Cell and Molecular Flashcards
(278 cards)
1
Q
What is the cell cycle clock?
A
Network of interacting proteins in the nucleus that recieve signals from outside and inside the cell, integrates them and decides the cell’s fate (i.e. proliferate or quiescence?)
2
Q
What are the 5 stages of the cell cycle?
A
G1 phase S phase G2 phase Mitosis (prophase, prometaphase, metaphase, anaphase, telophase) Cytokinesis
3
Q
What are the two major control factors of the cell cycle?
A
- Cyclins drive cycle forwards
2. Checkpoints stop the cycle
4
Q
What are the CDK and cyclin pairings throughout the cell cycle?
A
G1: CDK4/6 --> D cyclins G1 after R point: CDK2 --> E cyclins S: CDK2 --> A cyclins (although later CDK2 replaced by CDC2) G2: CDC2 --> B cyclins M: CDC --> B cyclins G0 to G1: CDK3 --> C cyclin
5
Q
Why are D-type cyclins an exception?
A
They are controlled by extracellular signals such as growth factors and integrin-mediated ECM attachment unlike other cyclins which have intracellular signals that coordinate with the cell cycle, activating complexes of the subsequent phase and inhibiting those active in previous phases.
6
Q
What is the role of CKIs?
A
They inhibit and regulate CDKs
7
Q
Where are the three checkpoints and what does each one do?
A
G1 checkpoint: checks cell size, environment and DNA damage
G2 checkpoint: checks DNA replicated, cell size and environment
Metaphase checkpoint: checks all chromosomes are aligned on the spindle
8
Q
What are the processes in the G1/S restriction point progression?
A
The CDK4/D causes phosphorylation of pRb. CDK2/E causes further phosphorylation. pRb releases E2F TF which causes gene transcription.
9
Q
What are the three places where DNA damage is detected and acted upon to stop the cell cycle?
A
G1
Entry into S-phase
Entry into mitosis
10
Q
What processes occur when DNA damage is detected and what happens after this is G1?
A
ATM/ATR get activated and associate with the site of DNA damage. ATM/ATR will activate other kinases to block the cell cycle. p53 is stabilised and turns on p21 (a CKI).
p21 renders G1/S-CDKs and S-CDKs inactive and repairs DNA. If this is not possible cell will undergo apoptosis.
11
Q
What extracellular signals determine if a cell will move past the restriction point?
A
If the serum and growth factors are removed in the final hour of G1 then the cell will proceed. If they are removed before they will revert back to G0.
12
Q
What is the process of DNA replication in S phase to ensure it is only replicated once and is done so accurately?
A
In G1 phase, inactive helicases are loaded onto replication origins forming a PreRC. This is called licensing and happens in G1 phase therefore it can only occur once. The helicases are activated in S phase and DNA is unwound and replicated. M phase triggers chromosome segregation.
13
Q
What are homologous chromosomes and how do they relate to chromatids?
A
Chromosomes that have the ‘same’ genes arranged in the same order - one inherited from the father, 1 from the mother. Chromatids are the newly copied DNA strands that are still joined to each other via a centromere.
14
Q
Why is yeast used a genetic model for the cell cycle?
A
It divides rapidly <1hr
Its control genes are highly conserved
It can be grown as haploids or diploids therefore mutations can be maintained from haploid to diploid as only one parent cell
15
Q
Why is Xenopus laevis used as a biochemical model for the cell cycle?
A
Easy to collect eggs
Rapid division rate (~30 mins)
Large size makes purification of proteins easier (lots of cytoplasm)
Can be manipulated by injection of RNAs or chemicals into the oocyte
16
Q
What genetic tricks can you use to identify mutations?
A
Diploids can be used to maintain mutations that are studied as haploids i.e. if the haploid cell dies, the diploid will still have the mutation and can be observed
Temperature sensitive mutations allow growth at permissive temperatures but mutation will show at restrictive temperatures
17
Q
What does M-Cdk cause?
A
Assembly of mitotic spindle
Each sister chromatid is attached to an opposite pole
Chromosome condensation
Breakdown of the nuclear envelope
Rearrangement of the actin cytoskeleton and Golgi
18
Q
How does M-cyclin trigger entry into mitosis?
A
M-cyclin levels increase through G2 and M (by increase of Cyclin B expression) to create a pool of inactive M-Cdk complex. This is activated by CAK and Cdc25. The active M-Cdk acts in a positive feedback loop.
19
Q
What is APC and what are its targets?
A
Anaphase-Promoting Complex is a ubiquitin ligase.
It targets S+M cyclins to make sure the previous complexes are inactive
It targets securin (protects protein linkages holding sister chromatids together) by activating a protease which separates the sister chromatids (anaphase)
20
Q
What is loss-of-heterozygosity and hemizygosity and why could this cause problems?
A
Loss-of-heterozygosity is where both chromosomes will have the same allele e.g. both mutant.
Hemizygosity is where there is a loss of one allele.
This can be a problem as if one allele has a mutation there is no healthy allele to replace its function.
2-hit hypothesis suggests most genes need two mutations to cause a phenotypes.
21
Q
What is chromosome non-disjunction and when would it occur?
A
Chromosomes ending up in the wrong daughter cell and occurs in anaphase by a lagging chromosome. Results in too little or too many chromosomes.
22
Q
What is the structure of the mitotic spindle?
A
Interpolar microtubules (spindles that overlap) Kinetochore microtubules (attach to chromosomes at kinetchores/centromeres) Astral microtubules (contact cell cortex to position the spindle by connecting to plasma membrane) Centrosome (centriole surrounded by pericentriolar matrix, this acts to nucleate microtubules)
23
Q
How are inappropriate attachments sensed?
A
By trial and error. Tension is generated when there is a correct attachment. If the attachment is incorrect there will be lower tension, an inhibitory signal which loosens the microtubule attachment site and it attemps to attach again.
24
Q
What can cause loss-of-heterozygosity?
A
Nondisjunction
Mitotic recombination
Gene conversion
25
What are the processes of meiosis in the ovary and testes?
Repeated mitotic division of diploid cells with growth an differentiation to produce oogonium (ovaries) and spermatogonium (testes).
Meisosis I the primary oocyte/spermatocytes (4n) divide into secondary oocytes/spermatocytes (n).
Meisosis II they divide into (one) mature egg or (four) spermatids (n).
26
What happens during meiosis I?
Pairing up of duplicated maternal and paternal homologs (prophase I)
Homolog pairs line up on the spindle. (metaphase I)
Segregation of homologs at anaphase I as one complete chromosome (2 chromatids) pulled to separate poles (telophase I)
27
What happens during meiosis II?
The sister chromatids line up and sperate in Anaphase II.
28
When do homologues pair up and why is this important?
In meiotic prophase I.
Alligns the chromosomes up ready for anaphase (along with the formation of the synaptonemal complex)
It allows for genetic recombination between paternal and maternal DNA on the same chromosome
29
What is the synaptonemal complex?
Proteins which facilitate pairing by bringing them 400nm apart.
The axial core (proteins that bind chromatin via cohesin) are cross-linked by transverse filaments.
30
What is the main difference between meiosis and mitosis and what allows for this difference?
Mitosis = sister chromatids separate
Meiosis = homologs separate
--> In meiosis, both kinetochores attach to the same spindle pole. This is done by protein complex removed after meiosis I by crossing over and during anaphase I the cohesin is removed from the arms but still connects the kinetochores
31
How is crossing over regulated?
At least 1 crossing over per bivalent but no more than 4
| Crossover interference where once one forms it inhibits others close by
32
What are two chromosome abnormalities?
Abnormalities in chromosome number (aneuploidy, can be monosomy, trisomy or polyploidy)
Chromosome structural rearrangements
33
How does nondisjunction in meiosis I compare to in meiosis II?
Meiosis I there will be a pair of homologous chromosomes in one daughter cell and a lack of that chromosome in the other. All gametes will be abnormal.
In meiosis II there will be a pair of sister chromatids in one daughter cell and a lack of this chromatid in the other. Half of all gametes will be abnormal.
34
What are the names for types of numerical chromosomal abnormalities?
Polyploidy --> triploid
Aneuploidy (autosomes) --> nullsomy (missing a pair of chromosomes), monosomy (missing one chromosome), trisomy (one extra chromosome)
Aneuploidy (sex chromosomes) --> additional sex chromosome or lacking a sex chromosome
35
What are syndromes arising from nondisjunction?
Trisomy 22 --> usually die before birth
Trisomy 18 (Edwards syndrome) --> usually die before birth
45 X Turner's syndrome (monosomy) --> 1% survive
36
Why are syndromes arising from nondisjunction lethal?
Haploinsufficiency i.e. the gene is usually expressed from both alleles with dose mattering. If inactivation of one allele then dose not enough and lose expression.
