Talk:2014 Group 2 Project

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2014 Projects: Group 1 | Group 2 | Group 3 | Group 4

  1. Do not remove this notice {{2014 Project discussion}} from the top of the discussion page.
  2. Newest student comments should be entered at the top of this current page under the subheading "Student Discussion Area" (you cannot edit the sub-heading title).
  3. All comments should begin with your own signature button, that will automatically enter student number date/time stamp.
  4. Do not use your full name here in discussion, if absolutely necessary you may use first names only.
  5. Do not remove or edit other student comments.
  6. Use sub-headings if you want to add other draft information, images, references, etc.
  7. Only your own group members should edit this page, unless directed otherwise by the course co-ordinator.

Group Assessment Criteria

  1. The key points relating to the topic that your group allocated are clearly described.
  2. The choice of content, headings and sub-headings, diagrams, tables, graphs show a good understanding of the topic area.
  3. Content is correctly cited and referenced.
  4. The wiki has an element of teaching at a peer level using the student's own innovative diagrams, tables or figures and/or using interesting examples or explanations.
  5. Evidence of significant research relating to basic and applied sciences that goes beyond the formal teaching activities.
  6. Relates the topic and content of the Wiki entry to learning aims of cell biology.
  7. Clearly reflects on editing/feedback from group peers and articulates how the Wiki could be improved (or not) based on peer comments/feedback. Demonstrates an ability to review own work when criticised in an open edited wiki format. Reflects on what was learned from the process of editing a peer's wiki.
  8. Evaluates own performance and that of group peers to give a rounded summary of this wiki process in terms of group effort and achievement.
  9. The content of the wiki should demonstrate to the reader that your group has researched adequately on this topic and covered the key areas necessary to inform your peers in their learning.
  10. Develops and edits the wiki entries in accordance with the above guidelines.


17 April 2014

Before the next practical class (after the mid-semester break) the following items must be completed:

  1. You have written draft text in the section(s) that you have been assigned by your group.
  2. Your text should include source references clearly identifying original research from review articles.
  3. Your section(s) must include at least one research/review image or student drawn image related to the section topic.
  4. You have clearly identified the work you have contributed on the project discussion page.

Projects will be presented by your group to the rest of the class at the beginning of the next practical.

--Z3223095 (talk) 16:49, 20 March 2014 (EST)

--Z3421035 (talk) 16:50, 20 March 2014 (EST)

--Z5011413 (talk) 16:49, 20 March

Topics that each group member will focus on to find 4 articles

3421035- introduction 3223095- diseases 5011413- protein complexes (TOM complex)

Multiple pathways for sorting mitochondrial precursor proteins: http://onlinelibrary.wiley.com/doi/10.1038/sj.embor.7401126/full

Assessment

Images

Media

<mediaplayer>http://www.youtube.com/watch?v=gL4c3dhjKxE</mediaplayer>





Articles

This article explores one of the mechanisms that the mitochondria uses to transport proteins or preproteins through its outer and inner membranes. From other research articles, it has been found that there are protein complexes embedded in the membranes which aid the transportation of charged preproteins. There are differing groups for the inner and outer membranes. One such group of complexes are called the TIM complexes, which contain various subgroups with the main ones being TIM17, TIM23 and TIM44. These proteins help the preprotein cross the inner membrane. TIM17 and TIM23 have been recognised as integral proteins which span across the inner membrane and are said to be structural members. TIM44 is loosely associated with TIM23 and has been discovered to have a matrix heat shock protein 70 (mtHsp70) binding area. Dekker and his team identified that a complex, of approximately 90K containing TIM17 and TIM23, was the major preprotein import site. Through various techniques, they were able to suggest that the import channel is able to hold a translocating chain. [1]

This next article investigates the cleaving mechanism used to separate preproteins, such as Oxal, from TOM complexes in order to transport them through the TIM complexes and into the cytosol of the mitochondria. It has been identified that the inner membrane potential and the matrix heat shock protein 70 (mtHsp70) enable the release of Oxal from the TOM complexes in the outer membrane. The article suggest that there is a close interaction between TOM complexes and translocases, such as the TIM complexes, of the inner membrane. One of the experiments done by Frazier and her team, examined whether mtHsp70 was only required for the transportation of Oxal or was it involved in other preprotein transport across the inner membrane of the mitochondria. They found that the mtHsp70 was required to release Oxal from the TOM complex in the outer membrane. Through various experiments, they concluded that the mitochondria has more than one pathway for translocation of hydrophobic proteins.[2]

