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From CellBiology

Useful Links: PubMed | Course Timetable | 2015 Group 5 Project | Paraformaldehyde

Lab Attendance

Individual Assessment

Lab 1

A series of electron micrographs of bacteria from a snow surface found at Dome C, Antarctica with increasing magnification from a) to d).[1]

Lab 2

Super-resolution microscopy reveals LINC complex recruitment at nuclear indentation sites[2]

Lamins are proteins found in intermediate filaments of cells, which form the nuclear lamina. There are two types of lamins within the nuclear lamina, A-type and B-type. The A-type in particular is found to have a correlation with tissue stiffness and matrix elasticity. The nuclear lamina communicates with peripheral cytoskeleton via physical connections called LInkers of Nucleoskeleton and Cytoskeleton (LINC). These interactions between the nucleus and the cytoskeleton are currently kickstarting new studies as to how extracellular cues can affect cellular processes such as gene expression. To add emphasis on this, recent findings show that the nucleus responds to these external cues. In endothelial cells, recent studies show that apical and central actin stress fibres have a mechanical role regarding cell and nuclear shape. And that forces from these fibres affect nuclear homeostatic balance and internal chromatin structure. This article aims to explore the molecular interactions of these physical links at nuclear indentation sites using Structured Illumination Microscopy (SIM), a kind of super-resolution microscopy. To do this, they used human umbilical vein endothelial cells (HUVECs) and stained for actin, Lamin-A and C, Syne-2 to see the effects of the physical link between nucleus and cytoskeleton, as well as the response of the nuclear lamina to external stress.

They tested the effect on nuclear shape by microcontact printing. External force led to reorganisation of central and apical actin fibres. Central stress fibres were arranged in circular bundles for circular cells and straight cables in rectangular cells, parallel to its long axis. Apical stress fibres arranged themselves randomly in circular cells and thin fibres in rectangular cells, parallel to its long axis. Using SIM, they studied the response of the nuclear lamina to apical and central compressive forces in both rectangular and circular endothelial cells. Elongated cells, in response to compressive forces, showed signs of mechanical indentations parallel to its long axis. Whereas in circular cells, there were little to none. These indentations are a result from the compressive forces of apical stress fibres on top of the nucleus. Also, using SIM, concentration levels of LINC were found to be twice as high in sites of indentations. Meaning, apical stress fibres hold the nucleus in place. Based on this observation, apical actin fibres are responsible for nuclear positioning; whereas, central actin fibres are responsible for cell polarity, division, and motility.

Lab 3

<pubmed>18675790</pubmed> Laminin plays a major role in adhesion in the basement membrane. Human embryonic stem cells (hESCs) express integrin alpha6beta1, which are binding types for laminin, specifically laminin-111, -332, and -511/-521. These laminin isoforms could possibly be used as a culture substrate for hESCs, while keeping it in it’s undifferentiated state. This discovery shows the potential use of laminin as a means of cultivating hESCs for regenerative medicine.

<pubmed>16505007</pubmed> Defects in laminin (Ln) chains could result to ocular diseases. Ln isoforms have a role in the development of the human eye. The Ln-1 chain is present only during the early embryogenesis and is absent in adult tissues. The Ln-alpha 1 chain is present in ocular tissue during the early stages and gradually disappeared from the corneal epithelium at 12 weeks of gestation (wg). This evidence suggests that Ln-alpha 1 chain may have a role in the polarisation of epithelial cells. Ln-alpha2 chain was present in extra ocular muscles at 10 wg. Ln-alpha3 chain was present in the basement membrane of corneal epithelium at all ages and may have a relation with Ln-5, which has a role in formation of hemidesmosomes. Hemidesmosomes allow basal cells to adhere to the basement membrane. There are several more isoforms of laminin that are present in the basement membrane of the developing human eye. The presence of different isoforms in the basement membrane suggest that one isoform can replace the role of another isoform in case there is a deficiency of that isoform.

<pubmed>24830794</pubmed> The regenerative ability of human adipose tissue derived stromal cells (hADSCs) is currently being investigated in the field of regenerative medicine (e.g. rats with compression injury in the spine are subjected to hADSCs). hADSCs deposit human laminin at the site of lesion, which recruits neural precursors. In this experiment, the introduction of hADSCs reduced the lesion area, as well as the expression of glial scar marker GFAP, in the spinal cord of rats. Regenerating fibres also increased in number.

