WHOLE MODULE Flashcards
What is meant by the term isoform
An isoform is a protein variant that differs based on posttranscriptional modifications
What process is used to create different protein isoforms
Alternative splicing
What regions of the immature mRNA transcript are removed during splicing
Introns
Splicing means that different proteins can be created from the same gene, T or F
T
All eukaryotic genes contain introns and exons, T or F
F – yeast do not contain introns
What components of genes allow for alternative splicing to occur
Optional introns and exons, mutually exclusive exons and internal splice sites
40% of the Drosophila genome is alternatively spliced. What percentage of human genes are also spliced
0.75
Other than splice donor and acceptor sites within gene transcripts, what other features of the mRNA allows for alternative splicing
Other sequences contained within the mRNA and the secondary structure also affects the choice of splice sites
How is splicing regulated
By RNA binding proteins
Give an example of a gene that undergoes alternative splicing
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
What are the three key genes in determining sex in Drosophila and what are their roles
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.
How are male Drosophila determined using alternative splicing and the interactions between the three sex determining genes
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.
How are female Drosophila determined using alternative splicing and the interactions between the three sex determining genes
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.
Give an example of how polyadenylation can act as a regulation of gene expression
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.
What is meant by the term leaky scanning
Sometimes the first AUG codon can be missed by the ribosome
Sequences around the start codon help to initiate translation, T or F
T
What is meant by the Kozak sequence
The Kozak sequence is the optimal translation initiation sequence that contains the start codon and ideal bases adjacent.
Recall the Kozak sequence
ACCAUGG
How can leaky scanning lead to the production of different protein products
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
High levels of what translation cofactor increase the probability that the first start codon will be recognised
eIF-4F
How does the HIV virus make use of regulated nuclear transport to influence gene expression
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.
Signals within which regions of mRNA transcripts target them to particular parts of the cell
3’ and 5’ untranslated regions
How are 3’UTRs recognised by cellular proteins which lead to the sequestration of RNA in one part of the cell
Intermolecular base pairing within the 3’UTRs form stem loops which are recognised by cellular proteins
What is the role of ferritin in the regulation iron availability
Ferritin is a protein that stores iron inside the cell leading to a decrease in Fe availability
What is the role of transferrin in the regulation iron availability
Transferrin is a receptor that imports iron into the cell leading to an increase in available Fe
How does aconitase interact with ferritin and transferring mRNA
Aconitase binds to stem loops in the 5’UTR of ferritin mRNA and in the 3’UTR of transferrin mRNA
Aconitase can also bind to iron itself, T or F
T
Explain the role of aconitase in increasing Fe availability when iron levels are low
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]
Explain the role of aconitase in decreasing Fe availability when iron levels are high
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
What co-factor of the translation machinery is required for all mRNA translation
eIF-2
When a cell is in the G0/quiescent phase of the cell cycle, globally, translation is turned down. How is this achieved
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.
eIF-2 with its bound GTP binds to Met-tRNA to start ribosome scanning, T or F
T
What are IRES sequences and how can they influence gene expression
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.
IRES translation initiation is an effective way of encoding a second reading frame within RNA, T or F
F – this is not a very effective method with less than 10% of second ORF translation
Which translation initiation cofactor is required to bind to the IRES stem loop and initiate translation independent of the 5’-Cap and PolyA tail
eIF-4G
Where are IRES sequences commonly found
IRES sequences are often found in viral transcripts
How can viruses use IRES sequences to promote translation of their transcripts
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
What is the significance of eIF-4G cleavage in apoptosis
eIF-4G is cleaved during apoptosis to prevent its binding to eIF-4E and hence prevent any more translation
RNA stability is defined by the half-life of the different mRNAs and varies greatly, T or F
T
How does the polyA tail change over time and act as a clock to determine mRNA age
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
How can mRNA half-lives be extended
Re-adenylation of the polyA tail
Often factors that promote translation also promote mRNA degradation to prevent overexpression, T or F
F – de-adenylating nuclease (DAN) is responsible for de-adenylation of polyA tails and competes with eIF-4E binding to the mRNA cap.
What technique is used to visualise protein localisation within a cell or tissue
Antibody staining
What technique is used to visualise gene transcription inside cells and tissues and is quick and inexpensive
In situ hybridisation
GFP and transgenics are techniques used to visual gene expression and protein localisation, T or F
T
Which method is often the first port of call to visualise RNA expression due to its cheap costs and short time
In situ hybridisation
Explain how an expression plasmid is made in the process of making an antibody
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.
Why are expression plasmid promoter regions made to be inducible
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
Explain the purpose of an epitope tagging system when creating expression plasmids in antibody synthesis
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.
Explain the process of antibody-affinity purification
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.
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
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.
What is the name of the region of a protein to which an antibody binds to
Epitope
How are antibodies visualised once they are bound to a target protein
Tagging antibodies with dyes or enzymes to allow determination of where proteins are localised
What is meant by antibody sandwiches and why are they used to visualise protein localisation
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
How are tagged secondary antibodies produced
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
Give two examples of commonly used enzyme conjugates in antibody detection
Alkaline phosphatase – turns the substrate blue. Horseradish peroxidase – turns the substrate blue
To stain a cell/tissue with a tagged antibody, the animal/cells must be chemically fixed, how is this achieved
A fixative, usually formaldehyde is introduced to cross-link proteins together
What are the two types of antibody staining and what are they used for visualising
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.
