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Fission yeast mitochondria are distributed by dynamic microtubules in a motor-independent manner.

Li T, Zheng F, Cheung M, Wang F, Fu C - Sci Rep (2015)

Bottom Line: Intriguingly a chimeric molecule carrying an actin binding domain and tom22p results in mitochondria associated with actin filaments at the actomyosin ring during mitosis, leading to cytokinesis defects.These findings suggest that the passive motor-independent microtubule-based mechanism is the major contributor to mitochondria distribution in wild type fission yeast cells.Hence, we establish that attachment to microtubules, but not kinesin-dependent movement and the actin cytoskeleton, is required and crucial for proper mitochondria distribution in fission yeast.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry [2] HKU-Shenzhen Institute of Research and Innovation, The University of Hong Kong, Shenzhen, China.

ABSTRACT
The cytoskeleton plays a critical role in regulating mitochondria distribution. Similar to axonal mitochondria, the fission yeast mitochondria are distributed by the microtubule cytoskeleton, but this is regulated by a motor-independent mechanism depending on the microtubule associated protein mmb1p as the absence of mmb1p causes mitochondria aggregation. In this study, using a series of chimeric proteins to control the subcellular localization and motility of mitochondria, we show that a chimeric molecule containing a microtubule binding domain and the mitochondria outer membrane protein tom22p can restore the normal interconnected mitochondria network in mmb1-deletion (mmb1∆) cells. In contrast, increasing the motility of mitochondria by using a chimeric molecule containing a kinesin motor domain and tom22p cannot rescue mitochondria aggregation defects in mmb1∆ cells. Intriguingly a chimeric molecule carrying an actin binding domain and tom22p results in mitochondria associated with actin filaments at the actomyosin ring during mitosis, leading to cytokinesis defects. These findings suggest that the passive motor-independent microtubule-based mechanism is the major contributor to mitochondria distribution in wild type fission yeast cells. Hence, we establish that attachment to microtubules, but not kinesin-dependent movement and the actin cytoskeleton, is required and crucial for proper mitochondria distribution in fission yeast.

No MeSH data available.


Related in: MedlinePlus

A chimera of klp3 motor domain and tom22p can drive mitochondria movement in vivo.(a) Quantification of the patterns of mitochondria distribution in the indicated cells. Compared to mmb1∆ cells, none of the motor-deletion mutants, either alone or in combination, caused significant mitochondria aggregation defects. (b) Schematic diagram depicting the subcellular localization of klp3(MDo)-TagRFP-tom22. The motor domain (a.a. 1–335) of klp3p is fused to TagRFP tagged tom22 at its N-terminus. (c) Maximum projection images of wild type cells expressing GFP-atb2p and klp3p(MDo)-TagRFP-tom22 (from an ase1p promoter). Cells were treated either with DMSO or with MBC for 10 min before imaging. Klp3p(MDo)-TagRFP-tom22 colocalized with microtubules and exhibited amorphous cytoplasmic localization upon microtubule depolymerization with MBC. Scale bar, 5 μm. (d) Maximum projection images of wild type cells expressing GFP-cox4 and klp3p(MDo)-TagRFP-tom22. Klp3p(MDo)-TagRFP-tom22 colocalized with mitochondria in the cytoplasm. Scale bar, 5 μm. (e) Maximum projection time-lapse images of a cell expressing GFP-atb2p and klp3p(MDo)-TagRFP-tom22. A divided mitochondrion (red arrowhead) marked by klp3p(MDo)-TagRFP-tom22 rapidly moved along the microtubule lattice towards the cell tip (red arrows). Green and red dash lines mark microtubule and mitochondria tips, respectively. Montage images were constructed using the region indicated in the analyzed cell. Scale bar, 5 μm. (f) A microtubule fragment (green dash lines) was slid away from the cell tip and towards the cell center (green arrows) by a stationary mitochondrion. Montage images were constructed using the region indicated in the analyzed cell. Scale bar, 5 μm
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f2: A chimera of klp3 motor domain and tom22p can drive mitochondria movement in vivo.(a) Quantification of the patterns of mitochondria distribution in the indicated cells. Compared to mmb1∆ cells, none of the motor-deletion mutants, either alone or in combination, caused significant mitochondria aggregation defects. (b) Schematic diagram depicting the subcellular localization of klp3(MDo)-TagRFP-tom22. The motor domain (a.a. 1–335) of klp3p is fused to TagRFP tagged tom22 at its N-terminus. (c) Maximum projection images of wild type cells expressing GFP-atb2p and klp3p(MDo)-TagRFP-tom22 (from an ase1p promoter). Cells were treated either with DMSO or with MBC for 10 min before imaging. Klp3p(MDo)-TagRFP-tom22 colocalized with microtubules and exhibited amorphous cytoplasmic localization upon microtubule depolymerization with MBC. Scale bar, 5 μm. (d) Maximum projection images of wild type cells expressing GFP-cox4 and klp3p(MDo)-TagRFP-tom22. Klp3p(MDo)-TagRFP-tom22 colocalized with mitochondria in the cytoplasm. Scale bar, 5 μm. (e) Maximum projection time-lapse images of a cell expressing GFP-atb2p and klp3p(MDo)-TagRFP-tom22. A divided mitochondrion (red arrowhead) marked by klp3p(MDo)-TagRFP-tom22 rapidly moved along the microtubule lattice towards the cell tip (red arrows). Green and red dash lines mark microtubule and mitochondria tips, respectively. Montage images were constructed using the region indicated in the analyzed cell. Scale bar, 5 μm. (f) A microtubule fragment (green dash lines) was slid away from the cell tip and towards the cell center (green arrows) by a stationary mitochondrion. Montage images were constructed using the region indicated in the analyzed cell. Scale bar, 5 μm