The following article explores the structural organisation of the TIM17.73 complex. It identifies the sub-components of the complex, which are TIM17, TIM23 and TIM44 and states that these components are found in equimolar amounts. TIM44 is said to be a peripheral protein and therefore is assoicated with the matrix side of the membrane and the TIM17.73 complex. Through immunodepletion, Moro and his team were able to identify that all TIM23 complexed together with TIM17 to form the TIM17.73 complex. TIM44 was found to associated with this complex at the matrix side of the inner membrane. [3]

In this last article, it was noted that various compounds, such as steroid hormones, are transported into the mitochondrial cytosol though various proteins within the outer and inner membranes. One such hormone which was investigated was cholesterol. A complex called the transduceosome which is where steroidogenic proteins interact with outer mitochondrial membrane translocator proteins and voltage-dependent anion channels. It has been suggested that the cholesterol transfer happens through the specialised interaction sites between the inner and outer membranes, which are composed of voltage-dependent anion channels and the adenine nucleotide translocase of the inner membrane. In this article, Rone and her team set out to identify the mechanisms in which cholesterol is transferred from the outer membrane to the inner membrane of the mitochondria where the cytochrome P450 enzyme, CYP11A1, is located. Through various experimental technqiues, they were able to conclude that several mitochondrial and cytosolic proteins are needed to aid the transportation of cholesterol from the outer membrane to the inner membrane. The outer membrane protein, translocator protein, is needed to bind and separate the cholesterol, the mitochondrial-targeted steroidogenic acute regulatory protein is required to initiate the transportation of cholesterol into the inner membrane. The outer membrane voltage-dependent anion channel anchors the translocator protein and the steroidogenic acute regulatory protein. Finally, the transfer of cholesterol CYP11A1 occurs after the formation of the AAA domain-containing protein 3A, which forms a bridge, connecting the outer membrane to inner membrane. [4]

Articles for user z5011413 on Tom Complex

  • Materials for potential import into the mitochondria are recognized by a specific receptor on the surface of the mitochondria that delivers them into a protein import channel in the outer membrane. The receptor and channel include several non-identical subunits and are referred to as the Tom Complexes (“Transport across the Outer Membrane”). The receptor consists of at least four non-identical integral membrane proteins named (after their approximate molecular masses in kDa) Tom70, Tom37, Tom22, and Tom20. This articles goes through the functions of these proteins but only Tom20 is essential for protein transport. [5]
  • The two receptors, Tom22 and Tom20, are critical for the assembly of Tom40 channel units and have distinct roles as organizers of Tom40 dimers into larger TOM structures. Based on biochemical and electron microscopy studies of the TOM complex isolated from yeast mitochondria, this article discovered the molecular reason for the different number of channel-like structures. The TOM complex from wild-type yeast contains up to three stain-filled centers, while from a mutant yeast selectively lacking Tom20, the TOM complex particles contain only two channel-like structures. From mutant mitochondria lacking Tom22, native electrophoresis separates an ∼80 kDa subcomplex that consists of Tom40 only and is functional for accumulation of a precursor protein. The article concludes that while Tom40 forms the import channels, the two receptors Tom22 and Tom20 are required for the organization of Tom40 dimers into larger TOM structures. [6]
  • In this article Rapaport obtained knowledge about the TOM complex through studying Saccharomyces cerevisiae and Neurospora crassa that contain homologous components to higher organisms like mammals and plants. The receptors Tom20 and Tom22 are involved in the translocation of most protein precursors, especially those with N-terminal targeting signals. Tom70, forms a binding site for a more bound set of molecules that will become proteins, most notably the mitochondrial carrier family. The subunits Tom40, Tom22, Tom7, Tom6 and Tom5 are embedded in the outer membrane where they form the Tom core complex, also called "the general insertion pore". Tom40 forms the protein-conducting channel, whereas Tom22 and Tom5 probably link the receptors to the pore (this is not concluded yet). In conclusion, the article found that Tom40 is essential for the viability of yeast and N. crassa cells. Although Tom22 was also thought to be essential, it has recently been reported that, under special genetic conditions, a very ‘sick’ tom22Δ strain can be isolated. None of the other Tom subunits is essential for viability in yeast or N. crass. [7]
  • In the article, “Biogenesis of Porin of the Outer Mitochondrial Membrane Involves an Import Pathway via Receptors and the General Import Pore of the Tom Complex”, the mitochondira from Neurospora crassa and Saccharomyces cerevisiae were used to analyze the import pathway of porin (voltage dependent ion channel) which is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins were explored in this article. [8]

