- 1 Lab attendance
- 2 Lab 1
- 3 Lab 2
- 4 Lab 3
- 5 Lab 4
- 6 Lab 5
- 7 Lab 6
- 8 Lab 7
- 9 Lab 8
- 10 Lab 9: Peer assessment
- 10.1 Group 1 - Testosterone signalling
- 10.2 Group 2 - Vascular Endothelial Growth Factor
- 10.3 Group 3 - Extrinsic Apoptosis
- 10.4 Group 4 - Notch Signalling
- 10.5 Group 6 - Insulin Signalling
- 10.6 Group 7 - GPCR: Beta Adrenergic Receptors
- 10.7 Group 8 - Leukocyte Extravasation
- 10.8 Group 9 - p53 Signalling
- 11 Lab 10
- 12 Lab 11
- 13 Lab 12
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This is my first lab in cell biology.
Internal Link example
External link example
<pubmed>19547746</pubmed> In E.coli, chemotaxis is used as the basis for signal processing however previous studies with normal microscopy have not shown how the transmembrane receptors form large sensory clusters of proteins, what controls the cluster size and density and how the location of these clusters are maintained in cells that are growing.
Greenfield et al, 2009 demonstrates how these clusters are formed and provides evidence for a stochastic (random) mechanism of self assembly using super resolution microscopy.
Chemical fixative chosen: Methanol SDS
- Clear and colourless liquid
- Mild characteristic alcohol odour
- 100% volatile
- Soluble in water
- Boiling point = 65°C
- Melting point = -98°C
- Flash point = 11°C (cc)
- Highly flammable
- Toxic irritant by inhalation, contact with skin or if swallowed
- Experimental teratogen
- Affects the central nervous system
- Damage to optic nerve may occur with chronic or high level exposure
<pubmed>16793760</pubmed> This review article provides a concise overview of the Wnt/Beta Catenin Signalling which can used in the introduction.
<pubmed>15372092</pubmed> There are a number of clearly labelled diagrams in this article that can be used as a starting point for the student drawn image.
<pubmed>18673238</pubmed> This article provides an historical overview of the key events that shaped our understanding of Wnt/Beta Catenin signalling and hence can greatly aid the history section.
<pubmed>17081971</pubmed> This review also contains many simplified diagrams that can be used to support the student drawn image.
Musashi (Msi) is a group of evolutionarily conserved RNA binding proteins. Musashi-1 is 362 amino acids long and is approximately 39kDa Protein information. It is often preferentially expressed in the nervous system. The Drosophila Msi was first identified followed by mammalian orthologs - human and mouse Musashi-1 (Msi1). This protein is strongly expressed in fetal and adult neural stem/progenitor cells and contributes to their self renewal via translational regulation of several target mRNAs.
Primary antibody: Anti-musashi 1
Host species: Rabbit polyclonal
Reacts with: mouse and human
Applications: immunocytochemistry/immunofluorescence and western blot
ICC/IF - Use at a concentration of 1 µg/ml.
WB - Use at a concentration of 1 µg/ml. Detects a band of approximately 39 kDa.
Secondary antibody: Alexa Fluor® 488 goat anti mouse IgG antibody
Dilution for immunofluorescence: 1:500
In-class exercise sheet
Exercise 1: Analysis of morphological phenotypes of Tm4 overexpressing B35 neuroepithelial cells
Do you see a difference in phenotype (morphology) between Tm4 overexpressing and control cells?
- There were higher percentages of pronged and stumped phenotypes for the overexpressing Tm4 cells, which had an increase of approximately 12% and 9% respectively. This may imply the importance of Tm4 overexpression in increasing the degree of branching in the phenotype.
- The broken fan phenotype increased by 16% in the control which means Tm4 may have an inhibitory effect on this phenotype.
- In addition, pygnotic and stringed phenotypes depicted small increases in the control sample, which may imply that Tm4 was not actively involved.
- The fan phenotype was observed in the Tm4 overexpressing cells only.
How could Tm4 overexpression lead to this difference?