Mentions: Although both neurons and fission yeast utilize microtubules to distribute mitochondria, they accomplish this by apparently different approaches. In axons, an active mechanism is employed, with kinesins and dynein responsible for anterograde and retrograde transport of mitochondria, respectively10. However, in the fission yeast cells, it appears that mitochondria are distributed passively through attachment to dynamic microtubules9. It has been implicated that the conventional kinesin-1 klp3p has no role in mitochondria distribution in fission yeast11. However, there are six kinesins (i.e. klp2p16, klp3p11, tea2p17, klp5p1819, klp6p1819, klp8p20) and one dynein21 present in the cytoplasm of fission yeast, and therefore they may be potential players in mitochondria distribution. Among these motor proteins, klp2p and dynein are minus-end-directed while klp3p, tea2p, klp5p, klp6p, and klp8p are plus-end-directed motor proteins. To test if these motor proteins are involved in regulating mitochondria distribution, our first attempt was to systematically examine the patterns of mitochondria distribution in single kinesin- and dynein-deletion mutant strains. As shown in Fig. 2a, only klp3∆, tea2∆ and klp8∆ mutant cells showed a very small degree of mitochondria aggregation, comparable to mal3∆ cells the absence of which has been known to result in short microtubules, thus indirectly causing mitochondria aggregation22. These data extended previous findings that kinesins and dynein did not play a major and direct role in fission yeast mitochondria distribution. To reinforce this conclusion, functional redundancy of the motor proteins should be considered. As the motor proteins have multiple roles in regulating mitosis and meiosis, it remains a technical challenge to knock out all six kinesins and dynein to assess the effect of combined deletions on mitochondria distribution. Instead, we sought to simultaneously knock out motor proteins with the same directionality. Since klp8p is localized to the actomyosin ring at the cell cortex20, it is unlikely that klp8p is involved in mitochondria distribution. In addition, klp5p and klp6p function as a heterodimer19, and it is therefore conceivable that deletion of either one would lead to identical phenotypes. As shown in Fig. 2a, the double and triple deletion mutants displayed comparable patterns of mitochondria distribution to the single deletion mutant. Hence, we conclude that kinesins and dynein play a negligible role in mitochondria distribution in fission yeast.