Explain the process of in situ hybridisation
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
Explain the differences seen in bicoid mRNA and protein localisation seen by using antibodies and in situ hybridisation
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
How is bicoid mRNA localised anteriorly in the Drosophila embryo
Contains within its 5’UTR a region that localises it at the anterior region of the cells
Explain how GFP works to provide fluorescence
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
Fluorescence always gives off an emission wavelength that is greater than the excitation wavelength and a lower energy, T or F
T
Explain how GFP is used in gene fusion to create transgenic lines
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.
What does GFP gene fusion specifically allow that reporter constructs done
Because a functional protein of interest is produced the subcellular localization of the protein once can be studied
How does the incorporation of GFP still lead to the production of a functional protein
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
Once GFP constructs have been created how are these then used to visualise protein expression
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
What is the name given to the representation of the differences between species and their evolution from common ancestors
Phylogenetic tree
It has been shown that most of the genes across kingdom Animalia have been relatively conserved. How has this been achieved
BLAST analysis of protein structures to determine regions of significant homology
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
Changes in expression of a common set of genes
For great apes and man the change in the nucleotide sequence is about 1% every 10million years, T or F
T
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
0.5% difference – 1 in 200 nucleotides
Firstly, molecular data is used to distinguish specices which is then backed up by morphological data, T or F
F – vice versa
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
FOXP2 is a highly conserved protein which only shows differences at a few positions in the amino acid sequence
Which positions in the amino acid sequence does the FOXP2 vary
80, 303 and 325
Mice and chimps both have a threonine residue at position 303, what therefore can be inferred about the common ancestor of these species
Their common ancestor must have also had a 303T
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
Aspartate
When sorting animals into phyla, programmes similar to BLAST consider all possible relations between animals. How are they then sorted
The tree is assembled based on the simplest model with the fewest changes
What is meant by the term parsimony
The idea that you always assume the simplest model
Convergent evolution goes against the parsimony model. How does convergent evolution account for differences in the amino acid sequence
Changes in the amino acid sequence of two animals occur independently of each other
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
T
Although there are only 4 families of vertebrate FGF receptors, how many distinct receptors are there
22
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
The common ancestor also had only 4 FGF receptors
What can explain the other 16 FGF receptors present in vertebrates that aren’t in sea squirts
These will likely have arisen due to genome duplication both locally and by ploidy events
What is the name given to a duplicated gene present in the genome
Paralogue
Chromosome duplications or duplication mutations are seen frequently when comparing genomes, T or F
T
When a gene is first duplicated, what is its role
It is redundant
Over time duplicated genes evolve allowing a refinement of function or for a new function to development, T or F
T
What ways can the extra copy of a gene change
Can change the pattern of gene expression or the structure/function of the protein
Big changes to protein structure are caused by what type of mutations
Domain swapping
Smaller changes in the protein structure of a duplicated gene are caused by nonsense mutations, T or F
F – missense mutations
What is considered to be the common driving force for morphological evolution of animals
Changes in expression of genes
What changes in the transcription machinery present within the DNA explain why expression pattern changes have a major role in morphological evolution
Enhancers can change easily
What process can account for bringing a new enhancer regions close to the coding sequence of a gene
Non-homologous recombination
Enhancers must occur upstream of a target gene, T or F
F – enhancers can occur anywhere in the gene sequence and their exact position is usually unimportant
Changes in protein structure have to be precise so as not to introduce a variety of negative effects. Give some examples of these effects
Introduction of a stop codon, change in the reading frame, interference with protein folding or a disruption in RNA splicing
Describe the differences seen in the development of appendages in the fruit fly and Crustacea
Crustaceans development limbs throughout their abdominal and thoracic segments whereas in Drosophila, no legs develop in the abdominal regions
Explain the interactions between distal-less and ultrabithorax in forming legs in Drosophila
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
How do Dlx and Ubx act differently in Crustacea to fruit flies
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
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
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
What would be the results of ectopic expression of Crustacean ubx in the abdominal segments of Drosophila embryos
Limb formation in the abdomen due to loss of inhibition of dlx expression
What is meant by the term signal transduction
The process by which extracellular signalling molecules cause changes in target cells
List some of the downstream effects of signalling mechanisms on the cell
Survival, death(apoptosis), migration, differentiation, division
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
Metabolic enzymes, gene regulatory proteins and cytoskeletal protein elements
What type of signalling molecules do cell surface receptors often interact with
Hydrophilic ligands
What kind of signalling molecules do intracellular receptors interact with
Hydrophobic/lipophilic signalling molecules
What kind of proteins are often the downstream targets of intracellular receptors
Transcription factors
What is the other name given to ligand-gated ion channels
Inotropic receptors
Give an overview of signalling from GPCRs
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
How many transmembrane domains are indicative of GPCRs
7 transmembrane domains
Describe the general structure and activation of enzyme-coupled receptors
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
Juxtacrine signalling is an example of a short-range signalling mechanism. Outline how this signalling mechanism acts
In juxtacrine signalling, two cells are in direct contact and the ligand is a membrane-bound signal molecule (transmembrane protein)
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
Paracrine
Describe what is meant by autocrine signalling
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.
Where in the body is autocrine signalling often used
Used a lot in the immune system
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
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.
