Question 11 pts From your General Cell Biology knowledge, what do Rab proteins d

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Question 11 pts

From your General Cell Biology knowledge, what do Rab proteins do?

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Chlamydia trachomatis are the leading cause of bacterially acquired sexually transmitted disease and preventable blindness worldwide.  Chlamydia replicate inside eukaryotic cells, inside membrane-bound vacuoles called inclusions. The current understanding of how Chlamydia hijacks the host cell was reviewed last month. This section is derived from important studies from 2003.

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The experiment above is investigating the source of the membrane that surrounds the Chlamydiainclusion (white arrowhead) in human cells in culture. In A, the human cells were incubated with phosphatidylethanolamine that was labeled with a tiny gold particle called “nanogold”. The labeled phosphatidylethanolamine incorporated into the plasma membrane.

In B, the plasma membrane proteins in the human cells were labeled with nanogold on the extracellular side. In both cases, cells were infected with Chlamydia and processed for TEM after 30 min-2 h.

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Question 21 pts

From your General Cell Biology, deduce why the phosphatidylethanolamine and plasma membrane proteins were labeled with gold and not a fluorescent antibody.

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Question 31 pts

What did the data show?

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Question 41 pts

What could be concluded from this data?

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Question 51 pts

What is the implication of this data?

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In the experiment above, a series of green fluorescent protein (GFP) fusion proteins were made. In the top panel, GFP alone was ectopically expressed in human cells. In the lower panels, GFP was fused to different Rab proteins and expressed exogenously in human cells.

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In the experiment above, the Chlamydia inclusions are marked with white arrowheads. In the top panel, GFP is diffusely localized throughout the cytoplasm, except the inclusion. In the middle panel, these GFP-Rabs are localized to the periphery of the inclusion. In the lower panel, these GFP-Rabs are not localized to the periphery of the inclusion.

Use the table below, and Rab trafficking figure to figure out with which trafficking pathways theChlamydia inclusions are associating. Consider protein production/receptor recycling/secretion as one category of pathways and endocytic/lysosomal as the other category of pathways.

TGN, trans Golgi network.

Above is a diagram of the trafficking vesicles in a typical eukaryotic cell. The left panel is from a review published summer 2014 showing the roles of different Rab proteins. On the right is a diagram we saw in class).

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Question 61 pts

Which trafficking pathways were associated with the Chlamydia inclusion?

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In the experiment below, the fusion of the Chlamydia inclusion with the lysosome was investigated. Human cells were infected with Chlamydia and incubated for 18 h, before fixing the cells for immunofluorescence. Below, the same specimen was visualized with aEB for Chlamydia and aLAMP for lysosomes. The presence of the inclusion is marked by the black arrowhead in the DIC image of the same specimen.

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Question 71 pts

What is the DIC image showing?

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Question 81 ptsSkip to question text.

Many Chlamydia inclusions were analyzed for lysosomal fusion. This analysis was also repeated withY. pseudotuberculosis bacteria, which are known to not recruit Rab proteins. Fusion with the lysosome was counted when the aEB and aLAMP immunofluorescence co-localized. This was expressed as percent fused and plotted in a bar chart in the right panel. What can you conclude from the data above?

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Chlamydia inserts transmembrane Inc proteins in the inclusion membrane, such that the carboxy terminus protrudes into the cytosol where it could associate with vesicle trafficking proteins. In the experiment below cells were infected and then fixed, permeablized and GFP-Rab11 was visualized through the green filter and Inc immunofluorescence was visualized through the red filter. Inclusions are labeled with white asterisks (*).

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Question 91 pts

What did the data above show?

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Question 101 pts

The results from above were enhanced when a Rab11 Q70L mutant was expressed (but we were not shown this data). This mutant has no GTPase activity. From your General Cell Biology, what can you deduce about Rab11 Q70L.

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Question 111 pts

What could be concluded from this mutant?

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Question 121 pts

Put all the data together. What are the implications for how Chlamydia might be interacting with host cell trafficking?

Explanation / Answer

Q- What do Rab proteins do?

A-They direct vesicle trafficking within the cell. They help in processes involving targeting the newly formed proteins to the plasma membrane through vesicles.

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