Fission yeast mitochondria are distributed by dynamic microtubules in a motor-independent manner.

Li T, Zheng F, Cheung M, Wang F, Fu C - Sci Rep (2015)

A chimera of klp3 motor domain and tom22p can drive mitochondria movement in vivo.(a) Quantification of the patterns of mitochondria distribution in the indicated cells. Compared to mmb1∆ cells, none of the motor-deletion mutants, either alone or in combination, caused significant mitochondria aggregation defects. (b) Schematic diagram depicting the subcellular localization of klp3(MDo)-TagRFP-tom22. The motor domain (a.a. 1–335) of klp3p is fused to TagRFP tagged tom22 at its N-terminus. (c) Maximum projection images of wild type cells expressing GFP-atb2p and klp3p(MDo)-TagRFP-tom22 (from an ase1p promoter). Cells were treated either with DMSO or with MBC for 10 min before imaging. Klp3p(MDo)-TagRFP-tom22 colocalized with microtubules and exhibited amorphous cytoplasmic localization upon microtubule depolymerization with MBC. Scale bar, 5 μm. (d) Maximum projection images of wild type cells expressing GFP-cox4 and klp3p(MDo)-TagRFP-tom22. Klp3p(MDo)-TagRFP-tom22 colocalized with mitochondria in the cytoplasm. Scale bar, 5 μm. (e) Maximum projection time-lapse images of a cell expressing GFP-atb2p and klp3p(MDo)-TagRFP-tom22. A divided mitochondrion (red arrowhead) marked by klp3p(MDo)-TagRFP-tom22 rapidly moved along the microtubule lattice towards the cell tip (red arrows). Green and red dash lines mark microtubule and mitochondria tips, respectively. Montage images were constructed using the region indicated in the analyzed cell. Scale bar, 5 μm. (f) A microtubule fragment (green dash lines) was slid away from the cell tip and towards the cell center (green arrows) by a stationary mitochondrion. Montage images were constructed using the region indicated in the analyzed cell. Scale bar, 5 μm
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f2: A chimera of klp3 motor domain and tom22p can drive mitochondria movement in vivo.(a) Quantification of the patterns of mitochondria distribution in the indicated cells. Compared to mmb1∆ cells, none of the motor-deletion mutants, either alone or in combination, caused significant mitochondria aggregation defects. (b) Schematic diagram depicting the subcellular localization of klp3(MDo)-TagRFP-tom22. The motor domain (a.a. 1–335) of klp3p is fused to TagRFP tagged tom22 at its N-terminus. (c) Maximum projection images of wild type cells expressing GFP-atb2p and klp3p(MDo)-TagRFP-tom22 (from an ase1p promoter). Cells were treated either with DMSO or with MBC for 10 min before imaging. Klp3p(MDo)-TagRFP-tom22 colocalized with microtubules and exhibited amorphous cytoplasmic localization upon microtubule depolymerization with MBC. Scale bar, 5 μm. (d) Maximum projection images of wild type cells expressing GFP-cox4 and klp3p(MDo)-TagRFP-tom22. Klp3p(MDo)-TagRFP-tom22 colocalized with mitochondria in the cytoplasm. Scale bar, 5 μm. (e) Maximum projection time-lapse images of a cell expressing GFP-atb2p and klp3p(MDo)-TagRFP-tom22. A divided mitochondrion (red arrowhead) marked by klp3p(MDo)-TagRFP-tom22 rapidly moved along the microtubule lattice towards the cell tip (red arrows). Green and red dash lines mark microtubule and mitochondria tips, respectively. Montage images were constructed using the region indicated in the analyzed cell. Scale bar, 5 μm. (f) A microtubule fragment (green dash lines) was slid away from the cell tip and towards the cell center (green arrows) by a stationary mitochondrion. Montage images were constructed using the region indicated in the analyzed cell. Scale bar, 5 μm
Mentions: Although both neurons and fission yeast utilize microtubules to distribute mitochondria, they accomplish this by apparently different approaches. In axons, an active mechanism is employed, with kinesins and dynein responsible for anterograde and retrograde transport of mitochondria, respectively10. However, in the fission yeast cells, it appears that mitochondria are distributed passively through attachment to dynamic microtubules9. It has been implicated that the conventional kinesin-1 klp3p has no role in mitochondria distribution in fission yeast11. However, there are six kinesins (i.e. klp2p16, klp3p11, tea2p17, klp5p1819, klp6p1819, klp8p20) and one dynein21 present in the cytoplasm of fission yeast, and therefore they may be potential players in mitochondria distribution. Among these motor proteins, klp2p and dynein are minus-end-directed while klp3p, tea2p, klp5p, klp6p, and klp8p are plus-end-directed motor proteins. To test if these motor proteins are involved in regulating mitochondria distribution, our first attempt was to systematically examine the patterns of mitochondria distribution in single kinesin- and dynein-deletion mutant strains. As shown in Fig. 2a, only klp3∆, tea2∆ and klp8∆ mutant cells showed a very small degree of mitochondria aggregation, comparable to mal3∆ cells the absence of which has been known to result in short microtubules, thus indirectly causing mitochondria aggregation22. These data extended previous findings that kinesins and dynein did not play a major and direct role in fission yeast mitochondria distribution. To reinforce this conclusion, functional redundancy of the motor proteins should be considered. As the motor proteins have multiple roles in regulating mitosis and meiosis, it remains a technical challenge to knock out all six kinesins and dynein to assess the effect of combined deletions on mitochondria distribution. Instead, we sought to simultaneously knock out motor proteins with the same directionality. Since klp8p is localized to the actomyosin ring at the cell cortex20, it is unlikely that klp8p is involved in mitochondria distribution. In addition, klp5p and klp6p function as a heterodimer19, and it is therefore conceivable that deletion of either one would lead to identical phenotypes. As shown in Fig. 2a, the double and triple deletion mutants displayed comparable patterns of mitochondria distribution to the single deletion mutant. Hence, we conclude that kinesins and dynein play a negligible role in mitochondria distribution in fission yeast.

