User:Z3240911

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Lab Attendance

--Z3240911 (talk) 15:56, 14 March 2013 (EST)

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--Z3240911 (talk) 15:11, 28 March 2013 (EST)

--Z3240911 (talk) 15:58, 11 April 2013 (EST)

--Z3240911 (talk) 15:12, 18 April 2013 (EST)

--Z3240911 (talk) 16:40, 2 May 2013 (EST)

--Z3240911 (talk) 16:51, 9 May 2013 (EST)

--Z3240911 (talk) 15:34, 16 May 2013 (EST)

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--Z3240911 (talk) 15:21, 6 June 2013 (EST)

Individual Assessments

Lab 1

Cytoskeletal comparison.gif

Cytoskeletal Comparison

A comparison of the equivalent cytoskeletal compenents in prokaryotic and eukaryotic cells in their dividing and non diving states. --Z3240911 (talk) 21:26, 19 March 2013 (EST)

Figure 2. Bacterial, archaeal, and eukaryotic cytoskeletons. Schematic representations are shown for a small number of model organisms from each of the three domains of life (A–C), showing the organization of the cytoskeleton in dividing and nondividing cells (right and left of each pair, respectively). Homologous filaments are colored similarly. Also shown is the possible organization of the cytoskeleton in the LECA (D), highlighting the ancestral families of microtubule motors. Bill Wickstead, Keith Gull The evolution of the cytoskeleton. J. Cell Biol.: 2011, 194(4);513-25 PubMed 21859859

[1] --Z3240911 (talk) 07:39, 26 March 2013 (EST)

--Mark Hill (talk) 10:27, 09 April 2013 (EST) I have taken a mark off for using an article that I have used in the actual lecture, I also prefer images not in the GIF format (though this has not affected mark).

Lab 2

Histochemical analyses and quantum dot imaging of microvascular blood flow with pulmonary edema in living mouse lungs by "in vivo cryotechnique" [2]

The author uses confocal microscopy to image the three dimensional (3-D) morphological features of normal mouse lungs and those under acute pulmonary hypertension in a cryopreserved in vivo state. Secondly, quantum dots are used monitor blood flow and serum proteins at different time points in different mice. Confocal microscopy allows for 3-D imaging in the form of z stacks and typically has a higher resolution due to unfocused light being removed by pinholes, making it an ideal technique for this article. The authors were able to obtain 41.73µm thick 3-D images of cryopreserved mouse lungs, this thickness and detail would not be acheived with a typical fluorescent microscope. It was also shown that when the lungs were under acute pulmonary hypertension the fluorescently tagged albumin tended to be localised in the alveolar spaces rather than along the alveolar capillaries, as in normal lungs. The 3-D imaging by use of z stacks and the resolution achieved from having pinholes made confocal microscopy a valid method for the article's aims.

Another method that could have been employed is multi-photon or two-photon microscopy. The section analysed could be thicker and the need for in vivo cryopreservation could be avoided. For example in "In vivo two-photon microscopy to 1.6-mm depth in mouse cortex" [3] the authors achieve imaging to a thickness of 1.6mm on a live, anaesthetised mouse via an intracranial window. It might be possible to alter this method to suit in vivo imaging of lungs.

--Mark Hill (talk) 10:29, 9 April 2013 (EST) A good paper on microscopy and the quantum dot technique combined and your comments regarding 2-photon are relevant.


--Z3240911 (talk) 08:50, 26 March 2013 (EST) --Z3240911 (talk) 07:33, 27 March 2013 (EST) --Z3240911 (talk) 11:46, 27 March 2013 (EST)


Lab 3

[4] Soonjoung Kim, Hongtao Yu Mutual regulation between the spindle checkpoint and APC/C. Semin. Cell Dev. Biol.: 2011, 22(6);551-8 PubMed 21439394

The article gives an in depth summary of the different mechanisms of inhibition and activation of the spindle-checkpoint and the Anaphase Promoting Complex (APC) and how the two regulatory components of cell division impact on one another. When the spindle-checkpoint is activated, it inhibits the activation of APC, thus keeping the cell from transitioning between metaphase and anaphase. The spindle-checkpoint is only inactivated when all kinetochores of sister chromatids are attached to microtubules stemming from opposite chromatids. The mechanism of the activation and inactivation of the spindle checkpoint is addressed with discussion of how a single unconnected kinetochore can activate the spindle checkpoint as well as how the spindle-checkpoint is silenced. Lastly, the authors address where research can go in future studies and outline questions that remain unanswered with respect to the mechanisms of the spindle-checkpoint and APC.


