The class V myosin motor protein, Myo2, plays a major role in mitochondrial motility in Saccharomyces cerevisiae

Abstract

The actin cytoskeleton is essential for polarized, bud-directed movement of cellular membranes in Saccharomyces cerevisiae and thus ensures accurate inheritance of organelles during cell division. Also, mitochondrial distribution and inheritance depend on the actin cytoskeleton, though the precise molecular mechanisms are unknown. Here, we establish the class V myosin motor protein, Myo2, as an important mediator of mitochondrial motility in budding yeast. We found that mutants with abnormal expression levels of Myo2 or its associated light chain, Mlc1, exhibit aberrant mitochondrial morphology and loss of mitochondrial DNA. Specific mutations in the globular tail of Myo2 lead to aggregation of mitochondria in the mother cell. Isolated mitochondria lacking functional Myo2 are severely impaired in their capacity to bind to actin filaments in vitro. Time-resolved fluorescence microscopy revealed a block of bud-directed anterograde mitochondrial movement in cargo binding-defective myo2 mutant cells. We conclude that Myo2 plays an important and direct role for mitochondrial motility and inheritance in budding yeast.

Figure 1.

Myo2 and Mlc1 are involved in mitochondrial morphology and inheritance. (A) Serial dilutions of wild-type (WT), TetO₇-myo2, and TetO₇-mlc1 cell suspensions were spotted onto plates with glucose-containing medium with or without 30 μg/ml Dox and incubated for three days at 30°C. (B) WT, TetO₇-myo2, and TetO₇-mlc1 cells expressing mtGFP were grown for the indicated time periods in liquid glucose-containing medium in the absence or presence of 30 μg/ml Dox. Actin was stained with rhodamine phalloidin in fixed cells and cells were observed by fluorescence microscopy. (left) DIC image. (second from the left) Mitochondrial morphology (mito). (second from the right) Organization of filamentous actin (a reversed fluorescence image is shown to better visualize faint actin cables). (right) Merged image of reversed actin fluorescence and mitochondrial fluorescence. The graph at the bottom is a quantification of phenotypes of 100 cells per time point. Bar, 5 μm. (C) WT, TetO₇-myo2, and TetO₇-mlc1 cells were grown to logarithmic growth phase in glucose-containing medium. TetO₇-myo2 and TetO₇-mlc1 cells were incubated in the presence of 30 μg/ml Dox for 15 h to deplete Myo2 or Mlc1. Ultrathin sections were analyzed by electron microscopy. All images are shown at the same magnification. Asterisks indicate the matrix space. (D) WT and TetO₇-myosin strains were grown in glucose-containing medium, cellular DNA was stained with DAPI, and cells were analyzed by DIC and fluorescence microscopy. Nuclei are seen as large fluorescent spots; mtDNA nucleoids are seen as small fluorescent foci. Bar, 5 μm.

Figure 2.

