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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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Motion analysis of vesicles and organelles. MARK2 is expressed inducibly in CHO cells under the control of the tetracycline responsive promoter. (a) Upon induction with 1 μg/ml doxycyclin for 12–18 h, HA-tagged MARK2 becomes detectable by the polyclonal rabbit HA-tag antibody in the cells (∼100% in the induced state). Because prolonged periods of MARK expression (24–72 h) destroy the microtubule network the motility assays were done at earlier time points where >90% of cells have normal microtubules and the MTOC is preserved. (b) Immunofluorescence of cells by anti-tubulin antibody DM1A showing that most cells are HA-MARK positive (a) and have a normal microtubule network. (c) The movement of an individual vesicle (arrowheads) was recorded during a period of 60 s (shown in 5-s intervals). TGN identifies the trans-Golgi network that was taken as a reference to distinguish between centripetal and centrifugal movements. (d) Single particle tracking of the vesicle shown in panel a during the 60 s with higher time resolution (1 s). The starting and end points are indicated by arrowheads. Note the reversal in direction at point R. (e) Instantaneous velocities of vesicle shown in panels c and d versus time. The diagram distinguishes between centripetal (toward the TGN, negative values) and centrifugal (away from the TGN, positive values) movements of the vesicle. Velocities below 0.3 μm/s (marked by horizontal lines) were counted as Brownian motion rather than motor-driven movement and not used in the subsequent statistical analysis.
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fig3: Motion analysis of vesicles and organelles. MARK2 is expressed inducibly in CHO cells under the control of the tetracycline responsive promoter. (a) Upon induction with 1 μg/ml doxycyclin for 12–18 h, HA-tagged MARK2 becomes detectable by the polyclonal rabbit HA-tag antibody in the cells (∼100% in the induced state). Because prolonged periods of MARK expression (24–72 h) destroy the microtubule network the motility assays were done at earlier time points where >90% of cells have normal microtubules and the MTOC is preserved. (b) Immunofluorescence of cells by anti-tubulin antibody DM1A showing that most cells are HA-MARK positive (a) and have a normal microtubule network. (c) The movement of an individual vesicle (arrowheads) was recorded during a period of 60 s (shown in 5-s intervals). TGN identifies the trans-Golgi network that was taken as a reference to distinguish between centripetal and centrifugal movements. (d) Single particle tracking of the vesicle shown in panel a during the 60 s with higher time resolution (1 s). The starting and end points are indicated by arrowheads. Note the reversal in direction at point R. (e) Instantaneous velocities of vesicle shown in panels c and d versus time. The diagram distinguishes between centripetal (toward the TGN, negative values) and centrifugal (away from the TGN, positive values) movements of the vesicle. Velocities below 0.3 μm/s (marked by horizontal lines) were counted as Brownian motion rather than motor-driven movement and not used in the subsequent statistical analysis.

Mentions: Our next aim was to monitor the movement of vesicles or organelles along microtubules and to record the parameters of speed, run length, changes in direction, and how they depend on kinases such as MARK. To test whether the phosphorylation of MAPs and their detachment from microtubules would lead to changes in mobility we expressed MARK2 in a controlled fashion in CHO cells under the inducible tet-on expression protocol (Gossen and Bujard, 2002) and chose early time points of MARK2 expression where the microtubule network was still intact. Fig. 3 a shows the punctate distribution of MARK2. The microtubule network is still intact, and there is no sign of colocalization of MARK2 with microtubules (Fig. 3 b).


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)

Motion analysis of vesicles and organelles. MARK2 is expressed inducibly in CHO cells under the control of the tetracycline responsive promoter. (a) Upon induction with 1 μg/ml doxycyclin for 12–18 h, HA-tagged MARK2 becomes detectable by the polyclonal rabbit HA-tag antibody in the cells (∼100% in the induced state). Because prolonged periods of MARK expression (24–72 h) destroy the microtubule network the motility assays were done at earlier time points where >90% of cells have normal microtubules and the MTOC is preserved. (b) Immunofluorescence of cells by anti-tubulin antibody DM1A showing that most cells are HA-MARK positive (a) and have a normal microtubule network. (c) The movement of an individual vesicle (arrowheads) was recorded during a period of 60 s (shown in 5-s intervals). TGN identifies the trans-Golgi network that was taken as a reference to distinguish between centripetal and centrifugal movements. (d) Single particle tracking of the vesicle shown in panel a during the 60 s with higher time resolution (1 s). The starting and end points are indicated by arrowheads. Note the reversal in direction at point R. (e) Instantaneous velocities of vesicle shown in panels c and d versus time. The diagram distinguishes between centripetal (toward the TGN, negative values) and centrifugal (away from the TGN, positive values) movements of the vesicle. Velocities below 0.3 μm/s (marked by horizontal lines) were counted as Brownian motion rather than motor-driven movement and not used in the subsequent statistical analysis.
© Copyright Policy
Related In: Results  -  Collection

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fig3: Motion analysis of vesicles and organelles. MARK2 is expressed inducibly in CHO cells under the control of the tetracycline responsive promoter. (a) Upon induction with 1 μg/ml doxycyclin for 12–18 h, HA-tagged MARK2 becomes detectable by the polyclonal rabbit HA-tag antibody in the cells (∼100% in the induced state). Because prolonged periods of MARK expression (24–72 h) destroy the microtubule network the motility assays were done at earlier time points where >90% of cells have normal microtubules and the MTOC is preserved. (b) Immunofluorescence of cells by anti-tubulin antibody DM1A showing that most cells are HA-MARK positive (a) and have a normal microtubule network. (c) The movement of an individual vesicle (arrowheads) was recorded during a period of 60 s (shown in 5-s intervals). TGN identifies the trans-Golgi network that was taken as a reference to distinguish between centripetal and centrifugal movements. (d) Single particle tracking of the vesicle shown in panel a during the 60 s with higher time resolution (1 s). The starting and end points are indicated by arrowheads. Note the reversal in direction at point R. (e) Instantaneous velocities of vesicle shown in panels c and d versus time. The diagram distinguishes between centripetal (toward the TGN, negative values) and centrifugal (away from the TGN, positive values) movements of the vesicle. Velocities below 0.3 μm/s (marked by horizontal lines) were counted as Brownian motion rather than motor-driven movement and not used in the subsequent statistical analysis.
Mentions: Our next aim was to monitor the movement of vesicles or organelles along microtubules and to record the parameters of speed, run length, changes in direction, and how they depend on kinases such as MARK. To test whether the phosphorylation of MAPs and their detachment from microtubules would lead to changes in mobility we expressed MARK2 in a controlled fashion in CHO cells under the inducible tet-on expression protocol (Gossen and Bujard, 2002) and chose early time points of MARK2 expression where the microtubule network was still intact. Fig. 3 a shows the punctate distribution of MARK2. The microtubule network is still intact, and there is no sign of colocalization of MARK2 with microtubules (Fig. 3 b).

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