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MARK/PAR1 kinase is a regulator of microtubule-dependent transport in axons.

Mandelkow EM, Thies E, Trinczek B, Biernat J, Mandelkow E - J. Cell Biol. (2004)

Bottom Line: The transport can be regulated through motor proteins, cargo adaptors, or microtubule tracks.This occurs without impairing the intrinsic activity of motors because the velocity during active movement remains unchanged.This transport inhibition can be rescued by phosphorylating tau with MARK.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck Unit for Structural Molecular Biology, 22607 Hamburg, Germany. mandelkow@mpasmb.desy.de

ABSTRACT
Microtubule-dependent transport of vesicles and organelles appears saltatory because particles switch between periods of rest, random Brownian motion, and active transport. The transport can be regulated through motor proteins, cargo adaptors, or microtubule tracks. We report here a mechanism whereby microtubule associated proteins (MAPs) represent obstacles to motors which can be regulated by microtubule affinity regulating kinase (MARK)/Par-1, a family of kinases that is known for its involvement in establishing cell polarity and in phosphorylating tau protein during Alzheimer neurodegeneration. Expression of MARK causes the phosphorylation of MAPs at their KXGS motifs, thereby detaching MAPs from the microtubules and thus facilitating the transport of particles. This occurs without impairing the intrinsic activity of motors because the velocity during active movement remains unchanged. In primary retinal ganglion cells, transfection with tau leads to the inhibition of axonal transport of mitochondria, APP vesicles, and other cell components which leads to starvation of axons and vulnerability against stress. This transport inhibition can be rescued by phosphorylating tau with MARK.

