Prof. Brennan L10-L14 Flashcards
1
Q
Actin cytoskleteon bidns to which junctions
A
TAF- Tight junctions, Adherens junctions, focal adhesions
2
Q
Intermediate filaments bind to which junctions?
A
Desmosomes and hemidesmosomes
3
Q
Septate junction
A
INvertebrate version of tight junctions. Lower down. below aderens!
4
Q
Tight junctions
A
3 TM proteins- Claudins, JAMs and occludin.
Claudin has 4 TMDs, the one that actually makes the junction. there are 24 claudins
Occludin- unique, just 1
JAM- 4 of them. Need to be there.
These attach to ZO-1,2 and 3 to attach to actin.
5
Q
Claudin 16
A
In tight junction in kidneys. Important for re-absorption of magneisum out of urine. Avoids hypomagnesia.
6
Q
Adherens junctions
A
Has Cadherins and nectins. 20 classical cadherins. Make homotypic interactions.
Cadherins attach to- alpha-catenin, beta-catenin, alpha actinin and vinculin.
Nectins attach to- alpha-cat, alpha act, vinculin as well as ADIP, ponsin and afadin.
7
Q
Cadherins
A
Make homotypic interactions.
Cadherins attach to- alpha-catenin, beta-catenin, alpha actinin and vinculin.
8
Q
Nectins
A
Nectins make homo and hetero interactions.
Nectins attach to- alpha-cat, alpha act, vinculin as well as ADIP, ponsin and afadin.
9
Q
Calcium dependent cadherin
A
Cadherins need ca2+ to make rigid dimer and then interdigitate like zip.
10
Q
Epithelial integrity
A
A function of cadherin. When cancer invads, loses cadherin and then can drop off tumour.
11
Q
Desmosomes
A
Contain NON-CLASSICAL cadherins!
Desmoglein and desmocollin families. These attach to plakoglobin and plakophillin_> attach to desmoplakin-> attach IMFs
12
Q
IMFs
A
can stretch without breaking. Tissues like skin and heart under stress. Good for desmosomes and hemidesmosomes.
13
Q
Pemphigus foliaceus
A
Autoimmune against desmoglein1-> skin layers blister constantly.
14
Q
Gap junctions
A
One connexin in each cell made up of 6 connexins. Can have many combos of connexins as there are >20 types and make heterotypic interactions. which ones determines property of gap junction.
15
Q
Pannexin
A
Flies have pannexins in gap junctions. we have 3 too.
16
Q
Connexin 43
A
Important in heart. Without connexin 43- get dyrhythmias
17
Q
dopamine in junctions
A
Dopamine closes gap junctions. Can see with fluorescent dye stays in one cell.
18
Q
hemidesmosome
A
alpha 6 and beta 4 integrins, plectin, BP180 and BP230.
intergrins attach to basement membrane by recognising laminin. BP230 attached to IMF.
19
Q
BP230
A
Part of hemidesmosome. attaches to imf
20
Q
Plectin
A
Part of hemidesmosome
21
Q
Basal lamina
A
basement membrane. ECM made of laminin and collagen 4.
22
Q
Parlecan and nidogen
A
cross links the laminin and collagen 4 in basal lamina.
23
Q
INtegrin alpha6beta4
A
IN hemidesmosome, links to basal lamina by recognising laminin.
24
Q
Epidermolysis bullosa
A
No hemidesmosomes. cant keep epidermal layer to dermis. No barrier. Die
25
Focal adhesions
alpha and beta integrins attach to basal lamina. Filamin, vinculin, talin and alpha-actinin attach to acin.
FAK and ILK are kinases-> signalling. Tell cell it's atttached so it doesnt kill itself.
26
Integrin
Have 18 alphas and 8 betas possible in focal adhesions. Type determines which ecm it sticks to.
27
Selectins
Adhere to cell surface carbohydrates. Recognise Lewis X-antigen (carbohydrate). Which is attached to PSGL1 protein.
