GFP chimeras of E-MAP-115 (ensconsin) domains mimic behavior of the endogenous protein in vitro and in vivo.

Cell Structure and Function
J C BulinskiW Chang

Abstract

E-MAP-115 (ensconsin) is a microtubule-associated protein (MAP) abundant in carcinoma and other epithelia-derived cells. We expressed chimeras of green fluorescent protein (GFP) conjugated to ensconsin's N-terminal MT-binding domain (EMTB), to study distribution, dynamics, and function of the MAP in living cells. We tested the hypothesis that behavior of expressed GFP-EMTB accurately matched behavior of endogenous ensconsin. Like endogenous MAP, GFP-EMTB was associated with microtubules in living or fixed cells, and microtubule association of either molecule was impervious to extraction with nonionic detergents. In cell lysates both GFP-EMTB and endogenous ensconsin were dissociated from microtubules by identical salt extraction conditions, and both molecules remained bound to a calcium-stable subset of Taxol-stabilized microtubules. These data show that microtubule association of ensconsin was affected neither by the absence of domains other than its microtubule-binding domain, nor by the presence of appended GFP. We took advantage of this finding to generate constructs in which additional GFP moieties were attached to EMTB, to obtain a more intensely fluorescent reporter of in vivo MAP binding. We show here that expression of...Continue Reading

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Citations

Jan 12, 2008·Journal of Cell Science·Andrey EfimovIrina Kaverina
Oct 6, 2017·Developmental Dynamics : an Official Publication of the American Association of Anatomists·Amrita RamkumarKassandra M Ori-McKenney
Dec 31, 2005·BioTechniques·Guillem GenovéAlison L Barth
Oct 20, 2018·Development·Mike R VisetsoukJennifer H Gutzman
Jan 25, 2018·BMC Biology·Nunu MchedlishviliBuzz Baum
Sep 5, 2020·The Journal of Cell Biology·Shubham KesarwaniMinhajuddin Sirajuddin
Apr 14, 2017·Cytoskeleton·Paul MooneyJesse C Gatlin
Jun 26, 2021·Neurology. Genetics·Merel O MolJohn C van Swieten

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