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Influence of MARK2 on the transport of vesicles and organelles. (a) Tracking pattern of GFP-VSVG–tagged vesicles in noninduced CHO cells (controls) and (b) in MARK2-induced CHO cells. Dots connected by a line identify consecutive positions of a vesicle (1-s time intervals, arrowheads indicate starting points). The coordinates are centered on the TGN (labeled strongly with GFP-VSVG) near the microtubule organizing center. Upon MARK induction the tracks become longer and straighter, indicating that fewer directional changes occur. (c–e) Quantification of GFP-VSVG–tagged vesicle movement in noninduced CHO cells (black bars) and MARK2-induced cells (gray bars). The data are based on the vesicle motion recorded from 30 cells, 150 vesicles were analyzed, error bars = SEM. (c) Velocities remain unchanged after the induction of MARK2 in either direction (inbound v∼0.9μm/s, outbound ∼0.7 μm/s; n > 500). (d) Reversal frequencies decrease strongly after MARK2 induction (∼0.04 s−1 with MARK2, ∼0.09 s−1 without MARK2, both for inbound/outbound and outbound/inbound reversals; n > 100). (e) Run lengths increase strongly (∼50%) upon MARK2 induction in both directions (n > 100). Note that both with and without MARK2 the inbound run lengths are ∼30% longer than outbound (4.6 μm inbound, 3.6 μm outbound without MARK2). (f and g) Motion of lysosomes. (f) This panel shows an overlay of 60 images separated by 1 s. Lysosomes move on linear tracks >1 μm (arrows). Particles that are immobile or show only short Brownian movements (≤0.3 μm and random direction) can also be observed (spots marked by arrowheads). (g) Velocities remain unchanged upon MARK2 induction (∼0.8 μm/s), but run lengths increase (from 3 to 5 μm). Reversal frequencies and directionalities cannot be determined because in CHO cells labeled with LysoTracker there is no internal reference indicating the cell's interior. (h) Endocytotic vesicles: CHO cells were transiently transfected with EGFP-labeled β2-adaptin, a marker of the AP-2 complex of endocytotic clathrin-coated vesicles. During the time of observation shown here (90 s) fluorescent paths are clearly visible. Linear or curved tracks longer than 1 μm represent actively transported vesicles (arrows), broader black or gray spots represent immobile particles and/or vesicles showing Brownian motion (arrowheads). The overexpression of GFP/β2-adaptin causes a strong background noise in the cytoplasm and the nucleus (N) becomes visible. The rather immobile fluorescent patches in the more peripheral regions of the cell are clathrin-coated vesicles or their precursors (arrowheads; Laporte et al., 1999). In the interior of the cell near the nucleus (N) an accumulation of the fluorescent signal can be observed suggesting that EGFP/β2-adaptin also colocalizes with the exocytotic and/or endosomal compartment. This is supported by the occurrence of fast moving vesicles entering or leaving this area. In contrast to the post-Golgi vesicles labeled by transient transfection of GFP-VSVG, the system here cannot provide reversal frequencies because of the strong background that shortens the time in which the particles are clearly seen in focus. (i) Velocities remain unchanged by MARK2 (∼1 μm/s), but run lengths increase almost twofold. The tracks illustrate three scenarios: the period of motion observed within the plane of focus is flanked by (1) periods of active transport out of focus, (2) resting states in focus and active transport out of focus, and (3) periods of pauses in focus. We only analyzed events of movement which were clearly in focus and which show at least two stop events in order to determine the run length. As in the case of lysosomes (g), the velocity of vesicles (i) remains roughly the same after MARK2 induction, but there is a notable increase in the run length. Error bars indicate SEM.
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fig4: Influence of MARK2 on the transport of vesicles and organelles. (a) Tracking pattern of GFP-VSVG–tagged vesicles in noninduced CHO cells (controls) and (b) in MARK2-induced CHO cells. Dots connected by a line identify consecutive positions of a vesicle (1-s time intervals, arrowheads indicate starting points). The coordinates are centered on the TGN (labeled strongly with GFP-VSVG) near the microtubule organizing center. Upon MARK induction the tracks become longer and straighter, indicating that fewer directional changes occur. (c–e) Quantification of GFP-VSVG–tagged vesicle movement in noninduced CHO cells (black bars) and MARK2-induced cells (gray bars). The data are based on the vesicle motion recorded from 30 cells, 150 vesicles were analyzed, error bars = SEM. (c) Velocities remain unchanged after the induction of MARK2 in either direction (inbound v∼0.9μm/s, outbound ∼0.7 μm/s; n > 500). (d) Reversal frequencies decrease strongly after MARK2 induction (∼0.04 s−1 with MARK2, ∼0.09 s−1 without MARK2, both for inbound/outbound and outbound/inbound reversals; n > 100). (e) Run lengths increase strongly (∼50%) upon MARK2 induction in both directions (n > 100). Note that both with and without MARK2 the inbound run lengths are ∼30% longer than outbound (4.6 μm inbound, 3.6 μm outbound without MARK2). (f and g) Motion of lysosomes. (f) This panel shows an overlay of 60 images separated by 1 s. Lysosomes move on linear tracks >1 μm (arrows). Particles that are immobile or show only short Brownian movements (≤0.3 μm and random direction) can also be observed (spots marked by arrowheads). (g) Velocities remain unchanged upon MARK2 induction (∼0.8 μm/s), but run lengths increase (from 3 to 5 μm). Reversal frequencies and directionalities cannot be determined because in CHO cells labeled with LysoTracker there is no internal reference indicating the cell's interior. (h) Endocytotic vesicles: CHO cells were transiently transfected with EGFP-labeled β2-adaptin, a marker of the AP-2 complex of endocytotic clathrin-coated vesicles. During the time of observation shown here (90 s) fluorescent paths are clearly visible. Linear or curved tracks longer than 1 μm represent actively transported vesicles (arrows), broader black or gray spots represent immobile particles and/or vesicles showing Brownian motion (arrowheads). The overexpression of GFP/β2-adaptin causes a strong background noise in the cytoplasm and the nucleus (N) becomes visible. The rather immobile fluorescent patches in the more peripheral regions of the cell are clathrin-coated vesicles or their precursors (arrowheads; Laporte et al., 1999). In the interior of the cell near the nucleus (N) an accumulation of the fluorescent signal can be observed suggesting that EGFP/β2-adaptin also colocalizes with the exocytotic and/or endosomal compartment. This is supported by the occurrence of fast moving vesicles entering or leaving this area. In contrast to the post-Golgi vesicles labeled by transient transfection of GFP-VSVG, the system here cannot provide reversal frequencies because of the strong background that shortens the time in which the particles are clearly seen in focus. (i) Velocities remain unchanged by MARK2 (∼1 μm/s), but run lengths increase almost twofold. The tracks illustrate three scenarios: the period of motion observed within the plane of focus is flanked by (1) periods of active transport out of focus, (2) resting states in focus and active transport out of focus, and (3) periods of pauses in focus. We only analyzed events of movement which were clearly in focus and which show at least two stop events in order to determine the run length. As in the case of lysosomes (g), the velocity of vesicles (i) remains roughly the same after MARK2 induction, but there is a notable increase in the run length. Error bars indicate SEM.

