TY - JOUR
T1 - Fine structure and dynamics of EB3 binding zones on microtubules in fibroblast cells
AU - Mustyatsa, V. V.
AU - Kostarev, A. V.
AU - Tvorogova, A. V.
AU - Ataullakhanov, F. I.
AU - Gudimchuk, N. B.
AU - Vorobjev, I. A.
N1 - Funding Information:
Experimental work and data analyses were supported by the Russian Foundation for Basic Research (Project Nos. 17-54-33009 to I.V. and 18-34-00944 to V.M.) and Grant AP05134232 from Ministry of Education and Science of the Republic of Kazakhstan to I.V. Computational modeling of tubulin and EB3 dynamics was supported by a Russian Science Foundation grant to N.G. (Project # 17-74-20152).
PY - 2019/8/1
Y1 - 2019/8/1
N2 - End-binding (EB) proteins associate with the growing tips of microtubules (MTs) and modulate their dynamics directly and indirectly, by recruiting essential factors to fine-tune MTs for their many essential roles in cells. Previously EB proteins have been shown to recognize a stabilizing GTP/GDP-Pi cap at the tip of growing MTs, but information about additional EB-binding zones on MTs has been limited. In this work, we studied fluorescence intensity profiles of one of the three mammalian EB-proteins, EB3, fused with red fluorescent protein (RFP). The distribution of EB3 on MTs in mouse fibroblasts frequently deviated from single exponential decay and exhibited secondary peaks. Those secondary peaks, which we refer to as EB3-islands, were detected on 56% comets of growing MTs and were encountered once per 44 s of EB3-RFP comet growth time with about 5 s half-lifetime. The majority of EB3-islands in the vicinity of MT tips was stationary and originated from EB3 comets moving with the growing MT tips. Computational modeling of the decoration of dynamic MT tips by EB3 suggested that the EB3-islands could not be explained simply by a stochastic first-order GTP hydrolysis/phosphate release. We speculate that additional protein factors contribute to EB3 residence time on MTs in cells, likely affecting MT dynamics.
AB - End-binding (EB) proteins associate with the growing tips of microtubules (MTs) and modulate their dynamics directly and indirectly, by recruiting essential factors to fine-tune MTs for their many essential roles in cells. Previously EB proteins have been shown to recognize a stabilizing GTP/GDP-Pi cap at the tip of growing MTs, but information about additional EB-binding zones on MTs has been limited. In this work, we studied fluorescence intensity profiles of one of the three mammalian EB-proteins, EB3, fused with red fluorescent protein (RFP). The distribution of EB3 on MTs in mouse fibroblasts frequently deviated from single exponential decay and exhibited secondary peaks. Those secondary peaks, which we refer to as EB3-islands, were detected on 56% comets of growing MTs and were encountered once per 44 s of EB3-RFP comet growth time with about 5 s half-lifetime. The majority of EB3-islands in the vicinity of MT tips was stationary and originated from EB3 comets moving with the growing MT tips. Computational modeling of the decoration of dynamic MT tips by EB3 suggested that the EB3-islands could not be explained simply by a stochastic first-order GTP hydrolysis/phosphate release. We speculate that additional protein factors contribute to EB3 residence time on MTs in cells, likely affecting MT dynamics.
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U2 - 10.1091/mbc.E18-11-0723
DO - 10.1091/mbc.E18-11-0723
M3 - Article
C2 - 31141458
AN - SCOPUS:85070850048
VL - 30
SP - 2105
EP - 2114
JO - Molecular Biology of the Cell
JF - Molecular Biology of the Cell
SN - 1059-1524
IS - 17
ER -