Imprinted genes i.e. monoallelic expression becomes lost
37
What is the karyotype?
The organised representation of all the chromosomes in a eukaryotic cell at metaphase
38
What is the structure of a chromosome and how does it differ during interphase?
A chromosome is a highly coiled fibre of chromatin. In interphase chromatin is uncoiled and nucleosomes are exposed.
39
What is the structure of the nucleosome?
DNA is wound round core histones. The histone is made up of 2 copies of 4 subunits and N-terminal tails from the 8 subunits project out of the nucleosome core and are free to interact with other proteins, facilitating regulation of chromatin structure and function.
40
What are the role of linker histones such as H1 and how do they function?
It stabilises the 30nm formation and facilitates the establishment of transcriptionally silent heterochromatin.
They strap DNA onto histone octamers and limit movement of DNA relative to the histone.
41
What are fractal globules comprised in interphase chromatin?
Dynamic globules within globules that can reversibly condense and decondense without becoming knotted.
42
Is transcriptional activation of a gene accompanied by movement from the periphery towards the centre or from the centre to the periphery of the nucleus?
From the periphery towards the centre of the nucleus
43
What are the different regions of the chromosome, where are they and what is their function?
Telomere - DNA sequences at the ends of linear chromosomes, maintain chromosomal integrity
Replication origin - any sites where replication can be initiated
Centromere - interior location but is not necessarily the midpoint, indirectly attaches to mitotic spindle and mediates chromosome segregation at mitosis and meiosis
Kinetochore - protein complex that binds centromeric DNA sequences and microtubules of mitotic/meiotic spindle
44
What is the structure of the kinetochore and chromatin formation during cell division?
Centromeres contain alpha-satellite DNA repeats that readily form condensed chromatin with histone octamers containing H3 variant.
The kinetochore inner plate proteins bind to the chromatin containing alpha-satellite DNA and the outer plate proteins bind to microtubules
45
What percentage of the DNA sequence of eukaryotic genomes encodes information for making cellular proteins?
1.5%
46
What is increasing biological complexity associated with in terms of DNA?
Increasing number of protein-coding genes
Increasing amounts of non-protein-coding DNA for regulating transcription and organising access to protein-coding genes (e.g. cis-regulatory which determines where and when in the body adjacent protein-coding genes are transcribed)
47
What are transposons and what are the 3 different types?
Mobile genetic elements that jump around the genome - also called 'transposable elements'. These take up almost half of the genome.
DNA transposons
Retroviral transposons
Non-retroviral polyA retrotransposons
48
How do DNA transposons integrate into the genome?
By a cut-and-paste mechanism without self-duplication. Transposase monomers (encoded in the transposon) cleave both ends of the DNA transposon element. The transposome becomes a central intermediate and is integrated into another chromosome.
49
How do retroviral transposons integrate into the genome?
Entry of the virus into the cell and RNA is released. Self-encoded reverse transcriptase makes DNA/RNA and then DNA/DNA double helix. The DNA is then copied and integrated into the genome, transcripted and translated to make new virus particles.
50
How do non-retroviral PolyA retrotransposons integrate into the genome?
By a copy-and-paste mechanism. RNA is synthesised from the DNA. Reverse transcriptase/endonuclease are synthesised and bind to the RNA. Cleavage of the first strand as DNA-primed reverse transcription occurs. The multistep DNA synthesis process produces the second DNA strand.
51
How is DNA always replicated?
In a 5' --> 3' direction by formation of phosphodiester bonds.
The leading strand is continuous whereas the lagging strand is discontinuous
52
What are key enzymes involved in converting Okazaki fragments into a continuous strand of DNA?
```
DNA primase - makes RNA primer
DNA polymerase - extended RNA primer but requires the primer-template junction
Ribonuclease H - removes RNA primer
DNA polymerase - extended across gap
DNA ligase - seals the nick
```
53
How is the processivity (= ability to catalyse consecutive reactions without releasing the substrate) of DNA polymerases enhanced?
By their association with a sliding clamp.
| It is positioned close to the primer:template junction by a clamp loader and helps move the DNA Polymerase forward.
54
What is the role of SSBs?
Single-stranded binding proteins expose single-stranded DNA in the replication fork, making it available for templating synthesis of the new DNA strand, easing replication fork progression and enhancing progressivity of DNA polymerase.
55
How do DNA topoisomerases prevent DNA from becoming tangled during replication and enhance processivity of DNA polymerase?
When helicase unwinds parental DNA strands at the replication fork, superhelical tension is introduced into the rest of the DNA helix which would cause tangling. DNA topoisomerases nick and reseal the backbone of the parental helix.
56
What are the two types of topoisomerases and how do they differ?
Type I - nicks and reseals one of the 2 DNA strands, does not require ATP
Type II - nick and reseal both DNA strands, ATP required
57
What is the origin of replication?
Specific DNA sequences where DNA replication starts by recruitment replication of initiator proteins
58
When do the two stages of biphasic initiation of DNA replciation in eukaryotes occur?
Replicator selection occurs in G1 phase
| Origin activation occurs in S phase
59
How is the Pre-Replicative Complex formed?
The origin recognition complex binds to the replicator sequence. Helicase-loading proteins Cdc6 and Cdt1 bind to the ORC. The Helicase Mcm2-7 binds to complete formation of pre-RC.
60
How does increased levels of Cdk in S-phase affect the pre-RC?
When Cdk activity is high, existing pre-RC is activated and formation of new pre-RC is inhibited
61
How does telomerase fill in the overhang where the RNA primer is removed to prevent chromosome shortening?
Telomerase adds TTAGGG repeats to 3' end to compensate for the loss of telomere sequences. This means DNA primase can bind and initiate new RNA primer synthesis.
62
What is the structure of telomerase and how does this allow for addition of multiple TTAGGG repeats?
Is a ribonucleoprotein with an intrinsic RNA component that acts as a template on which telomere repeat sequences are synthesised by the Telomerase Shuffle.
63
What are examples of things that can cause DNA damage?
```
Thermal degradation (heat/water)
Metabolic byproducts (oxidation)
Environmental substances (benzopyrene)
Radiation (UV, nuclear fission)
```
64
Which DNA bases are purines and which are pyrimidines? What are the differences between these?
The purines are Guanine and Adenine.
The pyrimidines are Cytosine and Thymine.
Purines have a double ring structure.
65
How many pairs of hydrogen bonds do each DNA bases have?
G - C have 3 pairs of H bonds
| A - T have 2 pairs of H bonds
66
How can deamination of DNA lead to damage?
Deamination of cytosine causes it to change to uracil. This will have higher affinity to adenine and can cause a transition mutation.
67
Why are transition mutations more likely to occur than transversions and which is less likely to result in amino acid substitutions?
Subsituting a double ring structure for another double ring structure is more likely than substituting a double ring for a single ring and vise versa.
Transitions are less likely to result in mutations.
68
How can frameshift mutations have a significant impact?
They generate missense proteins or could produce or get rid of stop codon
69
What can UV light induce (in terms of DNA damage)?
Formation of pyrimidine dimers (these are the primary cause of melanomas)
70
How is depurination (loss of purine base) resolved by the BER pathway?
The BER (base excision repair) pathway:
Glycosylase recognises and removes incorrect base but leaves backbone
AP endonuclease and phosphodiesterase remove sugar phosphate
DNA polymerase adds new nucleotide, DNA ligase seals the nick
71
How are pyrimidine dimers resolved by the nucleotide excision repair pathway?
Nucleotide excision repair pathway (NER):
The region around the pyrimidine dimer (~10 nucleotides long) is cut out by excision nuclease
DNA helicase peels away the region
DNA polymerase and DNA ligase fill in the complementary strand
72
How do translesional DNA polymerases aid replication when there is damage and why are they a last resort?
When sliding clamp and replicative DNA polymerase encounter DNA damage, the sliding clamp undergoes covalent modifications and loses affinity. They are both released from the DNA strand and translesional DNA polymerase is loaded by assembly factors. DNA is synthesised until covalent modifications are removed and normal DNA synthesis can continue.
Translesional DNA polymerases lack precision in template recognition and substrate base choice and exonucleolytic proof-reading activity. They are also a cause of most base substitution and single nucleotide deletion mutations.
73
How can a double strand break be repaired by non-homologous end joining?
The ends are recognised by Ku heterodimers and additional proteins DNA-PK and ATM PKs are added for processing of DNA ends. There is limited repair synthesis, only ligation therefore repaired DNA usually has a deletion of nucleotides
74
How can a double strand break be repaired by homologous recombination?
Nuclease digests the 5' ends of the break. The strands exchange by complementary base-pairing with another double stranded DNA molecule to form a Holliday junction. The undamaged DNA acts as template and can repair breakage by DNA polymerase. This invading strand is then released and DNA synthesis of other strand occurs. DNA is ligated and completely accurate DNA is repaired.
