From CellBiology

Group 6

Neuromuscular Junctions


Lab 1. Here, accidentally deleted signature

Lab 2. Here, accidentally deleted signature

--Mark Hill 09:10, 24 March 2011 (EST) Student accidentally deleted attendance for lab 1 and 2.

Lab 3. --Rega Tabbaa 11:35, 24 March 2011 (EST)

Lab 4. --Rega Tabbaa 08:40, 31 March 2011 (EST)

Lab 5. --Rega Tabbaa 09:08, 7 April 2011 (EST)

Lab 6. --Rega Tabbaa 08:39, 14 April 2011 (EST)

Lab 7. --Rega Tabbaa 08:48, 21 April 2011 (EST)

Lab 8.--Rega Tabbaa 08:21, 5 May 2011 (EST)

Lab 9.--User:z3255968--Rega Tabbaa 09:35, 12 May 2011 (EST)

Lab 10. --User:z3255968--Rega Tabbaa 09:24, 19 May 2011 (EST)

Lab 11. --User:z3255968--Rega Tabbaa 08:42, 26 May 2011 (EST)

Lab 12.--User:z3255968--Rega Tabbaa 09:14, 2 June 2011 (EST)

Lab Questions

Lab 1 Questions

1. What are the key cell biology journals?

Nature Cell Biology

Public Library of Science

The New England Journal of Medicine (NEMJ)

The Journal of Cell Biology (JCB)

Trends in Cell Biology


2.Which journals allow reuse of their published content?

The Journal of Cell Biology

BMC Cell Biology

Public Library of Science

Work Area

Here is some bold text

Here is some italic text

First Lab

Journal of cell biology

Lab 2 Questions

1. Which chromosomes contribute to the nucleolus?

The nucleolus is made up principally by rRNA genes. The chroosomes 13, 14, 15, 21 and 22, contain nucleolar organiser regions that are located in their short arms.

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

Studying NB-LRR Immune Receptor Localization by Agroinfiltration Transient Expression [1]

Advanced optical imaging in living embryos [2]



Topics for Group Project:

1. Cell apoptosis/necrosis

2. Microarray

3. PCR

Lab 3 Questions

1. Find the SDS information for chloroform and identify the hazards of this chemical.

Chloroform SDS

Hazards: This material causes cancer in laboratory animals, and is IARC listed as a probable human carcinogen. Inhalation and ingestion are harmful and may be fatal. May cause reproductive damage. Irritant. Exposure to alcohol may increase toxic effects. Prolonged or repeated skin contact may cause dermatitis. Typical TLV 50 ppm.



Lab 4 Questions

1. Identify a commercial supplier of an antibody that relates to Neuromuscular Junction

Santa Cruz Biotechnology, muscle-specific tyrosine kinase (MuSK)that are expressed on the postsynaptic membrane of the (NMJ)[3]

Muscle-specific tyrosine kinase (MuSK)is involved in the formation and maintenance of the NMJ, and is necessary for NMJ integrity. MuSK levels differ between adult skeletal muscles and influence postsynaptic plasticity. [4]

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

Cytochrome c:

-unlike the other cytochromes of the respiratory electron-transport chain, is a peripheral membrane protein

-encoded by CYCS (cytochrome c somatic)gene, which is located on chromosome 7 (7p15.3)

-soluble electron carrier in mitochondria

-trapped inside mitochondria by the outer membrane of mitochondria

-major energy source (provide ATP by oxidative phosphorylation)

-take electron from Cytochrome c reductase and donate electron for Cytochrome c oxidase

-the heme ligand is added inside the mitochondria and only when heme is added the protein is folded properly

-Cytochrome c is also involved in initiation of apoptosis

Lab 5 Questions

Lab 6 Questions

Difference in phenotype groups.jpg

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

Phenotype A: No change in cell numbers between control and TM4 group.

Phenotype B: TM4 group seems to be reduced by 15%, indicating that TM4 may inhibit this cell phenotype.

Phenotype C: There is a small increase in TM4 group about 3% which may not be count in the role that TM4 played in this particular phenotype.

Phenotypes D and E: TM4 groups expressed an increase about 11% and 9% respectively. This may indicate that TM4 is actively involved in the events of neurites growth for these types of phenotypes.

Phenotype F: No cells found for the TM4 group. This may indicate that TM4 entirely inhibits this phenotype.

2. What are the changes in phenotypes that you observe between group A and group B in the pictures?

Genotype A (TM4):

- More branching and processes appear

- This means that there is a considerable interaction between neorites

- The processors seem to be shorter, wider, fluoresce more cheerfully

- Most phenotypes shown were stringed, pronged and stumped

- They emerged in groups (clustered) rarely compared with genotype B

- The nuclei is round, shadowy pink-blue color

- Lamella stained yellow in its edges

- Pink cytoskeleton

Genotype B (control):

- Less branching and processes appear

- Less interaction

- Thinner and often crumbled together

- Most phenotypes shown were broken fan, stumped and stringed

- Very shiny blue nuclei

- The shape of the nuclei appeared in two forms: round and spindle (wide in the middle then tapering at both ends)

- Lamella stained red in its edges

- Red cytoskeleton

Obviously, the morphology of the cell in genotype B unfolded the opposite characteristics compared with the genotype A. This lead to a conclusion that TM4 expressed in genotype A is involved in motility of the cell. These cells showed more branching, processes and hence more interaction.