Uploading image

Import of Oxal through inner mitochondrial membrane.[9]


This article talks about how a mitochondrial dysfunction is a common characteristic in parkinson disease. The disease gene PINK1, results in deterioration of the functions of mitochondria. PINK1 is also found in the cytoplasm and when overexpressed enhanced cell motility. [10]


Mutations in PTEN-induced putative kinase 1 (PINK1) are a cause of autosomal recessive familial Parkinson's disease. [11] Attempts in studying the PINK1 pathway have been controversial because of its location in the cell and within the mitochondria. This study shows that this protein has a topology that the kinase domain faces the cytoplasm and the N-terminal tail is in the mitochondria. The results within this study rectify the location of PINK1 in mitochondria and may help in realising the normal and physiological function and normal physiological function and potential pathogenic role in Parkinsons disease. [12]


PINK1 is a serine/threonine kinase in the outer membrane of mitochondria. [13] It is referred to as the gene responsible of Parkinson's disease. In the cytosol is where PINK1's precursor is made and then transported into the mitochondria through the TOM complex. It discusses the fact that the import receptor Tom70 is essential for PINK1 import. It also states that the study observed that PINK1 has predicted mitochondrial targeting signal. Thus, the research results suggest that PINK1 is transported to the mitochondria by a pathway that is independent of the TOM core complex but crucially depends on the import receptor Tom70. [14]


PINK1 mutations are connected to autosomal recessive parkinsonism. PINK1 influences the shape and the function of the mitochondria. this paper describes a mechanism linking the dysfunction of the mitochondria and the changing of the mitochondrial shape that is related to PINK1. [15]

PINK1.png

Uploaded Image for user 5011413 on Tom Complexes

The Translocase of the outer mitochondria membrane(TOM)

The TOM complex contains the initial receptors Tom20 and Tom70 (yellow). These two receptors are loosely attached to the rest of the complex (the TOM core complex); using mild detergent, they purify with the other Tom components, but not under more rigorous conditions. The TOM core complex builds the protein-conducting pore and contains Tom5, Tom6, Tom7, Tom22 and Tom40. The names of the Tom subunits reflect their molecular weights. [16]

  1. <pubmed>9312000</pubmed>
  2. <pubmed>14560025</pubmed>
  3. <pubmed>10393182</pubmed>
  4. <pubmed>22973050</pubmed>
  5. <pubmed>8943210</pubmed>
  6. <pubmed>11866524</pubmed>
  7. <pubmed>19944477</pubmed>
  8. <pubmed>11266446</pubmed>
  9. <pubmed>14560025</pubmed>
  10. <pubmed>21177249</pubmed>
  11. <pubmed>18687899</pubmed>
  12. <pubmed>18687899</pubmed>
  13. <pubmed>23472196</pubmed>
  14. <pubmed>23472196</pubmed>
  15. <pubmed>19492085</pubmed>
  16. <pubmed>19944477</pubmed>


Extra Project Spoof

Mitochondrial transport of Calcium: http://www.ncbi.nlm.nih.gov/pubmed/11115368

Mitochondrial structure: http://www.ncbi.nlm.nih.gov/pubmed/?term=Electron+Tomography+of+Neuronal+Mitochondria%3A+Three-Dimensional+Structure+and+Organization+of+Cristae+and+Membrane+Contacts

TIM and TOM complexes: http://www.sciencedirect.com/science/article/pii/S1357272504001645