Have e-selectin, L-selectin and P-selectin
28
Site of infection
Site produces 2 proteins- IL1 and TNFalpha. Signal to endothelium which then expresses E-selectin and P-selectin. This allows the endothelium to interact with PSGL1 on the leukocyte. L-selectin on leukocyte is doing the same with psgl1 on endothelial too.
This is a weak interaction so the wbc is tumbling along not stopping yet.
Then 2nd signal- IL8 and PAF to leukocyte from endothelium. Activate integrins in leukocyte. AlphaLBeta2 and alphaMbeta2. This makes the leukocyte stop and then go through the cells. integrins attach to JAM proteins in tight junctions.
29
infection- integrins
Dont attach to Ecm in this case- exception to the rule. Attach to JAM proteins in tight junctions. WBC stops rolling and enters.
30
Enterocyte polarity
sodium-glucose symport at the top. Secretes through passive glucose carrier protein at bottom. Tight junctions keep them there. but need apical/basal polarity to send them there.
31
Tight junctions in polarity evidence
Tight junctions can show we need Par proteins etc. for polarity. Without these proteins get disrupted tight junctions and can see dye dribbling between cells.
32
Proteins settign up apicobasal polarity
Par3,Par6 and aPKC at apical membrane.
aPKC= a kinase and phosphorylates Par1 and Lgl, excluding them from apical membrane.
Lgl,scribble,Disc large and Par 1 are at basolateral membrane. Par 1 acts as a kinase here and phosphorylates Par3, excluding it from basolateral membrane.
33
How does Par3/Par6/aPKC get to apical membrane to start with?
Vertebrates- Recruited by Jam proteins which are found in tight junctions which are apical.
Invertebrates have septate junctions which are positioned differently.
So instead the proteins are recruited by Crumbs and Stardust. Crumbs= large TM protein which makes a homotypic interaction. On intracellular side it binds stardust. Stardust then binds Par6.
Cumbs and stardust are apical, above agerence junctions so complex is apical.
(vertebrates can use both methods but invertebrates must use crumbs and stardust because no tight junctions)
34
Where else is Pa3Par6 system used?
In neurones and migrating cells. Migrating cell needs Par3Par6 complex at front to define leading edge.
Neuones need par3par6 complex to define axons from dendrites. It's in axon.
35
How is planar cell polarity defined?
Needs flamingo, Frizzled and Strabismus
36
Planar cell polarity in drosophila wing
Flamingo= non-classical cadherin. Has 7 TMDs.
Flamingo forms homotypic interaction between cells. Once dimerized, bring in Frizzled and strabismus. Strabismus binds to Prickle which is on proximal side and suppresses actin pol. so you don't get a hair at proximal.
Frizzled binds Dishevelled. Promotes actin polymerisation to make hair on distal side.
37
4 neural defects and how they are caused
1. Occult spinal bifida. small part of skin at bottom of spine hasnt closed but get tuft of hair covering.
2. Spinal bifida- neural tube and skin have not come over on small part. Neural problems on lower part of body.
3. Anencephaly- head end fails to close. lack major portions of brain, skull and scalp. Die quickly.
4. Craniorachischisis- hasn't closed at all. Brain and spinal chord open. Most severe.
38
Vertebrates primary neurulation
Primary = top part: thoracic, neck and head.
1. Cuboidal epithelial turn columnar to form neural plate, dorsal side thickens.
2. Neural folds form and folds move towards midline.
3. Neural folds fuse to form tube, seperates away from epithelium.
39
Secondary neurulation steps in vertebrates
seconday= lumbar and sacral (bottom half)
1. Mesenchymal cells are dispered to start with. Then condense.
2. Formation of medullary and notocord.
3. Mesenchymal to epithelial transition (MET). Aquire polarity. So cavitation to form lumen- cells in middle dont become polar and die, leaving lumen.
40
MET in neurulation
Mesenchymal to epithelial transition.
41
Separation of neural tube from epithelium driven by
Driven by a change in cadherin expression. All of the initial ectoderm express e-cadherin. Cells in the neural tub express N-cadherin. This allows it to separate. Get all N-cadherin stuck together in the middle.
