Sélection Internationale Ens Ulm 2012, Cell Biology

Transcription

1 Sélection Internationale Ens Ulm 2012, Cell Biology Please read the whole subject before starting. Questions 1-8 and are general biology questions. In questions 9, 10, 11 and15, we ask you to interpret data from accompanying figures. Part 1. Cell differentiation, general (mammalians) During very early stages of embryogenesis, totipotent cells give rise to three primary tissue layers (also called primary germ layers) that will develop and form all tissues and organs of the body. Q1, Q2 At later stages of development, pluripotent progenitor cells can differentiate into a limited number of cell lineages. If these progenitor cells are isolated from the embryo and cultured in vitro in the laboratory, under specific conditions the different steps of differentiation can be recapitulated experimentally. To experimentally induce differentiation of progenitor cells into defined cell lines, specific diffusible growth factors have been extensively used in cell cultures in vitro. However, other environmental cues could also be involved in the differentiation process. For this reason, the role of the extracellular matrix and the nature of the support onto which cells are cultured has been explored. Q3, Q4 Cells interact with the extracellular matrix via trans-membrane "receptor" proteins located in the cell surface and called integrins. Q5 Integrins mediate incoming signals in cells, and the activated signalling pathways will result in an adaptative response of the cell. The final output will be changes in the adhesion forces of the cell to the matrix. Paxillin is an intracellular protein that associates with integrin in submembraneous complexes called focal adhesions. In the following experiment, paxillin was detected by use of a specific antibody. This was revealed by a secondary antibody coupled to a fluorescent green molecule, named FITC. Q6, Q7 Paxillin was detected by this technique in three different conditions of cell culture (Figure.1, A, B, C). The use of the intercalant agent DAPI in this procedure allowed to identify the cell nuclei in blue. The support onto which cells were cultured is not stained (and appears as black background). All images were taken with the same magnification and exposure conditions. Q8, Q9 1

2 Part 2. Cell differentiation, role of matrix stiffness (adapted from Engler et al., 2006) A way to explore the role of the extracellular matrix on differentiation is to modify the stiffness of the support onto which progenitor cells are cultured. In an article published by Engler and colleagues in 2006 in the journal Cell, the authors mimicked matrix elasticity in vitro with inert polyacrylamide gels. In these gels, the concentration of bis-acrylamide crosslinking sets the elasticity (light cross-linking = soft matrix ; heavy cross-linking = hard matrix). Adhesive properties of the matrix were provided by coating the gels with collagen (all gels, whatever their elasticity). Hence, Petri dishes were prepared with gels of different elasticity, coated with collagen, and the pluripotent mesenchymal stem cells (MSC) were isolated from young embryos and cultured for several days on these dishes. Figure 2 represents the experimental setup, and the results obtained in three ranges of matrix elasticity tested. Q10 In order to identify the cell types, detection of proteins specific of each cell type by antibodies was performed. Primary antibodies were revealed by a FITC-coupled secondary antibody (figure 3). Three proteins were considered : b3 tubulin, MyoD and CBFa1. In this figure, pictures in the second and third lines were taken with the same magnification, but pictures in line 1 were taken at a much lower magnification (see scale bars). Q11 The same primary antibodies were used to detect the proteins in Western Blots obtained from the cultures with different elasticity conditions (figure 4). The GL condition corresponds to «glass», i.e. cells cultured on top of glass coated with collagen. Q12, Q13, Q14 Table 1 shows the relative levels of a certain number of proteins (other than b3 tubulin, MyoD and CBFa1) assessed for the different experimental conditions by Western Blot. Q15 2

3 QUESTIONS Question 1: what is a totipotent cell? Question 2: what are the names of the three primary tissue layers? Question 3: what does «in vitro» mean? Same question for «in vivo», «in silico», «in situ» and «in utero.». Question 4: what is the extracellular matrix? Please cite 4 major components of it. Question 5: represent schematically the intracellular trafficking of a protein such as integrin, between the moment it has been translated, and the moment it reaches its final location. Question 6: please make a cartoon of the experimental procedure. What is the name of this technique? Question 7: name fluorescent proteins that can be used as tags in molecular biology? Question 8: what is an intercalant agent, such as DAPI? Question 9: what are the differences in the intensity of the fluorescent staining related to? What is your conclusion regarding A, B and C? Question 10: what do you observe? What can you conclude from this experiment, at the 96 hrs timepoint? Question 11: interpret figure 3. Question 12: briefly describe the Western Blot technique. Question 13: what is «actin» (one sentence)? Why probing actin in this experiment? Question 14: what can we learn from the GL condition? Question 15: considering figures 1 to 4, table 1 and your own scientific knowledge, make an hypothesis about the nature of the three cell types that were characterized. 3

4 Sélection Internationale Ens Ulm 2012, Cell Biology figures B C Figure 1 Figure 2 Scale bar = 20 µm

5 Figure 3 (kpa) Figure 4 Table 1 Scale bar = 5 µm