Mentions: Indeed, when MARK2 is induced by addition of doxycyclin in CHO cells there is a dramatic effect on vesicle behavior (Fig. 4). The predominant MAP in CHO cells is a variant of MAP4, one of the high-molecular weight MAPs that control microtubule dynamics (Bulinski and Borisy, 1980; Olson et al., 1995). If the endogenous MAP4 represents obstacles to vesicles on the microtubule track the detachment of MAP4 should facilitate movement. This is indeed seen in the representative traces of Fig. 4 a. The distances traversed during the observation time are normally small (a few micrometers) because there are many kinks where the vesicles change direction. However, induction of MARK2 leads to much more extended tracks (Fig. 4 b). The instantaneous velocities are not affected by MARK (∼1 μm/s; Fig. 4 c), but the reversal frequencies decrease by ∼50% (Fig. 4 d), and the run lengths increase by ∼50% in both directions (Fig. 4 e). Because MARK2 phosphorylates MAP4 or other MAPs at KXGS motifs in the repeat domain and detaches them from microtubules (Illenberger et al., 1996), these results argue that vesicle movement is facilitated and can indeed be regulated locally on the microtubule surface by MAP phosphorylation. This effect is opposite to the transport inhibition by elevated MAP expression (demonstrated in Fig. 1 by the clustering of mitochondria), so that MAPs and their kinases can be regarded as antagonistic with respect to microtubule-based vesicle trafficking.


MARK/PAR1 kinase is a regulator of microtubule-dependent transport in axons.

Mandelkow EM, Thies E, Trinczek B, Biernat J, Mandelkow E - J. Cell Biol. (2004)

