Cell polarity and its uses etc Flashcards
(80 cards)
1
Q
What are the 4 different Cell-Cell junctions?
A
- Tight Junctions
- Adherens Junctions
- Desmosome
- Gap Junction
2
Q
What are the two cell-matrix junctions?
A
- Hemidesmosome
- Focal Adhesion
3
Q
Describe appearance and function of tight junctions
A
- Branched and ribbon like
- form a barrier against the outside for e.g. water
- There is selective permeability for specific ions etc, which require specific claudins. E.g. Need to take in mg+, so need claudin 16. if not you will get hypomagnesia
4
Q
What are the main three proteins used in tight junctions?
A
- Claudins, transmembrane protein, drives formation of tight junctions between cells
- Occludin
- JAM proteins, 4 different ones, belong to immunoglobulin superfamily (contain a motif similar to immunoglobulin), have homotypic interactions.
5
Q
What transmembrane protein drives the formation of tight junctions?
A
Claudins
6
Q
What proteins make up tight junctions?
A
1) Claudins
- transmembrane protein, drives formation of tight junctions between cells
2) Occludin
- important for stability
3) JAM proteins
- 4 different ones, belong to immunoglobulin superfamily (contain a motif similar to immunoglobulin)
- Heterotypic and homotypic interactions, which is where one jam protein interacts with the same jam protein on the other call (JAM1 - JAM1) Homotypic
- Heterotypic is with a different Jam protein
7
Q
How is is paracellulasr water flow prevented?
A
By tight junctions. They act as a fence between the apical and basolateral services.
8
Q
How is polarity created across intestinal epithelial cells in respect to glucose absorption?
A
A gradient of glucose, as glucose is co-transported across with sodium.
- Sodium glucose transporter on the apical side
- Passive glucose carrier on the basal side
9
Q
What is homotypic?
A
When a protein will only interact with a protein of the same type. i.e E-Cadherins only interacting with E-Cadherins
10
Q
Describe the components of Adhererns junctions.
A
Adherens junctions
* Two seperate adhesion complexs:
Cadherins
* Homotypic
* E-cadherin, N-cadherin
* P- cadherins, VE-cadherins
* Interact with actin cyctoskeleton via beta catinin and alpha catinin, but can also do it via vinculin and alpha actinin
Nectin
* Form homotypic and heterotypic interactions
* Link to actin cytoskeleton via afidin
10
Q
Describe the components of Adhererns junctions.
A
Adherens junctions
* Two seperate adhesion complexs:
Cadherins
* Homotypic
* E-cadherin, N-cadherin
* P- cadherins, VE-cadherins
* Interact with actin cyctoskeleton via beta catinin and alpha catinin, but can also do it via vinculin and alpha actinin
Nectin
* Form homotypic and heterotypic interactions
* Link to actin cytoskeleton via afidin
11
Q
Cadherin is a component in Adherins junctions and can cause it adherens junction to form in cells that dont normally express them. What ion does it rely on?
A
Calcium
Ca2+
In low levels of calcium, the extracellular domains are really flexible. Addition of calcium causes them to homodimerize and form a stiff rod. further addition of calcium above 1mmol causes the homodimers to interact.
12
Q
What are the two main roles of cadherins?
A
1) Form adherens junctionss
- expression of cadherins causes these to form
- they form the part that interacts with the actin cyctoskeleton
2) Tissue seperating
- they are useful in cell sorting due to being homotypic
- E-cadherins only associating with E, so distinguish themselves from N-cadherins, also works with high concentration and low concentration.
13
Q
What is the link between cancer metastasis and cadherins?
A
Metastasis requires a loss of epithelial integrity.
This is associated with loss of cadherin expression.
14
Q
What is the purpose and structure of desmosomes?
A
Desmosomes
* Provide structural integrity
Transmembrane proteins desmoglein and desmocollin attach to complex containing plakogoblin, plakophilin and desmoplakin
15
Q
What kind of tissue are intermediate filaments found in and why?
A
Tissues that require to be able to resist high force/tearing.
This is because they have a good structural integrity.
16
Q
What is the function of Gap junctions?
What are they made up from?