Disease related to mitochondria dysfunction (Breast Cancer): http://www.ncbi.nlm.nih.gov/pubmed/?term=High+TIMM17A+expression+is+associated+with+adverse+pathological+and+clinical+outcomes+in+human+breast+cancer

Mitochondrial protein synthesis and import: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3512135/

Feedback

Group 2

Introduction

A descriptive introduction that links into the parts of the projects, and sets the basis to understanding the mechanisms of transport within the mitochondria and the proteins involved. Maybe a bit too much information is put into the Introduction, but that is my opinion and I still think it is a very good intro. Well referenced, and a related image. Well done.

- General Improvements:

o Possibly remove this sentence and place this in the Current/Future Research section. “It has puzzled researchers for years as to how RNA molecules from the nucleus makes its way through into the mitochondria mainly because the inner membrane is semipermeable and RNA molecules are not as flexible as proteins.”


Protein Transport into Mitochondria

Good text with relevant references. Probably needs to be expanded on slightly more. Images are definitely needed to show structure and possibly mechanisms of binding of TOM and TIM complex.

- General Improvements:

o What do the numbers signify after the TOM annotation? Is that a measure of molecular weight? Please alliterate onto how they are ordered.

o Images are essential here.


Pyruvate transport into Mitochondria

- General improvements

o Needs more information pertaining to pyruvate transport

o Images needed


Diseases

A lot of information is provided with relevant references. The only criticism that I have with this part is the usage of sentences. Try to connect the sentences or elongate them to provide a fluid reading to the audience. There are too many short sentences that can either be expanded or conjoined, and this would greatly increase the readability of this part.

- General improvements

o More images

o Good text, but needs to flow well


Current or Future Research

- General Improvements

o Maybe more future research?

o Images if needed. Otherwise, it is very good.

Overall

The structure is well thought out and the information is very detailed and relevant to the project in terms of mitochondrial transport. The only suggestion I could make is the use of more pictures, as it would help illustrate the text you are writing.



--Z3420257 (talk) 06:31, 15 May 2014 (EST)


The key points of the topic were clearly described. The introduction made good use of the funnel technique by introducing the role of the mitochondria and siting its special properties, ones that other organelles do not possess. It gave a brief understanding of how the process of transport works and a photo was also included to show the basic structure of the mitochondria.

The headings and sub-headings were wisely chosen. The way that the wiki was outlined made if much easier to understand. It was good to use the different macromolecules to look at things, although I can see that information is needed in the sections for fat and RNA transport.

The first section, which pertained to the transport of proteins into to mitochondria, was made easy to understand. You included all the definition in the paragraphs, as well as a colourful diagram relating to the topic. I think the diagram, being coloured, makes it more pleasant to the eye and attracts more attention/interest. I think you could add videos of how these TOM and TIM work to ease understanding.

The section on pyruvate transport was brief and precise. If you have more research papers that discuss it’s specific mechanism, maybe you could also add this. A photo/video could also be added to this section or maybe a photo of the 2 subunits responsible for its transport.

The section on disease was very interesting as it was on Parkinson’s, which is a disease familiar to all. It was made easy to understand. The only downside is that it could be more related to the topics and mechanisms discussed in the previous sections of the page. PINK was never mentioned in the subsequent sections. Maybe you could add a semi introduction of this in the section it is relevant to. The section on future research was also easy to understand. You should also add this to your glossary.

Overall, I think the topic was well outlined. It is easy to understand, which is important as they will be read mores by your peers than academes. More visuals could be incorporated to make the page more interactive and interesting, as mentioned before. The references were well sited and each piece of information was linked to the specific paper it came from. All you need to do is add more information on to topics you have yet to research.



Group 2 Peer-Review --Z3399239 (talk) 12:43, 15 May 2014 (EST)

Introduction: Good introduction and outline of mitochondria that shows development of knowledge beyond lecture material. Significant research is evident and well referenced/cited. Good use of imaging though possibly more descriptive diagrams could be used? Consider changing the first sentence from "The mitochondria are..." to just "Mitochondria are...". Make sure the image of the mitochondria has the relevant copyright clearance.

