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- 1 Sub-heading
- 2 References
- 3 Individual Assessments
- 3.1 Lab 1
- 3.2 Lab 2
- 3.3 Lab 3
- 3.3.1 Trans-endocytosis of CD47 and SHPS-1 and its role in regulation of the CD47–SHPS-1 system
- 3.3.2 Trans-Endocytosis of CD80 and CD86: A Molecular Basis for the Cell-Extrinsic Function of CTLA-4
- 3.3.3 β-Arrestin1 Mediates the Endocytosis and Functions of Macrophage Migration Inhibitory Factor
- 3.3.4 Reference
- 3.3.5 Copyright
- 3.3.6 Jak2 is a negative regulator of ubiquitin-dependent endocytosis of the growth hormone receptor
- 3.4 Lab 4
- 3.5 Lab5
- 3.6 Lab 6
- 3.7 Lab 7
- 3.8 Lab 8
- 3.9 Lab 9
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<pubmed limit=5>Sodium sulfate</pubmed>
Copyright: © 2011 Dempwolff et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Image of Nuclear membranes
© 2009 Garnier-Lhomme et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Super-Resolution Microscopy Strategies in Cell Biology Using a Spinning Disk Microscope
Hosny N.A. et al. (2013) present a comparative study of super-resolution microscopy strategies using two methods of super-resolution microscopy; Photoactivation Light-Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM), in conjunction with Spinning disk super-resolution imaging (SDSI) or Structured Illumination microscopy (SIM) and differing image analysis algorithms (RainSTORM, QuickPALM, GLRT, SOFI, 3B, Deconvolution-STORM (DeconSTORM), and Faster-STORM). Standard confocal microscopy has a resolution limit of 200nm, which has prevented further research into small molecular structures e.g. nuclear ultrastructure. Super-resolution microscopy has bypassed this resolution limit (described by Abbe's Law) and allowed for observation of structures as small as 30nm in size.
The study suggests that; 1) Multi-spectral SDSI can collect super-resolution images with good signal-to-noise (S/N), resolved in any selected axial plane within a cell. 2) PALM and STORM can both be used separately or in conjunction to produce super-resolution data. 3) SOFI has the best retention of image intensity information and provides the most accurate data reconstruction, in terms of spatially assigning all of the emission data found in the original images. 4) SIM was more appropriate for imaging 3D structures. 5) PALM/STORM SDSI could generate higher resolved data than SIM for single plane imaging dependent on the image processing algorithm used
Research on trans-endocytosis; Trans-endocytosis is a process whereby material created in one cell is incorporated into another cell through endocytosis.
Trans-endocytosis of CD47 and SHPS-1 and its role in regulation of the CD47–SHPS-1 system
<pubmed>18349073</pubmed> This article looks at trans-endocytosis of transmembrane proteins CD47 and SHPS-1. The study suggests that CD47 and SHPS-1 interaction initiates the transfer of CD47 from CD47-expressing cells to neighboring SHPS-1-expressing cells followed by the internalization of the ligand-receptor complex into the SHPS-1-expressing cells. SHPS-1 was found to undergo trans-endocytosis from SHPS-1-expressing cells to neighboring CD47-expressing cells, suggesting that trans-endocytosis of CD47 and SHPS-1 occurs bidirectionally. The study suggests that CD47 trans-endocytosis is implicated in the regulation of the CD47–SHPS-1 system.
This article is relevant to the sub-topic of trans-endocytosis as it describes the mechanism and physiological roles of endocytosis and gives a specific example of trans membrane protein trans-endocytosis.
Trans-Endocytosis of CD80 and CD86: A Molecular Basis for the Cell-Extrinsic Function of CTLA-4
Qureshi et al. (2011) investigate the cell-extrinsic mechanisms of Cytotoxic T lymphocyte antigen 4 (CTLA-4). CTLA-4 is thought to be an important factor in the prevention of autoimmune disease. Trans-endocytosis of CD80 and CD86 (ligands shared by both CLTA-4 and CD28, a stimulatory receptor) inhibits co-stimulation of CD28. The study suggests trans-endocytosis as a possible cell-extrinsic model of CLTA-4 function whereby co-stimulatory ligands are removed from antigen presenting cells (APCs).
This study is relevant as it outlines a mechanism whereby trans-endocytosis is used to inhibit co-stimulation of another cell. The study also shows that trans-endocytosis can play a regulatory role in autoimmune diseases.
β-Arrestin1 Mediates the Endocytosis and Functions of Macrophage Migration Inhibitory Factor
Xie et al. (2011) investigate the effects of Macrophage migration inhibitory factor (MIF) and β-Arrestin on MIF endocytosis. The study suggests that MIF utilises β-arrestin1 as a molecular scaffold to maintain integrity and specificity of signalling.
This study relates endocytosis to cytokines and chemical mediators involved regulating inflammatory and immune responses.
Image of Uptake of MIF via CPZ sensitive endocytosis
© 2011 Xie et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Jak2 is a negative regulator of ubiquitin-dependent endocytosis of the growth hormone receptor
Putters et al. (2011) investigate the ability of Janus kinase 2 (Jak2) to bind to the growth hormone receptor, preventing endocytosis of growth hormone receptor (GHR). The article shows that Jak2 specifically inhibits GHR endocytosis independent of its kinase activity and Growth Hormone-induced and constitutive endocytosis undergo the same mechanism of endocytosis.
This article is relevant because it shows that both growth hormone-induced and constitutive growth hormone receptor endocytosis depend on the same factors, therefore strongly suggesting that the modes of endocytosis are similar, if not identical.