[5] J D Reimann, E Freed, J Y Hsu, E R Kramer, J M Peters, P K Jackson Emi1 is a mitotic regulator that interacts with Cdc20 and inhibits the anaphase promoting complex. Cell: 2001, 105(5);645-55 PubMed 11389834

The article presents the discovery of a new early mitotic inhibitor, Emi1. Emi1 inhibits the Anaphase Promoting Complex (APC) by binding to Cdc20, a component which is essential for the activation of APC [6]. APC is an important regulatory complex in cell division as it triggers the transition between metaphase and anaphase, but there has been some unknown components surrounding the mechanism of its activation. The discovery of this inhibitor answers some of the questions about how APC is regulated and activated but there are questions left unanswered. Firstly, a question that may be asked with regards to destruction of the protein is what happens if the protein isn't destroyed after its interaction with Cdc20? The presence of nondestructable isoforms of the protein results in somatic cells not being able to commence through mitosis. The protein, after removal, is destroyed by proteolysis, mediated by the attachment of ubiquitin. Further mechanisms surrounding Emi1's destruction are being pursued by the authors. Another question that presents itself is are there any other inhibitory mechanisms or proteins interacting with Emi1?


[7] Annamaria Mocciaro, Michael Rape Emerging regulatory mechanisms in ubiquitin-dependent cell cycle control. J. Cell. Sci.: 2012, 125(Pt 2);255-63 PubMed 22357967

The article looks at particular mechanisms of regulation of the two E3 enzymes Skp1–cullin1–F-box complex (SCF) and the anaphase promoting complex (APC) with regards to ubiquitylation. As discussed in the summary of the above article [8],if certain components such as Emi1 are not destroyed by ubiquitin-dependent mechanisms, then the cell cannot progress through cell division. This reinforces the importance of ubiquitin-dependant destruction of inhibitory proteins and hence the importance of the enzyme cascade leading to ubiquitylation. The article discusses new signals and specific targeting of cell division regulators and groups together ubiquitylation enzymes that work in collaboration. Despite giving an overview of the current understanding of the regulation of SCF and APC, it was made clear that there is still a plethora of information to be uncovered in the topic in terms of cross-talk and mechanisms.


[9] Frank Stegmeier, Michael Rape, Viji M Draviam, Grzegorz Nalepa, Mathew E Sowa, Xiaolu L Ang, E Robert McDonald, Mamie Z Li, Gregory J Hannon, Peter K Sorger, Marc W Kirschner, J Wade Harper, Stephen J Elledge Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities. Nature: 2007, 446(7138);876-81 PubMed 17443180


The article addresses the interaction between the spindle-checkpoint and the progression of the cell cycle from metaphase to anaphase via the activation of the anaphase promoting complex. In particular the impact of a particular inhibitor, USP44, and its mechanism of inhibition is discussed


File:USP44_activity_is_cell-cycle_regulated_and_is_required_for_proper_spindle_checkpoint_function_and_anaphase_timing..jpg

Image deleted due to copyright.

Title: Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities Author: Frank Stegmeier, Michael Rape, Viji M. Draviam, Grzegorz Nalepa, Mathew E. Sowa et al. Publication: Nature Publisher: Nature Publishing Group Date: Apr 19, 2007 Copyright © 2007, Rights Managed by Nature Publishing Group

Lab 4

E-Cadherin Monoclonal Antibody(ECCD-2)- Life Technologies Link to Catalog

The E-Cadherin antibody catalog number 13-1900 is a monoclonal antibody raised in rat. The antibody recognizes mouse and has some reactivity with human E-Cadherin. This antibody can be utilized in western blotting, immunofluorescence, immunohistochemistry of parafin embedded sections, flow cytometry and immunoprecipitation.

The following journal articles have used this antibody for immunofluorescence:

Rubén G Contreras, Liora Shoshani, Catalina Flores-Maldonado, Amparo Lázaro, Alma O Monroy, María Luisa Roldán, Rosana Fiorentino, Marcelino Cereijido E-Cadherin and tight junctions between epithelial cells of different animal species. Pflugers Arch.: 2002, 444(4);467-75 PubMed 12136265

[10]

M Babich, L R Foti E-cadherins identified in osteoblastic cells: effects of parathyroid hormone and extracellular calcium on localization. Life Sci.: 1994, 54(11);PL201-8 PubMed 8107517

[11]

Lab 5

Done on sheet in class.

