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1
Q

What is meant by the term isoform

A

An isoform is a protein variant that differs based on posttranscriptional modifications

2
Q

What process is used to create different protein isoforms

A

Alternative splicing

3
Q

What regions of the immature mRNA transcript are removed during splicing

A

Introns

4
Q

Splicing means that different proteins can be created from the same gene, T or F

A

T

5
Q

All eukaryotic genes contain introns and exons, T or F

A

F – yeast do not contain introns

6
Q

What components of genes allow for alternative splicing to occur

A

Optional introns and exons, mutually exclusive exons and internal splice sites

7
Q

40% of the Drosophila genome is alternatively spliced. What percentage of human genes are also spliced

A

0.75

8
Q

Other than splice donor and acceptor sites within gene transcripts, what other features of the mRNA allows for alternative splicing

A

Other sequences contained within the mRNA and the secondary structure also affects the choice of splice sites

9
Q

How is splicing regulated

A

By RNA binding proteins

10
Q

Give an example of a gene that undergoes alternative splicing

A

Dscam in Drosophila is a very large gene that produces a massive mRNA transcript containing 100 exons. The final mature mRNA will contain one exon from 12 A exons, 48 B exons, 33 C exons and 2 D exons, creating 38,000 splice variants of the dscam gene product. This is indicative of its role in the Drosophila nervous system

11
Q

What are the three key genes in determining sex in Drosophila and what are their roles

A

Sex lethal (sxl), a RNA binding protein and splicing repressor, transformer (tra), a RNA binding protein that acts as a splicing activator and, doublesex (dsx) a transcription factor.

12
Q

How are male Drosophila determined using alternative splicing and the interactions between the three sex determining genes

A

Male Drosophila have one X chromosome and this acts as the default pathway for sex determination in fruit flies. The transcripts for sxl and tra are spliced to give rise to inactive proteins. The dsx transcript is also spliced but this gives rise to a male specific transcription factor that acts as a transcriptional repressor of female-specific genes.

13
Q

How are female Drosophila determined using alternative splicing and the interactions between the three sex determining genes

A

Female Drosophila have two X chromosomes and a sex chromosome to autosome ratio of 1. The presence of two X chromosomes results in the transient activation of an alternative sxl promoter sequence which leads to the production of the sxl transcript which is then spliced and translated to form a splicing repressor. The sxl protein produced binds to other sxl transcripts and represses splicing by blocking binding of U2AF. This feeds back to result in more production of functional sxl transcripts. The sxl protein also binds to the tra transcripts causing an alternative splice that produces a functional tra protein after translation. The functional tra protein is a splicing activator and causes splicing of the dsx transcript. Splicing of the dsx transcript produces the female dsx transcript which is translated to the female dsx isoform. The female dsx protein is a transcriptional repressor of male specific genes.

14
Q

Give an example of how polyadenylation can act as a regulation of gene expression

A

The site of polyadenylation within the mRNA can be regulated. B lymphocytes for example can produce two different isoforms of an antibody. The antibody gene for a specific antigen has two possible positions for cleavage and polyadenylation. This determines whether the antibody is to be secreted or to remain membrane-bound. To produce the membrane-bound antibody, the cell produces the long transcript of the antibody. In this case the first stop codon within the antibody mRNA transcript is spliced out. This results in the translation of the transmembrane domain. Once the protein is secreted it remains tethered to the membrane via the transmembrane domain. For an antibody to be secreted the short transcript is produced which results in the loss of a splice acceptor site. Thus, no splicing of the transcript occurs and the first stop codon isn’t lost. This results in a termination of translation at the first stop codon, prior to the transmembrane domain region. This means that when the antibody is secreted it isn’t tethered to the membrane by a transmembrane domain.

15
Q

What is meant by the term leaky scanning

A

Sometimes the first AUG codon can be missed by the ribosome

16
Q

Sequences around the start codon help to initiate translation, T or F

A

T

17
Q

What is meant by the Kozak sequence

A

The Kozak sequence is the optimal translation initiation sequence that contains the start codon and ideal bases adjacent.

18
Q

Recall the Kozak sequence

A

ACCAUGG

19
Q

How can leaky scanning lead to the production of different protein products

A

If the sequence is less than optimal the ribosome can miss the first start codon and begin at the second or third AUG. These proteins will all be produced in the same reading frame, differing only by the sequence in the N-terminus

20
Q

High levels of what translation cofactor increase the probability that the first start codon will be recognised

A

eIF-4F

21
Q

How does the HIV virus make use of regulated nuclear transport to influence gene expression

A

After integration of the HIV genome into the host cell, the whole genome is transcribed as one piece of mRNA. Alternative splicing allows for the many different protein products to be made. Although the full-length mRNA is needed to make new virions the unspliced mRNA containing the entire virus genome cannot leave the nucleus. One of the proteins encoded by the virus genome is the rev protein. During the early stages of infection when only the alternatively spliced viral mRNA can leave the nucleus, the rev transcript moves through the nuclear pore and is translated. The rev protein then interacts with the nuclear pore in late stage infection to allow the exit from the nucleus of the unspliced mRNA.

22
Q

Signals within which regions of mRNA transcripts target them to particular parts of the cell

A

3’ and 5’ untranslated regions

23
Q

How are 3’UTRs recognised by cellular proteins which lead to the sequestration of RNA in one part of the cell

A

Intermolecular base pairing within the 3’UTRs form stem loops which are recognised by cellular proteins

24
Q

What is the role of ferritin in the regulation iron availability

A

Ferritin is a protein that stores iron inside the cell leading to a decrease in Fe availability

25
Q

What is the role of transferrin in the regulation iron availability

A

Transferrin is a receptor that imports iron into the cell leading to an increase in available Fe

26
Q

How does aconitase interact with ferritin and transferring mRNA

A

Aconitase binds to stem loops in the 5’UTR of ferritin mRNA and in the 3’UTR of transferrin mRNA

27
Q

Aconitase can also bind to iron itself, T or F

A

T

28
Q

Explain the role of aconitase in increasing Fe availability when iron levels are low

A

Aconitase binds to stem loops in the 5’UTR of the ferritin mRNA and blocks translation by physically blocking the ribosome from moving along the transcript. Aconitase also binds to stem loops in the 3’ UTR of transferrin mRNA and blocks its degradation. Binding of aconitase stabilises the transferrin mRNA thus increasing transferrin synthesis. Decrease ferritin translation and increase transferrin stability and synthesis results in an increase in intracellular [Fe]

29
Q

Explain the role of aconitase in decreasing Fe availability when iron levels are high

A

Aconitase binds to iron itself in the cytoplasm which causes a change in its conformation. A change in aconistase comformation causes it to dissociate from the stem loops in the 5’UTR of the ferritin mRNA and the 3’UTR of the transferrin mRNA. The now unstable transferrin mRNA transcript is then degraded quickly and the ribosome is now free to move along and translate the ferritin mRNA. This leads to a decrease in Fe availability

30
Q

What co-factor of the translation machinery is required for all mRNA translation

A

eIF-2

31
Q

When a cell is in the G0/quiescent phase of the cell cycle, globally, translation is turned down. How is this achieved

A

Phosphorylation of eIF-2 causes its tight binding to eIF-2B. eIF-2B is usually required as a guanine nucleotide exchange factor but tight binding of eIF-2B to the phosphorylated eIF-2 prevents it’s recycling by GTP displacing the bound GDP. By preventing guanine nucleotide exchange, eIF-2 is prevented from initiating translation.

32
Q

eIF-2 with its bound GTP binds to Met-tRNA to start ribosome scanning, T or F

A

T

33
Q

What are IRES sequences and how can they influence gene expression

A

Internal ribosome entry sequences are stem loops contained within RNA that can initiation formation of the ribosome independent of the Cap/PolyA initiation complex by mimicking it.

34
Q

IRES translation initiation is an effective way of encoding a second reading frame within RNA, T or F

A

F – this is not a very effective method with less than 10% of second ORF translation

35
Q

Which translation initiation cofactor is required to bind to the IRES stem loop and initiate translation independent of the 5’-Cap and PolyA tail

A

eIF-4G

36
Q

Where are IRES sequences commonly found

A

IRES sequences are often found in viral transcripts

37
Q

How can viruses use IRES sequences to promote translation of their transcripts

A

Viruses favour translation of their transcripts by cleaving eIF-4G in a way that prevents it from binding to eIF-4E but can still bind to IRES stem loops

38
Q

What is the significance of eIF-4G cleavage in apoptosis

A

eIF-4G is cleaved during apoptosis to prevent its binding to eIF-4E and hence prevent any more translation

39
Q

RNA stability is defined by the half-life of the different mRNAs and varies greatly, T or F

A

T

40
Q

How does the polyA tail change over time and act as a clock to determine mRNA age

A

The polyA tail usually starts at around 200 residues in length but as time goes by this is gradually degraded by exonucleases. Once it reaches 30 nucleotides in length it is de-capped and degraded. Hence the number of adenines in the PolyA tail can determine the age of the mRNA transcript

41
Q

How can mRNA half-lives be extended

A

Re-adenylation of the polyA tail

42
Q

Often factors that promote translation also promote mRNA degradation to prevent overexpression, T or F

A

F – de-adenylating nuclease (DAN) is responsible for de-adenylation of polyA tails and competes with eIF-4E binding to the mRNA cap.

43
Q

What technique is used to visualise protein localisation within a cell or tissue

A

Antibody staining

44
Q

What technique is used to visualise gene transcription inside cells and tissues and is quick and inexpensive

A

In situ hybridisation

45
Q

GFP and transgenics are techniques used to visual gene expression and protein localisation, T or F

A

T

46
Q

Which method is often the first port of call to visualise RNA expression due to its cheap costs and short time

A

In situ hybridisation

47
Q

Explain how an expression plasmid is made in the process of making an antibody

A

The mRNA for the protein which you want to target an antibody for is extracted from a cell and converted to cDNA. This cDNA sequence is inserted into a vector containing a bacteriophage promoter. The cDNA is incorporated into the expression plasmid next to the bacteriophage promoter which is included as these promoters drive high levels of RNA synthesis and hence will produce large amounts of the protein. The promoters are also modified so that they are inducible either by chemical exposure or temperature changes to induce gene expression. These expression plasmids also contain an epitope tagging system to allow for rapid and efficient purification of the protein. These tag coding sequences are inserted in-frame and upstream of the protein of interest cDNA and are sequences to which antibodies are readily available for. These expression plasmids are then injected into bacteria which can then make the protein.

48
Q

Why are expression plasmid promoter regions made to be inducible

A

The problem with incorporating bacteriophage promoters is, due to their high levels of expression, the bacteria in which these vectors are introduced tend to die quickly due to exhaustion. By making the promoters inducible you can minimise the time spent synthesising protein to allow the cells to survive

49
Q

Explain the purpose of an epitope tagging system when creating expression plasmids in antibody synthesis

A

Epitope tags are short coding sequences integrated upstream of the cDNA in frame. These will be transcribed and translated with the desired protein and allow for the rapid and efficient purification of the protein. They code for peptides to which antibodies are readily available and already manufactured for.

