Step 7

Observations

Summary

Observations

Show an understanding of what you saw happening during your experiment. Describe the patterns and trends you saw emerge as you worked.

As you see in the projected model of signaling that is displayed in the image above, if RAP6 activates Rab5 (allowing Rab5 to go on in its function of fusing incoming vesicles together and forming the early endosome), then the localization of the PH domain of RAP6 to the nucleus may suggest that there could be an interaction between the PH domain of the entire RAP6 protein and Rab5 protein. When Rab5 is activated and goes on its business, it may have been also tagged for localization to the nucleus and have another function there. I observed a possible nuclear localization signal in the PH domain of RAP6 which may link Rab5 to the nucleus. The scope of my continuing research will be to understand what RAP6 could be doing in the nucleus.

Further Analyzations

Judges' TipExcellent observations will describe patterns or trends supported by the data (500 words maximum).

PH domain requires further characterization (not well represented)
Currently in the process of generating several PH domain constructs
- To understand specific sequence and position of nuclear localization signal
- If specific region of PH domain is implicated, can induce point mutations
- Mutations may determine key amino acids in nuclear localization function.

Based on the cell imaging of the PH Long domain above, it is evident that there is a stronger nuclear localization signal coming through.
This can be identified through the last column or MERGE, containing both green fluorescent protein expression and staining of the nucleus of fixed cells.
In these cells, once can see the tagged green fluorescent protein (GFP) being expressed or localized solely in the nucleus.
When sequenced, it is seen that the PH Short domain lacks key amino acids in nuclear localization function.
Therefore, the PH Long domain receives stronger nuclear localization functions and is now the region of interest.

Referring to the RAP6 constructs, the PH Long domain has also been proposed to be further subdivided into fragments A, B, and C.
The fragments A, B, and C, are based on nuclear localization from greater to lesser nuclear localization (witnessed above).
From the image, it is evident that the PH domain solely involves nuclear localization signals (NLS's).
As a result, for further research I intend to analyze the localization coming off of the three fragments in the dominant PH Long domain.
This further work would provide the ability to understand specific sequencing and positioning of the nuclear localization signals being received as well as if specific regions of the PH domain are implicated and causing point mutations, hence possibly determining key amino acids in nuclear localization function.

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