Cellular RhoGTPase activation assay
The invention relates to a cellular RhoGTPase activation assay based on the determination of changes in the actin cytoskeleton (actin cytoskeletal rearrangement assay); to the recombinant cell lines used to carry out this assay; to the constructs required to produce these cell lines; to the use of these recombinant cell lines for detecting effectors of Nogo receptor (NgR)-dependent signal transduction involving RhoGTPase, in particular for determining effectors of neuronal regeneration; and to assay methods using these recombinant cell lines.
1 . A recombinant cell line which expresses:
a) a functional intracellular factor which is involved in a signal pathway which induces a change in the cytoskeleton of the cell; and/or
b) a membrane-bound receptor which interacts with the intracellular factor.
2 . The cell line according to claim 1 , where the intracellular factor is a Rho GTPase, and/or the membrane-bound receptor comprises a functional neutrotrophin receptor p75 and a functional Nogo receptor (NgR).
3 . The cell line according to claim 2 , where
a) the functional RhoGTPase comprises an amino acid sequence as shown in SEQ ID NO: 16;
b) the functional p75 comprises an amino acid sequence as shown in SEQ ID NO: 9; and
c) the functional NgR comprises an amino acid sequence as shown in SEQ ID NO: 11.
4 . The cell line according to claim 1 , where cells are able to reorganize their actin cytoskeleton induced by the binding of a ligand to NgR.
5 . The cell line according to claim 4 , where the ligand binding induces an activation of Rho.
6 . The cell line according to claim 4 , where the ligand comprises Nogo-A or an NgR-binding fragment or derivative derived therefrom.
7 . The cell line according to claim 1 , derived from an adherent mammalian cell line with extended cell morphology.
8 . The cell line according to claim 7 , derived from a human epithelial or fibroblast cell line.
9 . The cell line according to claim 8 , derived from a human embryonic kidney (HEK) cell line.
10 . The use of a cell line according to claim 1 for determining effectors of NgR-dependent signal transduction.
11 . An assay method for determining changes in the cytoskeleton of a cell line to be investigated, where a recombinant cell line as defined in claim 1 , or a corresponding primary cell line is cultivated with a ligand which interacts with the cells and which, if appropriate, brings about the change in the cytoskeleton via induction of an intracellular signal pathway, and changes in the reorganization of the cytoskeleton of the cell lines treated in this way are determined.
12 . The assay method according to claim 11 , where the ligand interacts with a membrane-bound receptor.
13 . The assay method according to claim 11 , where the cell line is culivated in the presence or absence of an analyte which is suspected to comprise an effector which modulates the effect of the ligand on the cell.
14 . The assay method according to claim 13 , where the cultivation with the analyte takes place simultaneously or sequentially in relation to the cultivation with the receptor-binding ligand.
15 . The assay method according to claim 11 for determining effectors of NgR-dependent activation of the GTPase Rho, where the recombinant cell line or a corresponding primary cell line is cultivated in the presence of an NgR-binding ligand and in the presence or absence of an analyte suspected to comprise the effector, and differences in the reorganization of the cytoskeleton of the cell lines treated in this way are determined.
16 . The assay method according to claim 11 , where the average of at least one of the following parameters is determined for a representative cell population:
a) cell circumference
b) cell length
c) cell width
d) cell area
e) length of the cell projections
f) cell density
g) cytoplasmic area
h) cytoplasm vertices
i) refractive index
j) cell-free area
k) area covered with cells
l) a mathematical linkage of at least two of parameters a) to k), or a parameter which can be derived from at least one of the parameters a) to k), such as, in particular, the ratio of cell length to cell width, or the ratio of cell circumference squared to cell area; or
m) an appropriate reciprocal value of one of the parameters a) to 1).
17 . The assay method according to claim 11 , where the determination takes place by means of an automated method such as, in particular, by means of an automatic image analysis system.
18 . The use of an assay method according to claim 11 for determining effectors of neuronal regeneration.
19 . The use according to claim 18 , where the effector partly or completely inhibits Rho activation.
20 . The use according to claim 18 , where the effector promotes neuronal regeneration.
21 . The use according to claim 10 , where the effector partly or completely neutralizes a disease- or injury-related inhibition of axonal regeneration or a disease- or injury-related collapse of the neuronal growth cone.
22 . The use according to claim 18 , where the effector is an NgR antagonist.
23 . The use according to claim 18 in a screening method, in particular a high content screening method for effectors of neuronal regeneration.
24 . A vector comprising a nucleic acid sequence coding for functional RhoGTPase or comprising nucleic acid sequences coding for functional p75 and functional NgR.
25 . A method for producing a recombinant cell line according to claim 1 , where a wild-type cell line is transformed with at least one coding nucleic acid sequence which codes for a functional RhoGTPase and, if appropriate, for p75 and NgR.
26 . The method according to claim 25 , where the cell line is transformed with at least one vector comprising a nucleic acid sequence coding for functional RhoGTPase or comprising nucleic acid sequences coding for functional p75 and functional NgR.
27 . A high content screening method for determining changes in the cytoskeleton of a cell line to be investigated and in particular for determining effectors of NgR-dependent activation of the GTPase Rho, where an assay method according to claim 11 is used.
28 . An apparatus for carrying out a method according to claim 27 .
29 . A test kit comprising a recombinant cell line according to claim 1 or a corresponding primary cell line, and a ligand for the cellular receptor.