The chromosome Passenger complex:
importance
Ensures accurate chromosome segregation.
Prevents aneuploidy and cell division errors.
Dysregulation is linked to cancer and chromosomal instability.
CHROMOSOME SEGREGATION
The mitotic spindle:
formed from
- The mitotic spindle is formed from microtubules (MTs) that grow out from centrosomes (MTOCs microtubules organizing centres) that rom the spindle poles
CHROMOSOME SEGREGATION
The mitotic spindle:
action
- Kinetochore MTs nucleate at the centrosomes and extend by polymerization of alpha and beta tubulin heterodimers
The growing (+ve) end captures one of the sister chromatids via interaction with the kinetochore
- The other sister is captured by a MTV originating romo the opposite pole
This aligns the chromosomes on the metaphase plate
CHROMOSOME SEGREGATION
The mitotic spindle:
sister chromatids are
aligned on the metaphase plate before being separated in anaphase
CHROMOSOME SEGREGATION
The mitotic spindle:
key components
Centrosomes (Spindle Poles) - Microtubule-organizing centres (MTOCs) located at opposite ends of the cell
Microtubules (MTs) - Hollow protein filaments made of tubulin dimers
Kinetochore - Protein complex on chromosomes where MTs attach
Motor proteins (e.g. dynein, kinesin) - Drive spindle assembly and chromosome movement
CHROMOSOME SEGREGATION
The mitotic spindle:
⚙️ Mechanism of Chromatid Movement
Kinetochore MTs depolymerize at the + end (near kinetochores), pulling chromatids toward poles.
Motor proteins walk chromosomes along MTs.
Spindle poles also move apart (spindle elongation).
CHROMOSOME SEGREGATION
Mitotic chromosome segregation at anaphase
- Chromosome separation at anaphase can be divided into two stages
1. The separation due to shortening of the kinetochore MTs
- Pulling apart by separation of the poles as a prelude to the formation of two daughter cells
CHROMOSOME SEGREGATION
Mitotic chromosome segregation at anaphase:
1. Chromosome separation due to shortening of the kinetochores
- Sister chromatids are pulled apart by shortening of kinetochore MTs
- MTs are depolymerized at the +ve end by a kinesin
- The shortened MT is recaptured by CEMP-E
CHROMOSOME SEGREGATION
Mitotic chromosome segregation at anaphase:
1. Chromosome separation due to shortening of the kinetochores
Fluorescent recombinant proteins
- GFP (green fluorescent protein) is a protein that emits green fluorescent light when excited by ultraviolet light
- GP can be fused to any protein of interest and its intrinsic fluorescent properties would allow the visualization of this recombinant protein
- GFP was originally isolated from the jellyfish Aequorea Victoria although many other marine organisms express similar proteins
CHROMOSOME SEGREGATION
Mitotic chromosome segregation at anaphase:
1. Chromosome separation due to shortening of the kinetochores
experiment demonstrating
Experiment demonstrating that shortening of the kinetochore MTs results in movement of the chromosomes towards the poles
- Based on the use of fluorescent tubulin injected into fibroblasts
- This was incorporated into the MTs at metaphase
- At early anaphase a region of the MT was “marked” with a laser
- As anaphase progressed the MTs shortened and free tubulin was detected
- The position o the photobleached region of the MT revealed that the depolymerization occurred at the MT +ve end at the kinetochore
CHROMOSOME SEGREGATION
Mitotic chromosome segregation at anaphase:
1. Chromosome separation due to shortening of the kinetochores
- as anaphase progresses
- As anaphase progresses the poles are pulled apart thereby extending the spindle
- This combined with the depolymerization of the kinetochore MTs results in the chromosomes moving further apart
CHROMOSOME SEGREGATION
Mitotic chromosome segregation at anaphase:
2. Pulling apart by separation of the poles
movement is mediated by motor proeins that “walk” along the MTs
(-) end directed Dynein pulls the aster MTs
(+) end directed kinesin binds anti-parallel polar MTs. silding movement along these pushes the MTs apart
CHROMOSOME SEGREGATION
Mitotic chromosome segregation at anaphase:
2. Pulling apart by separation of the poles
linking
After sister chromatids are separated (Step 1: shortening of kinetochore microtubules), the next key event is:
Pole Separation (Anaphase B)
Spindle poles move further apart, increasing the distance between the two sets of chromosomes.
This elongates the entire spindle, helping complete the physical separation of chromatids.
How It Happens
Polar microtubules (from opposite poles) overlap and slide past each other, pushed by motor proteins like kinesin-5.
Astral microtubules pull on spindle poles via dynein motors anchored at the cell cortex.
Purpose
Ensures each daughter cell receives a full and equal set of chromosomes.
Helps establish the two sides of the cell for eventual cytokinesis.
CHROMOSOME SEGREGATION
Cohesin – a link with down’s syndrome?
- Oocytes initiate meiosis in the embryo
- Meiotic arrest prior to metaphase I
o Chromosomes held together by chiasmata and cohesion) - Resumes in adult women, metaphase I is completed at ovulation
- Metaphase II only completed following fertilization
CHROMOSOME SEGREGATION
Cohesin – a link with down’s syndrome?
Errors in chromosome disjunction lead to down’s syndrome
- Human (female) are a notable exception to the “accuracy rule”
- In humans ~20-30% o fertalized eggs have an incorrect number of chromosmes
- This increases dramatically with maternal age
- ~33% o miscarriages are aneuploid at 40 years old
- Increased risk of down’s syndrome
CHROMOSOME SEGREGATION
Cohesin – a link with down’s syndrome?
Cohesin defects may lead to…
meiotic non-disjunction in older women
homologous chromosomes linked by a chiasma + cohesin distal exchanges are “unstable”
chromosomes will now segregate at random at the 1st meiotic division
CHROMOSOME SEGREGATION
SUMMARY
- Centromere organisation
- The kinetochore
- Chromosome segregation in mitosis and meiosis
- The cohesion complex
- A possible link between cohesion and chromosome disjunction (segregation) errors in human female
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Chromosome and Chromatin Organisation
Eukaryotic chromosomes are highly compacted DNA molecules packaged with histone proteins into nucleosomes and higher-order structures (10nm fibre → 30nm fibre → chromatin loops), allowing for both efficient storage and regulated accessibility within dynamic chromosome territories in the nucleus.
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Chromosome Segregation in Higher Eukaryotes
During mitosis and meiosis, chromosome segregation is coordinated through centromeres, kinetochores, cohesins, and spindle microtubules to ensure accurate distribution of sister chromatids or homologs into daughter cells.
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Key Factors for Accurate Segregation
Accurate segregation relies on the kinetochore–microtubule interface, tension-sensing mechanisms, cohesin-mediated chromatid cohesion, and regulation by the anaphase-promoting complex (APC/C) and chromosome passenger complex (CPC).
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Consequences of Erroneous Segregation
Errors in chromosome segregation can lead to aneuploidy, miscarriages, and conditions like Down’s syndrome, particularly due to cohesin degradation in ageing oocytes.
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Methodologies to Study Segregation
Techniques such as G-banding, FISH, chromosome painting, live-cell imaging with GFP, and laser-based microtubule tracking are used to study chromosome structure, segregation, and dynamics.
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Human Genome Organisation
The human genome consists of 23 pairs of chromosomes organized into gene-rich euchromatin and gene-poor heterochromatin, with spatial organisation into territories that support efficient gene expression and genome maintenance.