75
What are some examples of human diseases caused by failures in DNA repair systems?
```
Breast and ovarian cancer (e.g. BRAC1 and 2)
Colon cancer
Skin cancer (e.g. XP variant)
```
76
What are characteristics variations involved in communication?
The number of cells which see and then actually respond to signal
The duration of signal
The method of signal
How far signal can travel
77
What are the different types of signals?
Contact-dependent
Synaptic
Paracrine
Endocrine
78
How do cells respond to an extracellular signal molecule?
Receptor protein detects signal
Intracellular signalling proteins (signalling cascade)
Effector proteins e.g. metabolic enzyme, gene regulatory protein, cytoskeletal protein)
Effect (e.g. altered metabolism, althered gene expression, altered cell shape or movement)
79
Why is signalling important and what are two examples of when signalling can go wrong?
If signalling goes wrong it can lead to diseases such as cancers
1. Mutation JAK2 V617F of JAK2 means its signalling is more active leading to myeloproliferative neoplasms (blood cancers)
2. Mutation BCR-Abl causes sustained Abl tyrosine kinase expression leading to chronic myeloid leukaemia
80
How do positive and negative feedback loops regulate signalling and what is an example of each?
Positive feedback works to increase the response quickly and to the max response (i.e. A --> B --> A --> B etc, switch mechanism). e.g. Notch pathway lateral inhibition
Negative feedback has a slower response so max response can be reached before it feeds back to inhibit the pathway (i.e. A --> B --| A, button mechanism). e.g. JAK/STAT pathway down regulation by SOCS negative regulator
81
What are two examples of modifications that can act as switches on existing proteins and what enzymes modulate these?
Phosphorylation - protein phosphatase (OFF) and protein kinase (ON)
GTP-binding - GAP (OFF) and GEF (ON)
82
What are two major groups of protein kinases in eukaryotes?
```
Serine/threonine kinase (S/T)
Tyrosine kinases (Y)
```
83
What are the 4 types of kinase inhibitors and how does each of them function?
Type I - binds to the active conformation of kinase with aspartate residue of the DFG motif pointing into the ATP-binding pocket
Type II - bind and stabilise inactive conformation of kinase with flipped aspartate residue facing outward of binding pocket
Type III - occupy an allosteric pocket that is adjacent to the ATP-binding pocket but does not overlap with it
Type IV - binds to an allosteric pocket remote to the ATP-binding pocket
84
What are key features that differ between ion channels?
Gating mechanism
Ligand or voltage
Ion selectivity of pore (defined by physical size of filter and amino acids lining the pore)
85
What is the P loop of an ion channel and what is it important for?
A hydrophobic loop between two TMs that does not go all the way through membrane.
Important for selectivity as acts as a filter
86
How does the voltage-gated ion channel become inactivated?
An inactivation peptide can swing in and block the open pore
87
What do TRP channels sense and which channel type is the structure similar to?
Chemicals and physical (e.g. temperature) stimuli.
| Structure similar to voltage-gated ion channels
88
What are ligand-gated ion channels gated by and what are different types (including how their structural components differ)?
Chemical transmitters (intercellularly generated or extracellular).
Cys-loop type - pentameric assembly e.g. nAChR, GABAa, 5-HT3
Ionotropic glutamate type - tetrameric assembly e.g. NMDA
P2X type - trimeric assembly e.g. P2XR
89
How do voltage-gated ion channels and ligand-gated ion channels work together to control the excitability and function of muscle and neurons?
Voltage-gated ion channels are responsible for AP generation and release of NT.
Ligand-gated ion channels detect NTs leading to the response in the cell.
90
What are three families of glutamate receptors caused by different genes, alternative splicing and RNA editing?
AMPA
Kainate
NMDA
91
What happens when nAChR is mutated or lost e.g. by autoimmune diseases?
nAChR receptors are present on skeletal muscle therefore will cause myasthenia gravis (muscle weakness).
They are also present in the CNS therefore overstimulating mutations can cause ADNFLE (epilepsy)
92
What are structural features of GPCRs?
The characteristic 7TMs (alpha helices) of all GPCRs are packed in a similar way
TM3 is centrally located next to binding pocket as is crucial for transduction of ligand binding
Other TMs and extracellular N' terminus also contribute to ligand binding (extended N' terminus = big peptide recognition)
93
What are PAR receptors activated by?
Protease cleaving (e.g. thrombin)
94
What are the basic principles of GPCR signalling?
Resting (three inactive components)
Activation by ligand binding causes conformational change, movement of TMDs opens cleft for alpha subunit to bind and exchange of GDP for GTP
GTP binding reduces affinity for beta and gamma subunits so they dissociate and begin intracellular signalling
95
How is duration of trimeric G protein signalling regulated?
By rate of GDP hydrolysis by Ga
96
How is the local concentration of 2nd messengers (e.g. 3'5'-cyclic AMP and 3'5'-cyclic GMP) determined?
Rate of production (e.g. by adenylyl cyclase)
Rate of removal (e.g. by phosphodiesterase)
Rate of diffusion from site of production
97
What are examples of effectors of G proteins?
Adenylyl cyclase (inhibit or stimulate)
Phosphodiesterase
Rho (or other small molecule proteins)
Phospholipase
--> enzymes which create 2nd messengers and ion channels
98
What is desensitisation?
Where the agonist may still be present but the receptor will not respond. Prolonged or frequent activation by stimulus can induce receptor desensitisation.
99
How is the beta 2 adrenoreceptor for regulation of metabolism in liver and skeletal muscle activated and what are its effects?
Stimulated by a ligand e.g. epinephrine or norepinephrine. Receptor activates trimeric GTPase and this activates adenylyl cyclase. This converts ATP to cAMP which activates PKA. This phosphorylates PK which is then activated by Ca2+. PK phosphorylates phosphorylase which makes glucose-6 P.
100
What are some mechanisms which can switch off signalling?
```
Agonist dissociating from receptor
GTPase activity of Gas
cAMP breakdown by phosphodiesterase
Dephosphorylation of enzymes
Negative feedback via PKA, B-arrestin, GRK
```
101
What are examples of activating mutations in GPCRs that can cause disease?
Parathyroid Ca2+ sensor --> hypoparathyoidism
Rhodopsin --> night blindness
Thyroid hormone receptor --> hyperthyroidism, thyroid cancer
102
How are lipid-derived second messengers produced?
Receptor regulated lipases (e.g. GPCRs activate PLC) target membrane lipids. Two types of 2nd messengers can be generated:
1. water soluble and so can diffuse through cytoplasm e.g. IP3
2. hydrophobic so remain in membrane e.g. DAG
Lipid kinases then add phosphate groups to lipids
103
What are PKCs, what activates them and what occurs when they are activated?
Protein Kinase C
These are Ser/Thr kinases and are activated by DAG (C1 domain) and Ca2+ (C2 domain - not present in all forms).
Substrate binding site becomes exposed as DAG causes dissociation of intramolecular pseudosubstrate domain from active site. Can then bind to ser/thr and transfer terminal phosphate of ATP.
104
What is an example of a downstream effect of GqPCRs (involving Munc13)?
GqPCRs and synthesis of DAG in plasma membrane recruits Munc13 (a C1 domain containing protein) to the membrane and stimulates secretory vesicle docking to the plasma membrane, preparing it for fusion
105
How does mutations in signalling cause Lowe Syndrome?
Mutations in OCRL which codes for inositol polyphosphate 5 phosphatase alter PIP2 levels which are normally tightly regulated by many enzymes
106
How are calcium ER levels refilled and maintained?
When Ca levels fall below critical level it leads to conformational change in STIM (from dimer to oligomer) as it moves to the plasma membrane and joins with Orai channels. These open and Ca is pumped from the ECF into the ER.
107
How can desensitisation of G protein coupled receptors occur and what does this mean?
Phosphorylation of receptors, used for negative feedback, can contribute to desensitisation as become uncoupled and unresponsive to agonist.
108
What is adrenaline, where is it produced and what is its role?
Adrenaline is a water-soluble, non-steroid hormone produced by the adrenal glands (mostly).
It acts via adrenergic receptors to produce a wide range of physiological responses including the fight or flight response.
109
What are the two structures that play key roles in the maintenance of homeostasis and act as gateways linking the neuronal and endocrinological systems?
Hypothalamus and pituitary gland
110
What is cholesterol and what is it a precursor of?
A lipid with an -OH group therefore is an alcohol. It makes up around 30% of all cell membranes.
Is a precursor of a range of steroid hormones e.g. cortisol, estradiol, testosterone, vitamin D3
111
What is the structure of steroid hormones and what does this allow them to do?
They have both hydrophilic (-OH group) an hydrophobic (lipid) properties.