Cells of different types can respond to the same signal quite differently. Give an example of this
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
What is meant by primary transduction
Primary transduction is the process the converts the signal from the extracellular signalling molecule to an intracellular signal
Intracellular amplification of signals is a common feature of signal transduction, T or F
T
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
F – spreading is also seen
What is the role of scaffold proteins in intracellular signalling
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
What is often the result of phosphorylation of receptors once they become activated
Phosphorylation of receptors often creates recognition (docking) sites for downstream protein binding
What are the two main types of GTP binding proteins involved in intracellular signalling
Guanine Exchange Factors (GEFs) and GTPase activating proteins (GAPs)
What class of receptors are the receptor tyrosine kinases
Enzyme-linked receptors
What sorts of cell behaviours are RTKs involved in regulating
Proliferation, differentiation and migration
How many different families of RTKs are there
16 different families
Each ligand receptor pair involves one specific ligand and one unique receptor, T or F
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
Give some examples of RTK ligands
Ephrins, Nerve Growth Factor, Fibroblast Growth Factor, Epidermal Growth Factor
What is the result of stimulating the EGF receptor tyrosine kinase
Stimulation of proliferation
What is the result of stimulating the IGF receptor
Stimulation of carbohydrate utilisation and protein synthesis
What is the result of stimulation of the TrkA receptor by binding of NGF
Stimulation of survival and growth of some neurons
Most RTKs are monomers with one major exception, which receptor is this
The insulin receptor is an RTK which is present as a dimer
What can be said about the extracellular domains of RTKs throughout the family
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
Describe the structure of the intracellular domain of RTKs
The intracellular domains possess the kinase activity. These are present as a single domain or split into two
What can be said about the transmembrane domain in RTKs
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
Outline canonical RTK activation
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.
What are the effects of auto and cross-phosphorylation of the active RTK
Increased kinase domain activity, stabilisation of the receptor active state (ligand independent) and the creation of docking sites for target proteins
Explain the gain of function approach that can be used to investigate RTK signalling
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.
Explain the loss of function approach that can be used to investigate RTK signalling
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
SH2 domains that bind to phosphotyrosines in RTKs also recognise adjacent residues. What is the recognition sequence which they recognise
Phosphotyrosine-Glutamate-Glutamate-Isoleucine
What component of the extracellular matrix do RTK ligands often form complexes with
Heparan sulphate proteoglycans (HSPGs)
The FGF receptor-ligand complex can become activated in the absence of binding to components of the extracellular matrix, T or F
F – the receptor can only become activated when in a complex with HSPGs
HSPGs can be membrane tethered in two ways. Describe these
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
HSPGs can also be entirely secreted, T or F
T
What species attached to the proteoglycan backbones can be sulphated to trigger ligand binding to the FGF receptor
Glycosaminoglycans
HSPGs are important extracellular modifiers of cell-cell signalling, what is their role in the extracellular environment
They are important in organising the extracellular matrix into basal lamina
Give some examples of HSPGs
Glypican, Syndecan and Perlecan
Describe the structure of HSPGs
Consist of a proteoglycan core with glycosaminoglycan side chains
Other than Heparan Sulphate, give some examples of sugar side chains present on proteoglycans
Aggrecan, Betaglycan, Decorin and Perlecan
Modification by sulphation of GAGs can provide a code which creates binding sites for specific proteins and sequences that carry information, T or F
T
The pattern of sulphation acts as a code allowing specific HSPGs to interact with specific proteins, T or F
T
Describe the effects of HSPGs on the gradients of secreted molecules
HSPGs control the steepness of a secreted molecule gradient and how far a growth factor can diffuse through the extracellular space
Explain the role of HSPGs in FGF signalling
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
Give some examples or proteins that bind to RTKs
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
Explain how activation of RTKs leads to signal transduction by the Ras pathway
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.
How does the MAP kinase pathway rely the signal transduction further from activation of the Ras GTPase
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
What are the mammalian homologues of MAP-KKK, MAK-KK and MAP-K
MAP-KKK –> Raf, MAP-KK –> Mek and MAP-K –> Erk
What is the effect of having multiple stages in the MAP Kinase pathway
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
Cyclins are an example of downstream targets of MAP-Kinases, T or F
T
MAP-K is regulated by its phosphorylation by MAP-KK, describe how MAP-K is activated
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.
How long after RTK activation is gene transcription influence
Within minutes
Explain how Ras acts as a molecular switch in downstream RTK signalling
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.
Explain how the apparatus for fluorescent microscopy works
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
Why is a greyscale camera used to study fluorescence
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
Explain how fluorescent resonance energy transfer can be used to investigate protein interactions
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
What phenomenon is FRET said to rely on
Paired fluorescence
Leprechaunism is a disease caused by mutations in the insulin pathway. Describe the symptoms of this condition
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
Where is insulin produced
In the B-cells of the Islets of Langerhans in the pancreas
Explain the cleavage events that take place after insulin synthesis
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
The cysteine bonding that holds the insulin peptides together can only form outside the cell, why is this
These cysteine bonds can only form in an oxidative extracellular environment
What is the immediate effect of insulin signalling
Glucose uptake from the blood into muscle cells and adipocytes
What is the long-term effects of insulin exposure
Increased expression of liver enzymes that synthesise glycogen as well as enzymes involved in triacylglycerol synthesis in the adipocytes
Describe the structure of the insulin receptor
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
The insulin receptor is an RTK, what is unusual about its structure
The insulin receptors is present as a dimer in its inactivated state
Describe what happens following ligand binding to the insulin receptor
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)
Explain the role of IRS in insulin signalling
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.