50
Q

Explain the process of antibody-affinity purification

A

The crude extract is poured onto a column containing beads with antibodies for the epitope tag bound to them. A pH 7 buffer is then also loaded into the column. The crude extract then runs through the column and the protein of interest is retained by binding of the antibody covered beads to the epitope tag. The rest of the crude extract travels through the column and is removed. The column then undergoes a series of subsequent washes with the pH 7 buffer until no more protein comes out of the column. The pH is then reduced to pH 3 with another buffer which breaks the interaction between the antibody and the epitope tag and protein of interest. This results in elution of the pure protein from the column.

51
Q

Once the target protein, for which you want to create an antibody for, is isolated from the crude extract, how is this then used to make the specific antibodies

A

The purified protein is injected into an animal (i.e. rabbit, mouse, donkey) several times, typically once a month for a three-month period. After 3 months, the blood is taken from the animal and the antibodies are then purified.

52
Q

What is the name of the region of a protein to which an antibody binds to

A

Epitope

53
Q

How are antibodies visualised once they are bound to a target protein

A

Tagging antibodies with dyes or enzymes to allow determination of where proteins are localised

54
Q

What is meant by antibody sandwiches and why are they used to visualise protein localisation

A

Antibody sandwiches are produced by raising a secondary antibody that will bind to the first antibody. This produces an antibody sandwich of the primary antibody bound to the target epitope and then a secondary antibody bound to the first one. The secondary antibodies are usually tagged and this allows amplification of the signal. Because more than one tagged secondary antibody will bind to the primary antibody, a greater signal is produced

55
Q

How are tagged secondary antibodies produced

A

Secondary antibodies are raised against general antibodies from the original animal species in which the primary antibodies were raised. These secondary antibodies produced in a different species will bind to any antibodies from the other species. These are then conjugated with dyes that are fluorescent allowing for the detection of protein location using specific wavelengths

56
Q

Give two examples of commonly used enzyme conjugates in antibody detection

A

Alkaline phosphatase – turns the substrate blue. Horseradish peroxidase – turns the substrate blue

57
Q

To stain a cell/tissue with a tagged antibody, the animal/cells must be chemically fixed, how is this achieved

A

A fixative, usually formaldehyde is introduced to cross-link proteins together

58
Q

What are the two types of antibody staining and what are they used for visualising

A

Whole mount staining is used to visualise whole structures and tissues whereas staining on section involves antibody staining of slices creating cross section, this is often used in human samples.

59
Q

Explain the process of in situ hybridisation

A

Start with a purified vector containing the cDNA of interest known as the template. This cDNA is incubated with RNA polymerase to make an antisense RNA probe. The antisense RNA probe will have incorporated epitope tagged nucleotides, special nucleotides with epitope tags conjugated to them. The cells are then incubated with antisense probe which will hybridise with the endogenous mRNA. Excess probe is washed off. The epitope tags often involve alkaline phosphatase which allows the mRNA to be visualised

60
Q

Explain the differences seen in bicoid mRNA and protein localisation seen by using antibodies and in situ hybridisation

A

In situ hybridisation of bicoid mRNA will reveal its localisation in cells at the anterior region of the Drosophila embryo with defined borders. However the antibody staining for the bicoid transcription factor protein will show a different pattern. It would show a decreasing gradient of the bicoid protein, indicative of a morphogen

61
Q

How is bicoid mRNA localised anteriorly in the Drosophila embryo

A

Contains within its 5’UTR a region that localises it at the anterior region of the cells

62
Q

Explain how GFP works to provide fluorescence

A

GFP is excited by blue light with a wavelength of 475nm. Excitation of the GFP results in electrons within the protein increasing their energy level. The transition of these electrons back down to low energy states gives off energy in the form of light. For GFP, this energy emits is given off as green light with a wavelength of 510nm

63
Q

Fluorescence always gives off an emission wavelength that is greater than the excitation wavelength and a lower energy, T or F

A

T

64
Q

Explain how GFP is used in gene fusion to create transgenic lines

A

Firstly, you genetically engineer GFP onto the end of the last exon of the target protein by adding a GFP encoding sequence to the end of the last exon. This will be translated to a fusion protein contiaining the target protein and a fused GFP protein.

65
Q

What does GFP gene fusion specifically allow that reporter constructs done

A

Because a functional protein of interest is produced the subcellular localization of the protein once can be studied

66
Q

How does the incorporation of GFP still lead to the production of a functional protein

A

GFP doesn’t interact with the other protein and doesn’t impact its folding. Hence a functional protein is produced which allows for the study of where it is localised. This method only tells you were and when gene is expressed because no functional protein is produced other than GFP

67
Q

Once GFP constructs have been created how are these then used to visualise protein expression

A

Microinjecting a solution of the DNA into the one-cell zygote is followed by incorporation of the construct into the host genome. The DNA randomly integrates into the genome by the DNA repair machinery and leads to the creation of a transgene

68
Q

What is the name given to the representation of the differences between species and their evolution from common ancestors

A

Phylogenetic tree

69
Q

It has been shown that most of the genes across kingdom Animalia have been relatively conserved. How has this been achieved

A

BLAST analysis of protein structures to determine regions of significant homology

70
Q

It was determined that what makes members of the animal phyla different is not differences in the genetic sequence as such but what difference was seen

A

Changes in expression of a common set of genes

71
Q

For great apes and man the change in the nucleotide sequence is about 1% every 10million years, T or F

A

T

72
Q

If the human and great ape nucleotide sequence changes by roughly 1% every 10 million years and the common ancestor of humans and chimpanzees evolved 5 million years ago, how different are their genomes. Represent this as a fraction and as a number of nucleotides

A

0.5% difference – 1 in 200 nucleotides

73
Q

Firstly, molecular data is used to distinguish specices which is then backed up by morphological data, T or F

A

F – vice versa

74
Q

When assembling the phylogenetic tree a specific gene is used to determine relation, this gene is FOXP2. Why is FOXP2 used as a way of differentiating between species

A

FOXP2 is a highly conserved protein which only shows differences at a few positions in the amino acid sequence

75
Q

Which positions in the amino acid sequence does the FOXP2 vary

A

80, 303 and 325

76
Q

Mice and chimps both have a threonine residue at position 303, what therefore can be inferred about the common ancestor of these species

A

Their common ancestor must have also had a 303T

77
Q

Humans and chimps both have 80D in the FOXP2 gene meaning that the common ancestor between these species also possess an 80D. Which amino acid is denoted by D

A

Aspartate

78
Q

When sorting animals into phyla, programmes similar to BLAST consider all possible relations between animals. How are they then sorted

A

The tree is assembled based on the simplest model with the fewest changes

79
Q

What is meant by the term parsimony

A

The idea that you always assume the simplest model

80
Q

Convergent evolution goes against the parsimony model. How does convergent evolution account for differences in the amino acid sequence

A

Changes in the amino acid sequence of two animals occur independently of each other

81
Q

When assembling the phylogenetic tree, molecular phylogeny is compared with morphological phylogeny and fossil records to give us a deeper understanding of evolution, T or F

A

T

82
Q

Although there are only 4 families of vertebrate FGF receptors, how many distinct receptors are there

A

22

83
Q

The Ciona or sea squirt is a distant vertebrate ancestor that only contains 4 FGF receptors. What does this tell us about the common ancestor of Ciona and modern vertebrates

A

The common ancestor also had only 4 FGF receptors

84
Q

What can explain the other 16 FGF receptors present in vertebrates that aren’t in sea squirts

A

These will likely have arisen due to genome duplication both locally and by ploidy events

85
Q

What is the name given to a duplicated gene present in the genome

A

Paralogue

86
Q

Chromosome duplications or duplication mutations are seen frequently when comparing genomes, T or F

A

T

87
Q

When a gene is first duplicated, what is its role

A

It is redundant

88
Q

Over time duplicated genes evolve allowing a refinement of function or for a new function to development, T or F

A

T

89
Q

What ways can the extra copy of a gene change

A

Can change the pattern of gene expression or the structure/function of the protein

90
Q

Big changes to protein structure are caused by what type of mutations

A

Domain swapping

91
Q

Smaller changes in the protein structure of a duplicated gene are caused by nonsense mutations, T or F

A

F – missense mutations

92
Q

What is considered to be the common driving force for morphological evolution of animals

A

Changes in expression of genes

93
Q

What changes in the transcription machinery present within the DNA explain why expression pattern changes have a major role in morphological evolution

A

Enhancers can change easily

94
Q

What process can account for bringing a new enhancer regions close to the coding sequence of a gene

A

Non-homologous recombination

95
Q

Enhancers must occur upstream of a target gene, T or F

A

F – enhancers can occur anywhere in the gene sequence and their exact position is usually unimportant

96
Q

Changes in protein structure have to be precise so as not to introduce a variety of negative effects. Give some examples of these effects

A

Introduction of a stop codon, change in the reading frame, interference with protein folding or a disruption in RNA splicing

97
Q

Describe the differences seen in the development of appendages in the fruit fly and Crustacea

A

Crustaceans development limbs throughout their abdominal and thoracic segments whereas in Drosophila, no legs develop in the abdominal regions

98
Q

Explain the interactions between distal-less and ultrabithorax in forming legs in Drosophila

A

Distal-less (Dlx) specifies leg precursor cells in the fly embryo and is expressed in the abdominal and thoracic segments. However, ultrabithorax (Ubx) is also expressed in the abdomen where is represses Dlx expression. Thus, legs form only in the thorax of the fly

99
Q

How do Dlx and Ubx act differently in Crustacea to fruit flies

A

In crustaceans Dlx and Ubx are both expressed in the abdomen and thorax. However, Crustacean Ubx has an anti-repression motif that was lost in insects. Thus dlx expression is not repressed in Crustaceans and abdominal legs develop. These help crustaceans to swim

100
Q

It was found that the Ubx protein in flies lacked an anti-repression domain in the ubx protein. What was seen when comparing the two copies of the ubx gene

A

There was a dramatic change in the Ubx C-terminus in flies. Crustaceans have a motif that block repression. On the other hand, Drosophila contain a polyalanine amino acid sequence which is absent in Crustaceans

101
Q

What would be the results of ectopic expression of Crustacean ubx in the abdominal segments of Drosophila embryos

A

Limb formation in the abdomen due to loss of inhibition of dlx expression

102
Q

What is meant by the term signal transduction

A

The process by which extracellular signalling molecules cause changes in target cells

103
Q

List some of the downstream effects of signalling mechanisms on the cell

A

Survival, death(apoptosis), migration, differentiation, division

104
Q

Signalling pathways usually involve an extracellular signalling molecule and a receptor protein that leads to the activation of intracellular signalling. Give examples of the different downstream targets of intracellular signalling

A

Metabolic enzymes, gene regulatory proteins and cytoskeletal protein elements

105
Q

What type of signalling molecules do cell surface receptors often interact with

A

Hydrophilic ligands

106
Q

What kind of signalling molecules do intracellular receptors interact with

A

Hydrophobic/lipophilic signalling molecules

107
Q

What kind of proteins are often the downstream targets of intracellular receptors

A

Transcription factors

108
Q

What is the other name given to ligand-gated ion channels

A

Inotropic receptors

109
Q

Give an overview of signalling from GPCRs

A

The GPCR will interact with a ligand that allows binding of the receptor to a G-protein forming a complex which can then interact with enzymes

110
Q

How many transmembrane domains are indicative of GPCRs

A

7 transmembrane domains

111
Q

Describe the general structure and activation of enzyme-coupled receptors

A

Enzyme coupled receptors are transmembrane proteins that interact with ligands. This interaction then activates the intracellular enzyme domain or recruits an intracellular enzyme leading to signal transduction

112
Q

Juxtacrine signalling is an example of a short-range signalling mechanism. Outline how this signalling mechanism acts

A

In juxtacrine signalling, two cells are in direct contact and the ligand is a membrane-bound signal molecule (transmembrane protein)

113
Q

Which kind of short-range signalling involves ligand secretion from the signalling cell which acts as a local mediator on neighbouring cells, diffusing only a few cell diameters away

A

Paracrine

114
Q

Describe what is meant by autocrine signalling

A

Autocrine signalling involves local groups of neighbouring cells with both produce the receptor and the ligand for a particular pathway. The secreted factor activates its own receptor on the same cell and neighbouring cells often as part of a positive feedback loop.

