Open AccessReversible association with motor proteins (RAMP): A streptavidin-based method to manipulate organelle positioning
Author(s) -
Carlos M. Guardia,
Raffaella De Pace,
Aritra Sen,
Amra Sarić,
Michal Jarník,
David A. Kolin,
Ambarish Kunwar,
Juan S. Bonifacino
Publication year - 2019
Publication title -
plos biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 4.127
H-Index - 271
eISSN - 1545-7885
pISSN - 1544-9173
DOI - 10.1371/journal.pbio.3000279
Subject(s) - organelle , kinesin , streptavidin , biology , microtubule , motor protein , molecular motor , cytoplasm , microbiology and biotechnology , population , biophysics , biotin , biochemistry , demography , sociology
We report the development and characterization of a method, named reversible association with motor proteins (RAMP), for manipulation of organelle positioning within the cytoplasm. RAMP consists of coexpressing in cultured cells (i) an organellar protein fused to the streptavidin-binding peptide (SBP) and (ii) motor, neck, and coiled-coil domains from a plus-end–directed or minus-end–directed kinesin fused to streptavidin. The SBP–streptavidin interaction drives accumulation of organelles at the plus or minus end of microtubules, respectively. Importantly, competition of the streptavidin–SBP interaction by the addition of biotin to the culture medium rapidly dissociates the motor construct from the organelle, allowing restoration of normal patterns of organelle transport and distribution. A distinctive feature of this method is that organelles initially accumulate at either end of the microtubule network in the initial state and are subsequently released from this accumulation, allowing analyses of the movement of a synchronized population of organelles by endogenous motors.