IRS is known as a surrogate, what is meant by this
IRS binds to the signal docking site on the activated insulin receptor but then creates additional docking sites for downstream targets
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
PI-3 kinase pathway
Describe the structure of PI-3 Kinase
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
Explain the role of PI-3 Kinase in Ras-MAP-K independent signal transduction
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:
Give some examples of the downstream protein targets of activated PKB
GSK3, GLUT4, FOXO and Phosphoenolpyruvate Carboxykinase
What is the effect of activated PKB on glucose transport
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
What is the effects of activated PKB on glucose storage and how is this achieved
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
What are the effects of PKB activity on glucose synthesis
FOXO is a transcription factor that activates expression of PEPCK, a glucose synthesis gene. PKB phosphorylates and inactivates FOXO and prevent glucose synthesis
Insulin’s effects on glucose synthesis are known as the faster response, T or F
F – it’s a slower response
How does FOXO act at low blood insulin levels
When there are low blood insulin levels, Foxo binds to an IRS near to PEPCK to activate its transcription
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
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.
Why do the primers used in PCR to analyse the effect of insulin on gene expression need to be close together
Because PCR can only work for between 10-20kbps
What is the benefit of using a heat stable DNA polymerase in PCR
Allows you to repeat the cycle (heating wont denature the polymerase but can still break the hydrogen bonding between the strands
How many PCR cycles are usually carried out
30 cycles – producing >1x109 fragments
What is the effect of designing short primers that anneal to a specific gene or sequence of interest within a fragment
Primers trim down DNA sequence from the genomic dsDNA to only amplify the target sequence that lies between the two primers
To look at gene expression specifically using PCR, it is essential that you must use high quality tissue. Why is this
You need to extract all of the mRNA transcribed by the tissue
What is the name of the technique used to quantify gene expression from mRNA
Reverse Transcriptase PCR
Describe the process of promoter analysis/promoter bashing
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.
On average, how many people will have cancer in their lifetime
1 in 3 to 1 in 4
What is the most common type of cancer
Lung cancer
What are the three main causes of cancer
Physical carcinogen – UV and ionising radiation, chemical carcinogens – asbestos and tobacco smoke, biological carcinogens – infection from certain viruses, bacteria or parasites
What family of receptors do the human epidermal growth factor receptors belong to
Receptor tyrosine kinases
What are the different types of HER receptor
HER1, HER2, HER3 and HER4
What is unusual about the HER2 receptor
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
What is unique about the HER3 receptor
The HER3 receptor has lost its intracellular tyrosine kinase domain and so cannot phosphorylate target proteins
What happens to the HER receptors (apart from HER2) following ligand binding
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
Which conformations of HER receptor dimers signal the strongest and why
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
Outline the HER pathway of activation
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
Different combinations of receptors stimulate different signalling pathways and can lead to cell proliferation, survival and a prevention of apoptosis, T or F
T
HER proteins are tumour suppressor genes, T or F
F – they are proto-oncogenes
What causes HER proteins to become oncogenic
Overexpression or mutations
Which mutant form of HER2 was first discovered in rats the promote cancer aetiology
Neu
What process can be used to determine the copy number of a gene that has been amplified in tumorigenesis
Fluorescent in situ hybridisation (FISH)
Normal breast tissue contains around 20,000 copies of the HER2 receptor, how many copies can be found in cancerous tissues
2 million copies – 10-fold increase
HER2 is a negative prognostic marker for several cancers, what is meant by this
Overexpression of HER2 correlates with poor survival rates of breast cancer patients
How do the median survival rates of HER2 positive and HER2 negative breast cancers compare
HER2 positive – 3 years, HER2 negative – 6-7 years
Up to what percentage of breast cancers are believed to be HER2 positive
0.25
Herceptin is a monoclonal antibody used to target HER2 overexpression, what is meant by a monoclonal antibody
A massive population of antibodies derived from a single cell and that all specifically recognise a single epitope
How are monoclonal antibodies made for HER2
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
What is the problem with using mice to produce HER2 antibodies
Murine antibodies are not well tolerated in humans, the immune response from these will kill
How is the problem of immune response to foreign antibodies overcome
Humanisation
What was the three different pieces of evidence for the use of the 4D5 monoclonal antibody in the treatment of HER2 positive breast cancer
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
Explain how immunoglobulins are humanised
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
What is the other name for the hypervariable domain involved in antigen recognition by immunoglobulins
Complementary determining region (CDR)
Explain the serious complications that can occur because of Herceptin treatment
Because HER2 receptors are present in the heart, the inhibition as a result of Herceptin treatment can lead to decreased cardiac viability
What is the best way of using Herceptin to treat HER2 positive breast cancer
Combination with surgery, chemotherapy – 85% of patient’s cancer free for 5 years
Describe Herceptin’s mechanism of action
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
New versions of HER2 inhibitors also inhibit receptor dimerisation, T or F
T
What is the general role of lipids in the cell membrane
Provide flexibility and continuity
What is the general role of proteins in the cell membrane
Mediate interactions with the surrounding environment as well as playing a role in motility and the uptake of nutrients
What is the general role of carbohydrates in the cell membrane
Involved in cell tagging and protection
What methods are there for sugars to be attached to the cell membrane
Sugars can either be attached to the membrane via proteins or via lipids
Which side of the membrane are sugars usually attached
The extracellular surface
Outline the basic structure of a phospholipid
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
List the four major phospholipids present in cell membranes
Phosphatidylserine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin
Phospholipids are amphipathic, what is meant by this
The contain both hydrophilic and hydrophobic parts. A hydrophilic (polar) phosphate group and the non-polar hydrophobic fatty acid tails
Explain the different behaviours of single and double tailed phospholipids when placed in solution
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
What causes phospholipids to form sealed compartments when placed in water
The repulsion by water of the hydrophobic regions makes the lipid bilayer hide its edges and form a sealed compartment which is energetically favourable
One-legged (two tailed) lipid molecules form vesicles encapsulating water, T or F
F – two-legged phospholipids do
Unsaturated fatty acids provide flexibility to cell membranes, whereas saturated lipid are too rigid and not compatible with cell membranes, T or F
T
What makes fatty acid tails unsaturated
The presence of double bonds within the hydrocarbon tails
Which organisms produce omega 3 fatty acids
Sea plants, fish and nuts
Describe the nomenclature of omega fatty acids
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
Land plants do not contain the enzyme to insert the double bond in the carbon 3 position – to make ?-3, T or F
T
Describe the dynamic behaviour of phospholipids in the membrane
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
Explain what is meant by the hydration layer and its tole in the cell membrane
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
Describe the structure of cholesterol
Cholesterol has a very solid core of 4 carbon rings. Phospholipids and cholesterol together form functional cell membranes
What is the role of cholesterol in the cell membrane
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
Cholesterol doesn’t make the membrane less fluid as a whole but makes the membrane less deformable at the surface, T or F
T
What configuration are the double bonds that make up unsaturated fatty acids usually in
This cis conformation which introduces sharp kinks
Why is it that unsaturated fats allow the membrane to be flexible
Unsaturated fatty acid tails pack loosely together allowing the bilayer to remain fluid
What attribute of cholesterols structure allows it to stabilise phospholipid bilayers
Cholesterol contains a hydroxyl group, a tiny polar head group and a rigid hydrophobic tail that provides the rigidity
Which side of the membrane are sugars found attached to
The outside of the membrane
What is the role of the negative charge present on the intracellular side of the cell membrane
The negative charge repels intracellular molecules and vesicles
Which phospholipid is strictly found on the inner surface of the membrane
Phosphatidylserine is strictly found in the intracellular surface of the cell membrane because of its negative charge
What features of the phospholipid unique to the intracellular surface of the cell membrane make it suitable
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
What percentage of the intracellular membrane is made up of phosphatidylserine molecules and how does this account for its charge
4% - however this accounts for most of the intracellular negative charge
Why do phosphatidylethanolamine and phosphatidylcholine have no overall charge
Because the negative phosphate head group charge is cancelled out by positive side groups, ethanolamine and choline
Explain the significance of phosphatidylserine in apoptosis and cell death
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.
What is the role of phosphatidylserine exposure to the extracellular environment in an intact organism
Exposure of phosphatidylserine on the cell surface labels the dead cell and its remnants so that they are rapidly consumed by macrophages
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
As single ? helices spanning both leaflets, multiple transmembrane spanning ? helices, a rolled ? barrel or as an ? helix inserted into only one leaflet
Other than integral or transmembrane proteins, what other way is there of proteins integrating with the cell membrane
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
Why do disulphide bonds only form in the extracellular environment
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
What is the role of disulphide bonds in membrane fluidity
Disulphide bridges in the extracellular domains make the protein more rigid and resistant to degradation
Phosphatidylinositol is only a minor component of the cell membrane, what percentage composition does it make up
0.01
What is the role of phosphatidylinositol in the cell membrane
Plays an important role in cell signalling (cancer, vesicle traffic etc.). Phosphatidylinositol can be phosphorylated thereby increasing its negative charge
Which leaflet is phosphatidylinositol found in
The inner leaflet
Myristoyl anchors are used to link peripheral proteins with the cell membrane. What kind of linkage is present in these anchors
Amide linkage between the terminal amino group of a protein and myristic acid
Palmitoyl anchors are another way of linking peripheral proteins with the cell membrane. What kind of linkage is present in these anchors
Thioester linkage between cysteine in the protein and palmitic acid
Give an example of another type of fatty acid linkage used to associate peripheral proteins with the membrane other than palmitoyl and myristoyl anchors
Farnesyl anchors – a thioester linkage between cysteine residue and a prenyl group
What is meant by the term lipid rafts
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
What is the proposed role of lipid rafts
Lipid rafts are thought to play a role in signalling and some forms of endocytosis
What is the role of sphingolipids in the formation of lipid rafts
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
Other than lipid rafts, what other ways are there of segregating proteins in the cell membrane
Caged behaviours, fenced domains and tight junctions all act to segregate proteins in the membrane
Both lipids and proteins are often tagged by complex sugars in the membrane, T or F
T
Give an example of where glycosylation is important
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
What is the significance of sugar modification in botulinum and tetanus toxins
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
What is the significance of carbohydrate modification of cell membrane constituents in organ transplantation
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.
Internal membrane compartments vastly outweigh the cell plasma membrane, T or F
T
Give an example of endocytosis in nutrient uptake
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
When are LDL receptors actively synthesised
When blood cholesterol is low
What is the name of the mechanism by which LDL is transported into cells
Receptor-mediate endocytosis
Give an example of a human disease characterised by defects in an endocytotic mechanism
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
Describe the structure of clathrin and how is causes membrane invagination
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.
