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Address correspondence to Department of Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, Netherlands. Tel.: (31) 20-566-5127. Fax: (31) 20-691-5519. E-mail: h.f.tabak{at}amc.uva.nl
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Abstract |
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Key Words: peroxisome inheritance; Vps1; fission; actin cytoskeleton; Myo2p
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Introduction |
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Peroxisomes are small single-membrane-bounded organelles containing a set of enzymes allowing them to participate in cellular metabolism (van den Bosch et al., 1992). Loss of peroxisomal function is the cause of a number of diseases in man, ranging from relatively mild single enzyme deficiencies to severe syndromes in which the biogenesis of the whole organelle is compromised (Purdue and Lazarow, 1994). The severe peroxisomal biogenesis disorders (PBDs)* are caused by a loss of protein import into peroxisomes, marking most of the PBDs as "protein-trafficking" diseases. The identification of a large number of proteins required for trafficking of peroxisomal proteins now forms the basis for a detailed molecular analysis of the import process (Hettema et al., 1999; Subramani et al., 2000). In contrast, not much data are available as to how peroxisomes multiply and are inherited upon cell division. Current opinion holds that peroxisomes multiply by growth and subsequently divide by fission, and also that they constitute a branch of the autonomous organelle family and are segregated from mother to daughter cell (Lazarow and Fujiki, 1985). More recently, however, observations suggest that a peroxisomeER connection exists, indicating that the ER might contribute to the biogenesis of peroxisomes. It has been postulated that peroxisomes can be formed from ER membranes (Erdmann et al., 1997; Titorenko and Rachubinski, 1998; Tabak et al., 1999). Although in many cell types peroxisomes are present in appreciable numbers and a stochastic segregation principle could suffice, all the evidence obtained thus far indicates that the proper distribution of all primary organelles between mother and daughter cells depends on specialized segregation processes involving the cytoskeleton and motor proteins (Catlett and Weisman, 2000).
In fungi, two cytoskeletal systems involved in the organization, maintenance, and segregation of cellular components have been identified: microtubules and the actin cytoskeleton. Microtubules are organized by the spindle pole body (SPB), the functional homologue of the microtubule organizing center of higher eukaryotic cells. Since S. cerevisiae performs a closed mitosis without breakdown of the nuclear envelope, microtubules can be classified in nuclear microtubules and cytoplasmic astral microtubules (Byers and Goetsch, 1975; Byers, 1981). Nuclear microtubules are involved in assembly of a bipolar spindle and segregation of the chromosomes (Jacobs et al., 1988; Straight et al., 1997), astral microtubules function to position, move, orient, and finally segregate the nucleus between mother and daughter cell (Palmer et al., 1992; Sullivan and Huffaker, 1992; Carminati and Stearns, 1997; Shaw et al., 1997; Tirnauer et al., 1999; Hoepfner et al., 2000). Whereas polarized vesicular movement was also shown to be dependent on astral microtubules in animal cells (Rogalski et al., 1984; Schliwa, 1984; McNiven and Porter, 1986; Vale et al., 1986; Rindler et al., 1987) and filamentous fungi (Howard and Aist, 1980; Howard, 1981; Steinberg, 1998), no such involvement could be demonstrated so far in S. cerevisiae (Jacobs et al., 1988; reviewed by Bretscher et al., 1994).
The actin cytoskeleton in yeast consists of two different structures, patches and cables. Patches are cortical actinrich structures that generally cluster near regions of active secretion and therefore mark sites of growth (Adams and Pringle, 1984). Cables are long bundles of actin filaments that can span the whole cell (Adams and Pringle, 1984). Both types of actin structures behave in a cell cycledependent manner (Kilmartin and Adams, 1984; Ford and Pringle, 1991; for review see Madden and Snyder, 1998). Transport along the actin cytoskeleton of cargo such as mitochondria, vacuoles, and late Golgi compartment elements is dependent on myosin motor proteins (for review see Sellers, 2000). Myosins move in a defined directionality along the actin cytoskeleton, the polarity of which is apparent by the localization of the patches. So far, only plus enddirected myosins toward the patches have been described in S. cerevisiae.