A
- It allows ions and signalling molecules like cyclic amp, ip3 etc through
- electrical coupling of cells.
- Gap junctions allow neighbouring cells to coordinate/communicate.
- Especially important in places like the heart.
- You can see its importance in the heart by removing connexins, which means the electrical signal cant pass through the myocytes
- is made from 2 connexons, each containing 6 connexins
- pore size is determined by the connexins
17
Q
What is an easy way of determining pore size?
A
Use dyes of different particle sizes.
18
Q
How do gap junctions aid our vision?
A
They help us to see in the dark.
- Gap junctions open in the dark
- This allows light to be picked up from a large number of neurons at once
- Means we can see in low light but is fuzzy
In the light, dopamine is present and we can pick up light from individual neurons, better quality.
Dopmaine controls if Gap junctions are open or closed.
19
Q
What is the function of hemidesmosomes and describe their structure.
A
A cell matrix junction
- link to intermediate filaments
- Provide strong adhesion, in particular epithelial tissues; stratified (skin), psuedostratified (oesophagous) and transitional.
Structure
Extracellular
* Alpha 6, beta 4 integrin pair and Bp180
Intracellular
* Plectin and BP230
20
Q
How do Hemidesmosomes attach to the basement membrane?
A
Hemidesmosomes provide strong attachement to basal lamina. They do this via the integrins attaching to the laminin.
The basal lamina consists of a laminin network and a collagen network. The Hemidesmosome interact with the laminin network which allows recruitment of bp230 and plectin.
- Lamanin network
- Collagen 4 network
- Linked with perlecan and nidogen
- Lacking any of this will mean breaks between epithelium and bit below that, means no epithelium, means no disease prevention or permeability barriers
21
Q
What determines what type of matrix that a focal adhesion adheres to?
A
The integrins.
- Alpha1beta1 and a2b1 integrins interact with collagen
- A6b1 will interact with lamanin
- A4b1 a5b1 interact with fibronectin
22
Q
What are the two functions of focal adhesions?
A
1) Interact with specific ECM via specific integrins
- maintaining tissue structure
- motility
2) Interact with signaling molecules.
- Promote ECM assembly
- When interacting with the correct matrix, they release signals that promote cell survival and cell death if on the wrong matrix
23
Q
What change needs to occur to focal adhesions in order for cell to metastasize?
A
Need to change the integrins they express, so that they can bind to different types of matrix and not release cell senescence signals.
24
Where are selectins found? What is their purpose?
Endothelium, leukocyte, platelet.
They recognize carbohydrates in adjacent cells. (instead of proteins like cell adhesion molecules). This is particularly important for leukocytes recognising sites of infections.
E-Selectins for endothelium
L selectins for leukoctyes
P Selectins for platelets.
25
How do selectin enable to leukocytes to to target sites of infection?
They recognise carbohydrates on the endothelium of the blood stream. At first attach weaklya nd role before attaching more firmly and squeezin between the gaps in the cells or even straight through the middle of the endothelial cell.
The intial capture is dependent on the selectins. Leukocytes have L-selectins, which interact with PSGL-1 in the enodthelial cells.
Cells near sites of infeaction produce give of two signals, interleukin 1 (IL1) and TNF alpha. This causes nearby endothelium to express PSGL-1, E selectin and P selectin creating adhesion.
* adhesion because PSGL-1 interacts with the L-selectins on leukocytes
* and E/P selectins interact with the PSGL-1 on the leukocytes.
* Second step, endothelium produces platelet activating factor and IL 8
* Activates integrins that interact with JAM1 and JAM 2 (proteins found in tight junctions)
JAM proteins causes firm sticking
26
Why is polarity required in enterocytes?
Apical/basal polarity
* Directed absorption and secretion
* Absorb nutrients from gut lumen and secrete into surrounding tissue fluid and on to blood stream
* Glucose brought in via sodium glucose symport
* Diffuses out via passive glucose carrier protein
Apical basal polarity required for this, for the localisation of symport and carrier proteins
27
How is polarity set up? (in epithelial cells)
Apical
* Par3, Par6, aPKC form an apically localised complex
* aPC phosphorylates Par1 and Lethal giant larvae (Lgl)
* Phosphorylated forms cant interact with the membrane on apical side, so Par1 and Lgl cant go near apical side
Basolateral
* however on the basolateral side they are not phosphorylated so form a complex along side disc large and scribble.