115
Q

Where in the body is autocrine signalling often used

A

Used a lot in the immune system

116
Q

In addition to paracrine, autocrine and juxtacrine signalling, there is another type of short range cell signalling. What is this signalling mechanism and how does it act

A

Short range signalling can also occur through gap junctions. Gap junctions act to physically connect cells together and allows them to share small molecules and ions. This acts to metabolically and electrically couple cells together and allows the transfer of species such as cAMP and Ca2+ between cells.

117
Q

Cells of different types can respond to the same signal quite differently. Give an example of this

A

The response of different cells to acetylcholine can vary in different tissues. In the heart, release of acetylcholine causes the relaxation of cardiac muscle whereas in skeletal muscle it causes contraction. Similarly, in glandular tissue, acetylcholine release causes secretion of vesicles

118
Q

What is meant by primary transduction

A

Primary transduction is the process the converts the signal from the extracellular signalling molecule to an intracellular signal

119
Q

Intracellular amplification of signals is a common feature of signal transduction, T or F

A

T

120
Q

Integration steps often occur in intracellular signalling where different pathways can affect the same components. In contrast, spreading of the signal is never seen, T or F

A

F – spreading is also seen

121
Q

What is the role of scaffold proteins in intracellular signalling

A

Scaffold proteins play an important role in anchoring numerous components in place to respond to the activated receptors. By holding components in place, scaffold proteins effectively increase the effective concentration

122
Q

What is often the result of phosphorylation of receptors once they become activated

A

Phosphorylation of receptors often creates recognition (docking) sites for downstream protein binding

123
Q

What are the two main types of GTP binding proteins involved in intracellular signalling

A

Guanine Exchange Factors (GEFs) and GTPase activating proteins (GAPs)

124
Q

What class of receptors are the receptor tyrosine kinases

A

Enzyme-linked receptors

125
Q

What sorts of cell behaviours are RTKs involved in regulating

A

Proliferation, differentiation and migration

126
Q

How many different families of RTKs are there

A

16 different families

127
Q

Each ligand receptor pair involves one specific ligand and one unique receptor, T or F

A

F – whilst some ligands are specific for one receptor and vice-versa, some ligands and receptors can be promiscuous and bind to various other components

128
Q

Give some examples of RTK ligands

A

Ephrins, Nerve Growth Factor, Fibroblast Growth Factor, Epidermal Growth Factor

129
Q

What is the result of stimulating the EGF receptor tyrosine kinase

A

Stimulation of proliferation

130
Q

What is the result of stimulating the IGF receptor

A

Stimulation of carbohydrate utilisation and protein synthesis

131
Q

What is the result of stimulation of the TrkA receptor by binding of NGF

A

Stimulation of survival and growth of some neurons

132
Q

Most RTKs are monomers with one major exception, which receptor is this

A

The insulin receptor is an RTK which is present as a dimer

133
Q

What can be said about the extracellular domains of RTKs throughout the family

A

The extracellular domains vary greatly along with the ligands. They do however share features such as Ig-like and fibronectin-like domains and often contain several repeating units

134
Q

Describe the structure of the intracellular domain of RTKs

A

The intracellular domains possess the kinase activity. These are present as a single domain or split into two

135
Q

What can be said about the transmembrane domain in RTKs

A

The transmembrane domain is said to lack structure and be very simple. It is short and string like, consisting of between 25 and 38 amino acid residues

136
Q

Outline canonical RTK activation

A

Following ligand binding, either as a dimer or monomer, the monomeric RTK receptor will dimerise by recruitment of the other receptor monomer. Similarly, ligand binding may also reorientate existing receptor oligomers. Activation of the RTK causes a change in conformation of the receptor dimer. This starts with the extracellular and transmembrane domains and is then translated to the intracellular kinase domain. This change in conformation of the intracellular domain unmasks the tyrosine kinase domain and exposes important residues for this process. The activated receptor then undergoes auto and crossphosphorylation. This increases the activity of the kinase domains, stabilises the active state of the receptor and causes the kinase domain to phosphorylate other tyrosines in the receptor to create docking sites. These kinase domains are now able to phosphorylate target proteins that bind to the docking site to transduce the signal.

137
Q

What are the effects of auto and cross-phosphorylation of the active RTK

A

Increased kinase domain activity, stabilisation of the receptor active state (ligand independent) and the creation of docking sites for target proteins

138
Q

Explain the gain of function approach that can be used to investigate RTK signalling

A

Genetically engineer DNA to generate a gene encoding an RTK whose extracellular ligand binding domain has been replaced with a homodimerization domain. Expression of this gene in an organism at high levels by incorporation of a transgene will result in the production of an RTK capable of dimerising in the absence of ligand binding. This receptor tyrosine kinase will be activated independently of the ligand and known as constitutively active. By expressing this transgene at high levels there is no need to interfere with the other endogenous gene.

139
Q

Explain the loss of function approach that can be used to investigate RTK signalling

A

Genetically engineer DNA to generate a gene encoding an RTK whose intracellular kinase domain is mutated. This will lead to a loss of kinase activity and thus no auto and crossphosphorylation. Hence the RTK will be unable to activate in response to ligand binding. This DNA can then be expressed at high levels to result in a dominant negative or antimorphic mutation whereby the mutant RTK will poison the endogenous receptor

140
Q

SH2 domains that bind to phosphotyrosines in RTKs also recognise adjacent residues. What is the recognition sequence which they recognise

A

Phosphotyrosine-Glutamate-Glutamate-Isoleucine

141
Q

What component of the extracellular matrix do RTK ligands often form complexes with

A

Heparan sulphate proteoglycans (HSPGs)

142
Q

The FGF receptor-ligand complex can become activated in the absence of binding to components of the extracellular matrix, T or F

A

F – the receptor can only become activated when in a complex with HSPGs

143
Q

HSPGs can be membrane tethered in two ways. Describe these

A

HSPGs can be tethered to the cell membranes either by a transmembrane domain within the proteoglycan backbone or through a lipid modification such as GPI anchors

144
Q

HSPGs can also be entirely secreted, T or F

A

T

145
Q

What species attached to the proteoglycan backbones can be sulphated to trigger ligand binding to the FGF receptor

A

Glycosaminoglycans

146
Q

HSPGs are important extracellular modifiers of cell-cell signalling, what is their role in the extracellular environment

A

They are important in organising the extracellular matrix into basal lamina

147
Q

Give some examples of HSPGs

A

Glypican, Syndecan and Perlecan

148
Q

Describe the structure of HSPGs

A

Consist of a proteoglycan core with glycosaminoglycan side chains

149
Q

Other than Heparan Sulphate, give some examples of sugar side chains present on proteoglycans

A

Aggrecan, Betaglycan, Decorin and Perlecan

150
Q

Modification by sulphation of GAGs can provide a code which creates binding sites for specific proteins and sequences that carry information, T or F

A

T

151
Q

The pattern of sulphation acts as a code allowing specific HSPGs to interact with specific proteins, T or F

A

T

152
Q

Describe the effects of HSPGs on the gradients of secreted molecules

A

HSPGs control the steepness of a secreted molecule gradient and how far a growth factor can diffuse through the extracellular space

153
Q

Explain the role of HSPGs in FGF signalling

A

FGF and its receptor forms a complex with heparan sulphate proteoglycans. HSPGs provide an extracellular scaffold for FGF and presents it to the receptor after it has oligomerised on the HSPG

154
Q

Give some examples or proteins that bind to RTKs

A

GTPase-activating proteins (GAPs) that function in the Ras/MAP kinase pathway, phospholipase C-? (part of the inositol lipid pathway) and PI-3 kinases that act as regulatory subunits

155
Q

Explain how activation of RTKs leads to signal transduction by the Ras pathway

A

Ras is a smallGTPase that is present in the membrane of the cell. The activated RTK contains phosphorylated tyrosine residues in its intracellular kinase domain that have occurred because of autophosphorylation. These phosphotyrosines are recognised by proteins that contain an SH2 domain. In the Ras pathway, this protein is Gbr2 which binds to the activated receptor by its SH2 domain. Gbr2 then recruits another protein to the complex called sos by its SH3 protein-protein interaction domain. Sos is a guanine nucleotide exchange factor (GEF) that is bound to the Ras GTPase. Binding of Gbr2 to sos couples the activated RTK to the inactive Ras. Sos also promotes dissociation of GDP from Ras which is displaced by GTP. Now that it’s bound to GTP Ras dissociates from sos and phosphorylates MAP-KKK to transduce the signal further.

156
Q

How does the MAP kinase pathway rely the signal transduction further from activation of the Ras GTPase

A

Activated Ras phosphorylates MAP-KKK which then binds to and activates MAP-KK by phosphorylation. Activated MAP-KK then goes onto phosphorylate and activate MAP-K. Activated MAP-Kinase can then phosphorylate transcription factors and other proteins leading to the regulation of gene transcription

157
Q

What are the mammalian homologues of MAP-KKK, MAK-KK and MAP-K

A

MAP-KKK –> Raf, MAP-KK –> Mek and MAP-K –> Erk

158
Q

What is the effect of having multiple stages in the MAP Kinase pathway

A

One activated MAP-KKK can phosphorylate and activate several MAP-KK proteins which in turn can phosphorylate and activated multiple MAP-K proteins. This acts as an amplification step in signal transduction

159
Q

Cyclins are an example of downstream targets of MAP-Kinases, T or F

A

T

160
Q

MAP-K is regulated by its phosphorylation by MAP-KK, describe how MAP-K is activated

A

In order to be activated MAP-K must have both of its phosphorylation sites on threonine and tyrosine residues phosphorylated by MAP-KK. These amino acids are interspaced by only one residue and lie in close proximity.

161
Q

How long after RTK activation is gene transcription influence

A

Within minutes

162
Q

Explain how Ras acts as a molecular switch in downstream RTK signalling

A

Ras is a smallGTPase that functions as a molecular switch. Its nucleotide-binding site is formed by several protein loops that cluster one end of the protein. Inactive Ras is bound tightly to GDP which is displaced by GTP when Ras becomes active. Ras can toggle between two conformational states depending on whether GTP or GDP is bound. The switch 1 and switch 2 regions change conformation dramatically between to two Ras states and this conformational change allows other proteins to distinguish activate Ras from inactive Ras. Active Ras binds to and activates, downstream target proteins in the cell signalling pathways. Hydrolysis of GTP to inactivate Ras requires the action of Ras-GAP which binds tightly to Ras burying the bound GTP. Ras-GAP inserts an arginine side chain directly into the active site. This Inserted arginine with threonine and glutamine side chains of Ras itself, promotes the hydrolysis of GTP.