Tropomyosin 4 (Tm4) is an isoform of the tropomyosin family of actin-binding proteins and is often involved in the regulation of muscle contraction and the cytoskeleton of non-muscle cells where they confer stability. According to Had et al (1994), Tm4 expression is concentrated in the growth cones of cultured neurons, particularly areas of active neurite growth. This suggests the role of Tm4 in the motile aspects of neurite growth during development and perhaps synaptic plasticity in adulthood. It was also postulated that Tm4 could be involved in the stabilisation of actin filaments and the regulation of myosin proteins and actin (which is required in growth cone motility). This correlates with the result of this lab as Tm4 overexpression was depicted in the phenotypes which had more branching and neurite growth and interaction.
Exercise 2: Effect of Tm4 expression on db cAMP induced differentiation of B35 cells
Do you see a difference in phenotype (morphology) between Tm4 overexpressing and control cells?
Genotype A (Tm4 overexpressing cells):
- Increased appearance of branching and neurite formation
- Indicates the presence of some form of interaction
- Predominant phenotypes - pronged and stumped
- Increased clustering of cells
- Processes appeared to be shorter and wider
- Yellow stained lamellae
- Pink stained cytoskeleton
- Pink-blue round nuclei
Genotype B (control):
- Decreased appearance of branching and processes compared to Genotype A
- Therefore less interaction observed
- Thinner and longer processes
- Predominant phenotypes - broken fan and stringed
- Red stain on the edge of lamellae
- Red stained cytoskeleton
- Very blue nuclei
How could Tm4 overexpression lead to this difference?
db-CAMP induces the differentiation of B35 cells, causing more processes to form however it inhibits the expression of Tm4. This results in the decreased length and hence growth of processes in Tm4 overexpressiing cells. In contrast, the predominant phenotype in the control is the stringed phenotype with longer processes, indicating that db-cAMP did not affect it as much as the Tm4 overexpressing cells.
- Added content to History
- Uploaded two images (one was copyrighted and hence removed)
- Added content to Embryonic Development
- Updated Glossary section
Figure 1. Overall structure of the β-catenin/XAxin-CBD complex
History Figure 1. Overall structure of the β-catenin/XAxin-CBD complex
1973: A Drosophila melanogaster mutant lacking wings, Wingless (Wg), was described 
1982: Roel Nusse and Harold Varmus found that Int1, a mouse protooncogene was associated with MMTV-induced mammary gland tumours 
1987: Int1 was found to be the mammalian homologue of Wingless in Drosophila
1989: New Wnt pathway components were found in a screen of lethal mutations in Drosophila
1991: β-catenin was molecularly cloned
1992: Shaggy was found to be cytoplasmic mediator of Wnt signalling
1993: APC was found to directly interact with β-catenin
1994: Dishevelled was identified as an essential element in the Wnt pathway
1995: APC was found to regulate Β-catenin stability.
1996: β-catenin was found to directly interact with LEF-TCF transcription factors
1996: Frizzled, a seven span transmembrane receptor, was identified as the cell surface receptor of Wnt ligands
1996: Porcupine, a multi-transmembrane protein, was found to process Wnt ligands
1996: Nuclear accumulation of β-catenin was found in colorectal cancers
1997: The three-dimensional structure of β-catenin was determined
1997: Phosphorylation targets β-catenin to ubiquitylation, involving interaction with the E3 ligase B-TrCP, and to proteasome dependent degradation
1997: Identification of the homeotic gene Ubx as the first Wnt target gene
1998: Axin 1 and axin 2 were found to interact with β-catenin, GSK3β and APC and to promote GSK3β - dependent phosphorylation and degradation of β-catenin
1999: Casein kinase 1 (CK1) was found to regulate β-catenin function
1999: Protein phosphatase 2A (PP2A) interacts with the β-catenin destruction complex and modulates GSK3 β (Glycogen synthase kinase 3β) function
2000: The three-dimensional structure of the β-catenin–TCF complex was determined
2000: Arrow, LRP5 and LRP6 were identified as coreceptors of Frizzled
2001: LRP5 was found to transduce Wnt signals by recruitment of axin to the plasma membrane.
