10-14: Brennan Flashcards
State the order in which the different types of junction appear in cells (from apical to basal), and state the main roles of each
- Tight Junctions (Barrier and Polarity)
- Adherens Junctions (Cell-Cell Adhesion via actin)
- Desmosomes (Cell-Cell Adhesion via IFs)
- Gap Junctions (Passage of small molecules + Electrical Coupling)
- Hemidesmosomes and Focal Adhesions (Attachment to BM)
Describe the overall structure and function of Tight Junctions (Zonula Occludins)
They form a series of “Ribbons” around the cell, anastomising (branching) and wrapping around to bring the membranes within 10nm of each other
Name the three (mentioned) proteins found in tight junctions
CLAUDIN, occludins and JAMs
Describe the function of Claudins
Many (of the 24 members) are thought to act as the backbone of TJ strands
They are distributed differently depending on the tissue, and this determines electrical resistance and ionic selectivity (this distribution is controlled during development)
Combination with occludin is necessary for the paracellular barrier
Describe the function of occludins
Role in stability of tight junctions - exact function is disputed
Interact directly with F-actin (Wittchen et al 1999), as well as ZOs 1, 2 and 3
Name some of the other TJ proteins besides Occludin and Claudins, and their role
JAMs, CAR, P0, OAP-1 and PMP22 - all interact with ZOs and other TJ proteins, though many do not yet have clearly established roles
Describe the two main roles of Tight Junctions
Prevents paracellular water flow: (important in skin [body/air] and kidney [urine/blood]) - note that different claudins allow different things through, e.g. Claudin16 necessary in kidney to allow Mg2+ back into blood to prevent Hypomagnesia
Fence between apical and basolateral surfaces: allow different compositions of plasma membrane at apical and basolateral ends (very important in enterocytes in the gut - need different sodium and glucose transporters on GI side vs blood side
Describe the two sets of adhesion complexes found in Adherens Junctions
Cadherin -> ß-cat -> a-cat -> vin and a-act
Nectin -> afadin -> ponsin-vin or a-cat or ADIP-a-act
Describe the role of cadherins in adherens junctions
Ca2+-dependent intercellular interactions between homotypic cadherins form contacts, which are strengthened by clustering and lateral spreading (juxtemembrane domain/JDM involved, binds p120-catenin)
Cadherins bind to many intracellular receptors (esp. catenins) which control actin organisation, stabilise cadherins and control gene transcription
Name and give examples for the three types of Cadherins
Classical (E, P, N, R, etc - found in adherens junctions)
Non-classical (found in desmosomes)
Proto-cadherins (early development and morphogenesis)
Describe the role of cadherins in cell sorting
Cadherins only interact with other cadherins of the same type (e.g., E/E, N/N - homotypic)
This maintains epithelial integrity (and loss of it can lead to intraversion and metastasis -> cancer)
What do nectins do?
They are found in Adherens Junctions along with Cadherins, and play a similar role, but bind different adaptor proteins and have some different behaviours
Describe the general structure of Desmosomes
Non-classical cadherins (4 desmogleins, 3 desmocollins) link to cytoplasmic plaque proteins (see other FC), which link to IFs
Name some of the cytoplasmic plaque proteins involved in desmosomes
Armadillo proteins - plakoglobin, plakophillin
Plakin proteins - desmoplakin
Others with limited expression - Perp in stratifying epithelia, Corneodesmosin in upper epidermis + hair
Why do desmosomes provide SO MUCH strength to tissues?
Desmosomes link to IFs rather than MTs or AFs (which both snap more easily, while IFs can withstand more deforming force)
In which kinds of tissues are desmosomes most common?
