TY - CHAP
T1 - Targeting of ASH domain-containing proteins to the centrosome
AU - Verdier, Patricia
AU - Morthorst, Stine Kjær
AU - Pedersen, Lotte Bang
PY - 2016
Y1 - 2016
N2 - A growing number of studies have used new generation technologies to characterize the protein constituents of cilia and centrosomes. This has led to the identification of a vast number of candidate ciliary or centrosomal proteins, whose subcellular localization needs to be investigated and validated. Here, we describe a simple and inexpensive method for analyzing the subcellular localization of candidate cilium-or centrosome-associated proteins, and we illustrate the utility as well as the pitfalls of this method by applying it to a group of ASH (ASPM, SPD-2, Hydin) domain-containing proteins, previously predicted to be cilia-or centrosome-associated proteins based on bioinformatic analyses. By generating plasmids coding for epitope-tagged full-length (FL) or truncated versions of the ASH domain-containing proteins TRAPPC8, TRAPPC13, NPHP4, and DLEC1, followed by expression and quantitative immunofluorescence microscopy (IFM) analysis in cultured human telomerase-immortalized retinal pigmented epithelial (hTERT-RPE1) cells, we could confirm that TRAPPC13 and NPHP4 are highly enriched at the base of primary cilia, whereas DLEC1 seems to associate specifically with motile cilia. Results for TRAPPC8 were inconclusive since epitope-tagged TRAPPC8 fusion proteins were unstable/degraded in cells, emphasizing the need for combining IFM analysis with western blotting in such studies. The method described should be applicable to other candidate ciliary or centrosomal proteins as well.
AB - A growing number of studies have used new generation technologies to characterize the protein constituents of cilia and centrosomes. This has led to the identification of a vast number of candidate ciliary or centrosomal proteins, whose subcellular localization needs to be investigated and validated. Here, we describe a simple and inexpensive method for analyzing the subcellular localization of candidate cilium-or centrosome-associated proteins, and we illustrate the utility as well as the pitfalls of this method by applying it to a group of ASH (ASPM, SPD-2, Hydin) domain-containing proteins, previously predicted to be cilia-or centrosome-associated proteins based on bioinformatic analyses. By generating plasmids coding for epitope-tagged full-length (FL) or truncated versions of the ASH domain-containing proteins TRAPPC8, TRAPPC13, NPHP4, and DLEC1, followed by expression and quantitative immunofluorescence microscopy (IFM) analysis in cultured human telomerase-immortalized retinal pigmented epithelial (hTERT-RPE1) cells, we could confirm that TRAPPC13 and NPHP4 are highly enriched at the base of primary cilia, whereas DLEC1 seems to associate specifically with motile cilia. Results for TRAPPC8 were inconclusive since epitope-tagged TRAPPC8 fusion proteins were unstable/degraded in cells, emphasizing the need for combining IFM analysis with western blotting in such studies. The method described should be applicable to other candidate ciliary or centrosomal proteins as well.
U2 - 10.1007/978-1-4939-3789-9_2
DO - 10.1007/978-1-4939-3789-9_2
M3 - Book chapter
C2 - 27514913
SN - 978-1-4939-3787-5
T3 - Methods in Molecular Biology
SP - 15
EP - 33
BT - Cilia
A2 - Satir, Peter
A2 - Tvorup Christensen, Søren
PB - Springer
ER -