Image of the week - Moesin & siRNA from x Truf

2016-06-03   |   0 Comments
Citizen Science Image of the week Plasma membrane Project Discovery Subcell Atlas

Figure 1. Staining of MSN (green) with DNA (blue) and microtubules (red) in U-251 MG cells. S
Figure 2. siRNA knockdown experiment of MSN (green) with DNA (blue) in U-2 OS human osteosarcoma cells. Control cells with scrambled siRNA (A) show green expression while cells with siRNA targeting MSN (B) show significantly less green fluorescence indicating the antibody (HPA011135) is correctly labeling MSN. The inset box-plot shows a quantification of the protein fluorescence knockdown.

It's time for another HPA image of the week! This week's image was brought to us by citizen scientists in Project Discovery, and specifically by x Truf a member of the Signal Cartel in EVE online who found this image while playing Project Discovery.

The protein stained in Fig 1. is an image of Moesin (MSN) found in the plasma membrane of the cell. This sample shows a staining of MSN in U-251 MG human glioblastoma astrocytoma (brain) cells.

MSN is a member of the ERM family which provide a link between plasma membranes and actin filaments. This link was briefly discussed in a previous blog. Due to their linkage between the cytoskeleton and the plasma membrane, the ERM family of proteins plays a vital role in cell adhesion and migration (Arpin M. et al. 2011). This family of proteins, and MSN specifically, have been implicated in a number of diseases including Parkinson's disease (Jaleel M. et al. 2007). It has also been shown to be an important biomarker that is differentially expressed in some forms of breast cancer (Wang C.C. et al. 2012).

This sample was brought to our attention as an example of where the membrane has a large number of finger like extensions which may appear to be focal adhesions on first glance. In this case we can see more continuous structures along the cell border (Fig 1, white box) while focal adhesions typically are short disjoint marks both near the cell edge and under the cell body as seen in this example from the dictionary.

MSN has been localized to the plasma membrane in all three cell lines and for all four "sibling" antibodies, or antibodies that label the same protein. These siblings give us a high confidence that the antibody is binding the same thing each time and that we are accurately characterizing the MSN protein.

Another way we test that our antibodies are labeling the proteins they are supposed to is to add small interfering RNA (siRNA) to our sample (Fig 2). These siRNAs block production of our protein of interest (green). If the antibody is correctly targeting only the blocked protein, the cells treated with the siRNA targeting MSN (Fig 2 B) will show no green fluorescence because no MSN protein is present in those cells. The siRNA don't always make it into the cells, so some cells in the knockdown may still expresses MSN as you can see in the bottom center of Figure 2 (B). The degree of knockdown can be evaluated by the box plot (Fig 2 B inset) which is visible in a larger version on the siRNA page for MSN.

Together with GFP cells (discussed last week), these siRNA experiments provide an important tool for validating antibody specificity, something Nature recently called a "crisis" (Baker M 2015).

Thanks to the all the citizen scientists participating in Project Discovery and particularly to x Truf & the Signal Cartel for suggesting this image and for their contribution to science!

Devin Sullivan