2003: The tyrosine kinase receptor Derailed in Drosophila (RYK in mammals) was identified as an alternative Wnt receptor
2006: LEF1 mutations were associated with sebaceous gland tumours in humans, showing that Wnt–β-catenin signalling is inhibited in these tumours
2007: Dishevelled was found to polymerize at the plasma membrane and to recruit axin upon Wnt stimulation
The Wnt/β-catenin signaling pathway has been implicated as an important pathway in human fetal development. Through immunohistochemical staining, Eberhart and Argani (2001)localised nuclear beta catenin in fetal lung, placenta, kidney, cartilage, capillaries, adrenal glands and skin. This indicates that Wnt signaling regulates the development of specific set of organs and tissues. For example, Wnt genes such as Wnt4 regulate the conversion of mesenchyme to epithelial cells in kidney morphogenesis.
In addition, Wnt/β-catenin signaling is also involved in maintaining the pluripotency of human embryonic stem cells (hESCs). Wnt3a promotes the reprogramming of somatic cells to pluripotency in conjunction with the classical transcription factors, Oct4, Sox2 and Nanog. For example, Oct4 has the effect of repressing Wnt/β-catenin signaling in self renewing hESCs and is depressed during hESC differentiation. Hence, this suggests that Wnt/β-catenin signaling is involved in differentiation rather than self renewal.
Glossary of Terms
- canonical: standard and well accepted
- differentiation: the process by which cells become mature and specialised in structure and function
- glycolipoprotein: a protein with attached lipid and carbohydrate groups
- homeotic gene: genes involved in embryonic development, specifically controlled the anterior-posterior axis
- oncogene: genes which transform normal cells into cancerous cells
- pluripotency: the ability of stem cells to differentiate into ectoderm, mesoderm and endoderm
- proteasome: a large intracellular particle which degrades proteins
- self renewal: the capability to undergo numerous cell divisions and maintain undifferentiation
- ubiquitylation: the process of adding ubiquitin
Mammalian cell line: MDCK (NBL-2)
Original source: Kidney tissue from Canis familiaris (dog)
Original reference: <pubmed>5918973</pubmed>
Lab 9: Peer assessment
Group 1 - Testosterone signalling
- Introduction: Ideally the introduction should provide an overview of the whole signalling pathway with an explanation of what this page aims to do. This provides direction for the audience. Although I quite like the idea of introducing testosterone as a molecule, it would be more effective if you had a separate section explaining the properties of testosterone perhaps in a table format.
- History: The colour scheme of the table is very eye catching and the information is concise and well summarised. The history section could be improved by adding a couple of more entries after 1994 and expanding the history.
- Biosynthesis: The idea of having this section is great however the content is not very user friendly especially for people who are new to biochemistry and testosterone signalling. I suggest simplifying the steps and adding a student drawn image depicting the pathway. You could even have each step correlate to numbers on the student drawn diagram.
- Regulation: Again – another fantastic idea! What could really make this section shine is the addition of a diagram of the hypothalamic-pituitary-testicular axis. I think a good physiology textbook is a great source of inspiration.
- Signalling pathway: This section needs revision. I suggest focussing on the two pathways: classical and non-classical which could be presented as a diagram or in steps (like in the biosynthesis section). Introduce new sections for the proteins and receptor structure.
- Normal function: Well written and researched information – all this sections needs is more structure and organisation to make it more user-friendly.
- Abnormal function: I like the way this section has been organised into subheadings. Good job.
- Clinical uses: This section is very informative and well structured. The highlight of this page!
- Current and ongoing research: As a whole, this section demonstrates good research however the expression of ideas in a concise manner is lacking. At times, the sentences are a little confusing however this can be easily remediated with some editing. Another great component to incorporate in this section is the graphs and diagrams from the studies to back up the research.
Overall, a marvellous effort! Your referencing is consistent with a great range of resources being used however please revise your list before the final submission to take care of some broken links or incomplete references.
Group 2 - Vascular Endothelial Growth Factor
- Introduction: Very concise paragraph which provides clear direction to the audience. Perhaps you could have a couple of sentences on VEGF as a family of signalling proteins and then explain why you’re focussing on VEGF-A. The idea is that you start off in a broad sense and then get specific with the various components. Fantastic image choice – however it’s lacking a caption.