<pubmed>25392494</pubmed> In this study, Lama2 mice were used to study the role of laminin alpha2 in the blood-brain barrier (BBB). Lama2 mice do not express laminin alpa2, a subunit of laminin211 heterotrimer in astrocytes and parasites. With the use of circulating tracer, Lama2 mice showed evidence of a malfunctioning BBB as the circulating tracers leak out into the brain parenchyma. Vascular endothelium of Lama2 mice have an abnormal integrity and defective tight junctions. Laminin211 interacts with dystroglycan receptors to prevent these abnormalities. Non-expression of dystryglycan showed the same results as the Lama2 mice. Therefore, interaction between laminin and dystroglycan give rise to functioning astrocytes, endothelial cells and parasites with regards to maintenance of the BBB.

Role of Laminin in Carcinogenesis and Protection. Inflammation triggers the accumulation of p53, which then activates LMα1 together with LMα5. This aids in the reduction of inflammation, which serves as protection. However, in a carcinogenic setting, high levels LMα1 and LMα5 accelerate colitis-associated tumorigenesis.[3]

Lab 4

Knockout Methods worksheet completed.

Lab 5

This is a graph of the percentage of different phenotypes of wild-type cells vs cells that overexpressed Tm4.

Tm4 promotes the formation of neutrites, hence the shift of 'fan', 'broken fan', and 'stumped' phenotypes to 'pronged' and 'stringed' phenotype. Neutrites contain actin filaments and tropomysoin stabilises actin filaments. As an isoform of tropomyosin, the overexpression of Tm4 will result to more stabilised actin filaments and form neutrites.

Lab 6

1. Identify an antibody that can been used in your group's extracellular matrix project.

⁃ Anti-Laminin 5 antibody

2. Identify the species deriving the antibody.

⁃ Rabbit polyclonal to Laminin 5

3. Identify the working concentration for the antibody.

⁃ 50 µg at 1 mg/ml

4. Identify a secondary antibody that could be used with this antibody.

⁃ F(ab')2 fragment goat anti-rabbit Alexa Fluor 594

5. Identify a paper that has used this antibody.

⁃ <pubmed>25133673</pubmed>

Lab 9

Human T-cell leukaemia

Culture Medium: RPMI 1640 + 2mM Glutamine + 10% Foetal Bovine Serum (FBS)

RPMI-1640: Liquid, With sodium bicarbonate, Without L-glutamine, supplement with 0.3 gm/L L-glutamine

RPMI-1640

Mouse embryo fibroblast

Culture Medium: Basal Medium Eagle (BME) + 10% Foetal Bovine Serum (FBS)

BME: Earle′s salts and sodium bicarbonate, without L-glutamine

Basal Medium Eagle

Penicillin and Streptomycin are both used to kill bacteria. Streptomycin was the first treatment effective against tuberculosis.


Lab 10

Proteoglycans

Great introduction! You clearly stated that you will be focusing on SLRPs, particularly on decor, biglycan, fibromodulin, and lumican. Also, great work on your history section. It is evident that you have spent enough time researching about the history of proteoglycans, the changes, and future avenues for research. Good work on your function section. It’s very concise and well-written. Focusing on just a few SLRPs is definitely a smart move. It saves you time and allows you to write a more detailed description of each SLRP. I like how you separated each SLRP and included subheadings for each e.g. Structure, Function, etc. My criticism of this section is that I wish you had removed the small descriptions of the SLRPs and moved it into their own heading. And instead, just include a line that goes something like what you had written in your introduction. Also, remove the subheadings “Decorin and biglycan” and “Fibromodulin and Lumican”. I don’t see why you have to put this in (just keep the small bolded subheading of each SLRP).

Great abnormalities section! I like how you grouped the abnormalities into three sections: eyes, kidneys, and musculoskeletal. I can see the that it is well researched and the jargon used was just right for our level of understanding. I can see that you have notes to find photos, which is good. PMC articles are usually very good for photos (it doesn’t have to come from the papers you used, as long as you have a visual of the effects of the disease). So, definitely find more photos. My criticism for this section is that I see you’ve used a decent amount of papers for each disease but I don’t see any overlaps in information. Try to find papers that share information so that you’re sure that the information you have in your page is consistent with other papers. Since you have a photo of the effect of Ehlers-Danlos syndrome, why not write about it?

Regarding the photos, good selection of photos. I feel like they really aided in understanding the topic, especially with the structure and abnormalities. Most of the images were properly cited and included the necessary information for uploading, such as copyright and reference. Good work on student-drawn/made images. The only file that I have an issue with is the Congenital_corneal_dystrophy.png, I’m not sure if this is made by a group member or taken from a paper. If it was taken from a paper, include the copyright and the reference. If it was made by a student, include the copyright appropriate for student-made images. I also like your table. It was a good opportunity for you to include your student drawn images. However, if you could link it within your sections that would be great since I’m not entirely sure where it comes in. My guess is structure. Also, include a heading or a description as to what the table is about. Lastly, with referencing, I can see that you have repeating references. Try to fix this by using the format given in the Lab 1 Handout - One page Wiki Reference Card

Overall, great work! Your section looks almost done, just a few touch ups here and there.

