Cell Biology Online Final Exam = Questions 1 and 2 (30 points) while 3 to 6 (35 pts each) 1) Your cell biology research has detected a new protein associated with a mitochondrial-based disease

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Cell Biology Online Final Exam = Questions 1 and 2 (30 points) while 3 to 6 (35 pts each)

1) Your cell biology research has detected a new protein associated with a mitochondrial-based disease. You name this protein the “Mighty Mite” protein.

To begin your study:

• You first need to use protein sequencing techniques (MALDI TOF vs. ESI-MS) to determine the amino acid sequence of Mighty Mite. Given what you have learned, discuss the methods used as well as controls.
• You then use proteomics analysis using a SELDI Chip analysis to study the mitochondria disease from Mighty Mite, such as was done in the investigation of CDG disease. Outline the experiments, potential results, and controls of your study

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2) The data from the question 1 experiment suggest that the disease is due to a mutant version of the Mighty Mite protein. You know that in most cases, a mutant vs. the typical Mighty Mite is different and may not have the correct protein partner interactions to do its normal function.

• Using at least two protein interaction techniques from 2 Hybrid, GST Fusions Pull Downs, Far Western Protein Overlays and Partner Detections, and FRET vs. BRET techniques determine the differences, if any, between the mutant vs. wild-type version of Mighty Mite proteins.
• Outline the experiments, controls, and conclusions as well.

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3) In your analysis of the Mutant Mighty Mite diseased cells, you note that besides an altered mitochondrial function, these cells seem to have a more significant number of abnormal-looking lift rafts with fewer caveosomes vs. cells with normal mitochondrial function which have few lift rafts but many caveosomes.

• Based on your knowledge of lift rafts and caveosomes structure and function, outline your experiments to study these observations, with controls and conclusions to explain the differences in lipid rafts/caveosomes
• Purify these Rafts for a complete analysis.

 

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4) Given your knowledge of Mitochondria Protein Sorting to potentially 4 Mitochondrial Locations, multiple mechanisms do a Structure and Functional Testing of the Mighty Mite protein both the mutant and wild-type version by looking at protein sorting, protein location, control experiments, and conclusions.  

Early results suggest that 50% of mutant Mighty Mite protein never is found inside the mitochondria, and the other 50% is stuck in the Matrix. Normal Mighty Mite is 100% in the intermembrane space only. Feel free to use in vitro and or in vivo analysis. See diagram.

 

 

50% Mutant Mighty Mite Inside the Matrix

 

 

 

50% Mutant Mighty Mite Outside

 

 

 

100% Normal Mighty Mite Inside the  Intermembrane Space

 

 

 

 

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5) In your final testing in question 4 analysis, you note that 50% of the mutant mighty mite protein in the mutant disease cells is not in the mitochondria, finding its way to the lysosome instead.

Given your knowledge of vesicle transport, ER to Golgi, Golgi to Lysosome protein transport. Pathway of Lysosomal Transport of Lysosomal Enzymes, M6P Signal, and M6P Receptors, including Tay-Sachs and Pombe’s Lysosomal Storage Diseases and Mechanisms.

Propose a series of experiments with controls to track the Mighty Mite protein into and through the ER, Golgi, and finally into the Lysosomes.

6) The mutant disease cells also have a strange shape as well suggesting an altered cytoskeleton. Given your knowledge of the cytoskeleton, determine if these cells have altered cytoskeleton vs. wild-type cells. Use known examples in cancer cells as a guide for cytoskeleton studies.

• Test all three types of cytoskeleton for localization in mutant vs. wild type cells
• Determine function or lack of function by inhibitor studies or biochemical effectors.
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Normal Mighty Mite Cells

 

 

 

Mutant Mighty Mite Cells

Actin (red), and Microtubulin (blue), and the cell nuclei (green)