163
Q

Explain how the apparatus for fluorescent microscopy works

A

In addition to the light on the specimen, fluorescent microscopes also have a light of a specific wavelength shone onto the stage. This is either provided by a mercury lamp or a laser. The excitation filter of the microscope is then specific for the fluorophore used to stain particular regions of the specimen. A dichroic beam splitter mirror is used to reflect only short wavelengths hence allowing longer wavelengths to pass straight through. As fluorescent objects emit light of a longer wavelength than was shone onto it, the emitted light from the specimen passes through into the eyepiece. A greyscale camera then measures the light intensity and creates a greyscale image which can later be analysed

164
Q

Why is a greyscale camera used to study fluorescence

A

Colour cameras aren’t sensitive enough to produce high quality images. Instead, colour is added to the greyscale image later to create what is known as pseudocolour

165
Q

Explain how fluorescent resonance energy transfer can be used to investigate protein interactions

A

Recombinant fusion of proteins X and Y to separate fluorescent proteins that absorb and emit certain wavelengths of light allows you to determine if X and Y interact/bind. By correlating the wavelength emitted by the fluorescent protein attached to X with the wavelength of light needed for fluorescence of protein Y you can activate protein Y fluorescence if it is in close proximity to X (i.e. it is bound). I.e. if shining light needed for fluorescence in protein X leads to the appearance of light that is given off as a result of protein Y fluorescent you can determine that X and Y interact

166
Q

What phenomenon is FRET said to rely on

A

Paired fluorescence

167
Q

Leprechaunism is a disease caused by mutations in the insulin pathway. Describe the symptoms of this condition

A

Usually fatal within the first 2 years of life. Patients have an elfin-like facial appearance with protuberant ears and relatively large hands and feet. Also there is a decreased amount of subcutaneous fat and muscle mass is seen, and the skin is abnormal with increased hair growth

168
Q

Where is insulin produced

A

In the B-cells of the Islets of Langerhans in the pancreas

169
Q

Explain the cleavage events that take place after insulin synthesis

A

The full-length insulin protein is heavily modified after synthesis. These multiple cleavage steps leave the final product with only amino and carboxyl terminal peptides. These fragments are held together by cysteine bonds that can only form outside the cell

170
Q

The cysteine bonding that holds the insulin peptides together can only form outside the cell, why is this

A

These cysteine bonds can only form in an oxidative extracellular environment

171
Q

What is the immediate effect of insulin signalling

A

Glucose uptake from the blood into muscle cells and adipocytes

172
Q

What is the long-term effects of insulin exposure

A

Increased expression of liver enzymes that synthesise glycogen as well as enzymes involved in triacylglycerol synthesis in the adipocytes

173
Q

Describe the structure of the insulin receptor

A

The insulin receptor is also synthesised as a full length protein that is then cleaved into ? and ? subunits that are held together by disulphide bridges

174
Q

The insulin receptor is an RTK, what is unusual about its structure

A

The insulin receptors is present as a dimer in its inactivated state

175
Q

Describe what happens following ligand binding to the insulin receptor

A

Ligand binding brings the intracellular kinase domain together and results in autophosphorylation of the receptor. Autophosphorlyation creates a single docking site for the insulin receptor substrate (IRS)

176
Q

Explain the role of IRS in insulin signalling

A

The insulin receptor substrate contains a phosphotyrosine binding domain (PBD) similar to SH2 that binds to the phosphorylated site on the activated insulin receptor. IRS then acts as a docking site for other proteins such as GRB2 leading to the activation of the Ras pathway.

177
Q

IRS is known as a surrogate, what is meant by this

A

IRS binds to the signal docking site on the activated insulin receptor but then creates additional docking sites for downstream targets

178
Q

What is the name of the other main branch of signal transduction from RTKs such as the insulin receptor other than the Ras/MAP-K pathway

A

PI-3 kinase pathway

179
Q

Describe the structure of PI-3 Kinase

A

PI-3 Kinase is made up of two subunits; the P85 subunit which contains an SH2 domain and the P110 subunit which contains the kinase activity

180
Q

Explain the role of PI-3 Kinase in Ras-MAP-K independent signal transduction

A

Upon binding to IRS, PI-3 kinase phosphorylates phosphoinositol-4,5-bisphosphate and PI 4-phosphate to PI 3,4,5- trisphosphate and PI 3,4-biphosphate, respectively. This creates a docking site for protein kinase B which, once recruited to the membrane, is phosphorylated by membrane associated kinases such as 3-phosphoinositide-dependent kinase (PKD1). PKB then goes through a conformational change to become active and is released to effect numerous protein:

181
Q

Give some examples of the downstream protein targets of activated PKB

A

GSK3, GLUT4, FOXO and Phosphoenolpyruvate Carboxykinase

182
Q

What is the effect of activated PKB on glucose transport

A

GLUT4 is already synthesised in the cells but is present in vesicles beneath the membrane. Insulin binding and subsequent activation of PI-3 Kinase leads to the increase translocation and insertion of GLUT4 into the membrane hence increase sugar uptake from the blood

183
Q

What is the effects of activated PKB on glucose storage and how is this achieved

A

PKB phosphorylates GSK3 to inactivate it this allows glycogen synthase to be active and to promote the storage of sugars. Normally, GSK3 phosphorylates glycogen synthase to inactivate it and block the storage of sugars

184
Q

What are the effects of PKB activity on glucose synthesis

A

FOXO is a transcription factor that activates expression of PEPCK, a glucose synthesis gene. PKB phosphorylates and inactivates FOXO and prevent glucose synthesis

185
Q

Insulin’s effects on glucose synthesis are known as the faster response, T or F

A

F – it’s a slower response

186
Q

How does FOXO act at low blood insulin levels

A

When there are low blood insulin levels, Foxo binds to an IRS near to PEPCK to activate its transcription

187
Q

Microarrays are one way of measuring changes in gene expression as a result of insulin signalling, what is the other main method of measuring these changes and how does it work

A

Quantitative PCR - This allows you to quantify the levels of RNA and hence levels of gene expression. A dsDNA template is first denatured by heating to break the hydrogen bonding between complimentary base pairs. This can either be genomic dsDNA or produced from mRNA using reverse transcriptase. Primers are designed to bind to a known target gene and are introduced to the sample with DNA polymerase and dNTPs. Successive rounds of DNA replication are carried out leading to an exponential increase in the amount of DNA. This can be quantitively analysed by the incorporation of a fluorescent DNA dye into the reaction mixture which will allow you to determine the amount of DNA present after each PCR cycle. From this you can determine the amount of DNA present initially.

188
Q

Why do the primers used in PCR to analyse the effect of insulin on gene expression need to be close together

A

Because PCR can only work for between 10-20kbps

189
Q

What is the benefit of using a heat stable DNA polymerase in PCR

A

Allows you to repeat the cycle (heating wont denature the polymerase but can still break the hydrogen bonding between the strands

190
Q

How many PCR cycles are usually carried out

A

30 cycles – producing >1x109 fragments

191
Q

What is the effect of designing short primers that anneal to a specific gene or sequence of interest within a fragment

A

Primers trim down DNA sequence from the genomic dsDNA to only amplify the target sequence that lies between the two primers

192
Q

To look at gene expression specifically using PCR, it is essential that you must use high quality tissue. Why is this

A

You need to extract all of the mRNA transcribed by the tissue

193
Q

What is the name of the technique used to quantify gene expression from mRNA

A

Reverse Transcriptase PCR

194
Q

Describe the process of promoter analysis/promoter bashing

A

Promoter bashing is used to identify the actual DNA sequences that act as promoters and enhancers. You start with the promoter of interest and much of the sequence adjacent it. A transgene is then made that uses a quantifiable reporter gene such as luciferase, GFP, B-gal or HRP. Then make a series of deletions in the promoter and flanking sequences to test the responsiveness to insulin to find which sequences are critical in the activity after ligand binding Look at the sequences that were deleted that lead to decreased levels of activity to identify the sequences acting as promoters.

195
Q

On average, how many people will have cancer in their lifetime

A

1 in 3 to 1 in 4

196
Q

What is the most common type of cancer

A

Lung cancer

197
Q

What are the three main causes of cancer

A

Physical carcinogen – UV and ionising radiation, chemical carcinogens – asbestos and tobacco smoke, biological carcinogens – infection from certain viruses, bacteria or parasites

198
Q

What family of receptors do the human epidermal growth factor receptors belong to

A

Receptor tyrosine kinases

199
Q

What are the different types of HER receptor

A

HER1, HER2, HER3 and HER4

200
Q

What is unusual about the HER2 receptor

A

Unlike the other members of the HER receptor family, HER lacks the extracellular ligand binding domain. This means that it doesn’t require ligand binding to dimerise and hence is constitutively active

201
Q

What is unique about the HER3 receptor

A

The HER3 receptor has lost its intracellular tyrosine kinase domain and so cannot phosphorylate target proteins

202
Q

What happens to the HER receptors (apart from HER2) following ligand binding

A

HER proteins undergo a conformational change upon ligand binding that is essential for dimerization and signalling. Once the ligand binds, the receptor can either homo or heterodimerise which stimulates the receptor to signal

203
Q

Which conformations of HER receptor dimers signal the strongest and why

A

Homodimers don’t signal as strongly as heterodimers. The HER2:HER3 heterodimer signals the strongest as it activates both the Ras/MAPK pathway and the PI-3 Kinase pathway

204
Q

Outline the HER pathway of activation

A

Growth factor binding to a HER receptor allows dimerisation with other HER receptors. Dimerisation of the HER receptors allows for phosphorylation of the intracellular tyrosine kinase domain. This creates binding sites for signalling proteins such as PTEN PDK etc. This in-turn leads to the activation of PI-3 Kinase pathway which prevent the apoptosis of cancer cells. HER receptors can also signal through the activation of the MAP-K pathway to increase cancer cell proliferation

205
Q

Different combinations of receptors stimulate different signalling pathways and can lead to cell proliferation, survival and a prevention of apoptosis, T or F

A

T

206
Q

HER proteins are tumour suppressor genes, T or F

A

F – they are proto-oncogenes

207
Q

What causes HER proteins to become oncogenic

A

Overexpression or mutations

208
Q

Which mutant form of HER2 was first discovered in rats the promote cancer aetiology

A

Neu

209
Q

What process can be used to determine the copy number of a gene that has been amplified in tumorigenesis

A

Fluorescent in situ hybridisation (FISH)

210
Q

Normal breast tissue contains around 20,000 copies of the HER2 receptor, how many copies can be found in cancerous tissues

A

2 million copies – 10-fold increase

211
Q

HER2 is a negative prognostic marker for several cancers, what is meant by this

A

Overexpression of HER2 correlates with poor survival rates of breast cancer patients

212
Q

How do the median survival rates of HER2 positive and HER2 negative breast cancers compare

A

HER2 positive – 3 years, HER2 negative – 6-7 years

213
Q

Up to what percentage of breast cancers are believed to be HER2 positive

A

0.25

214
Q

Herceptin is a monoclonal antibody used to target HER2 overexpression, what is meant by a monoclonal antibody

A

A massive population of antibodies derived from a single cell and that all specifically recognise a single epitope

