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From CellBiology

Lab Attendance

--Elisabeth Karsten 10:42, 10 March 2011 (EST)

--Elisabeth Karsten 09:03, 17 March 2011 (EST)

--Elisabeth Karsten 09:10, 24 March 2011 (EST)

--Elisabeth Karsten 08:49, 31 March 2011 (EST)

--Elisabeth Karsten 08:40, 7 April 2011 (EST)

--Elisabeth Karsten 09:01, 14 April 2011 (EST)

--Elisabeth Karsten 08:40, 21 April 2011 (EST)

--Elisabeth Karsten 09:25, 5 May 2011 (EST)

--Elisabeth Karsten 09:49, 12 May 2011 (EST)

--Elisabeth Karsten 08:58, 19 May 2011 (EST)

--Elisabeth Karsten 09:12, 26 May 2011 (EST)

--Elisabeth Karsten 08:43, 2 June 2011 (EST)

Individual Assessments

Lab One

1. What are the key cell biology journals?

  • The Journal of Cell Biology
  • BMC Cell Biology
  • Nature Cell Biology
  • PLoS Biology
  • Cell


2. Which Journals allow reuse of their published content

  • The Journal of Cell Biology
  • BMC Cell Biology
  • Public Library of Science


Lab Two

1. Which chromosomes contribute to the nucleolus?

The nucleolus is the site of ribosomal (rRNA) gene transcription, processing, and ribosomal assembly. Chromosomes 13, 14, 15, 21, 22 contain clusters of rRNA genes that contribute to the nucleolus and its function.


2. Identify and add a link to your page of a recent cell biology article using confocal microscopy.


Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy

Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy


Lab Three

1. Find the SDS information for Chloroform and identify the hazards associated with this chemical.


Biological Limit Values

  • No biological limit allocated.


Engineering Controls

  • Avoid inhalation. Use in well ventilated areas. Where an inhalation risk exists, mechanical extraction ventilation is recommended.


Poison Schedule

  • S6 - Classified as a Schedule 6 (S6) Poison using the criteria in the Standard for the Uniform Scheduling of Drugs and Poisons (SUSDP).


PPE

  • Wear splash-proof goggles, viton (R) or PVA gloves and coveralls. Where an inhalation risk exists, wear: a Type A (Organic vapour) respirator. At high vapour levels, wear: an Air-line respirator.


Health Hazard Summary

  • Toxic - irritant. This product has the potential to cause adverse health effects. Use safe work practices to avoid eye or skin contact and inhalation. Over exposure may result in nerve, liver, kidney and lung damage. Central nervous system and cardiac depressant. Experimental teratogen. Chloroform is classified as possibly carcinogenic to humans (IARC Group 2B).


Eye

  • Irritant. Contact may result in irritation, lacrimation, pain, redness and conjunctivitis. May result in burns with prolonged contact.


Inhalation

  • Toxic - irritant. Over exposure may result in irritation of the nose and throat, coughing, nausea, headache, fatigue, loss of appetite and vomiting. High level exposure may result in dizziness, breathing difficulties, pulmonary oedema and unconsciousness.


Skin

  • Irritant. Contact may result in drying and defatting of the skin, rash and dermatitis. May be absorbed through skin with harmful effects.


Ingestion

  • Toxic. Ingestion may result in nausea, vomiting, abdominal pain, dizziness, fatigue and diarrhoea. Ingestion of large quantities may result in liver and kidney damage, and unconsciousness. Aspiration may result in chemical pneumonitis and pulmonary oedema.


2. You will need to upload an image and add it to your page, with the reference and copyright information with the image.

Lipid droplets as ubiquitous fat storage organelles.jpg

Lipid droplets as ubiquitous fat storage organelles in C. elegans.

Zhang SO, Trimble R, Guo F, Mak HY. BMC Cell Biol. 2010 Dec 8;11:96. [1]

Lab Four

1. Identify a commercial supplier of an antibody that relates to your group project topic.

Sigma-Aldrich produces 'Monoclonal Anti-Pinin antibody produced in mouse' which is a desmosome associated protein.

Monoclonal Anti-Pinin


2. In mitochondria, where is the gene located that encodes Cytochrome C and what keeps this protein trapped within the mitochondria?

It is encoded by genomic DNA, by CYCS gene.

Cytochrome C is localised within the crista of the mitochondria. This is because it is water-soluble and so cannot pass the lipid membrane.


Lab Six

Group 2 - Tropomysin Phenotype Chart.JPG

1. What are the changes in phenotypes that you observe between Group A and Group B?

Group A showed an increase in the percentage of "pronged" and "stringed" phenotype of the B35 neuro-epithelial cells This is opposed to Group B (the control) that showed a much higher percentage of rounder "stumped" lamella, also including "fan" and "broken fan" phenotypes. Group B tended to clump together, altering the morphology of the cells, while - due to the pronged and stringed phenotype - there was still significant cell-cell contact, it only occurred at the tips of the lamella.