They are therefore able to penetrate through the BBB
112
What are the two classes of steroid hormones, where are they typically made and what are the 5 subtypes?
Corticosteroids --> made in adrenal cortex
Sex steroids --> made in gonads or placenta
```
Subtypes: Glucocorticoids Mineralocorticoids
(--> corticosteroids)
Androgens
Oestrogens
Progesterones
(--> sex steroids)
```
113
What is the primary structure for nuclear receptors that detect steroid hormones?
```
N-terminal domain
DNA binding domain (encodes the zinc finger that contains 4x cystine residues which co-ordinate with a zinc atom to form a looped structure that is able to access the major groove of the DNA double helix)
Hinge region (controls movement of receptor to the nucleus)
Ligand-binding domain
C-terminal domain
```
114
What happens when a ligand binds to a nuclear receptor?
The ligand activates the protein (often coactivator proteins bind too) and it binds to DNA.
The receptor-steroid-hormone complexes activate primary-response genes induces synthesis of primary-response proteins.
The primary-response proteins shut off primary-response genes and turns on secondary-response genes.
These secondary-response proteins elicit cellular responses.
115
How does the release of cortisol (the most important glucocorticoid) occur?
The hypothalamus releases corticotropin-releasing hormone (CRH). This acts on the pituitary gland which releases adrenocorticotropic hormone (ACTH) into the bloodstream. It interacts with the adrenal gland which then triggers production and release of cortisol.
116
What are glucocorticoid class steroid hormones involved in and what will happen if there is too little cortisol (e.g. damage to the adrenal glands or lack of ACTH) or too much cortisol (e.g. by a benign adenoma in pituitary gland leading to increased ACTH production or steroid abuse)?
Released in response to stress and reduced blood sugar levels. It causes long term adaptations to stress and affects metabolism, immune system, electrolyte balance and memory.
Too little cortisol can lead to Addisons Disease. Symptoms include depression, flu-like symptoms, nausea, weight loss and Addisonian crisis (where cannot respond to severe stress e.g. accident, operation).
Too much cortisol can lead to Cushings Syndrome. Symptoms include weight gain, raised blood pressure, puffy face, hair growth.
117
What is the structure of the insulin receptor?
Hetero-tetramer linked by di-sulphide bonds between cystine residues. Tyrosine kinase activity on the intracellular side and causes phosphorylation of itself and of substrates.
118
What occurs when the insulin binds to its receptor and what are the downstream effects?
Ligand binding triggers conformational change which moves the two kinase domains closer together. The kinase domains trans-phosphorylate and insulin receptor substrate 1 (a docking protein) also becomes tryrosine phosphorylated.
p-Tyrosine sites onf the IRS allow binding of the lipid inase PI3K which synthesises PIP3 at the plasma membrnae.
This recruits the phosphoinositide-dependent kinase (PDK) which directly phosphorylates the Thr308 residue of AKT.
AKT phosphorylates a number of substrates at Ser/Thr residues. These include FOXO (glucose production), TSC2 which permits activation of mTORC1 which activates SREBP1 (lipid synthesis) and S6K (protein synthesis), GSK3beta (glycogen synthesis) and TBC1D4 (glucose uptake).
119
What could excess or lack of insulin lead to?
Excess = hypo-glycemia, this can lead to :
Liver and muscles being stimulated to take up glucose
Too little blood sugar
Brain can ONLY metabolise glucose
Unconsciousness and death
```
Lack of = hyper-glycemia, this can lead to:
Diabetic foot ulcers
Diabetic retinal damage/blindness
Hypertension
Neuropathy
Loss of sensitivity
Weight loss
```
120
How are type 1 and type 2 diabetes different and which is more common?
Type 1 is caused by destruction of beta cells - often as a result of auto-immune attack
Type 2 diabetes mellitus is due to dysregulation of carbohydrate, lipid and protein metabolism due to impaired insulin secretion, insulin resistance or combination of both.
Type 2 more common
121
What is the first drug of choice for the management of type II diabetes?
Metformin (an antihyperglycemic agent)
122
What are the two membrane trafficking pathways?
```
Secretory/exocytic (biosynthetic) pathway - ER to Golgi to PM to PM/endosome/lysosome
Endocytic pathway (recycling or degradative) - cell surface to endosome to Golgi/ER/lysosome
```
123
What is the pathway from where proteins are synthesised to secretion either in vesicles or by constitutive secretion?
Proteins are synthesised on bound ribosomes on ER membrane
Budding and fusion of ER-to-Golgi vesicles to form cis-Golgi (also Golgi-to-ER retrograde transport
Cisternal progression from cis-Golgi to medial-Golgi to trans-Golgi network where they are sorted to different destinations
124
When are proteins glycosylated and what is the purpose of this?
As they transit the ER and Golgi
They assist with folding of proteins
They act as ligands either for intracellular trafficking/sorting or outside the cell for interactions with extracellular matrix and with other proteins/sugars on cells (and pathogen binding)
125
What are advantages and disadvantages of using yeast as a model organism?
Advantages:
Amenable for genetic studies (can grow as haploid and diploid cells)
Entire genome sequence known
Cheap and easy to grow in large quantities
Limited gene diversity (helps identify genes)
Fundamental pathways conserved
Disadvantages:
Limited cell-cell contact so unlikely to be informative about multicellularity
Small so high resolution imaging studies of intracellular compartments is difficult
Has a cell wall which can preclude some types of studies e.g. microinjection
126
What and how did Novik and Schekman's Sec- screening in yeast identify components of the secretory pathway?
Cells were analysed for their ability to secrete invertase and phosphatase at permissive and restrictive temperatures. The secretory mutants at restrictive growth conditions could synthesise proteins but not secrete them. Electron microscopy showed accumulation of vesicles or aberrant membranous structures.
127
How many genes were identified in yeast that are involved in transport of proteins from the ER to the plasma membrane?
23
128
What is each class of sec genes' defective function?
Class A - transport to ER
Class B - budding of vesicles from RER
Class C - fusion of transport vesicles with Golgi
Class D - transport from Golgi to secretory vesicles
Class E - transport from secretory vesicles to cell surface
129
Why weren't all of the genes/proteins involved in the exocytic pathway identified by Novick and Schekman?
They only identified temperature sensitive mutants and not all genes when mutated will cause this phenotype
They only considered secretion to the plasma membrane so defects in transport to endosome or vacuole would not be identified
Any redundantly functioning genes would not be identified (though yeast has relatively low gene redundancy)
130
What is endocytosis and why is it important?
The process through which the plasma membrane invaginates into the cell resulting in the production of a vesicle that is then able to fuse with endosomes and enter the endo-lysosomal membrane system.
Is important for:
Retrieval of molecules that formed part of the secretory vesicle for recycling
Downregulation of signals
Remodelling cell surface lipid and protein composition
(is also a means of entry into cells for many pathogens and toxins)
131
What are the stages in endocytosis?
Plasma membrane to endocytic vesicle
Endocytic vesicle to early endosome
Early endosome to late endosome (MVB) or recycling to the plasma membrane
Late endosome to Golgi or vacuole
132
What did End- screens in yeast using lucifer yellow or a bound pheromone alpha-factor find and how many genes were detected?
Fluorescence mostly around the plasma membrane with some inside, likely to be on endosomes.
7 end- genes detected. 5 of these directly involved in the process of membrane invagination and scission.
133
How did they Vps screen in yeast identify genes and how many genes were identified?
Carboxypeptidase Y is normally transported to the lysosome having been trafficked through the ER and Golgi. By creating mutants and seeing which ones secreted CPY instead of transporting it, they identified over 60 vps genes.
134
What does analysis of biochemical process, fluorescence microscopy and electron microscopy of vps screens tell us?
Biochemical can look a stages of protein modifications e.g. CPY glycosylated or proteolytically cleaved, can tell us which proteins are involved in what processes.
Microscopy shows fragmented vacuoles/smaller endosomes organelles not like WT.
135
What are the four possible destinations of proteins from the Golgi?
```
To plasma membrane
To early endosome
To late endosome/MVB
To vacuole
(also back to Golgi)
```
136
How are vacuolar mutants divided into classes and why can it be complicated?
Divided depending on the stage at which they appear to block the route to the vacuole e.g. MVB fusion with vacuole. Can be complicated as proteins can take different routes.
137
How is the late endosome/MVB (CPY pathway) sorted?
CPY synthesised in a prepro form and transported through ER to Golgi.
In late Golgi CPY is specifically recognised by a receptor Vps10 (receptor mediated sorting).
Transport step requires cytoplasmic factors: clathrin and two adaptors called Gga1 and Gga2.
pH means CPY dissociates from Vps10 at late endosome/MVB and transported to vacuoles where it is cleaved to generate mature form.