Bottom Line: Intriguingly a chimeric molecule carrying an actin binding domain and tom22p results in mitochondria associated with actin filaments at the actomyosin ring during mitosis, leading to cytokinesis defects.These findings suggest that the passive motor-independent microtubule-based mechanism is the major contributor to mitochondria distribution in wild type fission yeast cells.Hence, we establish that attachment to microtubules, but not kinesin-dependent movement and the actin cytoskeleton, is required and crucial for proper mitochondria distribution in fission yeast.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry [2] HKU-Shenzhen Institute of Research and Innovation, The University of Hong Kong, Shenzhen, China.

ABSTRACT
The cytoskeleton plays a critical role in regulating mitochondria distribution. Similar to axonal mitochondria, the fission yeast mitochondria are distributed by the microtubule cytoskeleton, but this is regulated by a motor-independent mechanism depending on the microtubule associated protein mmb1p as the absence of mmb1p causes mitochondria aggregation. In this study, using a series of chimeric proteins to control the subcellular localization and motility of mitochondria, we show that a chimeric molecule containing a microtubule binding domain and the mitochondria outer membrane protein tom22p can restore the normal interconnected mitochondria network in mmb1-deletion (mmb1∆) cells. In contrast, increasing the motility of mitochondria by using a chimeric molecule containing a kinesin motor domain and tom22p cannot rescue mitochondria aggregation defects in mmb1∆ cells. Intriguingly a chimeric molecule carrying an actin binding domain and tom22p results in mitochondria associated with actin filaments at the actomyosin ring during mitosis, leading to cytokinesis defects. These findings suggest that the passive motor-independent microtubule-based mechanism is the major contributor to mitochondria distribution in wild type fission yeast cells. Hence, we establish that attachment to microtubules, but not kinesin-dependent movement and the actin cytoskeleton, is required and crucial for proper mitochondria distribution in fission yeast.

No MeSH data available.


Related in: MedlinePlus