Protein Transport Into Mitochondria: Well referenced, relevant material. The diagram was not very well explained in my opinion and could use further development in describing what it is illustrating. The information presented looks good. I was confused by the statement "Such subunits are TIM17, TIM23 and TIM44 which are found in a ratio of roughly 2:2:2" would the ratio be the same as 1:1:1 or are you making a point that they function as dimers?

Pyruvate Into the Mitochondria: Good referencing. Looks a little under-developed but that may be due to lack of current understanding. Diagrams would help greatly here.

What can go wrong with Transport into Mitochondria: Good referencing up until the last two paragraphs. In line 4 should it be "It is made in the cytosol and imported into the mitochondria via the (TOM) complex.". Consider revising "When disrupted and the mitochondrial is damaged and it loses its membrane potential," as the sentence structure is confusing. This section could also be further developed and the picture associated explained and referenced.

Current or Future Research: Needs further development and information as well as diagrams. Some referencing present.

Overall: Some good content and referencing. Knowledge and research shows understanding that goes beyond lecture material. Relatively accessible to peers though more diagrams with descriptions would be helpful. The Glossary was a nice addition and could be further expanded. There are a number of repeats in the reference list, namely "Sébastien Herzig, Etienne Raemy, Sylvie Montessuit, Jean-Luc Veuthey, Nicola Zamboni, Benedikt Westermann, Edmund R S Kunji, Jean-Claude Martinou Identification and functional expression of the mitochondrial pyruvate carrier.". Headings and sub-headings are appropriate and easy to follow.


Group 2 peer-review So far the project is looking nice and organized. However, here are a few tips:

Could you please add the Group projects hyperlinks that directly navigate to each project page i.e." UNSW ANAT3231 Course Coordinator Dr Mark Hill
2014 Projects: Group 1 | Group 2 | Group 3 | Group 4"

For the introduction, it would be best to split this section into ‘intro’, ‘structure’ ‘function’ and ‘recent research/discovery’ because the last half talks about the recently discovered processes.

It might be good to talk about the urea cycle (which is pyruvate is apart of), why protein is needed in the mitochondria (i.e. breakdown), - Add more pictures (acts least 1 picture to each section). Also maybe draw a simple flow diagram of the TOM complex. - Mention ATP synthase. Including the motor diagram

It is good that you mentioned the protein transport, however it might also be essential to include the transport of ATP, i.e. respiratory chain and oxidative phosphorylation. Describe the origin of pyruvate. And include diagrams/processes that occur specifically in the mitochondrial membrane

Some sections seem to be missing i.e. Rna + lipd transport into mitochondria. If this transport process is too complex, it is best to ask Mark if it is needed or not in the project.

The disease and research part seems fine but may require more detail and pictures.

Sorry, I feel as though am going of a tangent with this and that I might be describing process that occur in the matrix. Please ask Mark if these are essential at all for the project.

Also the referencing looks great.

Thanks and good luck. --Z3375490 (talk) 14:25, 15 May 2014 (EST)


Feedback --Z3378012 (talk) 18:44, 19 May 2014 (EST)

The introduction provides a clear understanding of the topic. There is good sentence structure and flow which then gives a good lead up to the next subsections. The rest of the project builds well on the introduction. Every section you have set as heading gives good detailed information. The only real improvement I would recommend is more images and maybe even a more broad outlook on the transport system. The quality of what you have so far is amazing but the amount of information is very select.



feedback --Z3372830 (talk) 11:22, 22 May 2014 (EST)

Introduction which covers mitochondria structure, yet it is a little long. I also didn’t really see until the protein what was going to be covered on the page. I would reposition certain details to be small introductions. Perhaps just create a sub heading of mitochondria structure under the introduction would be sufficient.

The assignment makes great use of headings separates information clearly and succinctly. However everything seems to just be covered under the one point “1. Transport from the Cytoplasm to the Mitochondria”, changing it to something along the lines of “2. Transport to the Mitochondria”, “3.What can go wrong with transport… ”, “4. Current and future studies” etc. Although this a small cosmetic issue it would be easier to navigate through assignment.