Integrins

You have a brief and concise introduction, which is good. Just don’t forget to mention everything that will be included in your page when you start doing the final touches of your page. I can see that your history section is still in the works. It’s a good start. My suggestion is to add in what CSAT and JG-22 stands for because it can be quite confusing (I had to read it a few times to understand.) It’s good that you plan to include future research and a timeline. Regarding the timeline, do it if you have enough dates and events to include, which you can turn into a visual timeline and I think that would really impress everyone.

As for structure, although not complete, It is well-written and understandable. There is a bit of overlap with function but that’s alright. The photo included really helped visualise and understand the structure of your topic. My only issue with this section is the amount of references included. So far, I can only see one. Try to use four to five more papers just to make sure that your information is consistent with other papers.

For function, great job! I really like this section and to whoever did this section, you should be proud. It’s well-written and straight to the point. I like how you linked the function of integrin with the other extracellular matrix components. I think it welds all the group projects together. Also, smart move on including a video. A few problems with this section though is the lack of connection with the photos (and video) and the text. If you could add a brief description about what is happening in the video, that would really be great! There is a good amount of references used in this section.

For abnormalities, very interesting section especially with the cancer part of it. It is nicely written and understandable considering the amount of information I know about integrins. Try to add in a few photos to aid the text. Also, “ tumour initiating cells, or TICs,” you don’t have to say “or TICs” just add in (TICs) right after tumour initiating cells. I can see that you’ve done it with the other terms so just try to keep consistency with your writing. Overall, it’s a good section and I would say it is a very good effort considering that integrins, as you have mentioned, does not have that many evidence relating to diseases yet.

Regarding your photos, I like that you included text to describe what the photo is about. Don’t forget to include a student copyright in your student-drawn image. “Copyright Beginning six months after publication, I (student number) grant the public the non-exclusive right to copy, distribute, or display the Work under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/ and http://creativecommons.org/licenses/by-nc-sa/3.0/legalcode” Speaking of copyright, you have two images that are protected by copyright and you can’t use them. This one Integrin_Structure_and_Function_schematic.jpg and Integrin_structure_and_components.jpg. Lastly, with referencing, I can see that you have repeating references. Try to fix this by using the format given in the Lab 1 Handout - One page Wiki Reference Card

Elastic fibres

Good start with introduction. Just don’t forget to mention everything that is included in your page when doing the final touches. It is obvious that “Structure and Components” section is very well-researched. I see overlaps in references, which is an indication that whoever wrote this section used other papers to confirm that the information they wrote is consistent with others. If you could, include a photo of each structure, that would really help with visualising and understanding the information written. The “Assembly of Elastic fibre” is very good! I like the student-drawn image and how the steps are included within the text. Don’t forget to include something like “refer to image a” before listing the steps and also add “Steps in elastic fibre assembly” in bold to make the steps stand out. One issue I have is the Role of MFAP-4 image. I don’t see how that relates with the information you included.

With function, I like how you discussed the function of elastin in different organs of the body. This is consistent with what you mention for introduction and structure. There is a good amount of references however, they’re not intertwined with the other references used. Try to merge your information together rather than have them in separates chunks. There are a few images but don’t forget to mention them in your text to justify how your photos relate to your information. Also, add descriptions under your photos. With your abnormalities section, well-written and easy to understand. You included a good description of each disease and their effects to the body. You have mentioned that elastic fibres plays a role in some of the diseases however, I find that you didn’t really explain how they cause or add to the effect of the disease. Just try to work on this. I find that using 3-4 papers for each disease is enough to get a good amount of information. If possible, add treatment.

Fibronectin

Good start with introduction. It’s very detailed and well-researched. Just don’t forget to include what is in your page when you’re doing the final touches. A small section on history but very detailed in terms of the year and the event. You can try making a visual timeline on this and that will definitely add quality to your page. As with structure, I can see that it’s also filled with information. It is well-written and understandable however, I feel like you should include a diagram of this structure of fibronectin just to aid the readers in visualising it. This is a good opportunity for you to include any student-drawn images. You also mentioned the isoforms of fibronectin. Is this FnI, FnII, and FnIII? If so, just add a sentence saying that these are the three isoforms of fibronectin. Good use of dot points regarding FnI, FnII, and FnIII. What I suggest is, if you can tabulate this information, put it in a table. Also, regarding the photo included, where does this relate? Don’t forget to add a description of the photo.

