SNAREs and cell polarity

Question 1

give some examples of membrane fusion/vesicle being used

Answer 1

NT release
Hepatocytes secrete albumin
Immune cells secrete antibodies and cytokines etc…
Epithelial cells secreting mucus
Intracellular transport of proteins between organelles

Question 2

what three methods were used to understand the mechanics of vesicles?

Answer 2

1. biochemical reconstitution
2. yeast genetics
3. cloning

Question 3

how many SNAREs are there in the human genome?

Answer 3

38

Question 4

how was biochemical reconstitution used to investigate membrane fusion/vesicles?

Answer 4

Jim Rothman - broke up cells and purified the organelles
Focused on the intra-golgi transport
Demonstrated that vesicles bud from the membrane of one golgi sac and fused with the membrane of another, using a viral protein that gets glycosylated in the golgi, but he used radioactive sugars

identified NSF also

Question 5

how were yeast genetics used?

Answer 5

Another dude (Randy Schekman) was looking at yeast genetics and had also discovered a SNARE binding protein, a SNAP and NSF (but named them different things - SEC1, SEC17 and SEC18)

First time releasing genes were conserved between yeast and humans

genetic screening methods to identify yeast mutants exhibiting defective protein secretion.

Then manipulated yeast strains to induce mutations in specific genes, observing the resulting impact on cellular protein secretion - named these these SEC mutants, and pinpointed key genes associated with vesicle trafficking using genetics mapping

Question 6

how was cloning used?

Answer 6

Richard Scheller was investigating the brain and was looking at the pacific electric ray
Identified VAMP (a v-snare) and syntaxin (main component of a t-snare) (by injecting ray vesicles into other species and looking at the antibodies produced or something)

Question 7

how did Jim Rothman pull together the pieces for membrane fusion?

Answer 7

took broken up cells and tagged NSF (an ATPase)
He added a form of ATP that cannot be hydrolysed, allowing him to purify a large complex with all these pieces together - VAMP, syntaxin and SNAP (ATP needs to be hydrolysed for the complex to disassemble)

Question 8

what are V-SNAREs? (one used by at neurons?)

Answer 8

V-SNAREs/VAMPs/R snares (as arginine residues play a key role)

Integral membrane proteins located on the vesicle membrane (transport vesicle).

Common v-SNAREs include proteins like synaptobrevin/VAMP (vesicle-associated membrane protein) in neurons and cellubrevin in non-neuronal cells

Question 9

what are T-SNARES?

Answer 9

T-SNAREs are integral membrane proteins located on the target membrane (plasma membrane or organelle membrane).

Also known as Q-SNAREs as glutamine is central residue of the coil structure
T-SNAREs consist of two different protein families: syntaxins and SNAP-25 (synaptosome-associated protein of 25 kDa).

Syntaxins are integral membrane proteins, and SNAP-25 is anchored to the membrane via acylation

(ff - bit like how Rabs have acyl tail)

Question 10

how do SNARE complexes from?

Answer 10

involves the assembly of v-SNAREs and t-SNAREs in a specific manner.
Typically, one v-SNARE forms a complex with three t-SNAREs, creating a stable four-helix bundle structure. This brings the vesicle membrane close to the target membrane.
Must be 3Q/t-SNAREs : 1R/v-SNAREs
Typically act with SNAREs from the appropriate membranes, however can show some promiscuity. Other molecules like Rabs contribute to specificity

Question 11

what is meant by coiled-coil structure?

Answer 11

The SNARE complex is characterised by a coiled-coil structure, where the helical regions of v-SNAREs and t-SNAREs (already coils) wind around each other, forming a stable complex.

In the t-SNARE, syntaxin provides one coil, SNAP-25 provides two coils.
v-SNARE provides on coil (hence 3:1)
This means the resulting structure is a ‘4-coil’ coil

If you change this ratio is doesn’t work pretty much

Question 12

explain the zippering mechanism?

Answer 12

The assembly of the SNARE complex is often referred to as the “zippering” mechanism, where the coiled-coil structure progressively zippers up along the helices, facilitating the bringing together and eventual fusion of the vesicle and target membrane.

The ‘fusion pore’ can then open

Question 13

what are three common features of SNARE proteins?

Answer 13

Small, 14-40 kDa
All have 1 coiled domain minimum
Generally anchored by the C-terminal

Question 14

what is the role of NSF?

Answer 14

Not required for fusion actually
Membrane fusion happens, SNARE complex forms and vesicle contents are released…

But now the SNARE complex needs to be disassembled in order for it to repeat the process with another vesicle.

NSF does the disassembling of the SNARE complex (using ATP hydrolysis). Without it vesicles will ‘bud’ but cannot then fuse with the target membrane

Question 15

Describe findings of David Suzuki’s flies?