- History: The history section has been well researched and summarised however I think it can definitely be expanded to include more entries, particularly in the large gap from 1993-2004.
- Normal function: Although the information in this section is good and relevant to your page, it clearly does not belong to the normal function section. I suggest focussing on what VEGF actually does rather than what influences its production – you can have that in another section about “factors influencing VEGF production and release”. In addition, the dot point on “oncogenes” can also be shifted to a more relevant section such as abnormal function where you could link VEGF to cancer perhaps. I do like the way you’ve linked the glossary words in the text itself – great idea!
- Signalling pathway: Now in this section, you’ve talked about VEGF receptors. Although that’s relevant, I don’t think it really belongs in the signalling pathway section. Perhaps you could have it under another section which specifically deals with receptors and proteins related to the VEGF signalling pathway. Ideally, this section should illustrate the actual pathway itself – using a good balance of text and images. This is a great opportunity to have a student drawn image which your page lacks at the moment. The diagram on the right is fantastic (needs a caption though) – perhaps you could work on explaining the image in text.
- Abnormal function: This is my favourite section! Fantastic table with accompanying images – I think this section can be perfected by just mentioning why you’re focussing on VEGF-A earlier in the page i.e. introduction.
- Research- therapeutic applications: This is another good section that clearly demonstrates your understanding of current therapeutic applications. I particularly like the table and the graph which adds a nice touch to your explanation.
As a whole, this page was an excellent and aesthetically pleasing read. There are a couple of sections that need to be edited and worked on, including the glossary. In addition, it’s very important that you nail the signalling pathway because it is the crux of this whole assignment. Good job Group 2!
Group 3 - Extrinsic Apoptosis
- Introduction: A great way of introducing your signalling pathway. I particularly like the way you’ve deconstructed the whole concept of apoptosis by talking about it in a broad sense and then explaining the specifics. This was my favourite sentence because you explained a scientific concept using an analogy: “in intrinsic apoptosis, internal cellular stimuli induce the process, and the cell is its own judge, jury and executioner.” It was very entertaining to read. My area of improvement for this section is to perhaps add an image or diagram to make it more visually appealing.
- History: This section needs revision - some of the entries were too detailed and need to be summarised. The lack of references is an issue that needs to be remediated once peer reviews are over. I suggest referencing as you write your material rather than leaving it to the end – in this manner, you won’t forget where you got information from.
- Signalling pathway: The list of proteins is very comprehensive but serves no real function in providing information to the reader. I also think they should be moved to another section that specifically deals with the proteins and receptors that are involved in extrinsic apoptosis. You could have a table format to make things more visual. What this section needs is a fantastic and detailed student drawn image that depicts the signalling pathway in extrinsic apoptosis with accompanying text.
- Function: The information in this section was good but was not written very well – as a result, this section lacks structure and perhaps relevance as well. What is the function of extrinsic apoptosis normally in the body? I think the discussion of caspase function can be structured under a sub heading.
- Current research: Well done! This section is fantastic – the information is well researched and there is an image! You can perfect this section with some structure e.g. subheadings, and of course referencing.
Your page is lacking some key components such as abnormal function, glossary, referencing and images. However, never fear! - There is time to fix it up and you’re definitely on the right track with some sections. Well done and good luck!
Group 4 - Notch Signalling
- Introduction: The paragraph is very short however the content is concise and simple – it’s good to see you have indicated an outline of what you’re going to discuss. Perhaps you could add an image to make this section more appealing.
- History: I think more research needs to be conducted on the history of Notch signalling to add more entries to the timeline – the timeline is not complete at the moment. The existing entries you have are well summarised and referenced. I also like the colour scheme of the table.
- Pathway: The information in this section has been synthesised and presented very well – however I think it could improved by altering the structure to a numbered list or bullet point form with an accompanying student drawn image or diagram. I also think more referencing needs to go in this section.
- Proteins and receptors: You’ve made a great start with this section but it’s very general at the moment. I suggest having a table format or diagram to explain the different proteins and receptors, and their respective properties.