* Par1 is also a protein kinase and can phosphorylate Par3
* this means Par3 and its complex cant go on the basolateral side so is restricted to the apical side.
28
How do the Par complexs required for cell polarity get localised in the first place?
The apical complex can be localized through the tight junctions.
The Par3 will interact with the JAM proteins found in the tight junctions (which are always apical)
* Alternatively, via Par6 interaction with crumbs stardust complex which is found slightly above the tight junctions
* Particularly important in invertebrate cells which dont have tight junctions
29
What are some examples of where polarity is needed?
* enterocytes (receptors)
* neurons (axons from dendrites)
* migrating cells (leading from lagging edge)
Planar polarity
* Fly wing, trichomes hairs point from proximal to dismal
* Inner ear stereocillia in mammals, directionality needed for hearing
30
What is planar polarity and how is it formed?
Refers to opposing polarity across the left/right planes of the cell.
* Fly wing, trichomes hairs point from proximal to dismal
* Inner ear stereocillia in mammals, directionality needed for hearing
How?
* Flamingo makes homotypic interactions between cells of the wing epithelium
* This allows frizzled and strabismus to interact and localised frizzled to the distal side of the cell
* Frizzled signals via dishevelled to re-organise the actin cytoskeleton and form the hairs
* Strabismus signalling via prickle prevents hair development in the proximal end of the cell
Meaning hair forms n the distal end but not the proximal
31
Describe primary and secondary neural tube formation.
Primary Neurulation
* Made anteriorly
* Development from the ectoderm
* Columnar neural plate forms from the ectoderm epithelial cells
* Neural plate bends to form a grove, which folds closer.
* Fuse together to make a tube
Secondary nuerulation
* Mesenchymal cells condense
* Form the medullary cord and notochord
* Mesenchymal epithelial transistion (Met) , forms a cavity creating the lumen of neural tube
32
Describe primary nueral tube formation.
33
Describe secondary nuerilation.
34
How is neurulation airded by shroom?
Shroom:
• Shroom localisation to the apical side enables the cells to be made columnar and allow constriction
* Over expression causes too much constriction
* Localisation of gamma-tubulin to the apical membrane
* Nucleates microtubules to grow and elongate the cell
• Stimulates Rho and Rac, contraction of the actin cytoskeleton and nucleation/maintenance of actin cytoskeleton
35
Describe how polarity used in neurilation.
Then explain it/how it works.
Cell polarity
* Cell shape changes
* Cuboid to collumnar then contracting to form pyrimdal
* Using cell polarity
Planar polarity
* relies on the flamingo, Fz, Stbm etc
* allows convergent elongation in the neural tube
* Defines a midline
When scribble is mutated, so polarity isnt established, neural plate doesnt close
(scribble is part of the basal Par1 complex)
Establishing apicobasal polarity localizes the actin binding protein Shroom to the apical membrane. * Shroom localisation to the apical side enables the cells to be made collumnar and allow constriction.
Over expression causes too much constriction.
36
Explain detail the process by which shroom aids neuralation.
* cell polarity is required for neuralation
* Using polarity shroom is localised to the apical side enabling constriction, allowing cells to be made collumnar.
1) After localisation to the apical side, shroom then localises gamma tubulin to the apical membrane, which enables constriction at the apical side of the cell
2) Shroom stimulates Rho and ROCK at the apical membrane which stimulates myosin light chain, causing it to contract.
37
What is the role of planar polarity in Neurilation?
Planar polarity is required in order to establish a midline for convergent evolution for the formation of the neural groove.
* relies on the flamingo, Fz, Stbm etc
In the absence of planar polarity and therefore convergent elongation, the neural groove cant form a V-shaped and is left as a U-shape. This means they cant circle round to fuse.
38
Explain how the neural tubes fuse.
Ephrin A5 and A7 used for adhesion between folds to form neural tubes.
Expressed at the tips.