Influence of MARK2 on the transport of vesicles and organelles. (a) Tracking pattern of GFP-VSVG–tagged vesicles in noninduced CHO cells (controls) and (b) in MARK2-induced CHO cells. Dots connected by a line identify consecutive positions of a vesicle (1-s time intervals, arrowheads indicate starting points). The coordinates are centered on the TGN (labeled strongly with GFP-VSVG) near the microtubule organizing center. Upon MARK induction the tracks become longer and straighter, indicating that fewer directional changes occur. (c–e) Quantification of GFP-VSVG–tagged vesicle movement in noninduced CHO cells (black bars) and MARK2-induced cells (gray bars). The data are based on the vesicle motion recorded from 30 cells, 150 vesicles were analyzed, error bars = SEM. (c) Velocities remain unchanged after the induction of MARK2 in either direction (inbound v∼0.9μm/s, outbound ∼0.7 μm/s; n > 500). (d) Reversal frequencies decrease strongly after MARK2 induction (∼0.04 s−1 with MARK2, ∼0.09 s−1 without MARK2, both for inbound/outbound and outbound/inbound reversals; n > 100). (e) Run lengths increase strongly (∼50%) upon MARK2 induction in both directions (n > 100). Note that both with and without MARK2 the inbound run lengths are ∼30% longer than outbound (4.6 μm inbound, 3.6 μm outbound without MARK2). (f and g) Motion of lysosomes. (f) This panel shows an overlay of 60 images separated by 1 s. Lysosomes move on linear tracks >1 μm (arrows). Particles that are immobile or show only short Brownian movements (≤0.3 μm and random direction) can also be observed (spots marked by arrowheads). (g) Velocities remain unchanged upon MARK2 induction (∼0.8 μm/s), but run lengths increase (from 3 to 5 μm). Reversal frequencies and directionalities cannot be determined because in CHO cells labeled with LysoTracker there is no internal reference indicating the cell's interior. (h) Endocytotic vesicles: CHO cells were transiently transfected with EGFP-labeled β2-adaptin, a marker of the AP-2 complex of endocytotic clathrin-coated vesicles. During the time of observation shown here (90 s) fluorescent paths are clearly visible. Linear or curved tracks longer than 1 μm represent actively transported vesicles (arrows), broader black or gray spots represent immobile particles and/or vesicles showing Brownian motion (arrowheads). The overexpression of GFP/β2-adaptin causes a strong background noise in the cytoplasm and the nucleus (N) becomes visible. The rather immobile fluorescent patches in the more peripheral regions of the cell are clathrin-coated vesicles or their precursors (arrowheads; Laporte et al., 1999). In the interior of the cell near the nucleus (N) an accumulation of the fluorescent signal can be observed suggesting that EGFP/β2-adaptin also colocalizes with the exocytotic and/or endosomal compartment. This is supported by the occurrence of fast moving vesicles entering or leaving this area. In contrast to the post-Golgi vesicles labeled by transient transfection of GFP-VSVG, the system here cannot provide reversal frequencies because of the strong background that shortens the time in which the particles are clearly seen in focus. (i) Velocities remain unchanged by MARK2 (∼1 μm/s), but run lengths increase almost twofold. The tracks illustrate three scenarios: the period of motion observed within the plane of focus is flanked by (1) periods of active transport out of focus, (2) resting states in focus and active transport out of focus, and (3) periods of pauses in focus. We only analyzed events of movement which were clearly in focus and which show at least two stop events in order to determine the run length. As in the case of lysosomes (g), the velocity of vesicles (i) remains roughly the same after MARK2 induction, but there is a notable increase in the run length. Error bars indicate SEM.
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fig4: Influence of MARK2 on the transport of vesicles and organelles. (a) Tracking pattern of GFP-VSVG–tagged vesicles in noninduced CHO cells (controls) and (b) in MARK2-induced CHO cells. Dots connected by a line identify consecutive positions of a vesicle (1-s time intervals, arrowheads indicate starting points). The coordinates are centered on the TGN (labeled strongly with GFP-VSVG) near the microtubule organizing center. Upon MARK induction the tracks become longer and straighter, indicating that fewer directional changes occur. (c–e) Quantification of GFP-VSVG–tagged vesicle movement in noninduced CHO cells (black bars) and MARK2-induced cells (gray bars). The data are based on the vesicle motion recorded from 30 cells, 150 vesicles were analyzed, error bars = SEM. (c) Velocities remain unchanged after the induction of MARK2 in either direction (inbound v∼0.9μm/s, outbound ∼0.7 μm/s; n > 500). (d) Reversal frequencies decrease strongly after MARK2 induction (∼0.04 s−1 with MARK2, ∼0.09 s−1 without MARK2, both for inbound/outbound and outbound/inbound reversals; n > 100). (e) Run lengths increase strongly (∼50%) upon MARK2 induction in both directions (n > 100). Note that both with and without MARK2 the inbound run lengths are ∼30% longer than outbound (4.6 μm inbound, 3.6 μm outbound without MARK2). (f and g) Motion of lysosomes. (f) This panel shows an overlay of 60 images separated by 1 s. Lysosomes move on linear tracks >1 μm (arrows). Particles that are immobile or show only short Brownian movements (≤0.3 μm and random direction) can also be observed (spots marked by arrowheads). (g) Velocities remain unchanged upon MARK2 induction (∼0.8 μm/s), but run lengths increase (from 3 to 5 μm). Reversal frequencies and directionalities cannot be determined because in CHO cells labeled with LysoTracker there is no internal reference indicating the cell's interior. (h) Endocytotic vesicles: CHO cells were transiently transfected with EGFP-labeled β2-adaptin, a marker of the AP-2 complex of endocytotic clathrin-coated vesicles. During the time of observation shown here (90 s) fluorescent paths are clearly visible. Linear or curved tracks longer than 1 μm represent actively transported vesicles (arrows), broader black or gray spots represent immobile particles and/or vesicles showing Brownian motion (arrowheads). The overexpression of GFP/β2-adaptin causes a strong background noise in the cytoplasm and the nucleus (N) becomes visible. The rather immobile fluorescent patches in the more peripheral regions of the cell are clathrin-coated vesicles or their precursors (arrowheads; Laporte et al., 1999). In the interior of the cell near the nucleus (N) an accumulation of the fluorescent signal can be observed suggesting that EGFP/β2-adaptin also colocalizes with the exocytotic and/or endosomal compartment. This is supported by the occurrence of fast moving vesicles entering or leaving this area. In contrast to the post-Golgi vesicles labeled by transient transfection of GFP-VSVG, the system here cannot provide reversal frequencies because of the strong background that shortens the time in which the particles are clearly seen in focus. (i) Velocities remain unchanged by MARK2 (∼1 μm/s), but run lengths increase almost twofold. The tracks illustrate three scenarios: the period of motion observed within the plane of focus is flanked by (1) periods of active transport out of focus, (2) resting states in focus and active transport out of focus, and (3) periods of pauses in focus. We only analyzed events of movement which were clearly in focus and which show at least two stop events in order to determine the run length. As in the case of lysosomes (g), the velocity of vesicles (i) remains roughly the same after MARK2 induction, but there is a notable increase in the run length. Error bars indicate SEM.
Mentions: Indeed, when MARK2 is induced by addition of doxycyclin in CHO cells there is a dramatic effect on vesicle behavior (Fig. 4). The predominant MAP in CHO cells is a variant of MAP4, one of the high-molecular weight MAPs that control microtubule dynamics (Bulinski and Borisy, 1980; Olson et al., 1995). If the endogenous MAP4 represents obstacles to vesicles on the microtubule track the detachment of MAP4 should facilitate movement. This is indeed seen in the representative traces of Fig. 4 a. The distances traversed during the observation time are normally small (a few micrometers) because there are many kinks where the vesicles change direction. However, induction of MARK2 leads to much more extended tracks (Fig. 4 b). The instantaneous velocities are not affected by MARK (∼1 μm/s; Fig. 4 c), but the reversal frequencies decrease by ∼50% (Fig. 4 d), and the run lengths increase by ∼50% in both directions (Fig. 4 e). Because MARK2 phosphorylates MAP4 or other MAPs at KXGS motifs in the repeat domain and detaches them from microtubules (Illenberger et al., 1996), these results argue that vesicle movement is facilitated and can indeed be regulated locally on the microtubule surface by MAP phosphorylation. This effect is opposite to the transport inhibition by elevated MAP expression (demonstrated in Fig. 1 by the clustering of mitochondria), so that MAPs and their kinases can be regarded as antagonistic with respect to microtubule-based vesicle trafficking.

