Difference between revisions of "User:Z3333421"

From CellBiology
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* self renewal: the capability to undergo numerous cell divisions and maintain undifferentiation
 
* self renewal: the capability to undergo numerous cell divisions and maintain undifferentiation
 
* ubiquitylation: the process of adding ubiquitin
 
* ubiquitylation: the process of adding ubiquitin
 +
 +
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http://www.atcc.org/ATCCAdvancedCatalogSearch/AllCollectionSearch/tabid/454/Default.aspx

Revision as of 15:13, 3 May 2012

Lab attendance

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Lab 1

This is my first lab in cell biology.

Internal Link example

Course_Timetable

External link example

JOVE

Lab 2

Collagen bundles.jpg

<pubmed>19547746</pubmed> In E.coli, chemotaxis is used as the basis for signal processing however previous studies with normal microscopy have not shown how the transmembrane receptors form large sensory clusters of proteins, what controls the cluster size and density and how the location of these clusters are maintained in cells that are growing.

Greenfield et al, 2009 demonstrates how these clusters are formed and provides evidence for a stochastic (random) mechanism of self assembly using super resolution microscopy.

Lab 3

Chemical fixative chosen: Methanol SDS

Properties:

  1. Clear and colourless liquid
  2. Mild characteristic alcohol odour
  3. 100% volatile
  4. Soluble in water
  5. Boiling point = 65°C
  6. Melting point = -98°C
  7. Flash point = 11°C (cc)

Hazards:

  1. Highly flammable
  2. Toxic irritant by inhalation, contact with skin or if swallowed
  3. Experimental teratogen
  4. Affects the central nervous system
  5. Damage to optic nerve may occur with chronic or high level exposure


<pubmed>16793760</pubmed> This review article provides a concise overview of the Wnt/Beta Catenin Signalling which can used in the introduction.


<pubmed>15372092</pubmed> There are a number of clearly labelled diagrams in this article that can be used as a starting point for the student drawn image.


<pubmed>18673238</pubmed> This article provides an historical overview of the key events that shaped our understanding of Wnt/Beta Catenin signalling and hence can greatly aid the history section.


<pubmed>17081971</pubmed> This review also contains many simplified diagrams that can be used to support the student drawn image.

Lab 4

Musashi (Msi) is a group of evolutionarily conserved RNA binding proteins. Musashi-1 is 362 amino acids long and is approximately 39kDa Protein information. It is often preferentially expressed in the nervous system. The Drosophila Msi was first identified followed by mammalian orthologs - human and mouse Musashi-1 (Msi1). This protein is strongly expressed in fetal and adult neural stem/progenitor cells and contributes to their self renewal via translational regulation of several target mRNAs.

<pubmed>22429745</pubmed>

Primary antibody: Anti-musashi 1

Supplier: ABCAM

Host species: Rabbit polyclonal

Reacts with: mouse and human

Isotype: IgG

Applications: immunocytochemistry/immunofluorescence and western blot

Dilutions:

ICC/IF - Use at a concentration of 1 µg/ml.

WB - Use at a concentration of 1 µg/ml. Detects a band of approximately 39 kDa.

Anti-musashi-1 data sheet


Secondary antibody: Alexa Fluor® 488 goat anti mouse IgG antibody

Supplier: Invitrogen

Dilution for immunofluorescence: 1:500

<pubmed>21233308</pubmed>

Alexa Fluor 488 data sheet

Lab 6

Z3333421 Lab 6 graph2.JPG

Exercise 1: Analysis of morphological phenotypes of Tm4 overexpressing B35 neuroepithelial cells

Do you see a difference in phenotype (morphology) between Tm4 overexpressing and control cells?

  • There were higher percentages of pronged and stumped phenotypes for the overexpressing Tm4 cells, which had an increase of approximately 12% and 9% respectively. This may imply the importance of Tm4 overexpression in increasing the degree of branching in the phenotype.
  • The broken fan phenotype increased by 16% in the control which means Tm4 may have an inhibitory effect on this phenotype.
  • In addition, pygnotic and stringed phenotypes depicted small increases in the control sample, which may imply that Tm4 was not actively involved.
  • The fan phenotype was observed in the Tm4 overexpressing cells only.

How could Tm4 overexpression lead to this difference?

Tropomyosin 4 (Tm4) is an isoform of the tropomyosin family of actin-binding proteins and is often involved in the regulation of muscle contraction and the cytoskeleton of non-muscle cells where they confer stability. According to Had et al (1994), Tm4 expression is concentrated in the growth cones of cultured neurons, particularly areas of active neurite growth. This suggests the role of Tm4 in the motile aspects of neurite growth during development and perhaps synaptic plasticity in adulthood. It was also postulated that Tm4 could be involved in the stabilisation of actin filaments and the regulation of myosin proteins and actin (which is required in growth cone motility). This correlates with the result of this lab as Tm4 overexpression was depicted in the phenotypes which had more branching and neurite growth and interaction.

<pubmed>7876361</pubmed>

Exercise 2: Effect of Tm4 expression on db cAMP induced differentiation of B35 cells

Do you see a difference in phenotype (morphology) between Tm4 overexpressing and control cells?