Lab 6

Questions for part 1 and 2:

1) Do you see any change in phenotypes between A and B?

2) If you see a difference, speculate about a potential molecular mechanism that has lead to the change? If no change is observed, speculate as to why that could be?


Part 1: Undifferentiated B35 Neuroblastoma cells

Lab 6.PNG

1) The control cells show diversity in the phenotypes. Broken fan and stumped were more commonly observed, but overall there was small variation in the percentage of each phenotype present. In the Tm4 overexpressing cells we see a shift in the phenotype to stringed, 47% of observed cells in comparison to 20.8% in the control. We also see a significant decrease in the occurrence of the fan phenotype, only 1.4% of cells had the fan phenotype in the Tm4 overexpressors, whereas the control had 12.5%.

2) A potential molecular mechanism could be that this particular tropomyosin could regulate the formation of parallel bundles of actin rather than crossed, sheet-like actin. This could result in smaller lamellipodia and more filopodial structures, hence we see more cells with the stringed phenotype. The control could have variable tropomyosins present that may regulate the formations of different actin populations, ie parallel bundles and crossed, sheet-like bundles. This could account for the variation observed across the different phenotypes.

Part 2: Differentiated B35 Neuroblastoma cells

1) The Tm4 overexpressors appear to have more processes and branches than the WT cells. While the Tm4 appear to commonly have 3 or more processes extending out from the cell body, the control cells have a lot where only 2 processes extend out. Tm4 also seems to exhibit more branches and numerous branches off a single process. While the control cells also show some branching, the general impression I get is that it's not as frequent as in the Tm4.

2)It's possible that the same impacts on actin organization result in the Tm4 having more processes and branches than the control. Ie the Tm4 leading to more parallel bundles of actin rather than sheet-like actin could give processes and branches a starting point to form.

Lab 7

BMIF Microscopy question sheet

Lab 8

Peer review of other groups pages

Group 2: Cytokinesis

Intro: Maybe need to define cytokinesis better, you have that it’s “the process that leads to the production of two daughter cells from one parent” but that’s cell division in general. Also there are no references for this section.

History: A good start but a few more point would be great, you’re probably coming back to that section anyway.

Body: There is a lot of text that could be complimented by pictures and diagrams, also some more referencing is needed in sections like contractile ring assembly and contractile ring formation. With the animal and plant cytokinesis section, a table might be good to highlight the similarities and differences between the two.

Current/Future research: It would be great to have some figures to break up the text.


Group 3: Golgi Apparatus

First thing I noticed was that the heading was in capital letters which is a bit unusual.

The history table looks good, it seems to cover a lot of information and has nice neutral colours.

The morphology and mechanisms section has a good use of images to support the information explained in the text.

Some sections appear to be a bit light on referencing, i.e. the section about the limitations of the different models.

Areas of future research could use a bit more information and images.


Group 4: Spindle Apparatus

The first picture is really good, I actually like how it’s a big picture with no text because it makes the person who is looking at the page focus on what the spindle apparatus is.

Although the introduction gives a good introduction to cell division in general, there could be more specifics of spindle apparatus and why it’s an important component of cell division.

The History table is good and gives a lot of relevant information, but there is text missing from the 1890’s box.

The section about structure has a good balance of text and supporting pictures, while the function section could use a few more images. I also noticed in these sections that some paragraphs had no references.

The current research and complications sections were interesting and informative.

Overall, it’s a good page and the images help to explain what you’re writing about.


Group 5: Nuclear envelope

Intro: Good comprehensive introduction to the nuclear membrane as well as its structure and function. Diagram also works well to complement the information

History: Starts from the discovery and description of the nuclear envelope and goes through until almost present so it seems to also be pretty comprehensive. Perhaps a picture from a historical paper would go well in this section also.

Structure: the outer nuclear membrane section looks to be unfinished. But if it is finished, does one sentence need a sub-heading? An electron micrograph picture might go nicely in this section.

Breakdown during cell division: Lots of good information here that could be supported really well by adding some pictures.

Overall: I think almost every sub-heading has “nuclear envelope” included. The page is about the nuclear envelope so I think you could get away with having headings such as “Structure” instead of being repetitive.


Group 6: Anaphase

First thing I noticed was that you have no contents box at the top with the headings/sub-headings listed which is odd.