Ones that are stretched or pulled a lot, e.g., skin and heart
Describe the roles of gap junctions
- Allow electrical coupling and passage of small molecules from cell to cell
- Allow co-ordination of behaviour (e.g., heart cells contracting together)
Describe the structure of Gap Junctions
Maculae/Plaques made up of Connexons, which are hexamers of connexin channel proteins
2 Connexons in adjacent cells dock to form a channel that small messenger molecules can pass through
Describe the role of gap junctions in heart function
Connexin43 allows Ca2+ to pass between heart cells and co-ordinate contraction (heterozygotes for Cxn43 show half conductance)
Describe the role of gap junctions in the Retina
In low light, gap junctions connect adjacent retinal neurones together to aid vision - in low light, they are sealed to allow clearer vision as each cell senses more independently
What are pannexins and innexins?
Innexins are the equivalent of connexins found in non-chordate gap junctions (but these are convergent)
Pannexins are found in chordates and ARE homologous with innexins (but have a less significant role in us)
Describe the roles of hemidesmosomes
Provide a strong attachment to the basal lamina by anchoring IFs to the Col4/Laminin network
Describe the components of Type I (classical) and Type II hemidesmosomes, and which types of epithelia contain them
Type I (stratified, transitional and p.stratified) - a6ß4, plectin, CD151, BPAG1e and BPAG2
Type II (simple) - a6ß4, plectin
Describe how hemidesmosomes are linked to intermediate filaments
Through two members of the Plakin family (same family as desmoplakin from desmosomes)
- Plectin
- BPAG1e
Describe how hemidesmosomes are linked to the basal lamina
a6ß4 links to laminin-332
BPAG2 (BP-180) binds directly to Col4/Laminin network
Why are type II hemidesmosomes found in the types of epithelia that they are?
Stratified, Transitional and Pseudostratified tend to get stretched and deformed a lot (e.g., skin, bladder, oes/trachea respectively)
Name the (metnioned) fatal disease associated with attachment to BM, and its cause
Epidermolysis bullosa -> mutations affecting laminins, Col4 or integrins can cause it
Describe how focal adhesions link IFs (and thus cells) to the ECM
ECM - Integrins - Talin or Filamin or a-Act - vin (note, FAK and ILK signals involved)
Name 3 things that focal adhesions are required for
- Cell migration
- ECM Assembly
- Transmitting signals for survival and proliferation
(Can demonstrate these with integrin knockout experiments)
What is the role of selectins?
Targeting leukocytes to sites of infection
What are the three (mentioned) connections involving Selectins?
E-selectin -> PSGL1 in leuko
L-selectin -> PSGL1 in endo
P-selectin in platelet -> PSGL1
Why is apical-basal polarity essential?
For directed absorption and secretion (many proteins are specifically localised to the apical and basolateral membranes)
Describe an experiment used to establish which proteins are required for apical/basal polarity
Plate either WT or mutant (e.g., Par mutant) cells to form a layer, then add dye on top, and record whether it remains on top of the layer (in which case A/B polarity IS properly established) or passes between cells
Describe the steps required to establish apical/basal polarity
1 - Par3, Par6 and aPKC form an apically localised complex
2 - aPKC phosphorylates and excludes Par1 and LgL from the apical membrane
3 - Par1 and LgL thus only associate with the lateral membrane (LgL also recruits scribble and Disc large)
4 - Par1 phosphorylates and excludes Par3 from the basolateral membrane
Now, polarity is established, and maintained by phosphorylation
Which of the complexes described in the above flashcard on A/B polarity is INITIALLY formed, and how?