Vps10 is retrieved to the late Golgi through a specific aromatic-based signal in its protein sequence (YSSL, FYVF)
138
What is the key molecule for ensuring vesicles fuse with the target membrane?
SNARES
| v- and t-
139
What are the three main types of coated transport vesicles?
Clathrin
COPI
COPII
140
What are two ways newly made proteins can be translocated into organelles?
Co-translational translocation - delivered into ER membrane at same time as being translated
Post-translational - occurs after protein has been translated (occurs a lot for import into mitcohondria and chloroplasts)
141
How is a mature soluble polypeptide chain produced in the ER lumen via co-translational translocation?
The signal sequence on the growing polypeptide chain in ribosome that is being translated from mRNA causes protein to be recognised and inserted into translocating machinery Sec61.
This allows newly translocated proteins to be fed into ER lumen and the ribosome is sat tightly on membrane so no leakage.
Signal peptidase cleaves signal peptide so translocon can open gating laterally into membrane and allow this signal out.
142
How is a mature single-pass transmembrane polypeptide chain produced and inserted into the ER lumen via post-translational translocation?
There is a start-transfer sequence which is recognised by translocon.
This allows protein to move through membrane into ER lumen until stop-transfer sequence/hydrophobic amino acids on the protein is reached.
Signal peptidase cleaves off the start-transfer sequence and the stop sequence will remain in the membrane, emerging from the translocon which will close.
143
What is the difference between type I and type II membrane protiens?
Type I has N-terminus in the lumen whereas type II has C-terminus in the lumen
144
What are the proteins responsible for ensuring proper protein folding in the ER, what is the most abundant one and how does it function?
Chaperone proteins
Most abundant is BiP. It remains associated to the protein until it is fully formed and can be secreted into vesicle.
145
What can occur if there are defects in protein folding and how does the cell respond?
Can give rise to disease e.g. CFTRdelta508 where CFTR protein does not reach surface membrane.
This stress results in the Unfolded Protein Response where the cell shuts down translation and protein synthesis but upregulates synthesis of chaperones.
146
What are three things essential for the coated transport vesicle formation and what are the specific examples of these in COPII vesicles?
GTPases --> Sar1 in COPII
Adaptor proteins --> Sec23/24 in COPII
Coat --> Sec13/31 in COPII
147
What is the cycle of Ras (founding member of small GTPase) including the enzymes involved and where the active/inactive forms are found?
GDP-bound is inactive and usually found in the cytosol
GEFs exchange GDP for GTP to activate
GTP-bound is active and is membrane associated
GAPs exchange GTP for GDP to inactivate
148
How is the COPII vesicle formed and what is its structure?
Sar I is activated by a GEF. These recruit adaptor proteins which recognise cargo to be transported. The exit signals on soluble proteins attach to cargo proteins/receptors in the membrane.
It is surrounded by a coat which provides structural rigidity to help the vesicle bud.
149
What is the structure of adaptor proteins and how does this relate to its function?
2 subunits - one (Sec24) interacts with the membrane cargo by recognising motifs in the cytoplasmic domains, the other (Sec23) interacts with activated Sar1
150
Why do vesicles have a high SA:V ratio?
Large membrane but low liquid to ensure not many ER-resident chaperones get trapped in
151
How can centrifugation be used to study COPII vesicles and the ER membrane?
ER membrane proteins along with cytosol are put into centrifuge tube with sucrose mixture.
The cytosol acts as a source of COPII components (Sec23/24, Sar1, Sec13/31) so vesicles will form.
After putting in centrifuge, the ER membrane will be lower in the tube compared to vesicles as they are more dense and both can be extracted.
152
How can reconstitution experiments be used to look at formation of COPII vesicles?
Using centrifugation and adding different components e.g. cytosol or ATP, GTP we can see whether COPII vesicles have been formed.
Using probes for Ribophorin (a resident ER protein) and p58 (a COPII cargo protein) and Western blot can tell us whether vesicles have been formed or whether the ER membrane is still intact.
153
How are inactive Sar1-GDPs recruited from the cytosol?
The amphiphilic helix allows it to transiently associate with the membrane.
Sar1-GEFs on relevant membrane replaces GDP with GTP and become activated.
154
How is Sar1 regulated?
When Sec23 binds it acts as a GAP for Sar1.
| The GAP activity is further enhanced when Sec13/31 coat is recruited as this is important for uncoating.
155
What effect will mutant GTPases often have and how is this caused by both GDP and GTP mutants?
Dominant negative effect (i.e. interferes with normal physiological effects)
GDP mutant sequesters (soaks up) GEFs and inhibits process
GTP mutant cannot hydrolyse GTP and is always signalling
156
What are the 4 common coated vesicles' coats, GTPases and cargo?
COPII coat - Sar1 - newly synthesised proteins
COPI - Arf1 - Retrieved and newly synthesised proteins
Clathrin (at TGN) - Arf1 - lysosomal proteins and regulated secretory proteins
Clathrin (at PM) - ?? - endocytosed material
157
How can a Sec23A mutation be identified in Cranio-lenticulo-structural dysplasia, a recessive disorder which involves abnormal endoplasmic-reticulum-to-Golgi-trafficking?
Using immunofluorescence you can see there is a mutation causing less vesicle formation and no recruitment of Sec31 to exit sites.
Using reconstitution experiments you can see mutatnts still recuit Sec23/24 therefore binding of this to liposomes is unaffected.
However there is reduced budding compared to the WT threrefore mutation must be affecting the formation of COPII coated vesicle.
158
What could be a reason for why only some tissues are affected e.g. bones in Cranio-lenticulo-structural dysplasia?
Packaging of large cargo such as collagen will have differences in the interactions and structure of the cage of vesicles.
Paralogues in other tissues unaffected even though both gene copies lost.
159
What are the two domains of the ER and what is each responsible for?
Rough ER - covered in ribosomes for protein synthesis
| Smooth ER - sites of lipid synthesis and roles in calcium storage
160
Where are intermembrane contact sites found?
Between organelles, particularly between the ER and other organelles as it is important for ER function
161
What is the difference in Ca concentration in the ER lumen, endosomes, the cytosol and the extracellular space?
```
Extracellullar space - ~1nM
Cytosol - ~100nM
Newly formed endosome - ~1nM
Late endosome - ~2.5microM
ER lumen - ~60-500microM (~5000x more than cytosol)
```
162
What is the ER of muscle cells and how does it help with contraction?
Sarcoplasmic reticulum.
Specialised ER for handling Ca2+ transients required for muscle contraction.
Is very close to T tubule/sarcolemma and ryanodine channels allow intra SR Ca release.
163
What is Stim 1 and how does it function?
It is a calcium sensor in the ER (usually located at the end of a tubule)
When Ca is released from the ER Stim 1 comes together into polygomeric complex.
It becomes associated with particular area in plasma membrane with phosphoinositides to form membrane contact site.
This associates with orai Ca channels allowing for Ca uptake into the ER.
164
What has EM and live cell imaging told us about the structure of ER contact sites and therefore what are the functions they provide platforms for?
Ribosomes are excluded from contact sites
Membranes are very close (3-15nm)
ER contacts can be short- or long-lived
ER tubule encircles mitochondrion
Calcium mobilisation
Lipid transfer
Signalling
165
What is the basic structure of membrane contact sites?
Protein components that are often extended, or have extended coil regions, to allow bridges to form
166
What is CLEM and how does it work?
Correlative light and electron microscopy
Mark where fluorescence is in cell and freeze it so you can process it.
Means you can localise fluorescently labelled proteins to membrane contact sites and follow dynamic behaviour of these.
167
How is a late endosome characterised?
Multivesicular bodies (these pinch off and form veiscles)
168
How could membrane contact sites be involved in the movement of epidermal growth factor receptors into multivesicular bodies from the plasma membrane?
Combination of:
Mechanical role i.e. the ER membrane forming around endosomes promoting inward invagination
Regulatory role i.e. protein tyrosine phosphotase 1B could regulate the phosphorylation to be downregulated and incorporated into the MVBs
169
What are three features of lipid transfer?
It is unidirectional
Non vesicular transfer of lipids occurs at contact sites
Lipid transfer proteins may use concentration gradients of lipid to promote lipid transfer
170
What is Niemann Pick Disease C and how does it occur?
Disease causing accumulation of sphingomyelin accumulation in lysosomes due to defect in NPC1/2 at contact sites.
Affects spleen, liver, lungs, bone marrow and brain.
171
What are the main roles of membrane contact sites?
```
Lipid transfer
Signalling
Ca transfer
Organelle fission
Stress response
Organelle positioning
```
172
What are three types of carcinogens and what are examples of each?
Physical - UV and ionizing radiation
Chemical - asbestos and tobacco smoke
Biological - infections from certain viruses, bacteria or parasites
173
What are HERs, how are they activated and what is the exception?
