Lecture 04: Dynamics of cell division
Cell division is not only a mechanical process that produces two daughter cells; it is tightly coupled to cell fate decisions. The final step of
division: abscission, the cutting of the cytoplasmic bridge (can be accelerated, delayed, or inhibited).
These variations influence:
• Pluripotency exit in stem cells
• Differentiation timing
• Germline development
• Tissue morphogenesis
• Cancer cell behavior
Thus, abscission acts as a regulatory checkpoint integrating mechanics, signaling, and fate transitions.
Mitotic Spindle Organization
The mitotic spindle is composed of microtubules organized into distinct populations:
• Kinetochore microtubules: attach chromosomes
• Astral microtubules: connect spindle to cortex
• Interpolar microtubules: overlap at the central spindle
During anaphase:
• Sister chromatids separate
• The spindle elongates
• The central spindle becomes a signaling platform
Key protein: PRC1 → bundles overlapping antiparallel microtubules at the central spindle
Chromosome Passenger Complex (CPC):
• The CPC relocates during mitosis:
o Early mitosis: on chromosomes
o Anaphase: translocates to the central spindle
• Core component: Aurora B kinase (active kinase of CPC)
• Aurora B activity depends on:
o Binding to CPC components (INCENP)
o Autophosphorylation within the activation loop
o ATP binding in the catalytic site
o
• At the central spindle, Aurora B:
o Phosphorylates KIF23 (MKLP1) of centralspindlin
o Promotes recruitment of cytokinesis machinery
o Regulates abscission timing
Cytokinesis: Actomyosin Ring Formation
Cytokinesis begins with formation of an actomyosin contractile ring at the equator.
Molecular cascade:
1. Centralspindlin accumulates at the equator
2. Activation of RhoA (active RhoA zone)
3. Actin polymerization (visualized with LifeAct)
4. Myosin II recruitment
5. Contractile ring constriction
CPC activity is required for centralspindlin recruitment and
proper RhoA activation.
1|Page
,Cytoplasmic Bridge Formation and Maturation:
After cleavage furrow ingression, daughter cells remain connected by a cytoplasmic bridge.
Structure:
• Narrow membrane tube
• Central dense structure: midbody
• Contains microtubules and many anaphase proteins (CPC remnants)
Division takes ~1 hour, but bridge resolution can take several hours.
Steps of Abscission:
Abscission requires sequential remodeling:
1. F-actin Disassembly:
• Actin must be cleared first
• Regulated by Cofilin-1
• Transient Arp2/3-dependent actin pools form at secondary
ingression
2. ESCRT Recruitment:
• The ESCRT machinery mediates membrane constriction.
• Hierarchy:
o ESCRT-0: target recognition
o ESCRT-I / II: assembly initiation
o CHMP6 / CHMP7: nucleation
o ESCRT-III: polymerization
o VPS4: remodeling ATPase
• ESCRT-III forms helical filaments that constrict and sever membranes.
3. Microtubule Severing:
• Spastin cuts microtubules
• Stable recruitment depends on ESCRT and actin remodeling cross-talk
Final sequence: Cofilin → Arp2/3 remodeling → ESCRT-III cone elongation arrest → Spastin recruitment → Microtubule severing → Membrane
scission
Stable Cytoplasmic Bridges Across Biology:
Stable bridges are evolutionarily conserved.
1. Germline Cysts (e.g., Drosophila):
• Oocyte connected to nurse cells via ring canals
• Metabolites and cytoplasm transferred to oocyte
• Bridges enable coordinated development
2. Mammalian Embryos:
• Blastomeres remain connected up to blastocyst stage
• Bridges dismantled before next division
• Role unclear but linked to fate coordination
3. Choanoflagellates:
• Chains form via incomplete abscission
• Inhibiting abscission causes multicellular aggregates
4. Plants (Plasmodesmata):
• Form during cytokinesis
• Allow cytoplasmic continuity and communication
5. Fungi:
• Hyphae connected by cytoplasmic tubes
2|Page
, Functional Roles of Stable Bridges:
• Communication → cytoplasmic exchange (shown by photobleaching
recovery experiments)
• Synchronization → possible cell cycle coordination
• Differentiation control → fate coordination in embryos
• Morphogenesis → structural support in colonies
• Microtubule organization → bridges act as MTOC-like structures in early
mouse embryos lacking centrosomes
Bridges and Pluripotency in Embryonic Stem Cells:
In mouse embryonic stem cells:
• Naïve pluripotent cells exhibit stable bridges
• Differentiating cells show faster abscission
• Bridge maintenance decreases at pluripotency exit
Key Experiments:
1. Inhibition of Abscission:
• siRNA against ALIX (ESCRT recruiter)
• Increased number of persistent bridges
• Delayed exit from pluripotency
2. ESCRT Disruption:
• Impairs abscission
• Slows differentiation
3. Bridge Ablation:
• Artificial cutting of bridges
• Accelerates exit from pluripotency
Conclusion: Stable bridges help maintain pluripotency; faster abscission promotes differentiation.
NoCut Checkpoint and Aurora B Regulation:
Abscission can be delayed by:
• Persistent chromatin in bridge
• Nuclear pore assembly defects
• Replication stress
• High membrane tension
All activate Aurora B kinase, triggering the NoCut pathway.
Aurora B targets:
• VPS4 (ESCRT ATPase)
• CHMP4C (ESCRT-III component)
Active Aurora B → ESCRT inhibition → delayed abscission. This mechanism is especially relevant in cancer cells.
Mechanics of Abscission: Snapping vs Dissolving
Microtubule clearance occurs via two modes:
1. Dissolving:
• Bridge shrinks gradually
• Faster abscission
• High cell density
• Low contractility
• Low Aurora B activity
• High MCAK (microtubule depolymerase)
3|Page
Cell division is not only a mechanical process that produces two daughter cells; it is tightly coupled to cell fate decisions. The final step of
division: abscission, the cutting of the cytoplasmic bridge (can be accelerated, delayed, or inhibited).
