2010 Lab 6

From CellBiology

Cytoskeleton Laboratory Exercise

Dr Thomas Fath and Prof Peter Gunning

--Mark Hill 00:28, 13 April 2010 (UTC) Student, please note that this laboratory will form part of your individual assessment.

Laboratory Aim

  • The aim of the laboratory is to evaluate the impact of these two tropomyosins on cell morphology.
  • You will be given photos of random fields of either control cells or cells making 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.

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:

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


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

Laboratory Aim

  • The aim of the laboratory is to evaluate the impact of Tm4 on cell morphology.
  • You will be given photos of random fields of either control cells or cells making 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 is a real experiment. There is no established answer. The results you produce in this laboratory will contribute to our understanding of the roles of Tm4 in regulating specific cell structures.

Description of B35 Phenotype Classifications

Phenotype images 6images-mod.jpg

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

B35 1-fan.jpg
  • 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

B35 2-broken-fan.jpg
  • 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

B35 3-stumped.jpg
  • 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

B35 4-pronged.jpg
  • 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

B35 5-stringed.jpg
  • 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

B35 6-pygnotic.jpg
  • 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


Images for Analysis - Group 2


Images for Analysis - Group 3



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.




Related Links

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

2009 Course Content

Lectures

Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cell Mitochondria | Cell Junctions | Cytoskeleton Introduction | Cytoskeleton 1 Intermediate Filaments | Cytoskeleton 2 Microtubules | Cytoskeleton 3 Microfilaments | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells | Stem Cells | Development | Revision

Laboratories

Introduction to Lab | Microscopy Methods | Preparation/Fixation | Immunochemistry | Cell Knockout Methods | Cytoskeleton Exercise | Confocal Microscopy | Tissue Culture 1 | Tissue Culture 2 | Microarray Lab visit

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