Answer 15

• He caused mutations in flies using chemical mutagenesis, resulting in mutations in genes for dynamin (needed to make endocytic vesicles, and NSF and a-subunit of a voltage gated sodium channel)
• The resultant proteins were thermosensitive, so in warmer temperatures the flies were paralysed/comatose - fell to the bottom of the jar.

This reversed when temperature was returned to normal
Microscopy revealed an accumulation of vesicles at the membrane at the ‘restrictive’ temperature

Question 16

Give an overview of the tetanus and botulinum toxins

Answer 16

• Both extremely potent, only require a few nanograms/kg to be fatal
• Tetanus = bloodborne, found in soil so can’t exactly be eliminated
• Tetanus = extremely intense muscle contractions strong enough to break bone

Botulinum = found in food, causes lack of muscle tone/hypotonia, infants most susceptible (milk to solids) ‘floppy baby syndrome’

Question 17

What are the three highly conserved domains of the TeNT and botulinum (a-g) toxins?

Include their roles and the result they have

Answer 17

Binding domain: recognises and binds to specific receptors on nerve endings at neuromuscular junctions (usually).

Translocation domain: internalisation of the toxin via endocytosis
Protease domain: Cleavage of SNARE proteins - cleaves specific proteins involved in vesicle fusion and neurotransmitter release

Inhibition of Neurotransmitter Release: By cleaving SNAP-25 (or other SNARE proteins), botulinum toxin interferes with SNARE complex, no ACh release at NM junctions, causing paralysis
Its temporary: lasts around a month, as new nerve projections form, and the original nerve ending can regenerate the cleaved proteins as the toxin is eliminated

Question 18

Which SNARE proteins are the targets of which toxins?

Answer 18

boNT-A, E and C = SNAP-25

boNT-B, D, F, G and TeNT= VAMP2

Question 19

How does tetanus cause tense spasms while botulinum causes floppy symptoms?

Answer 19

BonT-B causes floppy - as it works at neuromuscular junctions

teNT = spasms/contractions - not clear how but it gets into the neuron and transported to inhibitory neurons. It inhibits inhibitory neurons = uncontrolled transmission at NM junctions

Question 20

What are some commercial uses of botulinum toxins?

Answer 20

Most treatments based on SNAP-25 and BoNT-A
Cosmetic uses
Uses in spasms, overactive bladder, limb spasticity, GI tract disorders, cross eyes, drooling in conditions like Parkinson’s. Have potential in disorders related to neuronal hyperactivity

BoNT-B used to treat cervical dystonia (involuntary contraction of neck muscles)

Question 21

What were the outcomes in mice when SNARE genes were knocked out?
Why aren’t they all fatal?

Answer 21

For VAMP2, SNAP25, syntaxin 1B = death at birth due to loss of synaptic transmission (couldn’t breathe for example)

Most are fatal, but not all - Syntaxin 1A = no gross abnormalities, subtle defects in synaptic transmission (not the main isoform?)

Question 22

Rare SNARE related diseases – what happens with mutations in syntaxin 11?

Answer 22

Familial hemophagocytic lymphohistiocytosis

• a q-SNARE (t-SNARE) that is ‘immune specific’
• Very rare, mostly affecting children and infants
• Results in increased immune proliferation, cytokine storm (often fatal)

Syntaxin 11 issues can specifically cause defective ‘degranulation’ in cytotoxic T-cells (they can’t secrete their lytic granules into pathogens)

Question 22

What rare SNARE related disease is related to VAMP2 and why isn’t it fatal?

Answer 22

Babies had hypotonia
Mutations are usually occurring in coil domain
Neurodevelopmental disorder with autistic features
VAMP2 is not the main isoform

Question 23

Explain the experiment done to see whether the VAMP2 mutation was the cause of the symptoms being seen?

Answer 23

Used purified liposomes - artificial vesicles basically, you insert your VAMP2 into some and your Q SNAREs into others. You add a dye that, when diluted/becomes quenched and fluoresces. So when membrane fusion occurs, dye diffuses and is diluted across the two membranes, more fluorescence = more membrane fusion

Tested two of the specific mutations found in patients. One was basically same as WT, however this does not mean it isn’t involved in causing the disease, many other proteins are involved in regulating SNAREs and membrane fusion that aren’t present in the experiment, the mutation could perhaps block one of these interactions
The other mutation had significantly lower fluorescence = so was preventing proper formation of SNARE complex formation

Question 23

What is meant by cell polarity?

Answer 23

Refers to the organisation of proteins inside cells and at the plasma membrane. Different regions have distinct protein compositions, altering shape of cells (morphology) and function

Question 24

What are hemidesmosomes and desmosomes?

Answer 24

Basolateral domain remains in contact with the basement membrane via hemidesmosomes

Desmosomes form tight junctions between epithelial cells, acting as a barrier to diffusion of proteins and movement of lipids in the outer leaflet

Question 25

What technique is often used to view cell polarity?