Human Epidermal Growth Factor Receptors
When a ligand binds they activate (apart from Her2) and homo or heterodmerise
Her2 is always in open conformation
174
What do HER pathways stimulate?
Cell proliferation
Cell survival
Anti-apoptotic
--> different combinations of receptors stimulate different signalling pathways
175
How many copies of HER2 are present in normal breast tissue compared to cancer tissue?
Normal ~20,000 copies per cell
| Cancerous ~2 million copies per cell
176
Why are antibodies ideal for targetting HER2+ cancer?
They can be raised against nearly any proteins
They're very specific
They can interfere with receptor signaling
They can target cells for destruction by the bodies immune system
177
How are monoclonal antibodies produced?
Mice are immunised with HER2
Their spleen cells are fused with myeloma cells to form hybridomas
These are cultured in HAT medium and positive cells are selected so monoclonal antibodies can be harvested
Humanisation (keeping antigen binding sites but switch the scaffold) of antibodies as murine antibodies not well tolerated in humans
178
What are the effects of using 4D5 (anti-HER2) to target HER2 overexpressing cancer cells?
They bind to the extracellular domain of HER2 and inhibits their proliferation
It suppresses tumour growth in mice
Injected radio labelled 4D5 targets HER2+ breast cancer cells in women
179
How are 4D5 humanised?
The murine heavy and light chain cDNA encoding 4D5 is cloned
The murine CDRs of 4D5 are cloned into human IgG1 heavy and light chain plasmids
The humanised antibody is then made by transfecting CHO cells with light chain and heavy chain plasmids
180
What are five places where membrane fusion is occurring in our bodies?
Synaptic vesicle fusion e.g. between neurons and muscles
Secretory granule fusion e.g. endocrine and exocrine pancreas
Secretion of serum proteins e.g. albumin from hepatocytes and antibodies from plasma cells
Mucus secretion e.g. epithelial mucousal cells
Intracellular transport e.g. of proteins between organelles in all your cells
181
What approaches can be used to go from the microscopic anatomy of a cell to the molecular machinery of vesicle fusion?
Biochemical reconstitution
Yeast genetics
Cloning
182
How does the Intra-Golgi transport assay (Biochemical reconstitution) work and what can it show us?
The donor compartment has a mutation meaning they cannot glycosylate the membrane protein. When infected with a virus this is trafficked from ER to PM via Golgi.
The acceptor compartment has a WT Golgi and radioactive sugar therefore if radioactivity then this shows membrane protein has moved and been glycosylated in healthy Golgi.
You can then fractionate the cytosol e.g. by column purification to take apart system and work out machinery responsible for transport step.
183
What is NSF and how was it identified?
N-ethylmaleimide Sensitive Factor is and ATPase required to unwind the SNARE molecules after
Identified by when adding N-ethylmaleimide it inhibits the reaction. Also antibodies for NSF inhibits the reaction.
184
What is SNAP?
Soluble NSF Attachment Protein (factor required to bind NSF to SNARE complex)
185
What chemical methods were used to find proteins involved in vesicle fusion?
Isolation of sec mutants in yeast screens (NSF and alpha-SNAP)
Cloning of synaptic vesicle proteins and using antibodies to identify DNA
Clostridial neurotoxins tetanus and botulinum B showed they cleaved VAMP and therefore fusion of synaptic vesicles
Biochemical purification of SNAREs (when large compex dissembles when ATP is hydrolysed the proteins could be identified)
186
What was Rothman's SNARE hypothesis?
1. SNAREs for each transport step within the cell
2. SNAREs should provide specificity to vesicle transport
3. SNAREs should be sufficient to drive lipid bilayer fusion
4. Proposed that NSF and ATP hydrolysis catalyses membrane fusion (this is incorrect as NSF unwinds SNAREs after)
187
What is the structure of the neuronal SNARE complex?
Syntaxin, SNAP25 (both on target) and VAMP (on the vesicle) zipper together in a parallel coiled coil by hydrophobic interactions.
Cystine lipid anchors of SNAP25 hold this in the membrane.
188
What is the process of SNARE zippering and how does this help a energetically unfavourable process?
When zippered together, the SNARES form tight trans-SNARE complexes.
Hemifusion is where a fusion stalk and then transmembrane contact are formed.
The fusion pore opens and enlarges.
The energy of zippering brings the two layers together.
189
What is the difference between Q and R SNAREs and what is the strutcure of Q and R SNARE complexes?
R (VAMP) contains an arginine
Q (SNAP25 and syntaxin) contains glutamine
3Qs (2 SNAP25s) to 1R and form a salt-bridge in the middle of the coiled coil (this is conserved in all complexes)
190
Are SNAREs specific?
No, they show some specificity but mostly controlled by lots of additional machinery e.g. rabs, coat proteins and tethers
191
What are common features of SNARE proteins?
Generally small (14-40kDa)
All have at least 1 coiled-coil or SNARE motif
Generally C-terminally anchored (some can have lipid anchors rather than TMDs)
A lot of SNAREs have extra regulatory domains which can regulate function of SNARE complex
192
How can TIRF microscopy be used to image SNARE machinery?
Lipid bilayer suspended on glass support with SNAP25 and syntaxin incorporated.
Fluorescent dyes in the vesicle can measure the fusion into membrane by TIRF microscopy.
193
What effect does removing the regulatory domain on syntaxin have when Ca is added?
More fusion - as domain is inhibitory
194
What happens when you induce temperature-sensitive mutations into flies e.g. comatose (codes for NSF)?
At restrictive temperatures the flies will fall over and go into paralysis but will recover at lower temperatures.
There are also more docked vesicles as there will be no SNARE recycling.
195
What are some diseases associated with mutations in SNARE proteins e.g. VAMP2, SNAP25b, SNAP29, Syntaxin 11?
VAMP2 - neurodevelopmental disorder with hypotonia and autistic features with or without hyperkinetic movements
SNAP25b - neurodevelopmental disorder with seizures, intellectual disability, severe speech delay, and cerebellar ataxia
SNAP29 - cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma syndrome (CEDNIK)
Syntaxin 11 - familial hemophagocytic lymphohistiocytosis type 4 (FHL4)
196
What is the effect of mutation S75P in VAMP2 on liposome fusion?
It slows the rate of liposome fusion.
| It is a dominant-negative mutation so replaces the function when put with WT.
197
What is familial hemophagocytic lymphohistiocytosis?
A rare disease of the immune system where overproliferation of T cells, natural killer cells, B cells and macrophages.
Can be caused by a number of different mutations and can cause a range of conditions.
198
How do mutations in syntaxin 11 cause FHL4 and 5?
STX11 is an unusual Q-SNARE as it does not have a TMD.
| FHL4 and 5 patients have reduced STX11 (although in FHL5 this is caused by a mutation in Munc18-2)
199
What are two diseases caused by bacteria that cause cleavage of SNAREs and loss of function?
```
Clostridium tetani (tetanus)
Clostridium botulinum (botulism)
```
200
How are clostridial toxins taken up by neurons and how do Boulinum and Tetanus differ in action?
The toxins bind to the cell surface and are endocytosed.
Endosome acidification occurs and endopeptidase translocation.
The toxins cleave SNAREs - type of SNARE depends on type of toxin e.g. TeNT cleaves VAMP, BoNT/A (botox) cleaves SNAP25.
BoNT paralyses the motor neuron
TeNT is retrotranslocated back up the motor neuron and inhibits the inhibitory neuron - causes muscles to spasm
201
What are clinical uses of Botulinum neurotoxins and why can't tetanus be used?
```
Cosmetic uses
Strabismus
Blepharospasm
Hemifacial spasm
Cervical dystonia
Axillary hyperhidrosis
Over active bladder
GI tract disorders
Sialorrhea
Temperomandibular disorder
Limb spasticity
```
--> because everyone is vaccinated against the toxin.
202
Why do we need degradation in cells (autophagy)?
```
Homeostasis
Removing damaged components
Recycling nutrients
Signalling
Reprogramming cells
```
203
What are the three forms of autophagy and how does this compare to other pathways?
Macroautophagy
Chaperone-mediated autophagy
Microautophagy
Macroautophagy is a lysosomal, bulk digestion pathway that can remove whole organelles and molecules released can support metabolism.
Chaperone-mediated autophagy/microautophagy are lysosomal pathways with relatively low capacity. They turn over specific, generally long-lived proteins and only degrade individual proteins.
Proteosomes (not autophagy) are non-lysomal and degrade individual proteins. They are the major turnover route for short-lived proteins.
204
What are four functions of autophagy?
