TY - GEN
T1 - Method
T2 - In vitro analysis of pericentriolar material assembly
AU - Woodruff, Jeffrey B.
AU - Hyman, Anthony A.
N1 - Publisher Copyright:
© 2015 Elsevier Inc.
PY - 2015
Y1 - 2015
N2 - Centrosomes are major microtubule-organizing centers in eukaryotic cells and play a critical role in embryonic development and asymmetric cell division. Centrosomes comprise a pair of centrioles surrounded by an amorphous proteinaceous meshwork called the pericentriolar material (PCM). Robust deposition of PCM around the centrioles is essential for a centrosome to achieve full microtubule nucleating potential. Despite the wealth of information on PCM composition and function, the mechanism and regulation of PCM assembly have been difficult to ascertain, due in part to the lack of an in vitro system. Here, we describe methods to establish an in vitro system to study PCM assembly in Caenorhabditis elegans. Specifically, we describe (1) how to express and purify the C. elegans PCM proteins SPD-5, SPD-2, and PLK-1 from baculovirus-infected insect cells, (2) how to assemble these proteins into PCM-like structures in vitro, and (3) how to quantify this assembly process in a semiautomated fashion.
AB - Centrosomes are major microtubule-organizing centers in eukaryotic cells and play a critical role in embryonic development and asymmetric cell division. Centrosomes comprise a pair of centrioles surrounded by an amorphous proteinaceous meshwork called the pericentriolar material (PCM). Robust deposition of PCM around the centrioles is essential for a centrosome to achieve full microtubule nucleating potential. Despite the wealth of information on PCM composition and function, the mechanism and regulation of PCM assembly have been difficult to ascertain, due in part to the lack of an in vitro system. Here, we describe methods to establish an in vitro system to study PCM assembly in Caenorhabditis elegans. Specifically, we describe (1) how to express and purify the C. elegans PCM proteins SPD-5, SPD-2, and PLK-1 from baculovirus-infected insect cells, (2) how to assemble these proteins into PCM-like structures in vitro, and (3) how to quantify this assembly process in a semiautomated fashion.
UR - http://www.scopus.com/inward/record.url?scp=84943350235&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84943350235&partnerID=8YFLogxK
U2 - 10.1016/bs.mcb.2015.04.006
DO - 10.1016/bs.mcb.2015.04.006
M3 - Conference contribution
C2 - 26175448
AN - SCOPUS:84943350235
SN - 9780128024492
T3 - Methods in Cell Biology
SP - 369
EP - 382
BT - Methods in Cell Biology
A2 - Oegema, Karen
A2 - Basto, Renata
PB - Academic Press Inc.
ER -