Cytokinesis: Cell Separation and Division

• Cytokinesis (CYTK) is the process of separating cells after organelles have moved to opposite sides.
• Cleavage furrow forms during CYTK, with a contractile ring of actin and myosin forming midway between the two asters.
• Microtubules run through the contractile ring and must depolymerize for CYTK to occur.
• Severing of membranes: vesicles fuse into the membrane as it pinches off.

Summary of Cytokinesis

• Rho signaling promotes local assembly/nucleation of actin/myosin into the contractile ring.
• Contractile ring assembly occurs midway between the two microtubule asters
• ER segregates during interphase.
• Mitochondria multiply prior to separation and localize to the cleavage furrow to supply energy.
• Golgi fragments during M-Phase in mammals, and forms the cell plate in plants.
• Some cells reposition their spindle to divide asymmetrically, producing different progeny.

Microtubules of the Mitotic Spindle

• Astral stimulation model: signals move down microtubules to the middle of the plasma membrane where the furrow forms.
• Central spindle model: the spindle midzone sends a signal to the cell cortex, activating the Rho signaling pathway and forming the contractile ring.
• Astral relaxation model: relaxation of contractile forces occurs where microtubules meet the plasma membrane, leading to contractile force at the cleavage furrow.
• Cells may use a combination of these mechanisms in a cell type- and species-dependent manner.

Cell Polarity During Cytokinesis

• Cell polarity refers to spatial differences in cell shape and structure that enable unique functions.
• A specialized set of proteins (RhoA, Myosin, Actin) is recruited to the region between spindle poles by microtubules and actin filaments.
• This recruitment enables furrow assembly and ingression before cell division.
• After mitotic exit, the cytoskeleton of monopolar mitotic cells is initially radially symmetric.
• A symmetry-breaking reaction then simultaneously polarizes microtubules and the cell cortex.
• This process concentrates cleavage furrow markers into a cap on one side of the cell at the midbody.
• Remnants of the midbody remain on inner plasma membrane of each daughter cell.
• Midbody remnants orient the spindle, giving polarity to new microtubules.

Post-Cytokinesis Cell Polarization

• Cell polarity involves spatial differences in cell shape/structure, which enable unique functions.
• Apical-basal polarity: epithelial cells have an apical membrane facing the body's outside surface/lumen, and a basolateral membrane oriented away from the lumen.
• Cell-cell communication: neurons use dendritic terminals, somas, and axonal terminals to communicate in one direction.
• Cell migration: cells moving in one direction must have a defined front and rear.
• The nucleus, golgi, and microtubules work together to guide cell migration.
• Microtubules extend asymmetrically from the centrosome, pulling the nucleus behind
• Nuclear translocation and microtubule polarization define polarization in mesenchymal cells.

Epithelial Cell Polarity

• From apical to basal side:
• Actin filaments bundle into microvilli, increasing the cell surface area.
• These bundles anchor on adherent junctions, which connect adjacent cells.
• Intermediate filaments anchor actin in the cell cortex with desmosomes/hemidesmosomes, connecting the cell to other cells and the ECM.
• Microtubules polymerize from apical to basal side, providing directionality for intracellular component transport.

PAR Complex (Apical-Basal Polarity)

• Consists of Par3, Par6, atypical protein kinase C (aPKC), and Cdc42.
• Subunits are dynamic and can change.
• Par3 and Par6 are scaffold proteins with PDZ domains that bind the plasma membrane, and they bind to each other and to aPKC.
• The complex has binding sites for the small GTPases Rac and Cdc42.
• Rac and Cdc42 are Rho family members which regulate actin assembly.

Crumbs Complex (Apical-Basal Polarity)

• Consists of the Crumbs Complex (CRB), cytoplasmic proteins PALS1, and PALS1-associated tight junction protein (PATJ).
• CRB (or CRB3 in humans) is a transmembrane protein and tumor suppressor in mammalian epithelial cells.
• PALS1 and PATJ are scaffold proteins similar to PSD-95 (PDZ) domains.