1. Nutrient recycling - under starvation autophagy is upregulated
2. Cellular remodelling - only mechanism to degrade organelles e.g. erthropoiesis and removal of sperm-derived mitochondria by egg
3. Removal of damaged components - e.g. mechanical damage or damaged miochondria, ageing
4. Killing intracellular pathogens e.g. TB, MRSA, viruses
205
How does age affect autophagy and what does this mean for long-lived or highly metabolic cells?
Cells continuously acquire damage however lysosomal capacity decreases as we age.
Reduced autophagy is the major reason for age-related degeneration.
Long-lived or highly metabolic cells (high metabolism
causes most damage inside cells) e.g. neurons and muscle are most susceptible.
206
What is the dietary restriction hypothesis?
Starvation/exercise upregulates autophagy (by AMPK/SIRT1 andTORC1 pathways) which leads to damage repair.
207
How might we manipulate the autophagy pathways to stop disease?
Promote or inhibit if pathogens using autophagy to survive.
| Promote for damage and organ removal to help with ageing, muscular dystrophy, neurodegeneration and cancer.
208
How were the 15 autophagosome genes identified in yeast?
When nitrogen-starved, vesicles form from the vacuole.
| The genes were identified from these genetic screens to see which caused no accumulation
209
How is an autolysosome formed and how can it be identified by EM?
Initiation event triggers the formation and build up of the membrane of the phagophore. The autophagosome is formed when the membrane is closed and this fuses with the lysosome. This undergoes acidification and maturation to form an autolysosome.
Is the only double membrane organelle so can be identified in EM by this.
210
What are the four pathways/complexes that are involved in the auto lysosome formation?
UKL1 complex responds to signals
P13K/Vps34 complex initiates it
Ubiquitin reactions that allow delivery and expansion of membrane
SNAREs recognise the lysosomes, fuse them with phagosomes and mediate closure of final autophagosomes
211
How can autophagy be selecvtive?
Inside the phagosomes are Atg8 (or LC8 in humans).
Adaptor proteins have a AIM (Atg8 Interaction Motif) and a UBD (Ubiquitin Binding Domain). Proteins can be tagged with ubquitin and the adaptor proteins will transport them into the phagosome.
Additionally some proteins may have own AIMs and will not need adaptor proteins to bind.
212
Why are neurons particularly sensitive to autophagy?
Because they are long-lived, metabolically active and have long axons
213
What are the proteinopathies in neurodegenerative diseases such as Huntingtons, Parkinsons and Alzheimer's?
Big accumulation of ubiquitinated inclusions
In Huntington's these are Huntingin aggregates
In Parkinsons these are alpha-synuclein in the medulla
In Alzheimer's these are amyloid beta plaques
214
How is Huntington's disease caused and why does it onset with age?
Caused by polyglutamine (polyQ) expansion in Huntingtin protein. The more polyQs you have the more susceptible to the disease (Q<18 = healthy, Q>35 = disease-causing). Loss of normal function of protein.
With age, the lysosomal capacity decreases so their ability to remove proteins decreases. This could be by a toxic oligomer which damages proteosome or toxic aggresomes by sequestering binding proteins.
215
How is Parkinson's disease caused and what is the main neuropathology?
Loss of dopaminergic neurons however only 5-10% of cases familial.
The main neuropathology is aggregates of alpha-synuclein (Lewy Bodies).
216
What happens when alpha-synuclein mutations occur in Parkinson's (although this is rare)?
a-synuclein is normally degraded by chaperone-mediated autophagy (where the receptor recognises the AAs and proteins and transports into lysosomes - normally regulates long-lived proteins).
When a-syn is mutated it binds but cannot be translated into the lysosome causing aggregates and toxicity.
217
What mutations could cause mitochondrial dysfunction in Parkinsons and what effect may this have?
PINK1 - mitochondrial kinase (loss of function in 5-10% sporadic early-onset Parkinsons)
PARKIN - cytosolic E3 ubiquitin ligase (mutated in 50% of autosomal recessive 10-15% of sporadic early-onset Parkinsons)
When mitochondria depolarised, PINK1 activated which activates PARKIN which ubiquitinises mitochondria and it undergoes mitophagy.
Losing these genes leads to accumulation of damaged mitochondria, these release ROS which induces more oxidative damage, protein misfolding and damaged organelles (creates feedback loop).
218
How does autophagy activity change throughout development of cancer?
```
It is anti-oncogenic before tumour development as has important roles in:
Cell homeostasis
Damage removal
Reduced ROS/genotoxicity
Reducing inflammation
```
Once cancer forms, autophagy has role in helping cells survive by:
Survival during oxygen or nutrient shortage (i.e. before vascularisation)
Prevention of apoptosis
Survival during chemotherapy
219
What are potential strategies for autophagy therapeutics?
Blocking survival to metabolic stress with autophagy inhibitors (good for solid tumours but not cancers of blood)
Inhibit autophagy to increase apoptosis during chemo
Elevate autophagy to remove damage and prevent cancer
220
What is cell polarity and why is it important?
Organisation of proteins inside and at the surface of cells.
Means regions have distinct protein/lipid and other molecule compositions so cell can have different capabilities morphologies and functions.
Important for asymmetric cell division.
221
What do common features of pathways in diverse cell types that establish and maintain cell polarity lead to?
Assembly of key cytoskeletal elements (most often actin and myosin)
Directed protein transport/membrane trfficking to deliver both proteins and membrane
Ensuring mitotic spindle can be aligned appropriately so that material is partitioned appropriately during cytokinesis
222
What are key features of cell polarity in pathways such as migration, cell fate determination and epithelial polarity?
Key proteins such as small GTP proteins
Markers of different cells
--> conserved machinery and proteins but may have different roles in different cells
223
How do common protein complexes establish and maintain diverse shapes in cells?
They acts as scaffolds for small Rho GTPases on specific membranes. These control shape by regulating acto-myosin cytoskeleton and directing protein/vesicular trafficking.
224
How does budding yeast generate polarity to undergo asymmetric division and produce a daughter cell with a reset properties (i.e. can generate finite number of times whereas mother cell will be more restricted)?
Marking the site by cortical membrane protein
Decoding the site with signalling complex
Establishing the site by Rho-GTPase Cdc42 activated and organises cytoskeleton and trafficking pathways
Maintaining the site by feedback loops to make sure growth remains localised and continues
225
How do PAR proteins form the core of a cells polarity network?
Opposing and complementary membrane domains work antagonistically to maintain themselves and define a cell's axis of polarity.
226
How is the first asymmetric division in C. Elegans initiated and how are Par proteins involved?
Entry of sperm polarises the oocyte and position of entry defines the posterior end of the zygote (also P0 cell). This divides asymmetrically along the A-P axis to create one larger anterior cell (AB) which will form ectoderm and a smaller posterior cell (P1) which will form meso/endoderm germline.
The microtubules generated recruit Par1 and Par2 which antagonise anterior Par proteins. Par3/6/aPkc localise to anterior cortext, Par1/2 to posterior cortex and Par5 maintains the boundary.
Interactions between microtubules and the cortex results in pulling forces which cause spindle to be displaced toward posterior end.
227
How does Drosophila neuroblast cell division occur within the epithelial monolayer/ventral neuroectoderm?
When they delaminate from this position and undergo asymmetric cell division.
The smaller daughter cell is called a ganglion mother cell, which only divides once more to give rise to a glia cell and neuron, and a larger apical cell which continues to divide asymmetrically.
228
When the Drosophila neuroblast divides, how and when does the cell establish polarity for asymmetric division?
Whilst in the neuroectoderm, Par3 Bazooka/Par6 are found in a stalk that continues to extend into the epithelium.
After delamination, they continue to localise to the apical region.
Baz anchors another complex (Insc/Pins) at membrane in order to orient the mitotic spindle.
Scribble complex helps in spindle alignment.
229
What key roles do epithelial cells have in embryo morphogenesis and organ development?
Polarised actin cytoskeleton allows apical surface to constrict (important for gastrulation and tubulation)
Can orientate their mitotic spindle to allow division in that plane to increase their cell number or perpendicular to generate different daughter cells
Can lose and re-acquire epithelial phenotype (EMT and MET)
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What are the complexes that interact physically and genetically with PAR complex to establish epithelial polarity?
Crumbs/CRB complex at apical
| Scribbles/SCRIB complex at basal
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What processes are involved in EMT?
A conversion of epithelial apical-basal polarity axis into a migration axis with front-rear polarity
Is triggered by signals that lead to a loss of E-cadherin
Asymmetric activation of small Rho GTPases (Cdc42 and Rac1 at front and RhoA at rear)
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What is mechanobiology?
The study of how physical forces and changes in cell or tissue mechanics contribute to development, physiology and disease - is important for cellular homeostasis and interaction between different cell types
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What is mechanotransduction?
The conversion of a physical force to a biochemical response (aka mechanosignaling)
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What is mechanosensing?
