Marker for fenestrae
The invention relates to a plasma membrane marker for identifying fenestrae. The invention also relates to a method of visualizing fenestrae utilizing a plasma membrane marker and light microscopy. The invention also relates to a method of identifying a plasma membrane marker for fenestrae. In particular, the invention relates to the characterization of moesin as a component of fenestrae sieve plates. More particularly, the invention relates to the use of moesin as a plasma membrane marker. Moesin may be used as a plasma membrane marker for the identification of fenestrae or permeability of endothelial cells.
1 . A method of identifying a marker for fenestrae in an endothelial cell line comprising the steps of:
a) isolating plasma membranes from a batch of fenestrae induced endothelial cells and a batch of fenestrae un-induced endothelial cells.
b) running a two-dimensional electrophoresis gel on material isolated from the batch of fenestrae induced endothelial cells;
c) running a two-dimensional electrophoresis gel on material isolated from the batch of fenestrae un-induced endothelial cells;
d) determining fold differences between staining intensities of spots in the two-dimensional gel of the batch of fenestrae induced endothelial cells and the spots in the two-dimensional gel of the batch of fenestrae un-induced endothelial cells;
e) excising a spot; and
f) analyzing the spot against a database of fingerprints from theoretical tryptic digests of proteins so as to assign a particular protein identity to the spot.
2 . The method of claim 1 , further comprising the step of producing a characteristic mass fingerprint for the excised spot utilizing mass spectrometry.
3 . The method of claim 1 , further comprising the step of confirming the assigned protein identity of the spot.
4 . The method of claim 2 , wherein the step of confirming the protein identity of the spot comprises immunoblotting antibodies onto the relevant fractions of candidate proteins.
5 . The method of claim 2 , wherein the step of confirming the findings of the spot comprises immunofluorescence against a characteristic pattern of PV-1 in induced cells as a reference standard.
6 . The method of claim 1 , wherein the endothelial cell line is selected from the group consisting of a bEND5 endothelial cell line and a Py4.1 endothelial cell line.
7 . The method of claim 1 , wherein the endothelial cell line is a brain endothelioma cell line.
8 . The method of claim 1 , wherein the step of isolating plasma membranes comprises a silica isolation procedure.
9 . A method for visualizing fenestrae in an endothelial cell line, said method comprising the steps of:
a) staining an endothelial cell line with an anti-moesin antibody;
b) imaging the endothelial cell line utilizing a light microscope; and
c) visualizing fenestrae based on the level of the fluorescently labeled anti-moesin bound to moesin in the endothelial cell line.
10 . The method of claim 9 , wherein the anti-moesin antibody is fluorescently labeled.
11 . The method of claim 9 , wherein the endothelial cell line is selected from the group consisting of a bEND5 endothelial cell line and a Py4.1 endothelial cell line.
12 . The method of claim 9 , wherein the endothelial cell line is a brain endothelioma cell line.
13 . A marker for detecting fenestrae in an endothelial cell line, wherein the marker comprises a protein selected from the group consisting of moesin, paralemmin, radixin, cofilin, twinfilin, alpha-enolase, annexin II, musculin, putative RNA-binding protein 3, nucleoside diphosphate kinase B, and hnRNP K.
14 . The marker of claim 13 , wherein the marker is a plasma membrane marker for fenestrae.
15 . The marker of claim 13 , wherein the marker comprises a protein selected from the group consisting of moesin and radixin.
16 . A marker combination for detecting fenestrae, wherein the marker combination comprises moesin and PV-1.
17 . A method for identifying fenestrae in an endothelial cell line comprising the step of visualizing a marker combination comprising moesin and PV-1.
18 . The method of claim 17 , wherein the step of visualizing a marker combination comprising moesin and PV-1 comprises light microscopy.
19 . The method of claim 17 , wherein the endothelial cell line is selected from the group consisting of a bEND5 endothelial cell line and a Py4.1 endothelial cell line.
20 . The method of claim 17 , wherein the endothelial cell line is a brain endothelioma cell line.
21 . A method of identifying fenestrae in an endothelial cell line comprising the step of detecting PV-1 and paralemmin.
22 . A method of identifying a marker for fenestrae in an endothelial cell line comprising the step of performing subtractive proteomic analysis.
23 . The method of claim 22 , further comprising the step of performing evolutionary genomics.
24 . A method of identifying a marker for fenestrae in an endothelial cell line comprising the step of performing evolutionary genomics.
25 . A method of altering sieve plate composition in a fenestrated cell line comprising the step of administering a dominant negative version of moesin to the fenestrated cell line.
26 . The method of claim 26 , wherein the fenestrated cell line is selected from the group consisting of a NIH3T3cell line and a bEND5 cell line.
27 . The method of claim 26 , wherein the dominant negative version of moesin is a fusion protein.
28 . The method of claim 26 , wherein the dominant negative version of moesin is a fusion protein comprising an N-terminal domain of moesin fused to a Green Fluorescent Protein.