Genotype A (Tm4 overexpressing cells):

  • Increased appearance of branching and neurite formation
  • Indicates the presence of some form of interaction
  • Predominant phenotypes - pronged and stumped
  • Increased clustering of cells
  • Processes appeared to be shorter and wider
  • Yellow stained lamellae
  • Pink stained cytoskeleton
  • Pink-blue round nuclei

Genotype B (control):

  • Decreased appearance of branching and processes compared to Genotype A
  • Therefore less interaction observed
  • Thinner and longer processes
  • Predominant phenotypes - broken fan and stringed
  • Red stain on the edge of lamellae
  • Red stained cytoskeleton
  • Very blue nuclei

How could Tm4 overexpression lead to this difference?

db-CAMP induces the differentiation of B35 cells, causing more processes to form however it inhibits the expression of Tm4. This results in the decreased length and hence growth of processes in Tm4 overexpressiing cells. In contrast, the predominant phenotype in the control is the stringed phenotype with longer processes, indicating that db-cAMP did not affect it as much as the Tm4 overexpressing cells.

<pubmed>7954857</pubmed>

Lab 7

  • Added content to History
  • Uploaded two images (one was copyrighted and hence removed)
  • Added content to Embryonic Development
  • Updated Glossary section

CONTRIBUTIONS

Figure 1. Overall structure of the β-catenin/XAxin-CBD complex

History Figure 1. Overall structure of the β-catenin/XAxin-CBD complex

1973: A Drosophila melanogaster mutant lacking wings, Wingless (Wg), was described [1]

1982: Roel Nusse and Harold Varmus found that Int1, a mouse protooncogene was associated with MMTV-induced mammary gland tumours [2]

1987: Int1 was found to be the mammalian homologue of Wingless in Drosophila

1989: New Wnt pathway components were found in a screen of lethal mutations in Drosophila

1991: β-catenin was molecularly cloned

1992: Shaggy was found to be cytoplasmic mediator of Wnt signalling

1993: APC was found to directly interact with β-catenin

1994: Dishevelled was identified as an essential element in the Wnt pathway

1995: APC was found to regulate Β-catenin stability.

1996: β-catenin was found to directly interact with LEF-TCF transcription factors

1996: Frizzled, a seven span transmembrane receptor, was identified as the cell surface receptor of Wnt ligands

1996: Porcupine, a multi-transmembrane protein, was found to process Wnt ligands

1996: Nuclear accumulation of β-catenin was found in colorectal cancers

1997: The three-dimensional structure of β-catenin was determined

1997: Phosphorylation targets β-catenin to ubiquitylation, involving interaction with the E3 ligase B-TrCP, and to proteasome dependent degradation

1997: Identification of the homeotic gene Ubx as the first Wnt target gene

1998: Axin 1 and axin 2 were found to interact with β-catenin, GSK3β and APC and to promote GSK3β - dependent phosphorylation and degradation of β-catenin

1999: Casein kinase 1 (CK1) was found to regulate β-catenin function

1999: Protein phosphatase 2A (PP2A) interacts with the β-catenin destruction complex and modulates GSK3 β (Glycogen synthase kinase 3β) function

2000: The three-dimensional structure of the β-catenin–TCF complex was determined

2000: Arrow, LRP5 and LRP6 were identified as coreceptors of Frizzled

2001: LRP5 was found to transduce Wnt signals by recruitment of axin to the plasma membrane.

2003: The tyrosine kinase receptor Derailed in Drosophila (RYK in mammals) was identified as an alternative Wnt receptor

2006: LEF1 mutations were associated with sebaceous gland tumours in humans, showing that Wnt–β-catenin signalling is inhibited in these tumours

2007: Dishevelled was found to polymerize at the plasma membrane and to recruit axin upon Wnt stimulation

Protein Structure Function
Beta-catenin
Frizzled
Dishevelled
GSK-3B
APC
Axin
TCF/LEF
CK1

Embryonic development

The Wnt/β-catenin signaling pathway has been implicated as an important pathway in human fetal development. Through immunohistochemical staining, Eberhart and Argani (2001)localised nuclear beta catenin in fetal lung, placenta, kidney, cartilage, capillaries, adrenal glands and skin. This indicates that Wnt signaling regulates the development of specific set of organs and tissues.[15] For example, Wnt genes such as Wnt4 regulate the conversion of mesenchyme to epithelial cells in kidney morphogenesis.

In addition, Wnt/β-catenin signaling is also involved in maintaining the pluripotency of human embryonic stem cells (hESCs). Wnt3a promotes the reprogramming of somatic cells to pluripotency in conjunction with the classical transcription factors, Oct4, Sox2 and Nanog.[16] For example, Oct4 has the effect of repressing Wnt/β-catenin signaling in self renewing hESCs and is depressed during hESC differentiation.[17] Hence, this suggests that Wnt/β-catenin signaling is involved in differentiation rather than self renewal.

Glossary of Terms

  • canonical: standard and well accepted
  • differentiation: the process by which cells become mature and specialised in structure and function
  • glycolipoprotein: a protein with attached lipid and carbohydrate groups
  • homeotic gene: genes involved in embryonic development, specifically controlled the anterior-posterior axis
  • oncogene: genes which transform normal cells into cancerous cells
  • pluripotency: the ability of stem cells to differentiate into ectoderm, mesoderm and endoderm
  • proteasome: a large intracellular particle which degrades proteins
  • self renewal: the capability to undergo numerous cell divisions and maintain undifferentiation
  • ubiquitylation: the process of adding ubiquitin


http://www.atcc.org/ATCCAdvancedCatalogSearch/AllCollectionSearch/tabid/454/Default.aspx