When a protein or cellular structure responds to a physical cue to initiate mechanotransduction
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What are the key processes in mechanotransduction i.e. how does mechanosensing lead to a cellular response?
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Mechanosensing (cells test their environment by adhesion receptors/membrane proteins probing ECM)
Signal transduction (mechanical signal transduced along a linked network, cytoskeleton is often the force conduit)
Signal integration at nucleus (accumulation of signals over time, chromatin rearrangment or nuclear pore opening)
Cellular response (from microseconds to minutes, affects cell shape, fate, motility, growth)
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What happens to endothelial cells when they have been exposed to constant fluid flow?
Actin fibres become parallel instead of unstructured.
This has consequences e.g. arrangment of focal adhesions
Stress fibres anchored to ECM using focal adhesions
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What are two examples of mechanotransduction in action?
Blood pressure autoregulation and coronary artery disease
| Auditory mechanotransduction by hair cells
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How is trans-epithelial resistance and transport affected when using a liquid-liquid interface compared to an air lipid interface?
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Higher TER (faster forming epithelium and tight junctions) reached with ALI
Less transport in ALI
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What was found out by mimicking lung inflammation on an organ on chip?
When adding TNF or E. coli to the epithelium, neutrophils will bind as ICAM (an adhesion receptor) is expressed in response to pathogen. Neutrophils then migrate through pores to the epithelium.
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How does adding strain/mechanical stretching (i.e. mimicking breathing) affect the uptake of nanoparticles, ROS generation and expression of ICAM-1?
Strain increases the uptake of nanoparticles by translocating into endothelium (mimicking breathing in these i.e. in big cities)
ROS generation is increased (leading to further damage of cell)
ICAM-1 expression is also increased
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What are examples of mechanical regulators?
Fluid flow
Stretching of epithelial tissue
Stiffness
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What is stiffness and how do you work it out?
Stiffness (Pascal) = stress/strain
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Stress = the force applied on an area (F/A)
Strain = material property, how it responds to stress, (change in length/original length)
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What is shear stress?
Stress that acts parallel to area e.g. fluid flow
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What is compression?
Pushing force (N)
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What is tension?
Pulling force (N)
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Why is stiffness important for culturing cells and how can you change it?
Growing soft tissues e.g. brain or neurons in a petri dish is very different to how they would grow in vivo.
Hydrogel can be put on petri dishes and the stiffness of this can be changed e.g. Hydroxyethylacrylate (HEA) stiffness can be changed by changing the amount of AAM.
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What happens when growing human-MSCs on hydrogels of different elasticity?
When grown on low stiffness will become brain cells.
When grown on medium stiffness will become muscle cells.
When grown on high stiffness will become bone cells.
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How can measuring stiffness by elastogram help diagnose chronic liver disease?
Stages of CLD correlates with an increase in stiffness.
Assessing prognosis and candidacy for treatment in patients with CLD.
Elastogram is non-invasive and therefore easier than a biopsy
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How does a tumour develop its stiffness?
Build up of ECM around tumour cells.
Fibroblasts recruited and differentiate into myoblasts which contract and contribute to the ECM stiffness surrounding the tumour.
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How do mechanical force promote tumour aggression?
Disruption of cell-cell adhesion e.g. breast tissue leads to EMT
Increased cell-ECM adhesion changes actomyosin contractility which alters gene expression, increases ECM collagen deposition and upregulation of cross-linking enzymes
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What are techniques used to measure cellular mechanical forces?
Atomic force microscopy (push cantilever on cell and record force required to create certain indentation)
Micropipette aspiration (how much force needed to suck up a section of membrane)
Optical tweezers
Magnetic tweezers
Uniaxial stretcher
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What are Piezo ion channels and how many types are there?
Ion channels with 38 TMDs.
Activated by stretch/changing of membrane tension
2 types - PIEZO1 and PIEZO2
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What are the two models of how Piezo channels work and how does actin affect this?
Model 1:
Induces curvature in membrane and when force applied the channel opens and flattens out
Model 2:
In innate state is planar to the membrane and increased membrane tension pushes channel apart laterally
Smaller force required when actin present
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How do integrins act as mechanosensors?
When ligand binds and they are activated integrins form focal adhesion with ECM.
The binding site on Talin becomes exposed so Vinculin binds causing downstream signaling and regulating stiffness of cell.
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How does the structure of talin change?
When force is applied.
When low force, is inactive and unfolded.
When larger force, becomes stretched and binds Vinculin and stabilises focal adhesion.
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What are caveolae and how do they respond to shear stress/increase in membrane tension?
These are invaginations of the plasma membrane involved in membrane trafficking, in particular endocytosis.
When under stress, invagination stretched out/flattened and cavin complexes are released and can trigger downstream effects.
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What are key features and proteins of caveolae?
Cav1 is linked to the actin cytoskeleton which is in direct contact with the nucleus.
Cavins contribute to signal transduction
EDH2/Pascin2 also contribute to signal transduction
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What happens when focal adhesion switches to fibrillar adhesion?
Talin switches to tensin
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What are the processes involved in integrin-dependent adhesion and mechanotransduction of this signal?
Integrins sense stiffness of ECM and translate it into biochemical signaling
Focal adhesion kinases become activated and G-protein Rho (activates YAP/TAZ and main regulator of filamentous actin)
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What can inhibitors that perturb cell-ECM interactions and downstream pathway act?
To inhibit the increase of ECM (e.g. TGF-beta inhibitors, MMP inhibitors and Losartan)
To inhibit ECM stiffness increase (e.g. LOXL2 inhibitors)
Inhibitors of certain proteins and factors e.g. HA, FAK, Rho and integrin
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How do cells grow differently in small or larger adhesive areas?
Spreads more on a bigger area.
| --> geometry is a physical cue and can steer cell behaviour
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What are principle mechanisms involved in the Hippo pathway?
Mst1/2 phosphorylates Lats1/2.
This negatively regulates YAP/TAZ by phosphorylating.
YAP/TAZ can then be degraded or retained in the cytoplasm meaning it cannot enter the nucleus and activate TFs.
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What are cell or tissue properties that regulate the Hippo pathway?
Apicobasal polarity
Mechanotransduction
Cell-cell adhesion
Contact inhibition
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What are cellular functions regulated by the Hippo pathway?
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Proliferation
Cell survival
Cell competition
Stem cell maintenance
Metastasis
Regeneration
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What is the difference in YAP/TAZ localisation and expression when cells grown in different stiffness?
Less expression of YAP/TAZ when stiffness low.
| YAP/TAZ localised in nucleus under high stiffness.
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What is the difference ine YAP/TAZ expression when grown on different size areas and is this because of the geometry or contact area?
Where larger area, YAP is in present in the nucleus.
Geometry/shape as even when on micropillars/reduced contact there is still activation of YAP in nucleus
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How does depolymerisation of the actin cytoskeleton affect YAP/TAZ and what causes this?
Is inactivated.
Loss of contractility.
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How is YAP/TAZ involved in osteogenesis and adipogenesis?
Osteogenesis is YAP/TAZ dependent
YAP/TAZ inhibits adipogenesis
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What are the main roles YAP/TAZ plays in mechanotransduction i.e. how can it be activated/inactivated and what effects will this have on the cell?
Activated by:
Cell spreading
Stiff ECM
High contractile forces
Activation leads to:
Proliferation
Osteoblast differentiation
Inactivated by:
Confined cell adhesion
Soft ECM
Low contractile forces
Inactivation leads to:
Apoptosis
Growth arrest
Adipocyte differentiation
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What is the biological data science pyramid?
A model for data systems and their role in knowledge production
(Big) data --> information --> knowledge --> insight
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Where is big data gathered from?
DNA, RNA, protein molecules (transcriptomic, whole genome sequencing, proteomic and epigenomic data)
Cells
Tissue samples
Organisms
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What is big data used to generate knowledge about in biology?
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Development
Physiology
Disease pathobiology
Drug safety and efficacy
Relationships between genotypes, environmental factors and disease risk - epidemiology
Understanding of past events
Prediction of future risks
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What is a volcano plot?
Identified genes exhibiting statistically significant changes in transcript abundance caused by certain factors e.g. exposure to glucocorticoid or hypoxia.
Genes are either up or down regulated and significant increases and they move away from zero.
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What is associated with high number of SNPs?
Disease (may potentially play a causative role)
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What does a Manhattan Plot show?
SNP degree of association with disease on each chromosome
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What does proteomic data reveal?
Protein-protein interactions
| The dynamics of these interactions across time and in space
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What is epidemiology?
Identifies exposures and predispositions that affect health and aims to reduce disease burden by public health interventions that reduce exposures (i.e. reduces the risk)
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What is pathobiology?
Seeks to understand ow interactions between exposures and predispositions govern health and aims to reduce disease burden through more effective diagnosis and treatment