Images provided are good, however the one provided under diseases need information as to what it is and what is being conveyed. Need for images sub heading, as an index of diagrams and figure that have been used. Reference are from a myriad of sources. Glossary is a fantastic addition, provides further information for the reader with terms they may not necessarily know. Overall this wiki page seems to heading in the right direction.


Group 2:

Introduction:

- Instead of using the phrase, “this leads to the idea” in the first sentence, you should use “this shows that…” because it makes it sound like it’s more of a postulation and not 100% solid fact. - It’s great that you have differentiated between the structure of the inner and outer mitochondrial membrane. This gives a better insight to the different roles they play in transport and the various mechanisms. - However, I think there is too much emphasis on the structure and role of the mitochondria in the introduction when it should also include information on the transport from the cytoplasm to the mitochondria. - The use of the mitochondria image is good in letting the viewers observe the major features of the mitochondria.

Proteins used in transport:

- This section is really well written as it talks about the various proteins used in transport. - The use of the image highlighting the various proteins is great for understanding the mechanisms involved.

Pyruvate transport into the Mitochondria:

- This section seems is good as it incorporates the proteins and signalling molecules needed for the transport of pyruvate. However, there needs to be more paragraphs on the transport of other important molecules to show why mitochondrial to cytoplasmic transport is important. - There should also be images to show the transportation of various molecules.

Diseases and current research:

- This section is very well written and is easy to understand. The use of the image relates to the text and is easy to navigate. - The section on current research in incomplete, however judging on the content written so far, it is really good. - It would be a good idea to use images for the current research section

The use of the glossary list really helps viewers understand and locate words they otherwise wouldn’t have known. The references are also correctly listed with each section having 3-4 references for each paragraph which is good. However, there is definitely more that needs to be written on the pages such as the transport of molecules other than pyruvate to make the page look more complete. Also, maybe a subheading for history should be placed to make the page look even more detailed and interesting. The history could include dates to which the structure of the mitochondria was found, the various imaging techniques that enabled us to view the mitochondria in a molecular level and also the discovery of important proteins included in the transport from the cytoplasm to the mitochondria. All in all, I think group has so far done a great job but needs to work on the criticism listed above.



lab 9

Hypothesis:

Isolated cells grown in optimal tissue culture conditions will most likely undergo apoptosis once placed in a foreign environment.

Aim:

To determine whether the cell undergoes an intrinsic or extrinsic apoptotic pathway by observing the amount of caspase 8 activated.

Method:

Human adherent cells are used for the tissue culture. They are washed carefully and loose cells are isolated. Adherent cells are gently spun and labelled with FAM green fluorescence from the FLICATM kit before trypsinization. Cells are incubated with the FLICA at 37oC for roughly 60 minutes before washing. After the addition of apoptosis wash buffer, cells are placed on ice and protected from light. Observations are made by the analysis of cells with a flow cytometer.

Click ‘Protocol’ tab in the following link for more detailed instructions.

http://www.immunochemistry.com/products/apoptosis-assays/caspase-assay-kits/green-flica-caspase-8-assay-kit.html

Suppliers: Immunochemistry Technologies

Resource: Green FLICA™ Caspase 8 Assay Kit

Results:

If there is a high fluorescence reading observed, that means that there is a high amount of caspase 8 present and therefore, leads to an extrinsic apoptotic pathway. However, if there is a low fluorescence reading observed, there is less caspase 8 and hence, there is no extrinsic pathway activated.

feedback

At first glance the introduction appears to be very long, which isn’t a bad thing. However you may need to be cautious that you are not going into too much detail as it can make it very complicated for readers to understand. The assignment has great structure and format except there may be one thing you need to fix in relation to the formatting of headings and subheadings. At the top of the page where it says “contents”, everything is under “1. Transport from the Cytoplasm to the Mitochondria”, I think it would be better if you started a new heading for extra information (e.g. “2. What can go wrong…”, “3. Current or future research…”, “4. Glossary”). The “glossary” heading is excellent, allowing readers to identify a term that they’re not familiar with. The reference list is long which is good as you don’t seem to be sticking to one source, but getting information from many sources. Overall this assignment has great direction and the content is well written and accompanied by helpful images.