- Normal function: There is evidence of excellent research and synthesis in this section with the use of subheadings complimenting the information. I think you could expand more on the role of Notch signalling in the CNS and perhaps make reference to the diagram.
- External links: The use of videos is a great idea. I suggest adding external links to current labs who are investigating Notch signalling. I don’t think future research belongs here – have it as a new section and make sure you explain the content in the articles you’ve listed.
As a whole, good effort! The page is visually appealing most of the time and has generally been structured well. All that’s left to improve is the content which is incomplete in some sections. Don’t forget to add a section about abnormal function and current research! Great work and good luck!
Group 6 - Insulin Signalling
- Introduction: Although your introduction is well researched and summarised, I don’t think an introduction should include that much detail. Ideally your introduction should briefly provide an overview about insulin signalling and then have a paragraph briefly outlining what your page will cover. Alternatively you could revise some of the sentences and remove the sub headings – to present your introduction as paragraph format.
- Structure: I think this section should come after the history. The image is very relevant however it does not contain the correct copyright information or a caption. In addition, the content in this section is also very sparse – perhaps you could have more details on the properties of insulin or present a simplified student drawn image of its structure with appropriate labels (this is imperative to orient the reader).
- History: Great work on having clear and concise entries in the history however some entries need some explanation involving the research team and relevance to insulin signalling. The picture on the right is also very relevant and makes this section more interactive. My area of improvement is to perhaps add more entries after 1998 to expand the timeline.
- Insulin receptor: This section contains an impressive student drawn image! I like the subheadings and short paragraphs however I think more information on the receptor itself and its structural components i.e. the labels on the diagram is required. In addition, I suggest formatting the image to remove large white spaces is also important along with a caption.
- Signalling pathway: Very well researched content and the use of subheadings is effective however I think content which involves a sequential basis are best presented as a numbered list with a corresponding image. In addition, the content was at times very heavy on technical jargon and long compound names. This can be overwhelming for people who are not familiar with insulin signalling. Perhaps you could rectify this by simplifying some of the technical sentences. Well done though! – The amount of detail in this section is commendable.
- Normal function: The existing content in this part is great however does not really focus on the functions of insulin in great detail. Perhaps the subheadings could be fleshed out more with further research.
- Abnormal function: Fantastic and concise paragraphs! I liked this section very much but I think it can be perfected with some re-structuring, namely reorganising the information into a table with accompanying images.
- Current research: I think it was interesting how you explained the current research ideas through the research institutes themselves. Having external links to the actual websites of these institutes is strongly recommended because it will engage the audience more.
As a group, you’ve demonstrate active albeit incomplete progress in all sections and that’s something very impressive and admirable! All you need to do is finetune and flesh out the content and structure to make your page more engaging and user friendly. Exceptional effort Group 6!
Group 7 - GPCR: Beta Adrenergic Receptors
- Introduction: Wow! The amount of detail in this introduction is fantastic but overwhelming. I think your introduction would be most effective if you provided an overview of GPCRs and beta-adrenergic receptor and then a paragraph outlining the aims of your page to orientate your audience. The first three paragraphs under GPCRs are fantastic and exemplify the amount of detail you need in the introduction – it should be very general and broad. You can explain the specifics of GPCR signalling in subsequent sections. Beautiful student drawn image! Kudos to the creator!
- History: This is a fairly good and succinct timeline.
- Gene description: Great content – you could perfect this section by incorporating the information into a table format with images showing the genes themselves if possible.
- Receptor agonists: You’ve made a good start on this section but I don’t quite understand its purpose. Perhaps you could write a couple of sentences explaining the table.
- Receptor structure: Another wonderful image! The variation of subheadings, images, dot points and paragraphs was refreshing – good job.
- Pathway and normal function: I think you’ve done a great job of incorporating many student drawn images that are both relevant and visually appealing. Although the content in this section has been well researched, the structure of stimulatory and inhibitory pathway segments could be more user-friendly if presented in bullet points or a numbered list format. On the other hand, the regulatory mechanism segment has been structured beautifully and reflects an appropriate way of presenting information on signalling pathway.
- Abnormal function, diseases and treatments: It is evident that a lot of effort and research has gone through composing the content in this section. All it really needs is some editing and restructuring to fine tune the appearance.