* Eph - epherin signaling normally used in repulsion
* Also causes bi directional signaling with them both causing signaling pathways
* Neural cells express a non-functional EPhA7 instead so that no bi directional signal is sent
* Allows adherence (yellow color)
* Non funtional able to mediate adhesion not repelling
* Allows neural folds to fuse
39
Outline the cell cycle. Go on. Do it. I dare you.
Cell cycle
Interphase
* Replication
Prophase
* Nuclear breakdown
Prometaphase
* Chromosomes attached
Metaphase
* Chormosomes align
Anaphase
* Chromatid segregation
Telophase
* Chromatids reach poles
* Envelope formatin
Cytokenesis
* Actomyosin ring
* Cells divide
Interphase
* Chromosome duplication
* Cell growth
40
What determines the direction a cell divides in?
The spindles are rotating during metaphase and then when enters anaphase it stops and divides in that orientation.
41
What is the difference between symmetric and asymmetric division?
Symmetric
* produces two equal daughter cells
* used to spread a thin tissue layer
Asymmetric
* 2 different daughter cells, but chromosomes still divided equally
* Used to thicken a tissue layer and creates diversity
examples
* starts more symmetric in mammals and gets more asymmetric as it develops
* c.elegans first embryo division is asymmetric
* Drosphilla embryo undergo asymetric division
42
What is the hertwig rule?
That cells divide along their long axis.
But studies found that if the cell didnt have a long axis, then cell division was random.
Exceptions
* Not always along long axis in some cell shapes
* In polarised cells, can occur against it
43
What are the three types of microtubules in cell division spindles?
Astral microtubules
Kinetochore microtubules
Interpolar microtubules
44
What causes the hertwig rule to occur?
Astral microtubules
* Dynein walks minus end directed along the microtubules
* This pulls on the astral microtubules to position the spindle, generating cortical force
* The Cortical force, rotates the spindle
* Pulling strength is dependent on the length and density of astral microtubules
* Longer astral microtubules are found at the axis that the spindle orients along
* Longer the astral MT, the more dynein, the more pulling strength.
* Longer astral MTs found at the ends, not the corners
45
How does dynein link to the cell cortex?
Dynein linked to the cell cortex via Numa-LGN-G alpha i complex.
This complex recruits dynein.
46
Why does cell division not always occur to hertwigs rule?
Sometimes division occurs in the direction of the subtrate.
47
What decides if the cell division is symmetric or assymetric?
48
When kidney cells are grown on gel, what causes the cells astral spindles to be aligned parallel to the apical surface?
The increased expression of LGN laterally. LGN recruits dynein which pulls on the astral microtubules.
No LGN, reoriention of spindle, not aligning with apical domain, random orientation. Whereas artificially putting LGn on the apical membrane changes allignment by 90*
49
In what way does cell devision in mdck (kidney cells) rely on Par3?
They need polarity in order to localised LGN to their lateral side.
50
What happens when Par 3 is knocked out of mdck kidney cells?
WELL wouldnt u like to know
* They need polarity in order to localised LGN to their lateral side.
* LGN no longer expressed laterally, but is expressed everywhere instead, how strange
* Turns out Gai is not effected
well
* normally, although Gai is everywhere, LGN can only bind and form the complex in the basolateral domain, bcz in the apical domain it gets phosphorylated
* now without Par3, it LGN doesnt get phosphorylated so can bind to Gai everywhere, so the spindle moves randomly.
51
What 4 types of cells make up the small intestine?
* Consists of 4 cell types
Enterocytes:
* Majority of cells on villus
* Absorb nutrients from the gut lumen into the body
Goblet cell:
* Produce mucus, protective and lubricate
Paneth cell:
* Found at the base of the crypt
* Produce set of proteins called defensins which help keep the villi more sterile
Neuroendocrine cell:
* one per villus
* Secrete hormones to regulate peristalsis
* Enable you to feel satiety - feeling full
Turnover due to harsh environment of the gut
* Abrasion of food
* Toxins in food that damage cells
52
What happens to damaged cell
* Eliminated from the tip of villus via apoptosis
1) Chromsomes condense
2) Nucleus will break up
3) Cytoskeleton breaks down (rounds up brings in any projections)
4) Membrane starts to bleb and membrane to be pinched off, taking some cellular contents with it
The neighbours phagocytose the vesical debris of the dead cell
Lost cells are replaced via proliferative cells that are found in the base of the crypt but mainly wall of crypt
Getting rid of damaged cells is importnant to avoid proliferating of mutant cancerous cells.