Identify an antibody against an adhesion junction protein that is commercially available.
Anti-E Cadherin [HECD-1] antibody
Add a link to the original data sheet page and identify the type of adhesion junction.
 - Tight junctions (present in all cells but most prevalent in epithelial cells and also forms the blood brain barrier)
Include the following information: type of antibody (polyclonal, monoclonal), species raised in, species reacts against, types of application uses, and if available any reference using that antibody.
Type of Antibody: Monoclonal
Species raised in: Raised in humans
Species reacts against: Does not react with Mouse or Rat
Types of Application: Flow Cytometry, Immunocytochemistry, Immunoprecipitation, Immunohistochemistry, Immunohistochemistry (frozen sections), Immunocytochemistry/Immunoflourescence, Western Blot
References: <pubmed>23584474</pubmed> <pubmed>23752180</pubmed> <pubmed>23825587</pubmed>
 - Junctional Adhesion Molecule 1(present in tight junctions of epithelial and endothelial cells and is involved in the regulation of junctional integrity and permeability)
Type of Antibody: Monoclonal
Species Raised in: Rabbit
Species reacts against: Reacts with Rabbit, Mouse, Human
Types of Application: Western Blot, Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Types of Application
Group 2 Peer-Review
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..."
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,". This section could also be further developed and the picture associated explained.
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 3 Peer-Review
Nuclear Envelope: Referencing not complete yet and a very general overview of the topic. Lacks diagrams. Looks like its still a work in progress at this point. Some of the sentence structure does not flow well e.g. "The outer membrane faces the cytoplasm and is continuous and closely associated with the rough endoplasmic reticulum and has ribosomes attached to it.".
Nuclear Pore Complex (NPC): Good referencing. Well structured. Could use some diagrams. Explanation of what "FG-Nups" are (which comes later) at this point.
Nucleoporins: Good introduction to nucleoporins. FG-Nups section is well referenced and provides a good insight into the topic.
RanGTP and RanGDP: Well referenced. Good information. Diagram is useful and accessible.
Nuclear Transport Receptors: Well referenced. Good information.
Overall: Certain sections are well put together e.g. RanGTP and RanGDP, but many parts are still missing. There are some useful diagrams but more could be used to help illustrate concepts. Referencing varies throughout the project but in general is good. At this point the project shows that some key concepts are understood but there are some that still need to be researched and added to. Relatively accessible information that goes beyond lecture material. Glossary could be useful?
Group 4 Peer-Review
Introduction: No references or images. Looks as if still a work in progress.
Fast and Slow Transport: Bullet points of basic information. Good references. Accessible information. Also a work in progress.
Motor Proteins: Good use of table to show differences between Kinesins and Dyeins. Good references. Good description of role of different kinesins. Could use diagrams/images.
Overall: Some development of concepts beyond lecture material. Still needs a lot more information/diagrams and content. Reference list for certain parts are good. Error in reference list at the bottom of the page. Does not show an overall understanding of the topic area yet. Relatively accessible information in the Fast and Slow Transport sub-heading.
The normal mechanism through which cell death in the grown tissue culture occurs is apoptosis
We will measure apoptotic rate by subjecting the tissue culture to Flow cytometry.
Key Techniques & Procedures
Flow cytometry is used to detect the fragmented DNA
Hoecht staining of apoptotic nuclei (with Hoescht 33342 as a blue stain) to determine the condensation and fragmentation of the nuclei. Hoechst 33342 binds preferentially to adenine-thymine (A-T) regions of DNA. This stain binds into the minor groove of DNA and exhibits distinct fluorescence emission spectra that are dependent on dye:base pair ratios.
FLICA (flourochrome inhibitor of caspase) is a simple yet accurate method to measure apoptosis via caspase activity in whole cells. It applies the green fluorescent inhibitor probe FAM-VAD-FMK to label active caspase enzymes in the cell samples. FLICA probes are comprised of an inhibitor peptide sequence that binds to active caspase enzymes, a fluoromethyl ketone (FMK) moiety that facilitates an irreversible caspase binding event, and a fluorescent tag (either carboxyfluorescein or sulforhodamine B) reporter. The green poly caspase inhibitor FAM-VAD-FMK utilizes the fluorophore carboxyfluorescein and the multi-enzyme recognition sequence valine-alanine-aspartic acid (VAD). Upon encountering active caspases, the FLICA probe interacts with the enzymatic reactive center of the enzyme via the peptide recognition sequence, forming a covalent thioether adduct with the enzyme through the FMK moiety. After a simple wash step, the remaining green fluorescent signal is a direct measure of caspase activity at the time the probe was added.
Thermo Scientific Pierce Hoechst 33342 Fluorescent Stain is a high-quality solution of Hoechst dye for fixed- and live-cell fluorescent staining of DNA and nuclei in cellular imaging techniques
FLICA™, Fluorescent-Labeled Inhibitor of Caspases, . FLICA-FAM
The E-Gel® Low Range Quantitative DNA Ladder with ethidium bromide staining. The E-Gel® Low Range Quantitative DNA Ladder
Cell sample (non-adherent cells) → add necessary fluorescent dye (FLICA™)→ perform flow cytometry → with blebs present, apoptosis is proven to have occurred
If apoptotic blebs and DNA fragmentation are detected by flow cytometry, the results suggest that the cells died by apoptosis. The Hoechst stain will present the apoptotic nuclei with a blue stained DNA. The FLICA will present a green stain of the caspase that are involved with apoptosis i.e. Caspases 3, 8, 9. The E-gel will present fragmented DNA, from the apoptic cell, will present a DNA ladder in comparison to the unfragmented live cell.