Par3/Par6/aPKC are recruited to the apical membrane by JAM proteins in vertebrates (or Crumbs and Stardust in invertebrates)
JAM extracellular domains bind homotypically (like Occludins and Claudins)
Stardust is an intracellular protein that binds to the Par3/Par6/aPKC complex, and also binds to the Crumbs protein, whose extracellular domains bind homotypically
Where do we see localised expression of Par3 besides A/B polarised cells (although similar principle)
At the leading edge of migrating cells, and the axon terminal of neurons
What is the other important type of cell polarity besides A/B polarity? (Describe and example)
PLANAR CELL POLARITY - orientation of structures within the PLANE of the epithelium
E.g., all hairs grow in Proximal->Distal direction, and from the same corner of their respective cells
Describe the steps necessary to establish PLANAR polarity in the Drosophila wing
1 - Flamingo ECDs bind homotypically between cells of the wing epithelium
2 - Flamingo interactions allow Frizzled and Strabismus (Fz and Stbm) to interact, with Fz localised to distal side of one cell, and Stbm to the proximal side of the next
3 - Frizzled signals via Dishevelled to reorganise the actin cytoskeleton, leading to actin polymerisation on the distal side of the cell
- Meanwhile, to prevent hair formation on the PROXIMAL side of the next cell, Strabismus signals via Prickle to prevent hair development
Name four Neural Tube Defects, and briefly describe what causes such conditions
Spina bifida, occult spina bifida, anencephaly, craniorachischisis
Various parts of the neural tube can fail to close, due to defects in early embryogenesis
Note: 70% of neural tube defects can be prevented by folic acid supplementation
State the two processes necessary for neural tube formation in vertebrates, and where each one occurs
PRIMARY NEURULATION - from head to lumbar
SECONDARY NEURULATION - from lumbar to sacral
Describe the mechanism of Primary Neurulation
1 - From the cuboidal epithelial cells, a section of Columnar Neural Plate is specified
2 - The Neural Plate bends at the median hinge point to form Neural Folds, which then move towards the midline
3 - The neural folds fuse to form a single neural tube
Describe the mechanism of secondary neurulation
Unlike primary neurulation, this involves the mesenchymal cells, not the epithelial cells:
1 - Mesenchymal cells coalesce, with no hollow region at first
2 - Cells undergo Mesenchymal-to-Epithelial Transition (MET), forming adhesions and A/B polarity
3 - Cells in the middle that fail to undergo MET die via apoptosis, forming a lumen
4 - This tube fuses with that formed by primary neurulation, to form a single long neural tube from head to sacral
Describe the role of Cadherins in Neurulation
In Primary neurulation, separation of the neural tissue from the surrounding epithelium is driven in part by changes in cadherin expression:
As the neural plate forms, that area switches from E to N-cadherin
If you express E-cadherin in the neural tube region, it fails to separate properly
Describe the role of the cytoskeleton in formation and bending of the neural plate
Microtubules elongate to cause cuboidal cells to become columnar
Apical AF-bundles contract to narrow the cells at their apices, causing the plate to fold
*Some of these FCs - might need to look at the diagrams in the actual lecture for a refresher
Which type of polarity is essential for folding of the neural plate in neurulation, and how can this be demonstrated experimentally?
Apical-Basal polarity - necessary both for cells to ELONGATE and undergo APICAL CONSTRICTION
Disrupting Scribble -> Cells don’t transition to columnar -> Cells can’t localise actin -> Neural tube is exposed on outside
In Scribble mutants, cells are cuboidal rather than columnar, AND actin is found throughout the cell rather than apically polarised
What is SHROOM (and what is seen in shroom mutants or shroom overexpression)?
Shroom is an Actin-Binding Protein that is highly localised to the apical membrane, and is necessary for cell elongation and apical constriction:
Shroom mutant cells are LARGE and fail to apically constrict, while Shroom over-expressing cells are small due to extra constriction
How does Shroom induce cell elongation in the neural plate?
Shroom localised y-tubulin to the apical membrane, which then forms the MTOC and thus controls the elongation of cells by MT polymerisation
How does Shroom induce apical constriction in neural plate cells?
Shroom recruits ROCK to the apical membrane, which activates MyosinII
Which part of neurulation requires Planar Cell Polarity?
Neural Tube Closure:
In PCP mutants (e.g., Crash, Loop-tail, Frizzled, Dishevelled) the nervous system is exposed, as it has failed to close
ALSO PCP of Frizzled and Strabismus allows tissues on either side to know where the midline is when forming the neural crest
ALSO(!) convergence extension (see other FC)
Besides neural tube closure and midline awareness, what else is PCP necessary for in neurulation?
