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You are investigating the activity of different cadherins in cells. You use recombinant DNA technology to express GFP-E-cadherin (green) in some L-cells (which do not normally contain cadherin) and YFP-P-cadherin (yellow) in other L-cells. Explain results you expect in three cultures containing: (1) only cells expressing GFP-E-cadherin, (2) only cells expressing YFP-P-cadherin, and (3) a mixture of GFP-E-cadherin-expressing and YFP-P-cadherin-expressing cells. Also predict how (a) knocking down the ability of the cells to synthesize fibronectin (FN) or (b) adding EGTA, which depletes free Ca2+ concentration in the culture medium, might affect the results.

User Lorean
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Final answer:

In cultures containing either GFP-E-cadherin or YFP-P-cadherin, cells will likely adhere to each other, resulting in green or yellow fluorescence respectively. In a mixed culture, cadherins may show heterophilic interactions, leading to mixed fluorescence. Knocking down fibronectin or adding EGTA will disrupt cell adhesion and alter cell interaction dynamics.

Step-by-step explanation:

When investigating the activity of different cadherins in cells by expressing GFP-E-cadherin (green) and YFP-P-cadherin (yellow) in L-cells, we can predict the following results:

  • In the first culture containing only GFP-E-cadherin, we expect to see a homophilic interaction, where cells expressing GFP-E-cadherin adhere to each other, leading to a green fluorescence in the area of cell-cell contacts.
  • In the second culture with only YFP-P-cadherin, a similar scenario occurs, but with yellow fluorescence indicating the P-cadherin interactions.
  • In the third culture with a mixture of both types of cells, if E-cadherin and P-cadherin can engage in heterophilic interactions, you might observe areas where green and yellow cells come together, potentially forming an overlapping (green and yellow) fluorescence. However, if they do not, the two populations would likely segregate, displaying distinct green and yellow patches.

For the additional variables:

  • Knocking down fibronectin production may impact the cells' ability to adhere to the ECM, potentially resulting in more loose cell aggregates or altered cell morphology, as fibronectin interacts with integrins to facilitate cell-ECM adhesion.
  • Adding EGTA, which chelates free Ca2+ ions, would likely disrupt cadherin-mediated cell adhesion. Cadherins require calcium to maintain their structure and function, so depletion of Ca2+ ions would result in loss of adhesion and cell dissociation.

User Brian Nguyen
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