Scribble Complex (Apical-Basal Polarity)

• A potential tumor suppressor that is frequently lost in cancer.
• It consists of scribble (SCRIB), lethal (2) giant larvae homologue (LGL), and discs large homologue (DLG).
• LGL is a cytoskeletal protein capable of binding myosin II.
• DLG is a scaffold protein.

Mechanism of Apical-Basal Polarity

• PAR Complex (par3/6, aPKC, Cdc42) becomes active at the apical lateral membrane, promoting the formation/maturation of early adhesion sites.
• Crumbs (CRB) complex (CRB, PALS1, PATJ) establishes the apical membrane and tight junctions.
• Scribble (SCRIB) complex (SCRIB, LGL, DLG) works at the basal lateral membrane, defining the basolateral plasma membrane domain.
• The three complexes have antagonistic interactions, allowing them to function with spatial and temporal distinction.
• These complexes spatiotemporally regulate epithelial polarization through interactions with the cytoskeleton and adhesion proteins.

Neuron Structure and Polarity

• Axons and dendrites of neurons differ in their protein and organelle composition.
• Axons are typically long and thin, with a uniform width that branches at right angles.
• Dendrites are shorter and thicker near the cell body, becoming thinner distally, and undergoing Y-shaped branching.
• Axons contain synaptic vesicles and release neurotransmitters at axon terminals.
• Dendrites, especially dendritic spines, have receptors for neurotransmitters and signaling systems.

Neuronal Polarization

• Neurites are formed in 5 steps
• Initial polarization of neurites is driven by:
• Increase in plasma membrane (vesicle recruitment/fusion)
• Increase in local concentration and activation of signaling molecules (PI3K, Rho GTPases)
• Increase in actin polymerization
• Enhancement of microtubule (MT) polymerization
• Signals include:
• Laminin
• WNT
• Netrins
• Growth factors
• After a small amount of growth, the opposite reaction is induced, causing:
• microtubule catastrophe
• actin depolymerization
• enhanced endocytosis
• decreased membrane receptors
• Neurites release negative feedback signals to the cell body or metabolize molecules for axon specification.

Initiation of Feedback Loops in Neurons

• An external stimulus (ex. growth factor) activates a RTK which activates PI3K.
• PI3K then produces PIP3, which activates RAP1B, leading to a negative feedback loop that degrades RAP1B in other neurites.
• RAP1B activates CDC42 (a Rho family member).
• CDC42 activates the Par complex, which includes a Ras-GEF, thereby activates RAC1 to promote actin nucleation and polymerization.
• This process will further activate a positive feedback loop at the level of PI3K, increasing the pathway’s activity, ultimately leading to axon specification and elongation.

Spine Formation on Dendrites

• Par6 and aPKC separate from the Par3 complex, leading to Rho GAP activation.
• Rho GAP blocks actin/myosin contractility, resulting in local relaxation at the plasma membrane and a small protrusion can form.
• Par3 and TIAM1 (Ras-GEF) activate RAC1, which promotes actin polymerization and mature spines.
• Relaxation of actin/myosin contractility allows the small protrusion of the spine to form.

Neuronal Transport

• The cell body of a motor neuron has a 1m long axon.
• Electrical signals are sent down the axon while proteins and mitochondria are shuttled by motor proteins, which takes 1-10 days.
• Anterograde transport is away from the Golgi and is is towards the +end of MTs.
• Retrograde transport is towards the Golgi and is towards the -end of MTs.
• Kinesin accumulates at the site of a new axon for protein transport required for axon growth.

Exceptions of Typical Neuronal Structure

• Microtubules no longer polymerize directly from the MTOC/centrosome in Neurons, instead they are stabilized by accessory proteins.
• In neurons, MTs polymerize from the Golgi.
• The Golgi extends into dendrites but not into axons.
• Because there is no Golgi in the axon, proteins must be stored/modified (N-Glycosylation) in the soma and transported to the axonal terminal