215
Q

How are monoclonal antibodies made for HER2

A

Immunise mice with HER2 and then isolate the spleen cells containing the antibody-producing B memory cells. These cells are fused with myeloma immortal cells. This creates an immortal cell line that will continue to make huge amounts of the antibody

216
Q

What is the problem with using mice to produce HER2 antibodies

A

Murine antibodies are not well tolerated in humans, the immune response from these will kill

217
Q

How is the problem of immune response to foreign antibodies overcome

A

Humanisation

218
Q

What was the three different pieces of evidence for the use of the 4D5 monoclonal antibody in the treatment of HER2 positive breast cancer

A

4D5 binds to the extracellular domain of HER2 and leads to an inhibition of their proliferation. Injecting mice with the anti-HER2 monoclonal antibody supresses tumour growth in mice. Finally, injection of a radiolabelled 4D5 targets HER2 positive breast cancer cells in women

219
Q

Explain how immunoglobulins are humanised

A

The hypervariable domain involved in antigen binding is swapped into human antibodies. This enables the antibodies to be used in human patients without causing massive immune responses. It is achieved by cloning the murine heavy and light chain cDNA encoding 4D5 into the human IgG1 heavy chain and light chain plasmids. The humanised antibody is then made by transfecting Chinese Hamster Ovary (CHO) cells with light and heavy chain plasmids

220
Q

What is the other name for the hypervariable domain involved in antigen recognition by immunoglobulins

A

Complementary determining region (CDR)

221
Q

Explain the serious complications that can occur because of Herceptin treatment

A

Because HER2 receptors are present in the heart, the inhibition as a result of Herceptin treatment can lead to decreased cardiac viability

222
Q

What is the best way of using Herceptin to treat HER2 positive breast cancer

A

Combination with surgery, chemotherapy – 85% of patient’s cancer free for 5 years

223
Q

Describe Herceptin’s mechanism of action

A

Binds to extracellular domain of the HER2 receptor and inhibits the proliferation of human tumour cells through multiple mechanisms of action. Firstly, it blocks downstream signalling of HER2 receptor but it also marks cells for degradation by the immune system. Finally, it is believed to also exploit the internalisation of HER2 receptors

224
Q

New versions of HER2 inhibitors also inhibit receptor dimerisation, T or F

A

T

225
Q

What is the general role of lipids in the cell membrane

A

Provide flexibility and continuity

226
Q

What is the general role of proteins in the cell membrane

A

Mediate interactions with the surrounding environment as well as playing a role in motility and the uptake of nutrients

227
Q

What is the general role of carbohydrates in the cell membrane

A

Involved in cell tagging and protection

228
Q

What methods are there for sugars to be attached to the cell membrane

A

Sugars can either be attached to the membrane via proteins or via lipids

229
Q

Which side of the membrane are sugars usually attached

A

The extracellular surface

230
Q

Outline the basic structure of a phospholipid

A

Phospholipid consists of a glycerol molecule, phosphate group, lipid head group and two hydrocarbon tails. The two fatty acids are attached to the oxygen groups in the glycerol molecule via an ester bond

231
Q

List the four major phospholipids present in cell membranes

A

Phosphatidylserine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin

232
Q

Phospholipids are amphipathic, what is meant by this

A

The contain both hydrophilic and hydrophobic parts. A hydrophilic (polar) phosphate group and the non-polar hydrophobic fatty acid tails

233
Q

Explain the different behaviours of single and double tailed phospholipids when placed in solution

A

Single tailed phospholipids will form micelles when placed in water. Two tailed phospholipids will form bilayers in water due to geometric restriction of additional fatty acid tail prevent micelle formation

234
Q

What causes phospholipids to form sealed compartments when placed in water

A

The repulsion by water of the hydrophobic regions makes the lipid bilayer hide its edges and form a sealed compartment which is energetically favourable

235
Q

One-legged (two tailed) lipid molecules form vesicles encapsulating water, T or F

A

F – two-legged phospholipids do

236
Q

Unsaturated fatty acids provide flexibility to cell membranes, whereas saturated lipid are too rigid and not compatible with cell membranes, T or F

A

T

237
Q

What makes fatty acid tails unsaturated

A

The presence of double bonds within the hydrocarbon tails

238
Q

Which organisms produce omega 3 fatty acids

A

Sea plants, fish and nuts

239
Q

Describe the nomenclature of omega fatty acids

A

Furthest carbon away from the carboxyl group in a fatty acid is called the omega (?) carbon. The position of the first double bond determines the name of the ?-fatty acid

240
Q

Land plants do not contain the enzyme to insert the double bond in the carbon 3 position – to make ?-3, T or F

A

T

241
Q

Describe the dynamic behaviour of phospholipids in the membrane

A

The behaviour of phospholipid molecules in a lipid bilayer is extremely dynamic. Phospholipids can rotate or exchange and diffuse in the lateral plane of the membrane. Phospholipids can also flip-flop (transfer between leaflets), but this occurs slowly and is very rare

242
Q

Explain what is meant by the hydration layer and its tole in the cell membrane

A

The hydrophilic head group of the phospholipids usually have several water molecules surrounding it creating the hydration layer. The hydration layer presents a physical obstacle for random vesicle fusion with the plasma membrane and each other

243
Q

Describe the structure of cholesterol

A

Cholesterol has a very solid core of 4 carbon rings. Phospholipids and cholesterol together form functional cell membranes

244
Q

What is the role of cholesterol in the cell membrane

A

Cholesterol makes membranes less permeable. Its affects the way hydrophobic tails interact with each other and makes the membrane less permeable to water soluble molecules by packing between the phospholipids

245
Q

Cholesterol doesn’t make the membrane less fluid as a whole but makes the membrane less deformable at the surface, T or F

A

T

246
Q

What configuration are the double bonds that make up unsaturated fatty acids usually in

A

This cis conformation which introduces sharp kinks

247
Q

Why is it that unsaturated fats allow the membrane to be flexible

A

Unsaturated fatty acid tails pack loosely together allowing the bilayer to remain fluid

248
Q

What attribute of cholesterols structure allows it to stabilise phospholipid bilayers

A

Cholesterol contains a hydroxyl group, a tiny polar head group and a rigid hydrophobic tail that provides the rigidity

249
Q

Which side of the membrane are sugars found attached to

A

The outside of the membrane

250
Q

What is the role of the negative charge present on the intracellular side of the cell membrane

A

The negative charge repels intracellular molecules and vesicles

251
Q

Which phospholipid is strictly found on the inner surface of the membrane

A

Phosphatidylserine is strictly found in the intracellular surface of the cell membrane because of its negative charge

252
Q

What features of the phospholipid unique to the intracellular surface of the cell membrane make it suitable

A

Phosphatidylserine contains a negative phosphate group as well as a serine group containing both a positively charge amino group and a negatively charge carboxyl group. This means that the phospholipid has a net negative charge

253
Q

What percentage of the intracellular membrane is made up of phosphatidylserine molecules and how does this account for its charge

A

4% - however this accounts for most of the intracellular negative charge

254
Q

Why do phosphatidylethanolamine and phosphatidylcholine have no overall charge

A

Because the negative phosphate head group charge is cancelled out by positive side groups, ethanolamine and choline

255
Q

Explain the significance of phosphatidylserine in apoptosis and cell death

A

Phosphatidylserine flips to the outer surface only upon apoptosis which takes place during cell death. This acts as a marker of cell death with lipid asymmetry of the plasma membrane breaking down. This in turn causes the cell membrane to become permeable to small molecules.

256
Q

What is the role of phosphatidylserine exposure to the extracellular environment in an intact organism

A

Exposure of phosphatidylserine on the cell surface labels the dead cell and its remnants so that they are rapidly consumed by macrophages

257
Q

One type of protein often associated with the cell membrane is the integral or transmembrane protein. What four ways can these proteins integrate with the membrane

A

As single ? helices spanning both leaflets, multiple transmembrane spanning ? helices, a rolled ? barrel or as an ? helix inserted into only one leaflet

258
Q

Other than integral or transmembrane proteins, what other way is there of proteins integrating with the cell membrane

A

Peripheral membrane proteins can associate with the membrane by inserting via a covalently bound lipid modification. Similarly, they can interact with integral membrane proteins via interactions such as disulphide bridges. Finally peripheral proteins can directly bind to lipid present in the phospholipid bilayer

259
Q

Why do disulphide bonds only form in the extracellular environment

A

Cysteines present on the intracellular side of membrane proteins will be in their reduced form and will not form disulphide bonds. Cysteine exposure to the extracellular environment results in oxidisation and the formation of disulphide bridges

260
Q

What is the role of disulphide bonds in membrane fluidity

A

Disulphide bridges in the extracellular domains make the protein more rigid and resistant to degradation

261
Q

Phosphatidylinositol is only a minor component of the cell membrane, what percentage composition does it make up

A

0.01

262
Q

What is the role of phosphatidylinositol in the cell membrane

A

Plays an important role in cell signalling (cancer, vesicle traffic etc.). Phosphatidylinositol can be phosphorylated thereby increasing its negative charge

263
Q

Which leaflet is phosphatidylinositol found in

A

The inner leaflet

264
Q

Myristoyl anchors are used to link peripheral proteins with the cell membrane. What kind of linkage is present in these anchors

A

Amide linkage between the terminal amino group of a protein and myristic acid

265
Q

Palmitoyl anchors are another way of linking peripheral proteins with the cell membrane. What kind of linkage is present in these anchors

A

Thioester linkage between cysteine in the protein and palmitic acid

266
Q

Give an example of another type of fatty acid linkage used to associate peripheral proteins with the membrane other than palmitoyl and myristoyl anchors

A

Farnesyl anchors – a thioester linkage between cysteine residue and a prenyl group

267
Q

What is meant by the term lipid rafts

A

Cholesterol and sphingolipids can form microdomains called lipid rafts. These lipid rafts are thicker regions of the membrane that form under the influence of membrane proteins that drive this domain formation

268
Q

What is the proposed role of lipid rafts

A

Lipid rafts are thought to play a role in signalling and some forms of endocytosis

269
Q

What is the role of sphingolipids in the formation of lipid rafts

A

Fatty acid chains of sphingolipids are longer and straighter than other phospholipids these rafts are thicker than rest of plasma. Sphingolipids carry long saturated fatty acids and thus segregate with cholesterol into distinct lipid rafts

270
Q

Other than lipid rafts, what other ways are there of segregating proteins in the cell membrane

A

Caged behaviours, fenced domains and tight junctions all act to segregate proteins in the membrane

271
Q

Both lipids and proteins are often tagged by complex sugars in the membrane, T or F

A

T

272
Q

Give an example of where glycosylation is important

A

Sphingomyelin is often glycosylated forming gangliosides which are very important in neurons. They exhibit progressive structural complexity. Neural stem cells carry simple sugars whereas mature neurons carry highly branched sugars. The presence of gangliosides acts as a signal that the neuron has matured. Myelination of neurons by Schwann cells is determined by the sugar maturation. Defects in ganglioside synthesis can lead to a variety of neurological disorders

273
Q

What is the significance of sugar modification in botulinum and tetanus toxins

A

Tetanus and botulinum bacteria produce toxin which recognize the complex sugars. These toxins bind only to fast myelinated neurons and hence cause efficient muscle paralysis. BOTOX binds to the ganglioside on the neuronal membrane to cause muscle paralysis and will then release an enzyme which will proteolyze its SNARE target and prevent neurotransmission

274
Q

What is the significance of carbohydrate modification of cell membrane constituents in organ transplantation

A

Limitations in organ transplantation are caused by a simple sugar modification. Human cells carry beta galactose while other animals carry alpha-galactose. Humans produce antibodies against alpha-galactose and thus reject transplants.