Answer 25

Immunofluorescence – labelling protein markers – proteins localised at apical, lateral, basolateral domains etc…

Question 26

What are the intrinsic and extrinsic routes for cells dividing to produce different lineages?

Answer 26

Intrinsic route - Two proteins/molecules, are unevenly distributed, e.g. A localised at one pole and B at the other, so when division occurs the resulting daughter cells inherit different levels of each molecule, resulting in different properties/lineages

Extrinsic route -
Different environmental signals e.g. growth factors cause originally identical daughter cells to develop into different cell types

Question 27

What was shown/investigated by:

1. Whitman’s leeches, 1878
2. Conklin ascidina oocytes
3. C.elegans and drosophila?

Answer 27

1. Showed that in cell division the two daughter cells could have different protein compositions in development
2. Followed proteins that were distributed differently depending on the tissue type
3. used to understand polarity proteins (par proteins)
   Worm - has defined number of cells
   OG cell (progenitor cell) goes through many asymmetric divisions to give rise to different cell types

Question 29

What group of proteins are needed for asymmetric cell division?

Answer 29

par genes were discovered, proteins that, when defective, mess with this process
There are Par proteins 1-6, with a seventh one being an aPKC (atypical protein kinase C)

Question 30

How is asymmetry achieved by Par proteins and immediately upon fertilisation?

Include details on the MTOC

Answer 30

Symmetry of a cell and the distribution of things like par proteins is broken as soon as sperm enters
* where the sperm enters = now the posterior pole
* Sperm provides microtubule organising centre (MTOC) to the posterior pole
* This MTOC recruits some par proteins - par 1 and 2, that end up at the posterior end

Results in distinct localisation of the par proteins (they all have their own place), some recruited by the MTOC @ the posterior pole (par 1 and 2), others at the anterior end (par 3, 6 and 7 - the PKC) par 5 maintains the boundary between the two

Question 31

Define cortex in terms of a cell

Answer 31

plasma membrane-associated part of the actin cytoskeleton

Question 32

What is the role of phosphorylation in creating asymmetry within a cell?

Answer 32

• Kinases (some of which are par proteins) at the apical side phosphorylate components that are meant to go the basolateral side, preventing basolateral proteins from associating with the apical membrane, therefore ensuring they can go where they are meant to. THIS CAN GO BOTH WAYS

This phosphorylation can work in a feedback loop (apical proteins phosphorylate basolateral ones so that the baso ones don’t bind to the apical, allowing more apical proteins to accumulate on the apical side of the cell, phosphorylating more baso proteins etc…)

Question 34

What is the role of the cytoskeleton in regards to Par proteins?

Answer 34

• Interactions between microtubules and the cortex results in pulling forces on the mitotic spindle, which causes the spindle to be displaced TOWARD the posterior end of the cell.

The actin-myosin cytoskeleton is also essential in redistributing the Par proteins (and other cell fate determinants) as needed

Question 35

how are epithelia an example of the importance of cell polarity?

glucose absorption?

Answer 35

great barrier, limits ion flow with tight junctions, great to keep out bacteria in places like the gut
Ep cells have a trafficking function. Regulated secretion of different components

Transcellular transport of glucose -
An example of the importance of cell polarity. Different proteins are used on either side of the cell (we know this) and therefore these transport proteins must be the right way around

Question 36

how do bacteria demonstrate cell polarity when dividing?

Answer 36

Also show use of Par proteins and the actin cytoskeleton to segregate material

E.g. bacteria must try to ensure both daughter cells get some plasmids -
Par M - an actin homologue - interacts with par R which will associate with a plasmid, in order to get plasmids at different poles before division

Question 37

what is cell polarity like in cancer cells?

EMT?

Answer 37

Cell polarity has to be maintained a lot - there are lots of areas it can go wrong

Advanced tumours = loss of polarity = invasive/malignant properties

apical/basolateral polarity is essential for functional epithelia. EMT = epithelial to mesenchymal transition = a breakaway from neat layer, allowing epithelial cells to metastasise/move into the blood

Question 38

normal cells were compared with cancer cells to see if a certain protein (amotl) was involved in the cytoskeleton’s role in apical/basolateral polarity.

what would be a good idea to test/investigate?

Answer 38

1. is there difference in the expression of the protein in healthy vs tumour cells?

low levels of the protein were found in healthy cells and tumour cells that maintained polarity, but levels were high in tumours with lack of polarity

Step 2 - interfering with the protein to see if it’s function is actually affecting tumour progression or if it just happens to be there

looked at polarity by looking at apical marker protein

high levels of Amotl = apical marker protein not where it should be (trapped in vesicles instead)

Step 3 - Is this consistent in vivo? - looked at mice, put tumours cells in mice expressing amotl vs not, no amotl cells resulted in tumours with maintenance of polarity and reduced metastasis

consider controls that could be used e.g. cells with the protein in a non-functional form