I really like what you’ve done with your function section. It’s very easy to understand since they’ve been broken into different parts. You’ve managed to include what the role of fibronectin is for each function and how they do it. With abnormalities, I like that you have lots of diseases included. This definitely highlights the importance of fibronectin. I can see that you researched about a lot of different diseases related to fibronection. However, I can see an imbalance with the quantity and the quality. Try to use more papers, around 3-4 per abnormality if you can. Try to include the role of fibronectin with some of the diseases (e.g. how the lack of fibronectin causes this diseases). But I do like the fact that you included the therapeutics involving fibronectin. I checked all your images and they’re all properly referenced and safe to use. Also, fix your subheadings. Lastly, with referencing, I can see that you have repeating references. Try to fix this by using the format given in the Lab 1 Handout - One page Wiki Reference Card

Collagen

No introduction but that’s fine since introductions are best done at the end of the project. Just don’t forget to mention everything that you will be talking about in your wikipage. Good start with your structure section. It’s definitely discussed the basics of the the structure of type II collagen. However, I suggest that you use more papers. Regarding the biosynthesis of type II collagen, where are your references? Don’t forget to include them. You have a really good photo showing the structure of collagen, if only you could relate it back to your text by saying something like “as seen in image X”. Also, just keep an eye for spelling mistakes, e.g. alpha not alfa.

Great work on your function section! It’s well-researched, has good structure, and your photo definitely relates to your text. I like how you split its function into different sections, which highlights the significance of collagen. My only suggestion for this section is to use more papers to back up some of your information. With abnormalities, I can see that this section is still in the works. There is a heavy use of technical jargons but the information is still understandable. I’m a bit confused as to why the bulk of the text isn’t under Kniest Dysplasia. Overall, I hope that you get the bulk of it done before submission. Try to use around 3-4 papers per disease. I found that those are enough to write a concise and detailed text. As for current research, well done! I see that you only wrote about the aims and he results of the paper. Try to write about more papers if you only plan to write that much for each paper. It’s a good summary of each paper, I just don’t think it shows you’ve done enough work compared to your colleague’s sections.

Regarding the photos, most of them are properly cited and safe to use. The student drawn image has the appropriate copyright. I only have an issue with the image used for the structure of collagen. The file name has a typo, collaghen instead of collagen. Also, it doesn’t include any references or copyright. Lastly, with referencing, I can see that you have repeating references. Try to fix this by using the format given in the Lab 1 Handout - One page Wiki Reference Card


Basement Membrane

Great start to your page with the introduction and history. You obviously have put a bit of research into your introduction. Good section with the formation of the basement membrane. You’ve linked the different components of the extracellular matrix and how they play a role in the formation of the basement membrane. Looking at the amount of citations, I can see that this section is well-researched. It is very easy to understand however, I suggest that you try to link the photo you’ve included with the text by saying something like “as seen in image X”. Also, is it possible for you to include a step-by-step process of the formation of the basement membrane? This is also a good opportunity for you to include a student-drawn image. Your structure section is obviously still in the process of completion. Just keep an eye out for typos e.g. isoforms not isoformes.

As with function, good job on the research! It’s very detailed and still very easy to understand. I can see you’ve used a lot of papers and have merged a few of them together. My suggestion is to make subheadings under the “basement membrane functions” heading for example, in kidneys, in vasculature, in muscles, etc. I think that would help break off the huge chunk of text. I like the image you’ve included. I only wish you could have related it in the text just like what I suggested for structure. Incredible job to whoever wrote your abnormalities section. The dropdown table for additional info on the glomerulus is such as smart addition. Also, I like how you included how abnormalities in the basement membrane leads to these diseases (some teams seem to forget to include that part). I also like your additional info for the rare abnormalities.

Regarding your photos, I think they’re good choice and very relevant to your topic. However, there are some photos that don’t have to correct copyright. Just make sure that you have permission to use these photos. Lastly, with referencing, I can see that you have repeating references. Try to fix this by using this format “[4]” when putting in your in-text citations to prevent repetition.

Lab 12

<pubmed>25544356</pubmed> Target Next Generation Sequencing involves capturing and amplifying specific regions of the DNA, with a quicker and higher output compared to the previously used Sanger sequencing. This paper investigates an mutation in the LAMA2 gene and how this contributes to muscular dystrophy.

References

  1. <pubmed>25101779</pubmed>
  2. <pubmed>25482017</pubmed>
  3. <pubmed>25347196</pubmed>
  4. <pubmed>XXXXX</pubmed>

Group Assignment

Notes