3. How does TM4 mediate these changes?

Tropomyosin (Tm) is one of the two primary componenets of the actin filaments and the other component is the troponin. Tm contains two polypeptide chains and is a long thin molecule. Tm molecules are bound head to tail, forming a polymer that run over the actin subunits alongside the outer edges of the groove between the twisted actin strands. It binds to actin and acts as a molecular barrier. In relaxed muscle, Tm blocks myosin-binding sites of actin and prevents the crossbridge cycle from occurring. While, the Ca ions released, bind and cause a conformational change to troponin. This shifts tropomyosin’s position on the actin filament and expose the myosin- binding sites.

However, there are several Tm’s isoforms and one of them is Tm4. A striated muscle isoform of a Tropomyosin (TM-4) gene was characterized and found to be necessary for contractile function in embryonic heart. These results demonstrated that a striated muscle isoform of the TM-4 gene was expressed embryonically and was necessary for normal structure and function of the ventricle [5]. Additionally, TM-4 may be involved in the motile events of neurite growth and synaptic plasticity [6]. This is and what we observed for each phenotypes of group A(Tm4) and group B(control). Tm4 group have more processes branching like dendrides tree, which were near each other seeing from cell to cell, allowing more interaction, communications. Other results from new research indicated that Tm-4 plays a role in regulating adhesion structures of osteoclasts, most likely by stabilizing the actin microfilaments present in podosomes and the sealing zone [7].

Lab 7 Questions

Lab 8 Questions

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

Neural cell line: CRL-2765 [8] from ATCC [9]

Muscle cell line: CCL-198 [10] from ATCC [11]

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

Lab 9 Questions

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

Neural cell line: CRL-2765 [12] from ATCC [13]

Muscle cell line: CCL-198 [14] from ATCC [15]

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

Neural cell line: CRL-2765[16]

Species: Rattus norvegicus (rat)

Source: cell type-neuronal Schwann cell; spontaneous immortalization

Morphology: neuronal

Growth Properties: adherent

The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium. To make the complete growth medium, the following components should be added to the base medium: fetal bovine serum to a final concentration of 10%.

Growth condition:

  • Air 95%; carbon dioxide (CO2) 5%
  • 37.0°C (temperature)
  • Remove and discard culture medium.
  • Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
  • Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).

Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.

  • Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
  • Add appropriate aliquots of the cell suspension to new culture vessels.
  • Incubate cultures at 37°C.
  • A subcultivation ratio of 1:6 to 1:10 is recommended
  • Medium renewal, 2-3 times weekly
  • Preservation; freeze medium; complete growth medium supplemented with 5% (v/v) DMSO
  • Storage temperature: liquid nitrogen vapor phase
  • recommended serum:ATCC 30-2020

Muscle cell line: CCL-198 [17] from ATCC [18]

Species: Mus musculus (mouse)

Morphology: fibroblast

Growth Properties: adherent

Source: Tissue- skeletal muscle, Cell type- fibroblast

Growth condition:

  • To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.
  • A subcultivation ratio of 1:2 to 1:5 is recommended
  • Medium renewal - twice per week
  • Remove medium, add fresh 0.1% trypsin for about 10 minutes, and centrifuge to remove trypsin. Resuspend cell pellet in fresh medium and dispense into new flasks. Subculture every 7 days.

Peer assessment comments

Group 1 Peer Review

  • The good thing about this project is the table of the neurotransmitters.
  • The picture is a good opening but the intro doesn’t introduce the topics that are covered.
  • History is not referenced so it make difficult to verify or know more about the historic research itself.
  • There is no information about the proteins involved/cell that make this structure
  • The diseases were explained as a pathology format which loses a bit connection. Should be more focused on what happened in this junction, what genes are involved in the abnormalities of this junction.
  • More research needed to complete the function part and what is important about this junction.
  • Some grammatical errors are here and there.
  • Drawings look good.
  • Glossary need some work
  • References need to be added.
  • Overall, the text needs to be simplified and balanced with the pictures. This will help the reader for a better understanding of this junction.

Group 2- Gap Junctions

  • Good introduction.
  • The text is balanced well enough with the photos.
  • The only thing now is to get rid of empty spaces.
  • History is good and referenced properly.
  • Electron micrograph filtered image belongs to history or is something else??
  • Bold text draws the attention and the reader knows what is going next.
  • The table with other junction is good.
  • Structure needs more to be done.
  • Function is good.
  • Diseases part is good and well-linked with the abnormalities of the junction.
  • Glossary is short.
  • Good headings and sub-headings.
  • Overall,good work.

Group 3: Tight Junction

  • Introduction is more like a literature text material.
  • Good history.
  • Structure is good and understandable.
  • Function has a lot of text and makes the reader tired before reading it. OR if you add more photos it will smooth the look of this part.
  • Several diseases are mention and not enough pictures (Pictures are the main speakers).
  • A long table with the diseases, some are left out of it correlated with 2 pictures, not a good idea.
  • Glossary = very short.
  • References need to be uniform
  • The text and the pictures are not balanced properly.

Group 4

  • Obviously, introduction=not done.
  • At least a picture is needed to draw attention of the reader.
  • History good.
  • Structure needs to be fixed (add pictures, ordering the text, right subheadings).
  • Function is good.
  • “Knock out” is a good idea.
  • Regulation section maybe a table is needed.
  • Diseases are presented good (just fix the design).
  • Current research not finished.
  • References need to be fixed.
  • Overall, not enough text in many parts of this group project, not enough pictures, not tables etc, therefore this research is not completed yet.

Group 5

  • Introduction is a short literature text format so need to be changed
  • A picture for this particular junction needs to be added.
  • History is short, not finished.
  • Structure stands good with the drawn diagram.
  • Function is short and is not balanced visually (@ least one picture must be added).
  • A good presented table which describe other types of cell junctions.
  • Current researches good, however there is nothing on future studies.
  • Glossary not finished.

Lab 10 Questions

Lab 11 Questions

Lab 12 Questions