What are the three major forms of cell polarity?
- apico-basal polarity (top and bottom, complex of proteins that associate with tight and adherens junctions that regulate apico-basal polarity)
- asymmetric cell division (divisions of stem cells: one daughter cell will remain as a stem cell while the other will differentiate - there must be polarity between these cells)
- planar cell polarity (left-right polarity e.g. orientation of hair cells)
What happens when we grow epithelial cells on plastic vs in a collagen matrix?
- epithelial cells grown on plastic form a monolayer
- epithelial cells (MDCK: Madin-Darby canine kidney cells) grown in a collagen matrix:
- secrete basal lamina (rich in laminin) - basal surface of cells
- organise into clusters of cells around a cavity with apical structures (tight junctions, actin-rich apical microvilli)
- They have a spontaneous ability to polarise in response to extracellular matrix (ECM) signals
What are the key characteristics of apico-basal polarity?
Apico-Basal polarity requires:
- adhesive contacts: Cell-cell (adherens junctions, tight junctions), Cell-ECM
- polarised vesicle trafficking to deliver cargo to specific membrane domains
- memrbane domain-specific polarity proteins (apical vs basolateral scaffold proteins)
- polarised cytoskeleton (and machinery) to support adhesion, transport and membrane scaffolds
What is the extracellular matrix?
tissues are not made solely of cells - a variable part of their volume is extracellular space filled with ECM
- epithelial tissue - very little matrix (basal lamina)
- connective tissue - lots of matrix
ECM directly bears mechanical stresses of tension and compression
Basal lamina provides support and nutrient mechanism for epithelial cells
Connective tissue - also provides support and nutrient, but in addition provides great tensile strength. Has a lot of very strong collagen fribils interspersed within the matrix
How is the ECM organised?
Organised meshwork of proteins and polysaccharides:
- structural proteins include collagen and elastin
- cellular adhesion to the ECM depends upon:
- cell adhesive ligands: laminin, fibronectin
- cell anti-adhesibe ligands: tenascin, chondroitin sulfate, proteoglycan
What are glycosaminoglycans?
- GAG chains (e.g. Hyaluronan) occupy large amounts of space and form hydrated gels
- they have lots of negative charge, which attracts water
- swell with water and occupy space
- MW 8 x 10^6
- almost visible to the naked eye (with a microscope) - 300nm
GAGs:
- unbranched polysaccgaride chains
- composed of repeating disaccharide units
- one sugar is always an amino sugar (e.g. N-acetylglucosamine)
- second is usually uronic acid (e.g. glucuronic acid)
- side chains are highly negatively charged and strongly hydrophilic
Hyaluronan (or hyaluronic acid)
- simple GAG; no sulfated units
- tends to open up the matrix and so facilitates cell migration
- major component of joint fluid
What are proteoglycans?
- GAG chains often attach to proteins (proteoglycans) via a link tetrasaccharide (typically xylose-galactose-galactose-glucuronic acid)
- proteoglycans (particular sulfated forms) bind to secreted growth factors to enhance/inhibit their activities
What are collagens?
Collagens are major proteins of the ECM (structural fibrillar proteins)
- exist as triple-stranded helices composed of three alpha-chains (NOT alpha helices - left hand helix not right hand)
- provide tensile strength to ECM in tissues
- glycine, proline, hydroxyproline most common order of amino acids
- interactions between three strands provide great tensile strength
What is laminin?
Laminin is a major component of the ECM and basal lamina (adhesive protein)
- trimer, cross-shaped structure
- alpha, beta and gamma chain
- alpha can interact with receptors on the cell surface e.g. integrins, dystroglycan, perlecan (via alpha chain)
- multiple domains for interacting with ECM proteins and receptors (integrins)
What is the Basal Lamina?
- a specialised ECM that underlies all epithelia (also find it around muscle cells and the kidney glomerulus)
- has specific functions depending on the kind of cell that it is associated with
- important determinant of apico-basal polarity
- it is not inert - it is a structure that can change
- key component: collagen IV (meshwork type collagen), laminin, nidogen, perlecan
- protruding from the membrane of the cells are these integrin receptors: often interact with adhesive molecules such as the laminin
- multiple interactions between all the proteins in the basal lamina
What matrix receptors do cells have on their surface?
- integrins: actin binding, signalling. There are different integrins that will engage with different ECM molecules and that can confer signals from the basal lamina into the cell and a lot of this involves regulation of the cytoskeleton
- non-integrins: cell adhesion e.g. dystrogylcans
What molecules regulate A-B polarity?
- Par3, Par6: scaffold proteins that bind to each other and to
- aPKC (atypical protein kinase C_
- associate with tight junctions and serve as binding sites for Cdc42 and Rac (GTPases) - organisers of the actin cytoskeleton
- loss of tight junctions results in loss of A-B polarity in epithelial cells
- Loss of Par3 or Par6 –> loss of asymmetric division and polar cell growth
- Crumbs complex is one of the most apical complexes
- Scribble complex is more basolaterally localised (tends to associate more with adherens junctions)
What is Rac?
- Rac regulates A-B polarity
- block of Rac function –> MDCK cells develop inverted polarity, cells don’t know where to secrete the laminin… less is secreted and it often becomes apically deposited
- this can be overcome by growing cells in a matrix rich in exogenous laminin - suggests that integrins can also organise apico-basal polarity
- How are Rac and basal lamina/integrins linked?
