The microtubule-binding protein ensconsin is an essential cofactor of kinesin-1

Abstract

Kinesin-1 is a major microtubule motor that drives transport of numerous cellular cargoes toward the plus ends of microtubules. In the cell, kinesin-1 exists primarily in an inactive, autoinhibited state, and motor activation is thought to occur upon binding to cargo through the C terminus. Using RNAi-mediated depletion in Drosophila S2 cells, we demonstrate that kinesin-1 requires ensconsin (MAP7, E-MAP-115), a ubiquitous microtubule-associated protein, for its primary function of organelle transport. We show that ensconsin is required for organelle transport in Drosophila neurons and that Drosophila homozygous for ensconsin gene deletion are unable to survive to adulthood. An ensconsin N-terminal truncation that cannot bind microtubules is sufficient to activate organelle transport by kinesin-1, indicating that this activating domain functions independently of microtubule binding. Interestingly, ens mutant flies retaining expression of this truncation show normal viability. A "hingeless" mutant of kinesin-1, which mimics the active conformation of the motor, does not require ensconsin for transport in S2 cells, suggesting that ensconsin plays a role in relieving autoinhibition of kinesin-1. Together with other recent work, our study suggests that ensconsin is an essential cofactor for all known functions of kinesin-1.

FIGURE 1

Ensconsin is required for kinesin-1-dependent cargo transport in Drosophila S2 cells. S2 cells were plated on concanavalin A-coated coverslips in the presence of cytochalasin D. Cargoes were imaged each second for 60 seconds. (A) Motility of MitoTracker-labelled mitochondria. For each condition indicated, the first frame of a time-lapse sequence is shown above, and a merge of 60 frames in sequence, color-coded according to the bar at lower right, is shown below. Due to the superposition of multiple colors, stationary particles appear white, whereas moving particles appear in multicolored paths. Boxed regions are enlarged in insets. Scale bars are 10 μm in main panels and 2 μm in insets. (B) Quantification of cargo transport. Cargoes were tracked over 60 frames, and the distance each particle traveled was determined (see Supplemental Experimental Procedures). Motility of the indicated cargoes in RNAi-treated cells is shown relative to motility measured in the untreated control; for the purposes of comparing multiple cargo types, control motility is set to 1. Mean values from at least two independent experiments are shown. Error bars indicate SD. *, p < 0.05; **, p < 0.01; compared to control. (C) Ensconsin RNAi does not deplete KHC protein. Immunoblots of cell lysates after RNAi treatment, using antibodies against KHC and ensconsin. Coomassie staining, loading control.

FIGURE 2

Kinesin-1-dependent microtubule sliding requires ensconsin. (A) S2 cells expressing tdEos-tubulin imaged in the green channel before photoconversion (left) and in the red channel after photoconversion through a slit (middle). (B) Two frames of a time-lapse movie (obtained in the red channel) demonstrate the movement of fluorescent patches of microtubules away from the photoconverted region in control cells, and the drastic reduction of this movement in KHC- and ensconsin-depleted cells. Time is indicated at left. Dashed line indicates cell boundary. Scale bar is 10 μm in both (A) and (B). (C) Quantification of microtubule sliding. The sliding rate is obtained by determining the amount of fluorescence outside the photoconverted region as a function of time (see Supplemental Experimental Procedures for more detailed description). Mean values from at least three independent experiments are shown. Error bars indicate SD. ***, p < 0.001, compared to control.

FIGURE 3

Ensconsin is required for kinesin-1-dependent transport in Drosophila neurons. Drosophila embryos were injected with DNA encoding mCherry-SKL, which labels peroxisomes, before the occurrence of cellularization. (A) Cells cultured from stage 9-11 Drosophila embryos are positive for neuronal markers Futsch (22c10) and ELAV by immunolabeling, verifying neuronal identity. Scale bar, 10 μm. (B) Kymographs of peroxisome motility in control, khc null, and ens null neurons. Motility was measured once per second for 60 seconds. Arrowheads highlight motile particles. Scale bar, 2 μm. (C) Quantification of peroxisome motility. Mean values from at least two independent experiments are shown. Error bars indicate SD. **, p < 0.01; compared to control.

FIGURE 4

(A-B) Microtubule binding by ensconsin is not required for cargo transport. Cells stably expressing the designated ensconsin truncations were depleted of endogenous ensconsin, and mitochondria motility was analyzed by time-lapse imaging. (A) GFP-ensconsin constructs use in this study. Numbers indicate amino acid positions. (B) Quantification of mitochondria transport in ensconsin rescue experiments. (C-D) Unregulated kinesin-1 mutants restore cargo in cells depleted of ensconsin. (C) Schematic of KHC (top) and KHC mutant constructs used in this study. Numbers indicate amino acid positions. Cells stably expressing the designated KHC mutants were depleted of endogenous KHC (D) or both ensconsin and KHC (dual, E), and mitochondria motility was analyzed and quantified. For quantifications, mean values of at least 2 independent experiments are shown. Error bars indicate SD. *, p < 0.05; **, p < 0.01.