What is the role of adapter proteins in endocytosis
Adapter proteins such as adaptin link selected cargo to the clathrin lattice
What is the role of dynamin in endocytosis
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
What is seen in mutants in dynamin
Mutated dynamin cannot hydrolyse GTP and thus cannot pinch off endocytic vesicles
Which type of vesicle coat allows the budding of vesicles from the endoplasmic reticulum
COPII
From which organelle(s) does the COPI coat allows the budding of vesicles from
The Golgi cisterne (or cis-Golgi)
Other than the cell membrane, what structure do clathrin coats mediate the budding of vesicles from
The trans-golgi network
What different roles do the SER and RER have
The RER is involved in protein synthesis whereas the SER is responsible for lipid synthesis and the formation of vesicles
Describe the process of lipid synthesis in the SER membrane
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).
Compare the tendency of fatty acids to stay in the membrane
A single fatty acid sitting in the ER membrane can easily escape whereas two fatty acids cannot because they are bound much more strongly
Newly-synthesized ER lipids and proteins are packaged into COPI vesicles which snip off the SER, T or F
F – these vesicles are COPII coated
Phagocytosis is a different type of endocytosis, how is it classified and why
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
Give examples of cells that are phagocytotic
Neutrophils and macrophages
Outline the process of phagocytosis
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
What is autophagy and how does it work
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
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
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
Insulin release is an example of one type of exocytosis. Which type is it and how does it work
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
What is the problem that prevents the fusion of vesicles with the target membrane
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
SNARE proteins help to overcome the force the prevents membrane fusion, what are the two main types of SNAREs
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
Which subtypes of SNAREs are Syntaxin and SNAP25
Syntaxin and SNAP25 are t-SNAREs
Explain how SNAREs act to force membrane fusion
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
What is the role of Rab proteins in exocytosis
Rab proteins identify target membranes for fusion and Rab-GTPs play a role in tethering and docking of vesicles to the membrane
Which enzyme catalyses the dissociation of SNARE coils by hydrolysis ATP and forcing synpatobrevin or VAMP-1 away from the t-SNAREs
NSF enzyme
What are the effects of the tetanus and botulinum neurotoxins produced by some bacterial infections
They can result in complete neuromuscular paralysis
How is botulism and tetanus contracted
Botulism happens upon consumption of contaminated food whereas tetanus infections can happen after skin cuts, during childbirth or dirty needle injections
How do the botulinum and tetanus toxins lead to muscular paralysis
Botulinum and tetanus toxins attack SNARE proteins with high specificity. SNAREs are responsible for acetylcholine release at the neuromuscular junction
Explain the mechanism of action of the botulinum toxin
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
How long does the paralysis caused by botulinum toxin last for if untreated
4-6 months
Explain the use of botulinum toxin in medicine
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
Whereas membrane-bound and secreted proteins are made in the RER, where are nuclear and cytosolic proteins made
Nuclear proteins are made on the outer nuclear membrane whereas cytosolic proteins are made in the cytosol
What is the role of signal recognition particles in membrane and secreted protein synthesis
SRPs direct the delivery of the ribosome and protein to the membrane
Following protein synthesis which compartment do the proteins move into were lipids are made and the transport vesicles are formed
Smooth endoplasmic reticulum
What happens once proteins reach the Golgi
In the Golgi there is a final addition of sugars and then the proteins are sorted and concentrated into transport vesicles
Where in the protein sequence are the signal sequences often found
At the N-terminus
What enzymes remove signal sequences once proteins have been sorted
Signal peptidases
As well as being cleave out often signal sequences contains internal structures that remain part of the protein, T or F
T
What are signal patches
Signal patches are specific 3D arrangements of amino acids that are used to target proteins
What is characteristic of the signal sequence that directs a protein to the endoplasmic reticulum
Proteins going to the endoplasmic reticulum have a signal sequence of 5-10 hydrophobic amino acids
What is characteristic of the signal sequence that directs a protein to the mitochondria
Proteins destined for the mitochondria alternate between positive and hydrophobic amino acid residues
What structures recognise signal sequences that guide proteins towards their target destinations
Sorting receptors which recognise SRPs
Signal recognition particles guide signal peptide sequences to the target compartment where the ribosome can then assemble, T or F
F - Signal recognition particles guide ribosomes following binding to the signal peptide sequence
Describe an experimental technique used to separate vesicles from the SER and RER
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
Ribosomes associate with the membrane of the target compartment via a direct interaction, T or F
F - Electron microscopy has shown that ribosomes associate with the membrane via protein translocators present in the membrane
Explain how proteins are transported and insert into the ER membrane
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.
Explain what is meant by SRPs and their receptors acting as molecular matchmakers
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
Explain how proteins insert into the ER membrane once the SRP has bound to its receptor
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
Describe how transmembrane proteins insert into the ER membrane and how this differs from ER luminal proteins
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
How does insertion into the ER membrane differ for multipass transmembrane proteins
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
What can be said about the affinity of start and stop transfer sequences in ER membrane proteins
They are hydrophobic
By what other key method other than transmembrane domains can proteins become anchored into the ER membrane
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)
What is meant by the process of conformational maturation that occurs after signal sequence removal by signal peptidases
Conformational maturation is the process whereby the maturing proteins adopts a thermodynamically stable shape
What type of bonding often occurs during protein maturation and acts to solidify protein shape
Disulphide bridges
Which sequence is contained within ER-resident enzymes to direct their return to the ER and which amino acids are involved
KDEL sequence: lysine-aspartate-glutamate-leucine
What are the three reasons why glycosylation is important
Increase protein stability in the harsh extracellular environment, enables cell-cell recognition as well as cross species separation
What attribute of protein glycosylation is responsible for the limitations in xenotransplantation and anima donors
Humans contain B-galactose attached to proteins whereas animals use ?-galactose
Where does initial carbohydrate addition to proteins start
ER
What is the initial purpose of carbohydrate chains added to proteins
Quality control tags
Which amino acid residue is often used to add initial carbohydrate modifications
Asparagine
Trimming and growth of carbohydrate chains proceeds step-by-step in individual Golgi cisternae, T or F
T
Give an example of where defective protein glycosylation causes disease
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
Why is it that each glycosylation step requires separate Golgi compartments
This keeps specific glycosylation enzymes away from each other
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
Sphingomyelin and Band 3 protein
Which blood group is the universal donor
O
Which blood group is the universal acceptor
AB
Describe the differences in the terminal oligosaccharide in people with the A, B and O antigens
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.
