Week 1 Section Questions: Cell Biology Lectures 1-2

Lecture 1 and 2 Learning Goals:

• Explain why spatial segregation of biochemical reactions is important as cells get larger
• List the different ways in which spatial segregation of molecules is achieved in cells
• Describe differences between prokaryotic and eukaryotic cells
• Understand the pro’s and con’s associated with different imaging techniques
• Understand the variables that affect resolution of light microscopy
• Describe different techniques to localize proteins inside cells (immunofluorescence, GFP
fusion proteins)
• Describe techniques to isolate cellular components

Lecture 1 and 2 terms you should be familiar with:

Polar molecules Sorting Signals

Hydrogen bond Cytosol
Hydrophilic Nucleolus
Hydrophobic Cytosolic proteins
Lipids Nuclear proteins
Membranes Nuclear pores
Scaffolding molecules Fractionation
Prokaryotic cells Chromatography
Eukaryotic cells

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Using Microscopy to Localize Proteins

Part I: A geneticist has found an interesting mutant which is defective in nuclear export because
of a mutation in a gene called Expo1. The normal version of Expo1 encodes a 100 kDa protein.

a) First, she wants to localize normal Expo1 inside a live cell. She has the
following tools available (i-ii). Which of these tools would be appropriate for localizing this
protein? What other specific reagents/tools would she need besides the microscope itself?
Explain this method of localizing the protein in detail.

(i) Light microscope

(ii) Fluorescent Light Microscope

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Part II: The scientist tries the above approach but unfortunately, she has a problem. The
addition of the GFP/fuorescent protein tag to Expo1 results in Expo1 no longer working the way
it is supposed to inside the cell. So she needs a different approach to determine where the
protein localizes.

b) What other approach can she take, again using the same tool above that you had
discussed? Describe this approach in detail.

c) What is the major trade-off/con of this alternative approach compared to her earlier
approach?