CONVERGENCE EXTENSION - narrows the body axis and causes elongation (makes tissues longer and thinner)
In the absence of Convergence Extension (e.g., in a Loop-tail mutant), the floor plate is wider, preventing fusion of the two neural folds
Name the two proteins required for ADHESION between neural folds, in order to close the neural tube (and describe their function)
EphrinA5 and (non-functional) EphA7
These are both membrane proteins, which normally show Bidirectional, Repulsive signalling
However, in this case, a splice variant of EphA7 which lacks its RTK domain (and thus cannot send a repulsive intracellular signal) is required for adhesion and joining of the neural folds
Describe normal cell turnover in the small intestine
Cells move from the crypt to the villus over around 3-5 days, being lost and undergoing apoptosis due to damage
Lost cells are replaced by proliferation of stem cells in the crypt of Lieberkühn
Describe the cell types found in the small intestine
Enterocytes - most common, absorb nutrients from gut
Goblet cells - secrete mucus to protect and lubricate
Paneth cells - secrete defensins to prevent infection
Neuroendocrine (RARE, 1 per villus) - secrete hormones, e.g., for peristalsis and hunger
State 5 key morphological changes in cells undergoing apoptosis
Chromosome condensation
Nuclear fragmentation
Cytoskeleton breakdown
Membrane blebbing
Phagocytosis by neighbouring healthy cells
Describe how DNA damage or unreplicated DNA leads to apoptosis
DNA damage activates the BH3-only proteins PUMA and NOXA, which then cause conformational change in BAX (a pro-apoptotic Bcl2)
Bax then translocates to the mitochondria and forms pores in the OMM, releasing Cyt C (and other proteins between IMM and OMM, e.g., SMAC/Diablo)
Describe how the release of Cyt C from mitochondria results in apoptosis
- Cyt C clusters and activates APAF-1 and Caspase 9 (apoptosome)
- Caspase9 cleaves and activates Executioner Caspases (e.g., Casp3)
- Executioner Caspases carry out further cleavages that result in regulated cell death, FOR EXAMPLE:
- actin (cytoskeletal breakdown)
- lamin (NEBD)
- ICAD (DNA fragmentation)
Where is each type of cell found in the small intestine?
Stem and Paneth Cells in the base of the Crypt
Transit Amplifying Cells on the walls of the Crypt
Enterocytes, Goblet Cells and Neuroendocrine Cells on the villus
Describe the fate map of a stem cell in the small intestine
Stem cell divides (occasionally) to give one stem cell and one TAC
TACs divide more rapidly until terminal differentiation
TACs can give rise to either an absorptive lineage (eventually enterocytes) or a secretory lineage (goblet, paneth of neuroendocrine)
How can intestinal stem cells be (simply) identified?
They express several genes that allow their identification (e.g., Lgr5, achaete-scutue-like2, Olfactomedin4, Bmi1)
Describe the significance of Lgr5-expressing cells in the small intestine
Progeny of Lgr5-expressing cells includes ALL cell types of the small intestine
As such, a single Lgr5-expressing cell can produce an entire crypt/villus complex
How are stem cells maintained and their division regulated (IN GENERAL)?
When stem cells divide, one of the daughter cells will remain in the STEM CELL NICHE (a combination of specific ECM and signals), and will thus become a stem cell to maintain the stem cell population
The other daughter cell becomes a TAC
What creates the stem cell niche and what leads to TAC formation in the SMALL INTESTINE?