Myo2 is required for interaction of mitochondria with actin filaments in vitro. (A) Wild-type (WT) mitochondria were incubated with isolated actin filaments in the absence or presence of ATP. After centrifugation of mitochondria through a sucrose cushion, bound actin was detected by immunoblotting. The mitochondrial protein Tom40 served as a loading control. Lanes 1 and 2 show standard conditions; lanes 3 and 4 show mitochondria that were extracted with 1 M KCl before incubation with actin filaments; lanes 5 and 6 show salt-extracted mitochondria that were incubated with salt extract (SE) before incubation with actin filaments. (B) WT, TetO₇-myo2, and TetO₇-mlc1 cells were precultured in glucose-containing medium and incubated for 15 h in the presence of 30 μg/ml Dox before isolation of mitochondria. Binding of actin to mitochondria was analyzed under standard conditions in A. (C) Binding of actin to wild-type and TetO₇-myo2 mitochondria was analyzed as in A. Lanes 1 and 2 show wild-type mitochondria analyzed under standard conditions; lanes 3 and 4 show TetO₇-myo2 mitochondria analyzed under standard conditions; lanes 5 and 6 show TetO₇-myo2 mitochondria that were incubated with wild-type SE before incubation with actin filaments. (D) Binding of actin to wild-type mitochondria was analyzed as in A. Lanes 1 and 2 show standard conditions; lanes 3 and 4 show mitochondria preincubated with affinity-purified Mdm31 antibodies at a concentration of 120 ng per mg of mitochondrial protein; lanes 5 and 6 show mitochondria preincubated with the same amount of affinity-purified Myo2 antibodies. (E) Replicate actin sedimentation experiments were quantified by densitometry of actin immunoblots. In each experiment, the signal obtained with wild-type mitochondria in the absence of ATP was set to 100. Bars represent mean values; circles represent individual data points of all measurements that have been performed. Arbitrary units are indicated. (F) Mitochondria isolated from mtGFP-expressing cells were incubated with Alexa Fluor 568–labeled actin filaments and observed by fluorescence microscopy. Depletion of Myo2 and Mlc1 and antibody pretreatments were performed as in A–E. Displayed are merged images of GFP and Alexa Fluor 568 fluorescence. An image of a sample incubated in the presence of ATP is only shown for wild-type mitochondria. Bar, 5 μm. (G) The graph shows a quantification of the experiment in Fig. 2 E. 200 mitochondria were scored per sample.

Figure 3.

Specific mutations in the Myo2 cargo-binding domain produce mitochondrial morphology and inheritance defects. (A) Wild-type (WT) and myo2 mutant cells expressing mtGFP were cultured in glucose-containing medium overnight at 30°C. Cultures were then diluted with fresh medium and either kept at 30°C (left) or shifted to 37°C for 3 h (right). Cells were analyzed by DIC and fluorescence microscopy. Asterisks indicate characteristic mitochondrial partitioning defects. Bar, 5 μm. (B) The graph shows a quantification of mitochondrial phenotypes scored in the experiment shown in A. Displayed is an excerpt of data presented in Table I. (C) Wild-type, myo2-L1301P, and myo2-Q1233R cells were grown as in A and analyzed as in Fig. 1 B. Bar, 5 μm.

Figure 4.

Mutations in the cargo-binding domain of Myo2 impair binding of mitochondria to actin filaments in vitro. (A) Wild-type (WT), myo2-L1301P, and myo2-Q1233R cultures were grown in glucose-containing minimal medium at 30°C and shifted to 37°C for 3 h before isolation of mitochondria. Binding of actin filaments to mitochondria under standard conditions was analyzed as in Fig. 2 A. (B) Replicate actin sedimentation experiments were quantified as in Fig. 2 E. Bars represent mean values; circles represent individual data points of all measurements that have been performed. (C) Cultures of mtGFP-expressing cells were grown as in A and the binding of mitochondria to actin filaments was analyzed as in Fig. 2 F. An image of a sample analyzed in the presence of ATP is shown only for wild-type mitochondria. Bar, 5 μm. (D) The graph shows a quantification of the experiment in Fig. 4 C. 200 mitochondria were scored per sample.

Figure 5.

Mutations in the cargo-binding domain of Myo2 impair anterograde mitochondrial movement and partitioning of mitochondria to the bud. (A) Wild-type and myo2-L1301P cells expressing mtGFP were precultured in glucose-containing minimal medium at 30°C, shifted to 37°C for 3 h, and observed by confocal time-lapse microscopy at ambient temperature. The first image of each series is a bright-field image; mitochondria are shown as maximum intensity projections of several optical planes. One representative cell carrying a large bud and one representative cell carrying a small bud are shown for the wild type and myo2(L1301P). Time is indicated in minutes. Bar, 5 μm. (B) Wild-type and myo2 mutant cells expressing mtGFP were grown as in Fig. 3 A and buds were scored for the presence of mitochondria. 200 budded cells were analyzed for each strain at each temperature.