42
Evidence for cadherins in separation of neural tube
If you deliberately express N-cadherin in the wrong place, the Ns will stick together and the tube won't form properly or separate from the ectoderm.
43
For neural plate to bend, need elongation of cells and then constriction of actin filament bundles at top, what does this require? (+ evidence)
Apicobasal polarity must be established.
Scribble is usually found at basolateral membrane. In scribble mutants, clls stay cuboidal and actin doesnt condense at apical side. In these mutants neural tube does not close.
44
Shroom
Shroom= actin binding protein that is localised to apical membrane when apicobasal polarity is established in neural plate formation.
Shroom induces elongation and apical constriction.
If no shroom, nervous system doesnt close properly
45
Shroom mutant
Shroom needed for cell elongation and apical constriction in neural plate folding. IN shroom mutant, apical surface doesnt constrict.
In shroom over-expression- apical surface constricts loads.
46
How does shroom induce cell elongation and apical constriction?
By localising gamma-tubulin to apical membrane.
And recruits ROCK to the apical membrane which activates Myosin II.
It localises Rho and Rap1-> Rho activated kinase=ROCK.
47
Neural tube closure needs what polarity?
Needs apicobasal to localise shroom to elongate cell and cause apical constriction.
Also needs planar cell polarity for convergence extension which narrows body axis and causes elongation.
Absence of convergence extension leads to a widened neural floor plate so neural folds can't fuse.
48
EphrinA5 and EphA7
Needed for the adhesion between the neural folds to close neural tube.
Usually Ephrina5 and epha7 repel each other. This is the exception. They are ligand and receptor. Bidirectional signalling. Uses a non-functional Epha& to allow this adhesion.
49
4 cell types in small intestine?
1. Enterocyte- absorptive
2. Goblet cell- makes mucous
3 Paneth cell- at bottom of crypt. Make defensins.
4. Neuroendocrine cell- very rare. Secrete hormones.
50
What is always active in dying cells and shows apoptosis is happening?
Active caspase 3
51
Active caspase 3
Always active in dying cells and shows apoptosis is happening
52
5 steps in apoptosis
1. chromosomes condense
2. nuclear fragmentation
3. cytoskeleton breaks down
4. Membrane blebbing (small vesicles made)
5. Phagocytosis by neighbours
53
Syndactyl
Failure of webbed fingers to separate. (reason for apoptosis)
54
Which protein family does apoptosis require?
BCl-2 family.
| Family of proteins that govern mitochondrial outer membrane permeabilication (MOMP). Can be pro or anti- apoptotic.
55
Bcl-2 subfamilies
1. Anti-apoptotic- biggest proteins. Contain all 4 bcl-2 homology domains (Bh4, bh3, bh1, bh2 and then TMD).
- > bcl-2, bcl-xl, bcl-w
2. Pro-apoptotic- Bax and Bak contain 3 of the domains.
3. BH3- only are pro-apoptotic and is the biggest group. Puma, Noxa, Bim, Bid and Bad.
56
members of bh3-only subfamily of bcl-2
```
Puma
Noxa
Bim
Bid
Bad
```
57
Why is bh3 homology domain needed in the pro-apoptotic proteins of Bcl-2 family? extra reading
BH3 domain is needed for dimerization with other proteins of BCL-2 fmaily and crucial for their killing activity.
(thought its also found in some anti-apoptotic proteins liek bcl-2 and bcl-xl)
58
Bh3- only proteins function
Detect apoptotic stimuli. Sense problems so biggest family.
Different proteins sense different problems.
Then initiate apoptosis.
Dna damage-? Puma and noxa
Detachment from ecm-> Bid
Loss of growth signals-> Bad and Bim
59
How do BH3 only proteins promote apoptosis?
Cause translocation of Bax to mitochondria.
Bax generates pores in the mitochondria membrane:
this releases cytochrome c, SMAC/diablo and apoptosis initiating factor (aif) into cell cytoplasm.