Notch and Wnt signals from neighbouring Paneth cells are required (loss of either -> no stem cells)
If a stem cell divides, and one of its daughter cells LOSES CONTACT with the PANETH cells, that cell still receives the diffusible Wnt signal, but not the contact-mediated Notch signal, causing it to differentiate into a TAC
Describe how Wnt signalling affects TACs
Wnt signalling induces the expression of C-MYC in TACs, which promotes cell growth
No Wnt -> No C-Myc -> less growth
Describe (naming specific TFs and Signals) how differentiation of cell types in the small intestine is controlled
Different Transcription Factors control differentiation:
C-myc -> TAC
Math1 -> Secretory Cell Differentiation
Neurogenin3 -> Neuroendocrine
Gfi1 -> Goblet AND Paneth
Sox9 -> Paneth
Hes1 -> Enterocyte
Mutants of any of these result in fewer (or none) of that cell type being produced, and MORE of the other cell types
SIGNALLING:
- Notch induces Hes1 (enterocytes)
- Wnt induces C-myc (TACs), Math1 (secretory) and Sox9 (Paneth)
Overall: The balance of Notch and Wnt signalling controls differentiation of the small intestine epithelium
How is the POSITION of cells maintained in the Small Intestine (e.g., how are Stem and Paneth cells maintained at the bottom)?
Eph receptors and ephrin ligands!
The repulsive signals caused by the binding of these proteins is required to keep crypt and villus cells in the correct positions
Removing them disrupts the structure of the Crypt and Villus
How was it PROVEN experimentally that the Lgr5 promoter is active in STEM CELLS (and thus its expression can be used to label them)? Describe the experimental details and rationale
3 Components Needed:
- A Transgene with an Lgr5 promoter
-> in any cells where Lgr5 is expressed, then whatever you place downstream of it will also be expressed
-> in this case, we place CRE(er) downstream of it - A constitutive promoter (e.g., Rosa26) with a STOP Cassette surrounded by LoxP, and LacZ gene
-> The transcript will be expressed in all cells, but in most, the STOP Cassette will limit translation
-> However, in cells where CRE is expressed (due to Lgr5 expression), CRE will recognise the two LoxPs and delete the STOP Cassette
-> Therefore, Lgr5-expressing cells will appear BLUE as LacZ will now be transcribed - Tamoxifen (the time-regulation component)
-> Remember that this process is taking place in GENOMIC DNA, so any changes made to the genome of Lgr5-expressing cells (e.g., removal of STOP) will be passed on to progeny
-> If we do not time-regulate CRE activation, LacZ may be expressed too early in development, and more cells will be stained blue than desired
-> Using Tamoxifen, we can ensure that we only stain Lgr5-expressing cells of the ADULT gut, and their progeny
Note: Tam regulation works because CRE is bound to estrogen-receptor, so remains in the cytoplasm until Tam binds and releases it from hsp90
Describe the three possible experimental observations when carrying out the Stem Cell Staining experiment on a newly studied promoter, and what each possible observation would suggest
- Short, sharp label that disappears after a few days [IF chosen promoter is found in ENTEROCYTES - this would label all enterocytes, but disappear as they were lost and replaced]
- Stained cells make their way up through villus, ALL villus cells become stained, then eventually staining is lost [IF chosen promoter is found in TACs]
- Initially, a few cells at the bottom become stained, then stained TACs AND stained crypt, then whole villus permanently stained for months [IF successfully labelled the STEM CELLS
Scenario 3 was observed when Lgr5-expressing cells were labelled, confirming that these were indeed stem cells
Later, the experiment was repeated but using the Notch-responsive Hes5 promoter instead of Lgr5 - the whole villus was labelled yellow by YFP, showing that Notch is also active in stem cells
Why did the villus in an Lgr5-staining experiment eventually lose its colour, despite successfully labelling the stem cells?
- Occasionally, when stem cells divide, BOTH daughter cells can fall out of the nice, so are replaced by another, non-labelled stem cells
- This can be intentionally visualised by labelling all 4 stem cells with different colours (e.g., YFP, GFP, RFP, BFP)
-> One by one, three of the colours will disappear as the original stem cells fall out of the niche and are replaced - Eventually the whole villus will be formed from cells of just one colour, until this too disappears as the last stained stem cell is replaced