These variations influence:
• Pluripotency exit in stem cells
• Differentiation timing
• Germline development
• Tissue morphogenesis
• Cancer cell behavior
Thus, abscission acts as a regulatory checkpoint integrating mechanics, signaling, and fate transitions.
Mitotic Spindle Organization
The mitotic spindle is composed of microtubules organized into distinct populations:
• Kinetochore microtubules: attach chromosomes
• Astral microtubules: connect spindle to cortex
• Interpolar microtubules: overlap at the central spindle
During anaphase:
• Sister chromatids separate
• The spindle elongates
• The central spindle becomes a signaling platform
Key protein: PRC1 → bundles overlapping antiparallel microtubules at the central spindle
Chromosome Passenger Complex (CPC):
• The CPC relocates during mitosis:
o Early mitosis: on chromosomes
o Anaphase: translocates to the central spindle
• Core component: Aurora B kinase (active kinase of CPC)
• Aurora B activity depends on:
o Binding to CPC components (INCENP)
o Autophosphorylation within the activation loop
o ATP binding in the catalytic site
o
• At the central spindle, Aurora B:
o Phosphorylates KIF23 (MKLP1) of centralspindlin
o Promotes recruitment of cytokinesis machinery
o Regulates abscission timing
Cytokinesis: Actomyosin Ring Formation
Cytokinesis begins with formation of an actomyosin contractile ring at the equator.
Molecular cascade:
1. Centralspindlin accumulates at the equator
2. Activation of RhoA (active RhoA zone)
3. Actin polymerization (visualized with LifeAct)
4. Myosin II recruitment
5. Contractile ring constriction
CPC activity is required for centralspindlin recruitment and
proper RhoA activation.
1|Page
,Cytoplasmic Bridge Formation and Maturation:
After cleavage furrow ingression, daughter cells remain connected by a cytoplasmic bridge.
Structure:
• Narrow membrane tube
• Central dense structure: midbody
• Contains microtubules and many anaphase proteins (CPC remnants)
Division takes ~1 hour, but bridge resolution can take several hours.
Steps of Abscission:
Abscission requires sequential remodeling:
1. F-actin Disassembly:
• Actin must be cleared first
• Regulated by Cofilin-1
• Transient Arp2/3-dependent actin pools form at secondary
ingression
2. ESCRT Recruitment:
• The ESCRT machinery mediates membrane constriction.
• Hierarchy:
o ESCRT-0: target recognition
o ESCRT-I / II: assembly initiation
o CHMP6 / CHMP7: nucleation
o ESCRT-III: polymerization
o VPS4: remodeling ATPase
• ESCRT-III forms helical filaments that constrict and sever membranes.
3. Microtubule Severing:
• Spastin cuts microtubules
• Stable recruitment depends on ESCRT and actin remodeling cross-talk
Final sequence: Cofilin → Arp2/3 remodeling → ESCRT-III cone elongation arrest → Spastin recruitment → Microtubule severing → Membrane
scission
Stable Cytoplasmic Bridges Across Biology:
Stable bridges are evolutionarily conserved.
1. Germline Cysts (e.g., Drosophila):
• Oocyte connected to nurse cells via ring canals
• Metabolites and cytoplasm transferred to oocyte
• Bridges enable coordinated development
2. Mammalian Embryos:
• Blastomeres remain connected up to blastocyst stage
• Bridges dismantled before next division
• Role unclear but linked to fate coordination
3. Choanoflagellates:
• Chains form via incomplete abscission
• Inhibiting abscission causes multicellular aggregates
4. Plants (Plasmodesmata):
• Form during cytokinesis
• Allow cytoplasmic continuity and communication
5. Fungi:
• Hyphae connected by cytoplasmic tubes
2|Page
, Functional Roles of Stable Bridges:
• Communication → cytoplasmic exchange (shown by photobleaching
recovery experiments)
• Synchronization → possible cell cycle coordination
• Differentiation control → fate coordination in embryos
• Morphogenesis → structural support in colonies
• Microtubule organization → bridges act as MTOC-like structures in early
mouse embryos lacking centrosomes
Bridges and Pluripotency in Embryonic Stem Cells:
In mouse embryonic stem cells:
• Naïve pluripotent cells exhibit stable bridges
• Differentiating cells show faster abscission
• Bridge maintenance decreases at pluripotency exit
Key Experiments:
1. Inhibition of Abscission:
• siRNA against ALIX (ESCRT recruiter)
• Increased number of persistent bridges
• Delayed exit from pluripotency
2. ESCRT Disruption:
• Impairs abscission
• Slows differentiation
3. Bridge Ablation:
• Artificial cutting of bridges
• Accelerates exit from pluripotency
Conclusion: Stable bridges help maintain pluripotency; faster abscission promotes differentiation.
NoCut Checkpoint and Aurora B Regulation:
Abscission can be delayed by:
• Persistent chromatin in bridge
• Nuclear pore assembly defects
• Replication stress
• High membrane tension
All activate Aurora B kinase, triggering the NoCut pathway.
Aurora B targets:
• VPS4 (ESCRT ATPase)
• CHMP4C (ESCRT-III component)
Active Aurora B → ESCRT inhibition → delayed abscission. This mechanism is especially relevant in cancer cells.
Mechanics of Abscission: Snapping vs Dissolving
Microtubule clearance occurs via two modes:
1. Dissolving:
• Bridge shrinks gradually
• Faster abscission
• High cell density
• Low contractility
• Low Aurora B activity
• High MCAK (microtubule depolymerase)
3|Page