The status of this project page is very close to the finished product which is a great achievement to have at this point. As a group, your teamwork is evident through the progress you’ve made which means you can spend the next week simply tweaking and finetuning your page. Outstanding effort Group 7!
Group 8 - Leukocyte Extravasation
- Introduction: Great job on providing an overview of leukocyte extravasation. My area of improvement for this is to provide a couple of sentences that summarise the general structure of your page and what you aim to cover.
- Pathway: A fantastic image! If the image on the right is student drawn, make sure you indicate this on the image page. The effectiveness of the image would be increased by making it larger and having the steps in the text correspond to the numbers in the diagram. Don’t forget to add references!
- Normal function: This is an excellent section which demonstrates a clear understanding of the normal function of leukocyte extravasation. Aesthetically, this section would greatly benefit from the addition of a diagram showing the mechanism of leukocyte activation or microbial death, and the use of bullet points or numbered list to break up the long paragraphs. The first paragraph needs editing to fix up some grammatical errors.
- Abnormal function: I can see a lot of effort and research has gone into establishing this section and it’s incredible however not very visually appealing. In my opinion, the best way to present this information is in the form of a table with accompanying images.
- Proteins: The use of images and diagrams in this section is commendable however the large chunks of text next to it is not very appealing to the reader. Perhaps you could resolve this by using table formats or dot points. The referencing is also pretty scarce in this section so make sure that’s followed up.
Excellent work Group 8! As a group, you’ve clearly demonstrated a good command of scientific concepts and research skills. The most important area to improve is the structure and appearance of the page as well as the referencing! Also, make sure the history and current research sections are completed.
Group 9 - p53 Signalling
- Introduction: The first paragraph in this introduction was excellent because it was straight to the point and very relevant. I think the second and third paragraphs belong to subsequent sections on normal and abnormal function. My area of improvement is adding another paragraph which provides a brief outline of what your page aims to cover – this is important because it provides a clear sense of direction to the audience.
- Pathway: I think it’s very important to clearly distinguish the three pathways p53 is involved in through the use of subheadings, tables or diagrams. The information in this section has been well summarised and research however it is not very user friendly. Hence, to improve, you could have a student drawn image with all three pathways for comparison – a flow diagram would be very useful here. Also, don’t forget to complete referencing this section.
- History: Excellent range of entries that have been well referenced and compiled. I like the design of the table however I think the history section should placed after the introduction.
- Current research: You’ve made a great start to this section – have a couple of more examples of current research with accompanying images, diagrams or graphs.
- Normal function: A fantastic example of good structure and content! The use of subheadings is very user friendly and could greatly benefit from the addition of an accompanying flowchart or mind map that summarises all the functions of p53.
- Abnormal function: This section needs a lot of work – p53 is called the guardian of the genome which is why p53 mutations have been implicated in a wide range of diseases. I think the diagram is pretty relevant and can work really well with some text.
Overall, fantastic team effort! You’ve shown excellent research skills in the sections you have completed and the page is generally user friendly. To produce a complete page, simply finish the allocated sections, take care of referencing (you have the same reference repeated multiple times) and improve the visual aspects. Well done and good luck!
--Mark Hill 13:10, 17 May 2012 (EST) Good section-by-section review, with specific examples supporting your comments. There still seems to be group reviews missing?
In-class activity sheet
Complete the group project
Identify a current technique used in gene sequencing. Next Generation Sequencing
Identify a recent cell biology research paper that has used microarray technology.
<pubmed>22246450</pubmed> What aspect of the research findings were contributed by the microarray technique?
One of the causes of infertility in women of young age is attributed to diminished ovarian reserve (DOR). The microarray was used to profile the gene expression of corona radiata cells and identify the genes which are downregulated in infertile women when compared to the control. The microarray results identified 12 genes which are potentially involved in the pathogenesis of DOR. The genes most commonly downregulated in DOR patients were the FOXC1 and CXXC5 genes. The use of microarray was particularly crucial to this experiment because it identified important genes that may contribute to the development of infertility. As a result, this can be used to direct future treatments or prevention strategies.