2013 Lab 6
- 1 Cytoskeleton Laboratory Exercise
- 1.1 Overall Aim of the Laboratory
- 1.2 Lab 6 Individual Assessment
- 1.3 Honours Projects - Dr Thomas Fath
- 1.4 Background
- 1.5 Description of B35 Phenotype Classifications
- 1.6 Analysis Notes
- 1.7 Images for Analysis - Group 1
- 1.8 Images for Analysis - Group 2
- 1.9 Images for Analysis - Group 3
- 1.10 Effect of Tm4 expression on db cAMP induced differentiation of B35 cells; Groups 1-3
- 1.11 Related Links
- 1.12 References
- 1.13 2013 Course Content
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.
Lab 6 Individual Assessment
- Complete the in-class analysis of cell morphologies.
Honours Projects - Dr Thomas Fath
Please discuss potential honours projects with Dr Thomas Fath during the class laboratory period.
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
2. "Broken fan" phenotype
3. "Stumped" phenotype
4. "Pronged" phenotype
5. "Stringed" phenotype
6. “Pygnotic” phenotype
- Where neurites or lamella from 2 or more cells are overlapping and you can't distinguish them, exclude these cells from the analysis. If you feel confident to discern the morphology of both cells, you can go ahead with including them in the analysis. Important is, that you are consistent in doing this between the two experimental groups.
- 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.
- Vertebrate tropomyosins (TMs) are expressed from 4 genes, and at least 18 distinct isoforms
- Search Entrez Databases
- Wikigenes TPM4 tropomyosin 4
- Anti-TM4 Antibody
- 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
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 - Intermediate Filaments | Cytoskeleton - Microfilaments | Cytoskeleton - Microtubules | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells 1 | Stem Cells 2 | 2013 Revision | Development
Laboratories: Introduction to Lab | Microscopy Methods | Preparation/Fixation | Immunochemistry | Cell Knockout Methods | Cytoskeleton Exercise | Confocal Microscopy | Tissue Culture | Stem Cells Lab | Microarray Visit | Lab
Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G