53
What is apoptosis useful for and and what does it use to do it?
* elimination of damaged and potentially cancerous cells
* getting rid of unwanted cells e.g. webbing between fingers in development
* infected cells
etc
* uses cytotoxic t cell
* doesnt cause inflammation
54
Whats necrosis?
Necrosis is by accident, gets ripped open, causes inflamation in nearby cells
55
Differentiate between the two types of apoptosis.
* Intrinsic apoptosis is when the cell itself recognises that it is damaged, and causes apoptosis on itself
* Extrinsic is when a neighbouring cell like a cytotoxic t cell recognises that the cell is faulty and signalling to the cell, inducing apoptosis.
56
What proteins regulate apoptosis?
Regulated by Bcl-2 family
Three groups:
* Bcl-2 subgroup - anti apoptosis, homology domains conserved (BH1,2,3,4)
* Bak family - 3 of the homology domains, pro apoptosis
* BH3 only family - diverse, sense all types of damage, different types of apoptotic stimuli, feed into common mechanisms,
57
What is the role of the BH3-only group in the bh2 family?
Detect apoptotic stimuli and trigger downstream responses that culminate is caspase release and degradation of the cell.
58
How does BH3 and cytochrome c trigger cell apoptosis?
When there is DNA damage or unreplicated DNA, puma and noxa is activated, which:
* Induces conformational change in Bax
* Increases levels of Bax on mitochondria
* Bax creates a pore in the mitochondrial protein
Allows proteins to escape including Cytochrome C, apoptosis-inducing factor (APF) and SMACK
Cytochrome C
Cytochrome C
* Interacts with APAF1 (apoptosis activating factor) and causes a cluster with caspase 9 called an apoptosome
* Activates them, makes a hectomer makes the apoptosome
* Brings in pro caspase 9 which cleaves the pro domain and leaves fully activated caspase 9
* When caspase 9 is activated it cleaves downstream caspases like caspase 3 (activating them by removing their pro domain)
Caspase 3 then causes downstream responses
* Cleaves actin; cytockeleton breakdown
* Cleaves lamin; nuclear envelop breakdown
* Cleaves inhibitor of caspase activated Dnase; DNA fragmentation
59
Differentiate between initiator caspases and executioner caspases.
Initiator capsases include caspase 9. This is because it is involved in activating the executioner caspase.
An executioner caspase is caspase 3 as when activated;
Caspase 3 then causes downstream responses
* Cleaves actin; cytockeleton breakdown
* Cleaves lamin; nuclear envelop breakdown
* Cleaves inhibitor of caspase activated Dnase; DNA fragmentation
60
How does the cell stop apoptosis from happening at low levels of activation? How is this set up?
They create a threshold on the surface of mitochondria for cytochrome c release.
This is done via bcl-2 release. Bcl-2 sits on the mitochondrial membrane and inhibits Bax to create a threshold that needs to be exceeded to cause apoptosis
* If have too much Bcl-2 then can never cause apoptosis
Diseases such as b cell lymphoma
61
How is the supply of cells in the crypts maintained
Supply of cells maintained by division of stem cells
* Stem cell divides
* Second stem cell (transit amplifying cell) then makes lots of divisions
* Differentiates towards and enterocyte or secretory cell lineage
62
Where are the stem cells, paneth cells and transit amplifying cells? What is their purpose?
Stem cells proliferate into transit amplifying cells before differentiating towards enterocyte or secretory cell lineage2
63
Where are the stem cells, paneth cells and transit amplifying cells? What is their purpose?
Stem cells proliferate into transit amplifying cells before differentiating towards enterocyte or secretory cell lineage. Paneth cells secrete intestinal proteins.
64
How were the cells at the bottom of crypts in the small intestine shown to be stem cells?
* Stem cells identified by the genes expressed (lgr5 achaete, olfacto etc)
Experiment to show cell at bottom of the crypt are actually stem cells
1) Cell is marked that expresses Lgr5 (which we think is produced by stem cells)
2) Wait and see where that cell ends
65
How is progeny of stem cells followed in crypts?