- Rac is a small GTPase that activates proteins of the ARP complex - causes actin nucleation
- ARP also has an important role in formation of lamellipodia
- Rac activation is critical at the leading edge of a migrating neutrophil (i.e. increased polarisation) –> actin polymermisation and protrusion
- polarisation and migration are linked
- a polarised cytoskeleton is important for polarising a cell (and migration)
Why is the Par3/Par6 complex important?
- regulation of polarity is complex
- Par3/Par6/aPKC complex also regulates assembly of the apical Crumbs complex and the more lateral scribble complex
- both are essential for a correctly polarised epithelial cell
- many of these proteins are tumour suppressors
- mutation of scribble causes production of unpolarised cells that do not respond to normal controls on proliferation (MAPK cascade)
What are the two antagonistic polarity complexes?
- apical polarity regulators (Par3/Par6/aPKC complex and Crumbs complex) promote apical membrane identity
- Baso-lateral regulators (Scribble, Lethal Giant Larvae/Discs Large) promote basolateral membrane identity
How do these complexes work?
- Par6, Par3 and aPKC form a complex
- aPKC phosphorylates Par6; causes dissociation from Par3
- Par6 positions aPKC at apical membrane via Crb and Cdc42
- Par3 binds and stabilises AJ
- aPKC phosphorylates BLR proteins (e.g. Lgl); inhibits their apical accumulation (targets them to the proteasome)
- regulation not completely understood
- complexes can vary depending on cell type (and stage of development)
- in humans best documented role is in retina - Crb mutations associated with severe form of blindness (Leber’s Congenital Amaurosis)
- Mutations affect targeting of Crb to membrane; photoreceptors lose polarity, become disorganised and die
What is Crb?
- Crumb is a critical component of A-B polarity
- large protein that has multiple domains and functions
- Loss of Crb causes dfx in polarity; loss of apical membrane identity
- over-expression causes increased apical membrane domain
- multiple binding partners: PDZ-binding, FERM-binding, ECD-Notch (in cis)
- Multiple downstream signals: membrane, ECM, Cytoskeleton, Growth (JNK, Hippo)
- In mammals there are two: Crb1 and Crb2
How can a stem cell divide to produce daughters with different fates?
Environmental Asymmetry
- Whereby a stem cell is sitting within a particular niche
- It is the extracellular/stem cell niche that regulates whether the daughter cells from its division will either become another stem cell and stay within the niche
- if it leaves the niche, it is exposed to different signals from the environment and it becomes a terminally differentiated cell
Divisional Asymmetry
- segregates intracellular determinants to different parts of the cytoplasm
- quite often these determinants are membrane associated
- if the cell divides along the segregated plane the daughters have different contents which determine whether they remain stem cells or terminally differentiate
Give an example of divisional asymmetry of stem cells.
E.g. drosophila neuroblast apical complex plays an important role in asymmetric cell division
Apical Par6/PKC complex results in Miranda protein (binds Numb) moving to basal side
Numb is only incorporated into one daughter cell; affects fate of daughter cell (neuroblast or differentiated fate)
Ganglion mother cell inherits Numb and differentiates into neurons
Give an example of environmental asymmetry of stem cells.
- haemopoietic stem cells in bone marrow
- in close association with stromal cell in marrow
- stromal cell expresses Kit ligand while stem cell expresses Kit receptor
- While the SC is associated with the stromal cell and bound to Kit it remains a stem cell
- However once this stem cell divides the daughter cell now becomes isolated from the stromal cell, no longer has the activation of the Kit receptor
- the cell will now commit to differentiation, or if it fails to receive other traffic signals that will direct it to various lineages within the haemopoietic system, it will undergo cell death
How do the cortical neurons divide asymmetrically.
Cortical neurons are formed by asymmetric divisions in the Ventricular proliferative zone of the vertebrate neural trube
Neuroblasts have asymmetrically localised protein components (Numb, Notch1)
- division in the plane of the epithelium results in symmetric division
- division perpendicular to the plane of the epithelium results in asymmetric division (notch –> neuroblast –> differentiation)
What is planar cell polarity?
Some epithelial cells also exhibit a polarity within the plane of the epithelium (i.e. orthogonal to A-B polarity)
Hence known as planar cell polarity
- e.g. wild type epidermal cells in fly wing orient hairs in the same direction, same with sensory hair cells in mouse –> flamingo mutant, hairs no longer uniformly oriented
Describe polarity/PCP phenotypes in Drosophila and mammals
Drosophila wing hairs normally aligned in proximal-distal axis. In PCP mutant (Fz-/-) –> swirls and whorls
Mouse epidermal hairs have regular orientation along body axis. PCP mutant (Fz6-/-) –> whorls and irregular waves.
Mouse inner ear
Drosophila eye (ommatidia): loss of organisation and directionality
What are the PCP genes?
- Planar cell polarity results from accumulation of distal complexes and proximal complexes
- the membrane protein Frizzled (Fz) acts as a key signalling protein at the distal boundaries
- Loss of Frizzled causes loss of asymmetric accumulation of complexes
- Involves intercellular signalling - non cell autonomous effects!
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