lecture 9 Flashcards
Cell Polarity - cells have polarity (especially epithelial cells) - polarity is important for function and survival - structure and role of the ECM in polarity - role for the cytoskeleton Apico-Basal Polarity (Epithelia) - Par3/Par6 and Crumbs complexes Asymmetric Cell Division and Cell Polarity (Stem cells) - Environmental and intrinsic factors Planar Polarity (Epithelia) - Wnt/PCP signalling
1
Q
What are the three major forms of cell polarity?
A
- 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)
2
Q
What happens when we grow epithelial cells on plastic vs in a collagen matrix?
A
- 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
3
Q
What are the key characteristics of apico-basal polarity?
A
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
4
Q
What is the extracellular matrix?
A
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
5
Q
How is the ECM organised?
A
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
6
Q
What are glycosaminoglycans?
A
- 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
7
Q
What are proteoglycans?
A
- 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
8
Q
What are collagens?
A
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
9
Q
What is laminin?
A
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)
10
Q
What is the Basal Lamina?
A
- 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
11
Q
What matrix receptors do cells have on their surface?
A
- 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
12
Q
What molecules regulate A-B polarity?
A
- 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)
13
Q
What is Rac?
A
- 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)
14
Q
Why is the Par3/Par6 complex important?
A
- 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)
15
Q
What are the two antagonistic polarity complexes?
A
- 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