In this study, we investigated the mechanisms supporting faithful inheritance of peroxisomes using in vivo time-lapse microscopy in S. cerevisiae. It represents a highly polarized cell in which vectorial transport of organelles and vesicles is an important feature in rapidly growing cells. We observed peroxisomal fission events and identified a mutant impaired in this process. In addition, we observed specific dynamics during peroxisomal segregation which suggests that this is a well controlled process. Furthermore, we found that peroxisome segregation requires a polymerized actin cytoskeleton and depends on the actin-associated motor protein, Myo2p.
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Results |
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Together, these observations in wild-type and vps1 mutant cells imply that peroxisomes can divide in a Vps1p-dependent and -independent manner and that their distribution between mother and daughter cell is not a stochastic event, but instead depends on an as yet unidentified, highly controlled segregation machinery.
Peroxisomes move independently from microtubules
Since peroxisomes were reported to be microtubule-associated organelles in mammalian cells (Rapp et al., 1996; Wiemer et al., 1997), we tested whether peroxisomal movement depends on microtubules.
We first analyzed if peroxisomes colocalized in vivo with astral microtubules. Therefore, we used a GFP-Tub1 construct to label all microtubules (Straight et al., 1997) and YFP-PTS1 to highlight the peroxisomes (Monosov et al., 1996; Brocard et al., 1997). Although GFP and YFP could not strictly be separated by fluorescent imaging techniques, the two different structures were easily identified by their typical morphology. The GFP signal of astral microtubules was very weak, preventing masking of the intensely labeled peroxisomes by the GFP signal of a microtubule. We analyzed 50 cells in different cell cycle stages from log phase cultures, but no direct interactions of microtubules and peroxisomes were apparent (Fig. 4 A). To further verify that peroxisomal dynamics did not depend on astral microtubules, we analyzed spc72 cells which are unable to generate long astral microtubules (Chen et al., 1998; Knop and Schiebel, 1998; Souès and Adams, 1998). In spite of the absence of long astral microtubules and completely mispositioned and misoriented spindles, the early bud localization of peroxisomes and faithful segregation into mother and daughter cells persisted (Fig. 4 B). Initial time-lapse analysis did not reveal any characteristic alterations in peroxisome dynamics in spc72
cells compared with wild-type cells (unpublished data). Also, experiments with the microtubule inhibitors nocodazole or benomyl showed no effect on peroxisome movement (unpublished data). Together our data indicate that peroxisomes are not dependent on microtubules for vectorial movements in S. cerevisiae.
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We analyzed 50 Cap2-YFP, CFP-PTS1labeled cells in different cell cycle stages. Z-axis scans performed through the cells using Cap2-YFP as a cortical reference revealed almost exclusive cortical localization of peroxisomes; however, although the general distribution of actin patches and peroxisomes was very similar, no tight colocalization of patches and peroxisomes was apparent (Fig. 5, AC). Nevertheless, the cortical localization and relative distribution was indicative of association of peroxisomes with actin cables. To test this hypothesis we performed time-lapse analysis of cells expressing both Cap2-YFP and CFP-PTS1 in wild-type cells. Since myosin/actin cablebased cargo movements have only been reported to occur toward the patch-covered plus end (the barbed end) in S. cerevisiae (Brown, 1997), we hypothesized that long range peroxisome migration on actin cables would be directed toward actin patches. To test this hypothesis, we analyzed the movement of CFP-PTS1labeled peroxisomes relative to Cap2-labeled actin patches in 38 wild-type cells. Long range movement of actin patches (translocations >1 µm) in the mother cell body strictly followed the actin polarity (Videos 68, available at http://www.jcb.org/cgi/content/full/jcb.200107028/DC1; representative examples extracted from Video 6 shown in Fig. 5 D). Peroxisomes exclusively moved toward the selected bud site. In small budded cells, they generally moved toward the bud tip where they formed clusters (Fig. 5 D, 0'70'). Although we observed oscillations back and forth, long range retrograde movement only occurred after the isotropic switch and reorientation of the actin cytoskeleton toward the motherdaughter junction, before cytokinesis (Fig. 5 D, 70'-145'). When the subsequent bud site was selected at the distal pole, the observed reorientation of the actin cytoskeleton was always accompanied by a change in the direction at which at least several peroxisomes moved. (Fig. 5 D, 145'175').