There was also moderate colour change observed between the two. In group B, the nucleus was distinctly blue, surrounded by a red cytoskeleton, however in group A, the nucleus was barely discernible as a pink colour while a surrounding pink cytoskeleton. There was also a yellow colour present at the edges of the lamella.


2. How does Tm4 mediate these changes?

Tropomyosin is an integral coiled coil protein that attaches to actin and regulating most, if not all, of its function. There are a number of different isoforms of tropomyosin that are present in different areas of the cell and act on different actin isoforms. They help to confer structure and function to the actin and cytoskeleton. This particular isoform (Tm4) stimulated movement. Tropomyosin inhibits lamellipodium formation and encourages the development of lamellae while enhancing migration. However, the complete mechanism still remains unknown.


Lab Eight

1. Identify from one of the cell line repositories: a neural cell line and a muscle cell line.

Neuro-2a (CCL-131) - Neuroblastoma Neuroblastoma

L6 (CRL-1458) - Myoblast Myoblast


2. Identify the species and growth conditions for these cell lines.

Neuroblastoma

  • Mus musculus (mouse)
  • Base medium: fetal bovine serum to a final concentration of 10%
  • Atmosphere: air, 95%; carbon dioxide (CO2), 5%
  • Temperature: 37.0°C

Myoblast

  • Rattus norvegicus (rat)
  • Base medium: fetal bovine serum to a final concentration of 10%
  • Atmosphere: air, 95%; carbon dioxide (CO2), 5%
  • Temperature: 37.0°C
  • Growth Conditions: The myoblastic component of this line will be depleted rapidly if the cells are allowed to become confluent.

Peer Review

Synaptic Junctions

  • Some gramatical errors under “what is a synaptic junction”
  • What happens at each part of the synaptic junctions? Ie what happens at the synaptic cleft?
  • Need the basics before getting into different types of junctions ,
  • Lots of good information in “types of synaptic junctions” section, but there are many points without much elaboration. For example, if you mentioned two or three important discerning features of electrical synapses, it would be easier to read that 10 different points and minimal explanation
  • The chemical synapses section is a lot easier to read, if you follow a similar path for the electrical synapses (how not only what) it’d show your effort a bit better.
  • The links for ‘other synapses’ don’t lead to anything
  • Good table, it is succinct and information but regulated your use of capital letters
  • Need a section to explain function before getting into modulation and regulation, where they’re present etc (everywhere I know, but it would make it clearer to state it)
  • In ‘synaptic regulation and modulation’ section, the following paragraph doesn’t make sense – “Graded potentials can vary in size because they depend on the amount of neurotransmitter releasedThis is achieved through the regulation of free calcium inside the cell[16]. Axoaxonic synapses control calcium influx into the presynaptic terminal of . If more neurotransmitters bind to receptors, the amplitude of the depolarization increases. Summation to threshold can occur in two ways, or a combination of these two ways;”
  • Need to explain what axoaxonic synapses are
  • Neurotransmitter table looks really good
  • The abnormalities section looks really good and detailed, lots of information there
  • Fantastic pictures that have been drawn, they look really good and concise
  • There seem to be lots of spelling errors and gramatical errors throughout, I suggest you go through the whole thing and do a quick edit

Gap Junctions

  • Really good set out, nice and organized, making it easy to read
  • The history is really long and detailed. There’s a lot of really good information in it, but it could be a bit better if only the really important bits were outlined. Of course, this isn’t a very big criticism as you can’t really have too much information.
  • Good description of the structure. Could you note differences between human gap junctions and their homologues in plants?
  • Good incorporation of KO mice in explaining the function of Cx32
  • I like how you’ve done the locations of gap junctions, well structured
  • It’s a good idea to give a comparison with the other junctions. The table is nice, but since you’re not doing the other junctions do you need that much detail? You could always make links to the other pages or something
  • Good variety of abnormalities and nice, brief descriptions
  • Nice incorporation of the really recent research, show’s how up to date your information is
  • Overall a really good page. Nice work!

Tight Junctions

  • The history looks quite good, nice and concise
  • For the structure can we get a bit of a general overview? You seem to have gone straight into the specifics. Or maybe if you refer to the picture or something? There just doesn’t seem to be quite enough information to get my head around the precise structure.
  • The ‘molecular components’ section looks really good, well set out and informative, I found it quite readable
  • “barrier function” not “barrier funciton”; subheading in “function”
  • The “function” section was really good, the pictures there correlated nicely with the content
  • Clever integration of tight junction distribution
  • In the “classification of epithelia” section, could you break it up a bit? There is a whole lot of information in one go. Maybe put a few subheading? Dot points? Even another picture or so would help.
  • I really like how you’ve the table for disease. Really well set out, I like the idea of the links, but what exactly are they leading to? When I click on them I get some random NCBI page. Might want to fix that up a bit. If you could like it back to the page where those structures are discussed I think that’d be pretty useful.
  • Some sort of discussion of current research could also be good.