Lets out H+ ions which disturbs gradient so cell stops making atp from oxidative phosphorylation.
60
Cytochrome C
Cytochrome c clusters in a heptamer and activates APAF1 (apoptosis activating factor) and cascase 9.
This makes a complex-> the apoptosome.
With 7 x cytochrome c, caspase 9 and apaf1.
(procaspase 9 becomes caspase when apoptosome comes together)
61
Pro-caspase 9
When apoptosome comes together, procaspase 9 is cleaved to active caspase 9 which cleaves and activates downstream caspases----
pro-caspase 3 --> caspase 3.
62
Caspases in apoptosis
Caspase 9 in apoptosome with cytochrome c and APAF1.
Caspase 9 is an initiating caspase. cleaves and activates caspase 3.
Caspase 3= execution caspase.
Caspase 3- breaks down actin cytoskeleton, breaks down lamin in nuclear envelope. activates CAD which breaks down DNA.
63
Caspase 3
Caspase 3= execution caspase.
| Caspase 3- breaks down actin cytoskeleton, breaks down lamin in nuclear envelope. activates CAD which breaks down DNA.
64
Intestinal stem cells genes
Express several genes:
Lgr5
Achate-scute like 2
Olfactomedin 4
Bmi1
65
CRE
If you add Cre to specific cells you will get LacZ.
Normally you dont get the protein because a stop codon between promoter and LacZ exon.
If you add CRE it removes the lox stop lox so you get LacZ!
66
CRE labelling
Can label cells in small intestine with CRE.
If you label enterocytes will get lots of blue and then lose blue and none left after a few months.
Ifyou label stem cells with CRE- will get whole intestine blue after a while because all cells come from stem cell.
67
Simpler labelling of stem cells in intestine
Label Lgr5 cells. After a while all cells will be labelled because stem cell can make whole crypt/villus. so if it makes whole thing know it's a stem cell.
68
Stem cell requirements
lamin-rich environment
Notch signal and Wnt signal.
(Wnt and Notch comes from Paneth cells, remove these lose stem cells)
69
Transient Amplifying cells receive which signals
Lose the Paneth cell-derived Notch signal. Still get Wnt signal but not notch. so stop being stem cells.
Wnt induces expression of c-Myc in transit amplifying cells. Myc is needed for the TA cells to get bigger so they can divide.
70
c-Myc
Needed by Transient amplifying cells to proliferate- grow in bluk rather than divide. but need to grow in size before they divide.
71
Math1
required for secretory cell differentiation. Need Math1 to turn TAC into secretory projenitor.
72
Gfi-1
Needed for goblet and paneth cell differentiation.
| IN gfi1 mutant, goblet and paneth cells arent made but neuroendocrine cells are still there. Even more are made.
73
Sox9
Needed for Paneth cell differentiation.
74
Neurogenin 3
Needed for neuroendocrine differentiation
75
Hes 1
required for enterocyte differentiation.
Notch signalling induces Hes1 expression. Common in development to re-use the same signalling pathways. No notch- no hes1.
76
Notch and Wnt in differentiation
Wnt induces math1 and sox9 expression. Wnt-> ssecretory Notch-> enterocytes.
77
small intestine stem cell differentiation
hes1= enterocyte
math1 and gfi1= goblet and paneth
math1 and sox9= paneth
math1 and Neurogenin 3= neuroendocrine cell
Notch promotes hes1
Wnt promotes math1
78
Ephrin/eph in small intestine
Eph receptors in crypt
Ephrin ligands in villus
Repel each other. Keep enterocytes in villus and tac in crypt. without ephrin/eph get cells in the wrong place.
79
Maintained ephrin/eph signalling
Causes eary adenomas to grow within villus. Mutation causes constant wnt signalling-> continues to express eph. So always stay in crypt, in the end so many cells in crypt it will buckle and tissue will grow inside the villus. Cancer
Constitutive wnt signalling is the 1st step in colorectal carcinogenesis. Cells won't differentiate.