Bottom Line: The transport can be regulated through motor proteins, cargo adaptors, or microtubule tracks.This occurs without impairing the intrinsic activity of motors because the velocity during active movement remains unchanged.This transport inhibition can be rescued by phosphorylating tau with MARK.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck Unit for Structural Molecular Biology, 22607 Hamburg, Germany. mandelkow@mpasmb.desy.de

ABSTRACT
Microtubule-dependent transport of vesicles and organelles appears saltatory because particles switch between periods of rest, random Brownian motion, and active transport. The transport can be regulated through motor proteins, cargo adaptors, or microtubule tracks. We report here a mechanism whereby microtubule associated proteins (MAPs) represent obstacles to motors which can be regulated by microtubule affinity regulating kinase (MARK)/Par-1, a family of kinases that is known for its involvement in establishing cell polarity and in phosphorylating tau protein during Alzheimer neurodegeneration. Expression of MARK causes the phosphorylation of MAPs at their KXGS motifs, thereby detaching MAPs from the microtubules and thus facilitating the transport of particles. This occurs without impairing the intrinsic activity of motors because the velocity during active movement remains unchanged. In primary retinal ganglion cells, transfection with tau leads to the inhibition of axonal transport of mitochondria, APP vesicles, and other cell components which leads to starvation of axons and vulnerability against stress. This transport inhibition can be rescued by phosphorylating tau with MARK.

Show MeSH
Related in: MedlinePlus