How does the structure of the various blood type antigens account for the antibodies that those patients will possess
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.
Give an example of protein trimming in the process of maturation
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
Give an example of a disease caused by improper/defective protein cleavage
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
Give an example of where different cells can process the same peptide into different hormones
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
What are the four factors that influence cell shape
Adjacent cells, cell adhesions, extracellular matrix and the function of the cell
What subcellular activity also plays a role in defining cell shape
Migration, phagocytosis, transport and cytoskeletal dynamics
Why is the actin cytoskeleton not a rigid structure
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
Describe the four main components of the actin cytoskeleton
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
What are the two type of actin
Monomeric or globular/G-actin and polymeric or filamentous/F-actin
What can be used to cause the spontaneous polymerisation of monomeric actin in vitro
The addition of salts stimulates actin polymerisation
What can be said about the likelihood of actin polymerisation to occur
The initial step in actin polymerisation is energetically unfavourable and a very slow reaction
Describe the phases of actin filament assembly
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
Actin polymerisation occurs at both ends of the filament, T or F
F – actin tends to be added at one end (+ end) and subunits are removed at the other end (- end)
What is meant by actin filaments being referred to as polar
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
Profilin is an important actin-binding/accessory protein, explains its dual roles
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
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
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
Give an example of another important actin accessory protein and its role
Gelsolin – involved in capping existing actin filaments as well as capping and nucleation
GTPases are small monomeric proteins, T or F
T
What is the rough weight of a GTPase
21kDa
GTPases hydrolyse ATP, T or F
F – they hydrolyse GTP
What is the purpose of the post-translational lipid modifications often seen in GTPases
These hydrophobic lipid groups added to the proteins will target them to specific membrane sites
What is the name of the superfamily to which all small GTPases belong
Ras Superfamily of GTPases
In its inactive state, GTPases are bound to GDP, what is required to activate signalling
Displacement of the GDP by GTP activates the GTPase and initiates signalling
What is the result of the intrinsic nature of GTPases to hydrolyse GTP
Hydrolysis of the bound GTP by the GTPase releases a phosphate and switches it back to an inactive state
Nucleotide-free GTPases are extremely energetically favourable, T or F
F – its extremely unfavourable
What are GEFs and what is the role of these proteins in the cyclic nature of GTPase activity
Guanine nucleotide exchange factors stabilise GTPases in a transition state so that GTP can then bind after GDP release
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
GAPs are negative regulators of GTPase signalling as they promote the catalyses of GTP hydrolysis to the inactive GDP-bound form
What is the role of guanine nucleotide dissociation inhibitors
GDIs effectively pull the GDP bound GTPases out of the cycle and hold it in the cytoplasm to create a pool of inactive GTPases
What changes happen at the molecular level as a result of GTP nucleotide binding to GTPases
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
What are the three members of the Rho family of GTPases
RhoA, Rac1 and Cdc42
What is the role of the Rho family of GTPases
They coordinate actin cytoskeletal organisation, which in turn ultimately controls cell morphology, movement and polarity
What is the role of Cdc42
Cdc42 is a RhoGTPase that controls the polymerisation of actin filaments and the formation of actin spikes or filopodia
What is the role of Rac1
Rac1 controls the organisation of new actin filaments, particularly branched actin, into dynamic ruffling structures or lamellipodia
What is the role of RhoA
RhoA stabilises and consolidates actin filaments into a more rigid skeletal framework known as stress fibres
Describe a loss of function approach that can be used to elucidate the precise function of GTPases
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.
Describe a gain of function approach that can be used to elucidate the precise function of GTPases
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
What is the result of microinjection of constitutively active Rho into quiescent cells
Leads to the formation of stress fibres
What is the results of microinjection of dominant negative Rho into active cells
Leads to the loss of stress fibres
What is the result of microinjection of constitutively active Rac or Cdc42 into cells
Leads to the formation of membrane ruffles or filopodia respectively
What is the name of the specific 16 amino acid sequence which activated Rho proteins bind to within effector proteins
Cdc42/Rac1 Interactive Binding (CRIB) sequence
Rac stimulates the formation of new linear actin filaments whereas Cdc42 stimulates formation of branched actin filaments, T or F
F – vice versa
Active Rac activates WAVE proteins, what is the downstream effect of this activation
Activated WAVE proteins bind to Arp2/3 and lead to the formation of actin filaments associated with branching
What other protein family are activated by RacGTPases that go onto activate Arp2/3 and lead to actin filament formation
WASP
How does Rho activation lead to the formation of stress fibres
Rho activates Rho kinase which in turn phosphorylates myosin to increase its contractility which ultimately leads to the formation of stress fibres
Rac activation is required to precede Cdc42 activation, T or F
F – vice versa
What can happen as a result of a failure of cell migration
Cell, tissue and organism dysfunction and death
Give an example of where cell adhesion and migration play a vital developmental role
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
Give a classical example of where cell adhesion was studied
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
What is seen if the cells of embryonic tissues that have been separated are reintroduced
They also re-aggregate into distinct clusters consisting of cells of the same type
Larger re-aggregates of cells tend not to reorganise due to the number of different interactions, T or F
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
What are L cells
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
Transfection of L cells with cadherin family genes causes what
Homophilic binding between L cells expressing the same cadherins leading to adhesion
What is meant by graded sorting
By varying the degree and levels of cadherin family gene expression, you can influence the degree of sorting of the cells expressing these genes
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
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.