275
Q

Internal membrane compartments vastly outweigh the cell plasma membrane, T or F

A

T

276
Q

Give an example of endocytosis in nutrient uptake

A

The protein portion of LDL is recognised by LDL receptor on the cell surface which binds with high affinity to LDL. The adapter molecule called adaptin binds to the intracellular domain of the LDL receptor. Adaptin recruits clathrin molecules which binds to it and coats the inside of the membrane. Assembly of the clathrin coat causes the membrane to invaginate. This forms a vesicle that buds off the inside of the cell taking the LDL receptors and the bound LDL with it along with clathrin and adaptin. Inside the cell the clathrin coated vesicle uncoats and fuses with the endosome. The endosome has an acidic pH which causes the LDL receptors to release the LDL cargo. Empty LDL receptors are recycled back to the plasma membrane in recycling vesicles that bud off from the endosome. Meanwhile the endosomal content delivered to the lysosome where hydrolytic enzymes digest the LDL releasing cholesterol, amino acids and small peptides

277
Q

When are LDL receptors actively synthesised

A

When blood cholesterol is low

278
Q

What is the name of the mechanism by which LDL is transported into cells

A

Receptor-mediate endocytosis

279
Q

Give an example of a human disease characterised by defects in an endocytotic mechanism

A

Defects in endocytosis can cause atherosclerosis. This occurs due to mutations in LDL receptor account for familial cases of atherosclerosis. This results in an accumulation of lipoproteins in blood and the formation of atheromatous plaques which block arteries

280
Q

Describe the structure of clathrin and how is causes membrane invagination

A

Clathrin has a lattice structure that forms cage-like structures when in complexes. Clathrin itself is said to have a triskelia structure consisting of a tri-legged curved shape which forces membrane invagination. A molecule of clathrin consists of 3 heavy and 3 light chains.

281
Q

What is the role of adapter proteins in endocytosis

A

Adapter proteins such as adaptin link selected cargo to the clathrin lattice

282
Q

What is the role of dynamin in endocytosis

A

Dynamin is a large molecular weight GTPase that is required for coated pit scission to form coated vesicles during endocytosis. GTP hydrolysis allows the vesicle to bud off from the membrane

283
Q

What is seen in mutants in dynamin

A

Mutated dynamin cannot hydrolyse GTP and thus cannot pinch off endocytic vesicles

284
Q

Which type of vesicle coat allows the budding of vesicles from the endoplasmic reticulum

A

COPII

285
Q

From which organelle(s) does the COPI coat allows the budding of vesicles from

A

The Golgi cisterne (or cis-Golgi)

286
Q

Other than the cell membrane, what structure do clathrin coats mediate the budding of vesicles from

A

The trans-golgi network

287
Q

What different roles do the SER and RER have

A

The RER is involved in protein synthesis whereas the SER is responsible for lipid synthesis and the formation of vesicles

288
Q

Describe the process of lipid synthesis in the SER membrane

A

Fatty acid binding protein delivers a fatty acid to the plasma membrane. Here, CoA transferase allows synthesis of two fatty acids at the glycerol head group. A phosphatase then releases the phosphate bound to the glycerol molecule which allows for hydroxylation. This hydroxyl group is then replaced by the lipid head group (i.e. choline, serine, ethanolamine).

289
Q

Compare the tendency of fatty acids to stay in the membrane

A

A single fatty acid sitting in the ER membrane can easily escape whereas two fatty acids cannot because they are bound much more strongly

290
Q

Newly-synthesized ER lipids and proteins are packaged into COPI vesicles which snip off the SER, T or F

A

F – these vesicles are COPII coated

291
Q

Phagocytosis is a different type of endocytosis, how is it classified and why

A

Phagocytosis is an example of actin-driven vesicle formation. This is because actin drives membrane engulfment in phagocytosis by forming a pseudopod around the bacterium

292
Q

Give examples of cells that are phagocytotic

A

Neutrophils and macrophages

293
Q

Outline the process of phagocytosis

A

The microbe adheres to the phagocyte which forms pseudopods that eventually engulf the particle. The phagocytic vesicle is then fused with a lysosome forming a phagosome. The microbe within the vesicle is killed and digested by lysosomal enzymes within the phagosome leaving a residual body. The ingestible and residual material is removed by exocytosis

294
Q

What is autophagy and how does it work

A

Autophagy is the third pathway towards lysosomal digestion. It acts to help eliminate malfunctioning cell elements and occurs via vesicle fusion and engulfment of organelles

295
Q

Exocytosis is responsible for secretion of hormones, digestive enzymes, recycling of plasma membrane receptors and neuronal communication. What are the 2 exocytotic pathways and how do they differ

A

Constitutive exocytosis is always occurring with little to no regulation and occurs in non-polarised cells. In contrast, regulated secretion involves the accumulation and storage of a molecule before a signal-triggered release

296
Q

Insulin release is an example of one type of exocytosis. Which type is it and how does it work

A

Release of highly-concentrated insulin from pancreatic beta cells is an example of regulated exocytosis and happens only in response to high glucose. Insulin is tightly packaged inside secretory granules before secretion. Vesicles move from compartment to compartment with excess lipid material being removed and recycled back to the golgi. This acts to concentrate insulin so much that it is nearly crystalline. Upon release, insulin binds to its receptor and triggers delivery of glucose transporters into the plasma membrane of muscle cells

297
Q

What is the problem that prevents the fusion of vesicles with the target membrane

A

The target membranes and the membrane of the vesicle are negatively charged due to the presence of phosphatidylserine. This causes a repulsion between the two membranes that prevents fusion

298
Q

SNARE proteins help to overcome the force the prevents membrane fusion, what are the two main types of SNAREs

A

Vesicular or v-SNAREs such as synaptobrevin are found on the vesicle membrane. Target SNAREs or t-SNAREs are found on the target membrane of the compartment

299
Q

Which subtypes of SNAREs are Syntaxin and SNAP25

A

Syntaxin and SNAP25 are t-SNAREs

300
Q

Explain how SNAREs act to force membrane fusion

A

SNARE proteins form a tight 4-helical coiled-coil with hydrophobic faces (containing leucine, valine etc.) coming together away from the cytosol. This coiled-coil structure acts as a zipper all the way to the target membrane and the force produced by SNARE protein coiling into two opposing membranes is translated and forces their fusion

301
Q

What is the role of Rab proteins in exocytosis

A

Rab proteins identify target membranes for fusion and Rab-GTPs play a role in tethering and docking of vesicles to the membrane

302
Q

Which enzyme catalyses the dissociation of SNARE coils by hydrolysis ATP and forcing synpatobrevin or VAMP-1 away from the t-SNAREs

A

NSF enzyme

303
Q

What are the effects of the tetanus and botulinum neurotoxins produced by some bacterial infections

A

They can result in complete neuromuscular paralysis

304
Q

How is botulism and tetanus contracted

A

Botulism happens upon consumption of contaminated food whereas tetanus infections can happen after skin cuts, during childbirth or dirty needle injections

305
Q

How do the botulinum and tetanus toxins lead to muscular paralysis

A

Botulinum and tetanus toxins attack SNARE proteins with high specificity. SNAREs are responsible for acetylcholine release at the neuromuscular junction

306
Q

Explain the mechanism of action of the botulinum toxin

A

Botulinum binds first to gangliosides on neuronal membranes. It then enters the luminal space of recycling synaptic vesicles and following endocytosis, one subunit, the SNARE Protease escapes the vesicle and enters the synaptic cytosol. Here the SNARE protease subunit cleaves a specific SNARE protein. The cleaved SNARE protein cannot support the fusion of synaptic vesicles which results in a long blockade of neurotransmission

307
Q

How long does the paralysis caused by botulinum toxin last for if untreated

A

4-6 months

308
Q

Explain the use of botulinum toxin in medicine

A

Botulinum neurotoxin can be used in medicine for local muscle paralysis. Due to its long-lasting muscle inactivation (4-6 months), the neurotoxin became a successful medicine. BOTOX is being used to treat muscular spasms, dystonias, cerebral palsy, Parkinsonian symptoms, and many other muscular disorders

309
Q

Whereas membrane-bound and secreted proteins are made in the RER, where are nuclear and cytosolic proteins made

A

Nuclear proteins are made on the outer nuclear membrane whereas cytosolic proteins are made in the cytosol

310
Q

What is the role of signal recognition particles in membrane and secreted protein synthesis

A

SRPs direct the delivery of the ribosome and protein to the membrane

311
Q

Following protein synthesis which compartment do the proteins move into were lipids are made and the transport vesicles are formed

A

Smooth endoplasmic reticulum

312
Q

What happens once proteins reach the Golgi

A

In the Golgi there is a final addition of sugars and then the proteins are sorted and concentrated into transport vesicles

313
Q

Where in the protein sequence are the signal sequences often found

A

At the N-terminus

314
Q

What enzymes remove signal sequences once proteins have been sorted

A

Signal peptidases

315
Q

As well as being cleave out often signal sequences contains internal structures that remain part of the protein, T or F

A

T

316
Q

What are signal patches

A

Signal patches are specific 3D arrangements of amino acids that are used to target proteins

317
Q

What is characteristic of the signal sequence that directs a protein to the endoplasmic reticulum

A

Proteins going to the endoplasmic reticulum have a signal sequence of 5-10 hydrophobic amino acids

318
Q

What is characteristic of the signal sequence that directs a protein to the mitochondria

A

Proteins destined for the mitochondria alternate between positive and hydrophobic amino acid residues

319
Q

What structures recognise signal sequences that guide proteins towards their target destinations

A

Sorting receptors which recognise SRPs

320
Q

Signal recognition particles guide signal peptide sequences to the target compartment where the ribosome can then assemble, T or F

A

F - Signal recognition particles guide ribosomes following binding to the signal peptide sequence

321
Q

Describe an experimental technique used to separate vesicles from the SER and RER

A

Light and heavy vesicles can be separated by differential centrifugation. Firstly the cell is homogenised in a blender to form a homogenate containing various vesicles depending on whether they were derived from the SER or RER. This homogenate is then added to a tube containing a sucrose gradient. The tube is then centrifuged at high speed to separate the vesicles. Smooth ER microsomes have a low density so stop sedimenting and float at a low sucrose concentration. Rough microsomes have a high density so stop sedimenting and float at a high sucrose concentration. A hole can then be punched in the bottom of the tube and the various fractions can be separated and extracted drop by drop

322
Q

Ribosomes associate with the membrane of the target compartment via a direct interaction, T or F

A

F - Electron microscopy has shown that ribosomes associate with the membrane via protein translocators present in the membrane

323
Q

Explain how proteins are transported and insert into the ER membrane

A

Signal sequences direct the protein to the endoplasmic reticulum through the action of signal recognition particles (SRPs). SRPs bind to the signal sequence in a newly synthesised protein as it emerges from the ribosome. Protein synthesis then slows down until the SRP-ribosome complex binds to the SRP receptor on the endoplasmic reticulum membrane. The SRP is then released passing the ribosome to the protein translocation tunnel in the endoplasmic reticulum membrane.