Adherens Junctions

  • There isn’t too much to the history, check for some more recent work, ie after 2000.
  • You might want to rethink some of the wording in the intro, not a huge problem but it might help with the flow.
  • The pictures and info in the “structure” section is really good. I like the addition of the video, it keeps it interesting. Cool video.
  • Function is alright, I see you’ve said there isn’t much known about it, but can you maybe elaborate a bit more on the points you have made? Are there examples to show this? Any idea of the mechanism behind embryogenesis? etc
  • A couple of grammatical errors in “regulation” section
  • In regulation section, “growth factors, the peptide hormones and …etc.”. What exactly do you mean by “and…etc”? Come up with one more example and replace the etc, it would just seem more official.
  • Other than that, the information in the regulation section is quite good, I like the set out.
  • “Abnormalities in Disease” doesn’t quite make sense. Maybe “Abnormalities and disease”, “Abnormalities causing disease”? Something like that.
  • Abnormalities section is quite good, good overview of a few diseases, interesting.
  • The addition of the current research is really good
  • The table at the end is pretty good, nice overview of other junctions.

Neuromuscular Junctions

  • Good history, nicely detailed, but has there been anything since 2000? Surely there has.
  • For the mechanism of action, I see what you’ve done with the dot points, but it could be even more effective as a sort of flow chart? That’d be really fantastic.
  • A bit further down, I’m getting a bit confused with the format. I know we shouldn’t be commenting on it, but how you’ve structured your information is a bit inconsistent. Ie the dot points. If you could compile it into a succinct paragraph? Or at least elaborate a bit more into the dot points. They are good in a lecture setting (where more information is provided vocally to “fill in the blanks”) but I personally get confused without the complete sentences.
  • None-the-less, you’ve got some great information there
  • AWESOME table of components. Really good and helpful, especially with the addition of the pictures. I really liked that bit.
  • Nice microscopy section, clever addition with some nice pictures.
  • Some fantastic information in “skeletal muscle” section, but it’s not entirely applicable to NMJs. Focus more on the NMJ involvement in contraction, as opposed to the details about the structure of actin and myosin etc.
  • Interesting stuff about embryology, good work there
  • Nice table for the abnormalities, lots of good stuff there
  • The research area looks really good, and a clever input of potential future research
  • Looks like a pretty good project!

Project Page

Calcium

Calcium ion levels are integral in junction assembly. In vitro, extracellular calcium levels have been found to trigger the formation of the junctions. [2] After cells that have been incubated in low calcium medium, were switched into a medium containing standard calcium concentrations, the formation of desmosomes was seen as soon as 5 minutes. [3] The deregulation of calcium levels within cells have been associated with junctional defects. Hailey-Hailey disease and Darier's disease are caused by defects in Golgi and ER calcium pumps. [4] These both lead to a loss of integrity in skin epithelium, due to a lack of cell-cell attachment via desmosomes. [5] [6]

Desmoplakin

It has been suggested that regulation of desmosome assembly is closely associated with the assembly of adherens junctions. [7] Desmoplakin is the only protein, so far identified, that is common to both junctions. In vitro, cells that did not express desmoplakin were unable to localise the proteins specific for the adherens junctions and the desmosomes. [8] Thus, it has been suggested that it plays a role in segregating the proteins relative to each junction and so affecting assembly.

Phosphorylation

Phosphorylation has been found to be largely involved in activating and deactivating desmosome assembly. It has been suggested that protein kinase C (PKC) is involved in the regulation of this action. [9] PKC inhibition is required for desmosome assembly to occur, that is, dephosphorylation encourages formation. Phosphorylation onto serine of desmoplakin by protein kinase C can lead to the disassembly of the cytoskeleton, junctions, and a loss of cell-cell contact. An increase in level of serine phosphorylation has also been linked with an increase in junction solubility of the desmoplakin. This suggests that phosphorylation may play an integral role in the dissociation of the desmosomal plaque from the keratin filaments. [10] The outcome of plakoglobin (an important desmosomal protein) is also largely dependent on its phosphorylation. [11]

Images and Videos

Pathophysiology of Bullous Diseases - video

[http://www.pnas.org/content/early/2011/03/30/1019469108.full.pdf The three-dimensional molecular structure of the desmosomal plaque]

Work Area

Lab One

Here is some bold text.

Here is some italic text

Lab One

Journal of Cell Biology Homepage


Lab Two

PubMed


Lab Three

Leena Latonen Nucleolar aggresomes as counterparts of cytoplasmic aggresomes in proteotoxic stress. Proteasome inhibitors induce nuclear ribonucleoprotein inclusions that accumulate several key factors of neurodegenerative diseases and cancer. Bioessays: 2011, 33(5);386-95 PubMed 21425306


Nucleolar aggresomes [12] [13]

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