Second thing I noticed was the great immunofluorescent image of a cell in anaphase. Great image, but does it have copyright information?

Intro: There’s no references included in the introduction which is a problem, and there are a couple of spelling and grammatical errors.

History: The table is a great way of presenting history information, but there’s not a lot of content here.

Body: you might need to review the layout of your page, it doesn’t seem to flow very well and some sub-headings only have 1 sentence in them.

Research: This section is very interesting. Perhaps a couple of pictures would bring it up even more.


Group 7: Mitochondria

Intro: It seems good so far, but the text is so condensed and there’s not a lot breaking it up. Also, sentences like “But more on that later” don’t really make your intro flow well. It’s kind of a redundant statement because we already know that you are introducing what you’re going to talk about.

Structure/Function: There is a lot of very detailed information here which is good and diagrams are relevant.

History: There is nothing in the table after 1996, maybe you haven’t finished this section? On that note, I noticed that there is no section for current/future research. As mitochondria are an interesting topic, it would be nice to have some information about current developments.

During Cell division: This section seems to be a bit short considering that this should be the main focal point of the page. Pictures would be to your benefit here.

Physiological: This section is well-written and comprehensive. The information is interesting as well as being relevant.

Lab 9

Handout completed in class on stem cells.

Lab 10

Get that project done!

Lab 11

References

  1. Bill Wickstead, Keith Gull The evolution of the cytoskeleton. J. Cell Biol.: 2011, 194(4);513-25 PubMed 21859859
  2. Yurika Saitoh, Nobuo Terada, Sei Saitoh, Nobuhiko Ohno, Takashi Jin, Shinichi Ohno Histochemical analyses and quantum dot imaging of microvascular blood flow with pulmonary edema in living mouse lungs by "in vivo cryotechnique". Histochem. Cell Biol.: 2012, 137(2);137-51 PubMed 22124864
  3. Demirhan Kobat, Nicholas G Horton, Chris Xu In vivo two-photon microscopy to 1.6-mm depth in mouse cortex. J Biomed Opt: 2011, 16(10);106014 PubMed 22029361
  4. Soonjoung Kim, Hongtao Yu Mutual regulation between the spindle checkpoint and APC/C. Semin. Cell Dev. Biol.: 2011, 22(6);551-8 PubMed 21439394
  5. J D Reimann, E Freed, J Y Hsu, E R Kramer, J M Peters, P K Jackson Emi1 is a mitotic regulator that interacts with Cdc20 and inhibits the anaphase promoting complex. Cell: 2001, 105(5);645-55 PubMed 11389834
  6. E R Kramer, N Scheuringer, A V Podtelejnikov, M Mann, J M Peters Mitotic regulation of the APC activator proteins CDC20 and CDH1. Mol. Biol. Cell: 2000, 11(5);1555-69 PubMed 10793135
  7. Annamaria Mocciaro, Michael Rape Emerging regulatory mechanisms in ubiquitin-dependent cell cycle control. J. Cell. Sci.: 2012, 125(Pt 2);255-63 PubMed 22357967
  8. J D Reimann, E Freed, J Y Hsu, E R Kramer, J M Peters, P K Jackson Emi1 is a mitotic regulator that interacts with Cdc20 and inhibits the anaphase promoting complex. Cell: 2001, 105(5);645-55 PubMed 11389834
  9. Frank Stegmeier, Michael Rape, Viji M Draviam, Grzegorz Nalepa, Mathew E Sowa, Xiaolu L Ang, E Robert McDonald, Mamie Z Li, Gregory J Hannon, Peter K Sorger, Marc W Kirschner, J Wade Harper, Stephen J Elledge Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities. Nature: 2007, 446(7138);876-81 PubMed 17443180
  10. Rubén G Contreras, Liora Shoshani, Catalina Flores-Maldonado, Amparo Lázaro, Alma O Monroy, María Luisa Roldán, Rosana Fiorentino, Marcelino Cereijido E-Cadherin and tight junctions between epithelial cells of different animal species. Pflugers Arch.: 2002, 444(4);467-75 PubMed 12136265
  11. M Babich, L R Foti E-cadherins identified in osteoblastic cells: effects of parathyroid hormone and extracellular calcium on localization. Life Sci.: 1994, 54(11);PL201-8 PubMed 8107517

Copyright

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© 2011 Wickstead and Gull, 2011. Originally published in Journal of Cell Biology. doi: 10.1083/jcb.201102065


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