324
Q

Explain what is meant by SRPs and their receptors acting as molecular matchmakers

A

SRPs and SRP receptors function as molecular matchmakers as they connect the ribosomes synthesising proteins containing the endoplasmic reticulum signal sequence to available endoplasmic reticulum translocation channels

325
Q

Explain how proteins insert into the ER membrane once the SRP has bound to its receptor

A

The signal sequence in the protein opens the translocation channel. The signal sequence remains bound to the channel while the rest of the protein chain is threaded through the membrane as a large loop before it is then cleaved off and degraded. A protein plug then closes the translocation channel in the endoplasmic reticulum membrane

326
Q

Describe how transmembrane proteins insert into the ER membrane and how this differs from ER luminal proteins

A

Proteins that are meant to insert into the ER membrane contain stop transfer sequences that halt the transfer process into the endoplasmic reticulum lumen. Stop transfer sequences are released laterally into the lipid bilayer and form a membrane spanning segment anchoring the protein to the membrane

327
Q

How does insertion into the ER membrane differ for multipass transmembrane proteins

A

Mulitpass membrane proteins have internal signal sequences consisting of several stop and start pairs which help to stitch the protein into the membrane. Combinations of Start-transfer and Stop-transfer signals determine the orientation of multipass membrane proteins in the membrane

328
Q

What can be said about the affinity of start and stop transfer sequences in ER membrane proteins

A

They are hydrophobic

329
Q

By what other key method other than transmembrane domains can proteins become anchored into the ER membrane

A

Swapping the signal peptide for a lipid anchor in the ER can take place to continue membrane association. Lipid pairs can be added to the protein by different enzymes. These anchors are usually glycosylphosphatidylinositol anchors (GPI)

330
Q

What is meant by the process of conformational maturation that occurs after signal sequence removal by signal peptidases

A

Conformational maturation is the process whereby the maturing proteins adopts a thermodynamically stable shape

331
Q

What type of bonding often occurs during protein maturation and acts to solidify protein shape

A

Disulphide bridges

332
Q

Which sequence is contained within ER-resident enzymes to direct their return to the ER and which amino acids are involved

A

KDEL sequence: lysine-aspartate-glutamate-leucine

333
Q

What are the three reasons why glycosylation is important

A

Increase protein stability in the harsh extracellular environment, enables cell-cell recognition as well as cross species separation

334
Q

What attribute of protein glycosylation is responsible for the limitations in xenotransplantation and anima donors

A

Humans contain B-galactose attached to proteins whereas animals use ?-galactose

335
Q

Where does initial carbohydrate addition to proteins start

A

ER

336
Q

What is the initial purpose of carbohydrate chains added to proteins

A

Quality control tags

337
Q

Which amino acid residue is often used to add initial carbohydrate modifications

A

Asparagine

338
Q

Trimming and growth of carbohydrate chains proceeds step-by-step in individual Golgi cisternae, T or F

A

T

339
Q

Give an example of where defective protein glycosylation causes disease

A

Myelination of neurons requires complex gangliosides enriched in sialic acid. Deficiency in sialic acid causes severe psychomotor delay evident by the end of the first year of life

340
Q

Why is it that each glycosylation step requires separate Golgi compartments

A

This keeps specific glycosylation enzymes away from each other

341
Q

The blood group of an individual is determined by the structure of the oligosaccharides attached to which proteins and phospholipids in the red blood cell membrane

A

Sphingomyelin and Band 3 protein

342
Q

Which blood group is the universal donor

A

O

343
Q

Which blood group is the universal acceptor

A

AB

344
Q

Describe the differences in the terminal oligosaccharide in people with the A, B and O antigens

A

People with the A blood type antigen contain an acetylated terminal galactose (N-acetylgalactosamine. People with the B blood type antigen possess a non-acetylated terminal galactose. AB patients will possess red blood cells with both the acetylated and the non-acetylated terminal galactose whereas O blood types will have neither.

345
Q

How does the structure of the various blood type antigens account for the antibodies that those patients will possess

A

A antigen patients will possess antibodies for the B antigen and vice versa. O group blood people will have antibodies for both A and B antigens whereas if you are AB you would have neither.

346
Q

Give an example of protein trimming in the process of maturation

A

Protein trimming occurs prior to the secretion of inulin. The signal peptide is removed from preproinsulin which produces the still inactive proinsulin molecule. Proinsulin is then moved to the Golgi and passes through it. Then, in the final stage, several amino acids in the middle of the of protein (known as chain C) are removed. The cleavage of proinsulin produces mature insulin and the C-terminal peptide. Chain A and chain B are held together by disulphide bridges

347
Q

Give an example of a disease caused by improper/defective protein cleavage

A

A genetic type of Type 1 diabetes is caused by a mutation that causes the misfolding of proinsulin in the ER. This means that proteases cannot cleave off C-peptide from Pro-insulin in secretory vesicles. This in-turn leads to the secretion of a dysfunctional pro-insulin into bloodstream as well as triggering an immune response to kill off the pancreatic cells producing the dysfunctional insulin

348
Q

Give an example of where different cells can process the same peptide into different hormones

A

Cleavage of opiomelanocortin can give rise to a whole range of different proteins which varies from cell to cell. Opiomelanocortin can be cleaved in neurons to give, B-MSH, B-endorphin, ?-MSH and ?-lipoprotein. In contrast, opiomelanocortin can be cleaved in the pituitary to give corticotropin (ACTH) and B-lipotropin

349
Q

What are the four factors that influence cell shape

A

Adjacent cells, cell adhesions, extracellular matrix and the function of the cell

350
Q

What subcellular activity also plays a role in defining cell shape

A

Migration, phagocytosis, transport and cytoskeletal dynamics

351
Q

Why is the actin cytoskeleton not a rigid structure

A

Rigid structures are unstable whereas tensile cytoskeleton are much more robust. A tensile structure can temporarily adjust the application of forces to maintain its shape

352
Q

Describe the four main components of the actin cytoskeleton

A

Stress fibres – stretch across the cells to link anchor points and provide stability. Cortical actin – involved in amoeboid migration. Lamellipodium – branched network of actin filaments that push the membrane out. Filopodium – rod like structures that push the membrane out but are unstable due to their rigidity

353
Q

What are the two type of actin

A

Monomeric or globular/G-actin and polymeric or filamentous/F-actin

354
Q

What can be used to cause the spontaneous polymerisation of monomeric actin in vitro

A

The addition of salts stimulates actin polymerisation

355
Q

What can be said about the likelihood of actin polymerisation to occur

A

The initial step in actin polymerisation is energetically unfavourable and a very slow reaction

356
Q

Describe the phases of actin filament assembly

A

The initial phase, known as the nucleation or lag phage occurs when all actin is in the G-actin state prior to nucleation. Once the core is established it is elongated rapidly during the growth phage where actin monomers are added at either end. Then the actin polymerisation reaches a plateau at a the critical concentration where the equilibrium phase ensues. The critical concentration is where removal of actin monomers is preferred and polymerisation stops, here, the rate of addition equals rate of removal

357
Q

Actin polymerisation occurs at both ends of the filament, T or F

A

F – actin tends to be added at one end (+ end) and subunits are removed at the other end (- end)

358
Q

What is meant by actin filaments being referred to as polar

A

Actin filaments have specific ends. The + end or barbed end is the faster growing end of the filament where polymerisation is favoured. The – end or pointed end is the slower growing end where depolymerisation is favoured

359
Q

Profilin is an important actin-binding/accessory protein, explains its dual roles

A

Profilin binds to free monomeric G-actin and transports it to the correct end of the microfilament as well as catalysing the exchange of the bound nucleotides. This all acts to promote microfilament assembly

360
Q

Which actin-binding protein acts as a nucleator to promote the formation of both new actin fibres and the branching of existing fibres and how does it do this

A

Arp2/3 is a nucleator of actin filament formation that mimics and actin nucleus. It consists of two subunits which resemble actin monomers and promote new actin filament formation either de novo or on the side of existing microfilaments

361
Q

Give an example of another important actin accessory protein and its role

A

Gelsolin – involved in capping existing actin filaments as well as capping and nucleation

362
Q

GTPases are small monomeric proteins, T or F

A

T

363
Q

What is the rough weight of a GTPase

A

21kDa

364
Q

GTPases hydrolyse ATP, T or F

A

F – they hydrolyse GTP

365
Q

What is the purpose of the post-translational lipid modifications often seen in GTPases

A

These hydrophobic lipid groups added to the proteins will target them to specific membrane sites

366
Q

What is the name of the superfamily to which all small GTPases belong

A

Ras Superfamily of GTPases

367
Q

In its inactive state, GTPases are bound to GDP, what is required to activate signalling

A

Displacement of the GDP by GTP activates the GTPase and initiates signalling

368
Q

What is the result of the intrinsic nature of GTPases to hydrolyse GTP

A

Hydrolysis of the bound GTP by the GTPase releases a phosphate and switches it back to an inactive state

369
Q

Nucleotide-free GTPases are extremely energetically favourable, T or F

A

F – its extremely unfavourable

370
Q

What are GEFs and what is the role of these proteins in the cyclic nature of GTPase activity

A

Guanine nucleotide exchange factors stabilise GTPases in a transition state so that GTP can then bind after GDP release

371
Q

GTPase activating proteins are responsible for catalysing the hydrolysis of the GTP bound to GTPases, thus do they act as positive or negative regulators of GTPase signalling

A

GAPs are negative regulators of GTPase signalling as they promote the catalyses of GTP hydrolysis to the inactive GDP-bound form

372
Q

What is the role of guanine nucleotide dissociation inhibitors

A

GDIs effectively pull the GDP bound GTPases out of the cycle and hold it in the cytoplasm to create a pool of inactive GTPases

373
Q

What changes happen at the molecular level as a result of GTP nucleotide binding to GTPases

A

This causes a very small conformational change dictated by the presence of a final phosphate that changes the orientation of the switch 1 and switch 2 domains. This leads to an activation of signalling

374
Q

What are the three members of the Rho family of GTPases

A

RhoA, Rac1 and Cdc42

375
Q

What is the role of the Rho family of GTPases

A

They coordinate actin cytoskeletal organisation, which in turn ultimately controls cell morphology, movement and polarity

376
Q

What is the role of Cdc42

A

Cdc42 is a RhoGTPase that controls the polymerisation of actin filaments and the formation of actin spikes or filopodia

377
Q

What is the role of Rac1

A

Rac1 controls the organisation of new actin filaments, particularly branched actin, into dynamic ruffling structures or lamellipodia

378
Q

What is the role of RhoA

A

RhoA stabilises and consolidates actin filaments into a more rigid skeletal framework known as stress fibres

379
Q

Describe a loss of function approach that can be used to elucidate the precise function of GTPases

A

Create a dominant negative mutant GTPase with a point mutation in the nucleotide-binding site. This will results in a GTPase that is always off and inhibitory due to never binding to GTP. This can be achieved via substitution of the P-loop. The dominant negative effect of this mutant is due to its binding to, and mopping up of active GEFs to prevent their action on functioning GTPases. By binding to these inhibitory mutant GTPases, the GEFs are no longer available to activate other functions wild type GTPases.