It marks the stem cells using lacZ which turns blue with xgal.
* Constitutive promoter linked to lacZ, allows lacZ to be expressed anywhere in the body, and when given xgal with lacZ expressed, it turns blue
* To localise it ,use a loxP stop loxP casset. This means LacZ isnt translated and nothing turns blue.
* Have this in all cells but cant translate
* If CRE recombines out between the two loxp sites, CRE will recognise that and cause the bit of dna between the two loxp sites to be removed.
* Meaning lacZ translated wherever the CRE is
* To get CRE to be regulated it is fused to the oestrogen receptor so is kept in the cytoplasm until wanted
* Until give Tam (tamoxyphed) when its activated
66
What is a simple way of testing if the cells at the bottom of crypts are stem cells?
Growing the LGR5 cells out and seeing what cells they form.
67
What signals do stem cells require in order to be maintained?
Wnt and notch, both at the same time.
68
Why is more force generated at the posterior cortex?
Theres a higher concentration of LGN found in the cell posterior. This means increased LGN means more pulling on them spindle fibres so all gets shifted.
This also results in assymetrical cell division in c.elegans.
* LGN increase on posterior side casued by cell polarity
* More LGN in posterior where the Par3 complex is absent
* As LGN woudl be phosphorylised by aPKC and wouldnt be able to bind G alpha i
* So cnt generate any force
69
How does asymmetric cell division occur in c.elegans?
The first cell division in c.elegans is asymettric, with p-granules only one side.
* More cortical force at the posterior
* Found by [plasting the centrosome and seeing the posterior explodes more
* The LGN is responsible for increased force generated at the posterior
* The off centre division happens cuz increased LGN at posterior
* Increased LGN means more pulling on them spindle fibres so all gets shifted
70
How is polarity set up in the embryo?
When the sperm nters, it delivers a sperm centrosome. This inactivates Rho Gtpase. * Which allows relaxing of myosin and actin at posterior
* Actomysin network retracts and carriers the PAR proteins away
Creates a AB side and P1 side
This then sets up asymmetric distribution of diterminents which a progenitors for different cell types.
71
What are the potential neuroblast devisions?
* Neuroblast cells are stem cells for the fly nervous system
* But they divide assymetrically to produce the replacement neuroblast and the ganglion mother cell
72
In symmetrically dividing cells LGn numa gai is found laterally, so divides evenly down the middle.
Why is the Lgn complex found laterally in dividing neuroblasts?
INscuitable is bound to the PAr3 complex
Inscrutable then recruits LGN (known as partner of inscuitable PINS, in flies)
72
In symmetrically dividing cells LGn numa gai is found laterally, so divides evenly down the middle.
Why is the Lgn complex found laterally in dividing neuroblasts?
Inscuetable is bound to the Par3 complex
Inscrutable then recruits LGN (known as partner of inscuetable PINS, in flies)
73
What is the role of asymmetric and asymmetric division in the embryo skin development? (broad)
Asymmetric: Produces a different apical cell that goes on to differentiate and generate stratified layers of the skin
Symmetric: expands and maintains the cell population as the embryo grows
74
What fate determents from the neuroblast end up in the ganglion cell when they dividing asymmetrically?
* prospero: Transcription factor that activate pro-neural genes in GMC
* Brat: Tumour supressor that promotes cell cycle exit and differentiation
75
How does budding yeast divide?
Spindle forms inside the nucleus
Spindle pole bodies (MicroTubuleOrganisingCenters) are part of the nuclear membrane
* Bud grows
* Microtubules (black lines) extend into the bud
* Pulling on the microtubules pulls the spindle and nucleus
into the bud
Myosin 5 is responsible for pulling the actin filaments into the bud. It attaches to the microtubules by binding to TIP proteins at th eplus end of the MT.
Once in the bud, dynein which is attached to the plasma membrane via NUm1, pulls on the MTs, dragging one end of the nucleus into the bud.
76
Describe the structure of nuclear pores
77
What are the characteristics of nuclear localisation signals?
1-2 stretches of basic residues.
78
What recognises the NLS?
