Revision as of 09:41, 1 May 2013

Cytoskeleton Laboratory Exercise

Dr Thomas Fath

Student, please note that this laboratory will form part of your individual assessment.

Overall Aim of the Laboratory

The aim of the laboratory is to evaluate the impact of tropomyosins on cell morphology.
You will be given photos of random fields of either control cells (transfected with empty expression vector) or cells over-expressing Tm4.
You will score the morphological characteristics of Tm4 over-expressing cells and compare them with the controls.
You will find that the control cells show a number of morphologies and you will need to take this into consideration.
You will therefore have to identify and score the frequency of the major morphology types in the control cells and then compare this with the cells making the tropomyosins.
This will be a blind analysis, so you will receive the identity of the control and experimental group after you carried out the phenotypic characterisation.

Honours Projects - Dr Thomas Fath

Please discuss potential honours projects with Dr Thomas Fath during the class laboratory period.

Background

Tm Isoform Normal Cellular Distribution Overview

The actin microfilaments are composed of a two stranded polymer of actin and most filaments also contain polymers of the rod shaped tropomyosin running along the major groove in the filament.

As you heard in the lecture on Microfilaments, the different tropomyosin isoforms have the ability to direct the assembly of specific cellular structures.

Previous experiments have been performed in which the neuroblastoma derived cell line, B35 (Schubert D, etal., 1974) is given a gene construct driving the expression of a specific tropomyosin. Cells are then isolated which express the introduced tropomyosin and analysed for changes in phenotype.

Thus far we have shown that:

Tm5NM1/2 drives the formation of stress fibres and results in cells with arced membranes and completely lacking lamellipodia.
TmBr3 drives the formation of very broad lamella.
Tm3 drives the formation of filipodia.

We have now introduced a new tropomyosin into B35 cells, Tm4.

Description of B35 Phenotype Classifications

During today's laboratory it may be useful to download the above image and open on the desktop for comparison beside the images you are analysing.

Neurite Definition - a projection which is narrower than the smallest diameter of the nucleus for at least half it’s length and is greater in length than the widest diameter of the nucleus.

WT Undifferentiated B35 cells can be described as having the following types of phenotypes.

Phenotype Categorisation for B35 cells established by Nikki Curthoys.

1. "Fan" phenotype

Clear lamellum adjacent to cell soma and nucleus (a single DAPI stain)
lamellum extends in a circular perimeter around the cell soma
- such that a single continuous lamellum extends distally in all directions from the soma to encircle the nucleus and soma entirely.

2. "Broken fan" phenotype

As “Fan” phenotype, except that a single lamellum extends from the soma distally to encircle between 30% and 100% of the soma perimeter.
The cell may or may not extend a single neurite, not longer than 3x the widest diameter of the nucleus.

3. "Stumped" phenotype

Cell soma has multiple (i.e. two or more) stalks of lamella extending distally from the soma.
The longest distance between the most distal edge of lamella (i.e. the lamellipodia) and the nucleus is not more than 3x the width of the nucleus at its widest point.
In addition, soma may extend neurites, but not greater in length than 3x the widest diameter of the nucleus.

4. "Pronged" phenotype

The cell extends at least one neurite, and no one neurite is longer than 3x the width of the nucleus at its widest diameter.
A single (but not multiple) lamellum encircling less than 30% of the soma perimeter may or may not extend from the soma.

5. "Stringed" phenotype

At least one neurite (as defined above) extends distally from the soma.
And at least one neurite is a length exceeding that of 3x the diameter of the nucleus at its widest part.
Cell may or may not extend lamella in addition to this.

6. “Pygnotic” phenotype

Cells comprising a nucleus, and an immediate lamellal fan around the nucleus.
- lamellal fan does not extend distally at any point more than the length of the narrowest diameter of the nucleus.

Analysis Notes

Where neurites or lamella from 2 or more cells are indistinguishable (i.e. appear continuous), this presence will be used to score the phenotype of *both* cells.
Lamellipodia and neurites which appear to arise from 2 or more cells, and extend between two or more nuclei, will be counted as the length extending from the soma until the point equidistant between those nuclei.
Cells which are in any part occluded by the perimeter of the field of view will not be included, unless that cell is a stringed cell, and the part being occluded is the neurite which has at least a length of 3x the width of the nucleus at its widest part within the field of view.
When two nuclei are less than the distance of diameter of one nuclei apart, they will be considered as undergoing mitosis, and counted as one cell.

Images for Analysis - Group 1

Image A01
Image A02
Image A03
Image A04
Image A05
Image A06
Image A07
Image A08
Image A09
Image A10
Image B01
Image B02
Image B03
Image B04
Image B05
Image B06
Image B07
Image B08
Image B09
Image B010

Images for Analysis - Group 2

Image A01
Image A02
Image A03
Image A04
Image A05
Image A06
Image A07
Image A08
Image A09
Image A10
Image B01
Image B02
Image B03
Image B04
Image B05
Image B06
Image B07
Image B08
Image B09
Image B10

Images for Analysis - Group 3

Image A01
Image A2
Image A03
Image A04
Image A05
Image A06
Image A07
Image A08
Image A09
Image A10
Image B01
Image B02
Image B03
Image B04
Image B05
Image B06
Image B07
Image B08
Image B09
Image B10

Effect of Tm4 expression on db cAMP induced differentiation of B35 cells; Groups 1-3

The aim of this part of the excercise is to reveal any phenotypes in response to Tm4 over-expression in B35 cells that are induced for differentiation using dibuturyl cyclic Adenosin-monophosphate (db cAMP). For this purpose genotype "A" and genotype "B" will be compared and analysed for altered formation of processes.

Image A01 differentiated
Image A02 differentiated
Image A03 differentiated
Image A04 differentiated
Image A05 differentiated
Image A06 differentiated
Image A07 differentiated
Image A08 differentiated
Image A09 differentiated
Image A10 differentiated
Image B01 differentiated
Image B02 differentiated
Image B03 differentiated
Image B04 differentiated
Image B05 differentiated
Image B06 differentiated
Image B07 differentiated
Image B08 differentiated
Image B09 differentiated
Image B10 differentiated

References

Gunning PW, Schevzov G, Kee AJ, Hardeman EC. Tropomyosin isoforms: divining rods for actin cytoskeleton function. Trends Cell Biol. 2005 Jun;15(6):333-41. Review. PMID: 15953552 | Download PDF version

Schubert D, Heinemann S, Carlisle W, Tarikas H, Kimes B, Patrick J, Steinbach JH, Culp W, Brandt BL. Clonal cell lines from the rat central nervous system. Nature. 1974 May 17;249(454):224-7. PMID: 4151463

2013 Course Content

Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G

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