380
Q

Describe a gain of function approach that can be used to elucidate the precise function of GTPases

A

Create a constitutively active GTPase mutant that is always on and remains in the GTP-bound form. This can be achieved by the substitution of the catalytic glutamine in the switch 2 region which perturbs GTP hydrolysis and creates an always active GTPase

381
Q

What is the result of microinjection of constitutively active Rho into quiescent cells

A

Leads to the formation of stress fibres

382
Q

What is the results of microinjection of dominant negative Rho into active cells

A

Leads to the loss of stress fibres

383
Q

What is the result of microinjection of constitutively active Rac or Cdc42 into cells

A

Leads to the formation of membrane ruffles or filopodia respectively

384
Q

What is the name of the specific 16 amino acid sequence which activated Rho proteins bind to within effector proteins

A

Cdc42/Rac1 Interactive Binding (CRIB) sequence

385
Q

Rac stimulates the formation of new linear actin filaments whereas Cdc42 stimulates formation of branched actin filaments, T or F

A

F – vice versa

386
Q

Active Rac activates WAVE proteins, what is the downstream effect of this activation

A

Activated WAVE proteins bind to Arp2/3 and lead to the formation of actin filaments associated with branching

387
Q

What other protein family are activated by RacGTPases that go onto activate Arp2/3 and lead to actin filament formation

A

WASP

388
Q

How does Rho activation lead to the formation of stress fibres

A

Rho activates Rho kinase which in turn phosphorylates myosin to increase its contractility which ultimately leads to the formation of stress fibres

389
Q

Rac activation is required to precede Cdc42 activation, T or F

A

F – vice versa

390
Q

What can happen as a result of a failure of cell migration

A

Cell, tissue and organism dysfunction and death

391
Q

Give an example of where cell adhesion and migration play a vital developmental role

A

Formation of the early neural tube, the neural crest cells are required to detach from the dorsal neural tube, migrate, re-clump and then differentiate

392
Q

Give a classical example of where cell adhesion was studied

A

H.V. Wilson in 1907 identified that the cells of sponges can re-aggregate following dissociate from each other. Interestingly this was seen in a species dependent manner whereby cells of the same species would clump together sorting themselves into species

393
Q

What is seen if the cells of embryonic tissues that have been separated are reintroduced

A

They also re-aggregate into distinct clusters consisting of cells of the same type

394
Q

Larger re-aggregates of cells tend not to reorganise due to the number of different interactions, T or F

A

F – in fact large re-aggregates do show organisation into distinct clusters based on cell type. In addition, these clusters begin to show regionalisation where particular cells have stuck together

395
Q

What are L cells

A

L cells are a cell line used for investigating cell adhesions. This is because they do not express any of the cadherin family of cell adhesion molecules

396
Q

Transfection of L cells with cadherin family genes causes what

A

Homophilic binding between L cells expressing the same cadherins leading to adhesion

397
Q

What is meant by graded sorting

A

By varying the degree and levels of cadherin family gene expression, you can influence the degree of sorting of the cells expressing these genes

398
Q

Monoclonal antibodies have been instrumental in identifying the vast array of CAM variants due to their ability to their ability to distinguish between very similar proteins. Explain how monoclonal antibodies are made

A

Firstly, the specific antigen (protein or epitope) is injected into a different animal so that these animals mount an immune response. After a certain period the animals are euthanised and the lymphocytes are extracted from the spleen. These lymphocytes will be the cells synthesising antibodies for the target antigen and are fused with lymphoma cells to create hybridoma cells. Lymphoma cells are used as they are hyperproliferative and can survive indefinitely whereas the extracted lymphocytes dies quickly outside the body. They hybridoma cells are selected for due to their ability to survive in harsher mediums. Once isolated the hybridomas are separated into wells containing one cell each. These cells are then allowed to proliferate and give rise to a clonal population of cells all expressing exactly the same antibody targeted for a single epitope of the target protein.

399
Q

What are the two families of cell adhesion molecules

A

Ca2+-dependant and Ca2+ independent

400
Q

To which CAM family do cadherins belong and what are the other subfamilies are there

A

Cadherins belong to the Ca2+-dependent CAM family along with selectins and integrins

401
Q

Describe the basic structure of cadherins

A

Integral membrane glycoproteins that are 720-750 amino acids in length

402
Q

What happens following Ca2+ binding to cadherins

A

Causes a conformational change allowing interactions between cells

403
Q

Cadherin family proteins only contain one Ca2+ binding site, T or F

A

F – they contain multiple

404
Q

Give an example of where cell adhesion molecules such as cadherins have roles other that in cell adhesion

A

VE-cadherin inactivation due to mutations causes abnormal vascular development and apoptosis of endothelial cells, indicating that it has roles in signalling

405
Q

What is the significance of hypervariable protocadherins

A

These CAMs are thought to have roles in specifying synapses as wells as mediating neurite self-avoidance

406
Q

What class of intracellular signalling proteins important in development have been found to bind to the intracellular domain of cadherins

A

Catenins (B-catenin)

407
Q

Selectins are another type of cell surface membrane, Ca2+-dependant adhesion molecules. What attributes of cells do selectins recognise in order to elicit adhesion

A

Selectins recognise particular carbohydrate groups on proteins in neighbouring cells

408
Q

How do selectins bind to target cells

A

The lectin domain of selectin binds to sugar groups present on the surface of these cells

409
Q

Explain the role of selectins in neutrophil trapping

A

The amount of selectins can be changed in endothelial cells and expression of them is found to be increased in an immune response. Selectins act to catch lymphocytes near to the site of inflammation which ultimately leads to an increase in diapedesis (crossing of the lymphocytes over the vessel wall).Catching by selectins allows other CAM molecules also present in the lymphocytes to adhere with the corresponding CAMs of the endothelial wall. This allows the lymphocytes to cross the vessel wall into the site of inflammation

410
Q

Explain the significance of E and N-cadherin in the development of the early nervous system

A

Initially, only E-cadherin is expressed in the early embryo. However, newly formed mesodermal cells produced during gastrulation lose E-cadherin expression. Once the neural tube has formed this E-cadherin expression is replaced by N-cadherin

411
Q

Complex changes in selectin expression accompany the migration of neural crest cells, T or F

A

F – changes in cadherins do

412
Q

What is the major family of Ca2+-independent CAMs

A

Neural cell adhesion molecules (N-CAMs)

413
Q

What is significant about the number of genes involved in N-CAM expression

A

All of the N-CAM family of cell adhesion molecules are derived from a single gene. The original N-Cam transcript is subject to a vast array of alternative splicing and post translational glycosylation

414
Q

Explain the role of polysialic acid in N-CAM adhesion

A

N-CAMs show variations in the amount of polysialic acid present as a post-translational modification. More post-translational modifications and polysialic acid is found in immature neurons which results in less adhesion

415
Q

Integrins are a key class of cell adhesion molecules. Explain the role integrins

A

Integrins are extracellular matrix receptors present in the membrane of cells. They act to link the extracellular matrix to the cytoskeleton

416
Q

Describe the structure of integrins

A

Integrins consist of two, non-covalently associated glycoproteins each with an ? and ? subunit.

417
Q

How many different known varieties of integrins are there

A

24 known integrins (from 18? subunits and 8? subunits)

418
Q

Which termini make up the intracellular and extracellular domains of integrins

A

The C-terminus forms the intracellular regions of the proteins whilst the N-termini are found in the extracellular domain of the receptors

419
Q

What is the name of the specific motif that integrins bind to on target extracellular matrix proteins

A

RGD – arginine-glycine-aspartate

420
Q

To which cytoskeletal element do the majority of integrins bind link to

A

Actin filaments

421
Q

Binding of which cations effect the binding of the two integrin glycoproteins

A

Ca2+ and Mg2+

422
Q

Glanzmann’s thrombasthenia is a disease associated with integrins. Discuss the cause and symptoms of this condition

A

Glanzmann’s thrombasthenia is caused by a mutation in integrin ?IIb?3 that causes symptoms similar to haemophilia. The abnormal integrin impairs platelet function and causes easy bruising and increased likelihood of bruising

423
Q

What is meant by a focal adhesion

A

Focal adhesions are areas of temporary attachment of a cell to the medium it is moving through

424
Q

What is the role of focal adhesion kinase (FAK) in cell adhesion and integrins

A

FAK is a cytoplasmic protein tyrosine kinase present at cell-matrix junctions in association with the cytoplasmic tails of integrins. It is activated on formation of adhesions but may actually inhibit focal adhesions. FAK is also regulated by the concentration of intracellular Ca2+

425
Q

Focal adhesions act through integrins, T or F

A

T

426
Q

Focal adhesions act during necrosis, T or F

A

F – the act during anoikis or attachment-dependent cell death

427
Q

Mammalian cell motility is interlinked with adhesion and is mostly actin-based, T or F

A

T

428
Q

What part of the actin cytoskeleton involved in cell motility is responsible for membrane tension and the transmission of tension

A

Actin cortex

429
Q

Outline the process of traditional cell migration

A

The actin-polymerisation-dependant protrusion and the firm attachment of lamellipodium at the leading edge of the cells moves the edge forwards and stretches the actin cortex. Contraction at the rear end of the cell propels the body of the cell forwards to release some of the tension. Finally, new focal adhesions are made at the front and the old ones are disassembled at the back as the cell crawls forwards

430
Q

What is the name of the complex responsible for nucleation of the actin filament and what is the involvement of Rac

A

Arp2/3 – stimulated by Rac activity

431
Q

What three proteins are involved in the maturation of focal adhesions

A

Actin-binding proteins (ABPs), small G-proteins and myosin

432
Q

What are the two types of focal adhesion junctions

A

Low density adhesions and high density adhesions

433
Q

Which type of focal adhesion junctions tends to be immobile

A

Low density adhesions

434
Q

Which type of focal adhesion junctions are Rac1 and cdc42 dependant

A

Low density adhesions

435
Q

What is the proposed difference in timing of the two types of focal adhesions during their maturation

A

Low density adhesions that are immobile tend to appear earlier, whereas, high density adhesions appear later and are motile

436
Q

Which proteins and interactions are required for formation of the high density adhesions

A

RhoA and actin-myosin interactions

437
Q

Signals to dictate cell motility can be soluble or insoluble, give examples of both

A

Soluble – netrins, insoluble – CAMs

438
Q

Fibronectin is an extracellular matrix protein that binds to integrins, what sequence is responsible for this binding

A

RGD – arginine-glycine-aspartate

439
Q

What is the role of fibronectin in migration of the neural crest cells

A

Fibronectin trails allows neural crest cell migration.

440
Q

Explain the role of membrane cycling in cell motility

A

Membrane endocytosis is occurring constantly around the whole cell. Meanwhile, exocytosis occurs at leading edge leading to an addition of phospholipid bilayer and an increase in the surface area of the membrane. This corresponds to a net membrane addition which could provide a net motive force for cell motility. There is also a net rearwards flow of membrane as membrane is constantly added at leading edge which shifts existing membrane backwards. Therefore, if the membrane is fixed by focal contacts the cell will move forward

441
Q

What is the role of endocytosis in cell motility

A

Endocytosis of a secreted factor alternates the concentration of that factor. In addition, when integrins and other CAMs bind to ligands and extracellular matrix proteins they can be taken into the cell which alters the way the cell subsequently interacts with the extracellular matrix