What are the two families of cell adhesion molecules
Ca2+-dependant and Ca2+ independent
To which CAM family do cadherins belong and what are the other subfamilies are there
Cadherins belong to the Ca2+-dependent CAM family along with selectins and integrins
Describe the basic structure of cadherins
Integral membrane glycoproteins that are 720-750 amino acids in length
What happens following Ca2+ binding to cadherins
Causes a conformational change allowing interactions between cells
Cadherin family proteins only contain one Ca2+ binding site, T or F
F – they contain multiple
Give an example of where cell adhesion molecules such as cadherins have roles other that in cell adhesion
VE-cadherin inactivation due to mutations causes abnormal vascular development and apoptosis of endothelial cells, indicating that it has roles in signalling
What is the significance of hypervariable protocadherins
These CAMs are thought to have roles in specifying synapses as wells as mediating neurite self-avoidance
What class of intracellular signalling proteins important in development have been found to bind to the intracellular domain of cadherins
Catenins (B-catenin)
Selectins are another type of cell surface membrane, Ca2+-dependant adhesion molecules. What attributes of cells do selectins recognise in order to elicit adhesion
Selectins recognise particular carbohydrate groups on proteins in neighbouring cells
How do selectins bind to target cells
The lectin domain of selectin binds to sugar groups present on the surface of these cells
Explain the role of selectins in neutrophil trapping
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
Explain the significance of E and N-cadherin in the development of the early nervous system
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
Complex changes in selectin expression accompany the migration of neural crest cells, T or F
F – changes in cadherins do
What is the major family of Ca2+-independent CAMs
Neural cell adhesion molecules (N-CAMs)
What is significant about the number of genes involved in N-CAM expression
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
Explain the role of polysialic acid in N-CAM adhesion
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
Integrins are a key class of cell adhesion molecules. Explain the role integrins
Integrins are extracellular matrix receptors present in the membrane of cells. They act to link the extracellular matrix to the cytoskeleton
Describe the structure of integrins
Integrins consist of two, non-covalently associated glycoproteins each with an ? and ? subunit.
How many different known varieties of integrins are there
24 known integrins (from 18? subunits and 8? subunits)
Which termini make up the intracellular and extracellular domains of integrins
The C-terminus forms the intracellular regions of the proteins whilst the N-termini are found in the extracellular domain of the receptors
What is the name of the specific motif that integrins bind to on target extracellular matrix proteins
RGD – arginine-glycine-aspartate
To which cytoskeletal element do the majority of integrins bind link to
Actin filaments
Binding of which cations effect the binding of the two integrin glycoproteins
Ca2+ and Mg2+
Glanzmann’s thrombasthenia is a disease associated with integrins. Discuss the cause and symptoms of this condition
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
What is meant by a focal adhesion
Focal adhesions are areas of temporary attachment of a cell to the medium it is moving through
What is the role of focal adhesion kinase (FAK) in cell adhesion and integrins
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+
Focal adhesions act through integrins, T or F
T
Focal adhesions act during necrosis, T or F
F – the act during anoikis or attachment-dependent cell death
Mammalian cell motility is interlinked with adhesion and is mostly actin-based, T or F
T
What part of the actin cytoskeleton involved in cell motility is responsible for membrane tension and the transmission of tension
Actin cortex
Outline the process of traditional cell migration
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
What is the name of the complex responsible for nucleation of the actin filament and what is the involvement of Rac
Arp2/3 – stimulated by Rac activity
What three proteins are involved in the maturation of focal adhesions
Actin-binding proteins (ABPs), small G-proteins and myosin
What are the two types of focal adhesion junctions
Low density adhesions and high density adhesions
Which type of focal adhesion junctions tends to be immobile
Low density adhesions
Which type of focal adhesion junctions are Rac1 and cdc42 dependant
Low density adhesions
What is the proposed difference in timing of the two types of focal adhesions during their maturation
Low density adhesions that are immobile tend to appear earlier, whereas, high density adhesions appear later and are motile
Which proteins and interactions are required for formation of the high density adhesions
RhoA and actin-myosin interactions
Signals to dictate cell motility can be soluble or insoluble, give examples of both
Soluble – netrins, insoluble – CAMs
Fibronectin is an extracellular matrix protein that binds to integrins, what sequence is responsible for this binding
RGD – arginine-glycine-aspartate
What is the role of fibronectin in migration of the neural crest cells
Fibronectin trails allows neural crest cell migration.
Explain the role of membrane cycling in cell motility
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
What is the role of endocytosis in cell motility
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