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Peroxisomal movement is dependent on the class V myosin Myo2
Movement along actin cables is mediated by myosin motors. In S. cerevisiae five myosin genes have been identified, two of class I, one of class II, and two of class V (for review see Sellers, 2000). Among these three classes, class V myosins are the most likely candidates for involvement in peroxisome segregation. They consist of an NH2-terminal motor domain, a neck domain, a coiled-coiled domain involved in dimerization, and a COOH-terminal cargo binding domain (Cheney et al., 1993; for reviews see Hildebrandt and Hoyt, 2000; Reck-Peterson et al., 2000). The two class V myosins in S. cerevisiae are named Myo2 and Myo4p. Myo2 is an essential protein. Analyses of temperature-sensitive MYO2 alleles have shown that Myo2 is directly involved in vesicular transport, vacuolar inheritance, and nuclear spindle orientation (Johnston et al., 1991; Govindan et al., 1995; Hill et al., 1996; Santos and Snyder, 1997; Catlett and Weisman, 1998; Schott et al., 1999; Beach et al., 2000; Yin et al., 2000). Myo4 is required for transport of specific mRNAs from the mother cell to the daughter cell (Bobola et al., 1996; Long et al., 1997; Takizawa et al., 1997). We expressed GFP-PTS1 in wild-type MYO2, myo212, myo216, myo220, myo266, and myo4 mutants (Johnston et al., 1991; Schott et al., 1999).
myo266 cells already at the permissive temperature were shown to be defective in vacuolar dynamics (Catlett and Weisman, 1998). When analyzing the localization of peroxisomes in the myo266 cells we observed a striking delay in insertion of peroxisomes into the bud (Fig. 9). 87% of all small-budded myo266 cells lacked peroxisomes in the bud, whereas this was the case in only 12% of wild-type cells (n = 250). Analysis of the other myo2-ts and the myo4 cells did not show significant alterations in tip localization of peroxisomes.
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Discussion |
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The constant number of peroxisomes per cell, the correct segregation of peroxisomes to the buds, and the rather precise control over the number of peroxisomes retained by the mother and delivered to the bud suggest the existence of various components taking part in peroxisome partitioning. In this respect, peroxisomes resemble mitochondria. Here a "retention zone" in the mother cell distal from the bud was defined in which a subset of mitochondria was retained by interaction with the actin cytoskeleton (Yang et al., 1999). Precise partitioning is even more obvious in the vps1 mutant. In all cases we observed that the single remaining peroxisome was partitioned equally well between mother and daughter. Whether this is achieved by a remaining fission capacity of another protein than Vps1p or is in part an indirect, physical effect of pulling forces acting from opposite sites remains to be studied.
Moreover, during our time-lapse analysis it became apparent that in a few vps1 mutant cells early insertion of the peroxisomal compartment failed. These cells showed a striking delay in cytokinesis compared with the other cells in the time-lapse sequence. This observation is similar to those made in some mdm and vac mutants (Weisman et al., 1990; McConnell and Yaffe, 1993; Xu and Wickner, 1996). This suggests the existence of a surveillance mechanism preventing the onset of cytokinesis until all organelles are properly partitioned. The conditional dependency of S. cerevisiae cells on peroxisomes and the reduced number of peroxisomes in the vps1
mutant could provide a valuable basis for genetic screens to identify putative components of such an organelle checkpoint mechanism.
We have studied the interaction of Vps1p with peroxisomes using a Vps1-YFP fusion protein and CFP-marked peroxisomes. Microscopic analysis of the double-labeled cells showed moderate cytoplasmic localization of the Vps1-YFP protein with additional spotted accumulations, most likely in the Golgi and post-Golgi compartments as suggested in the literature (Rothman et al., 1990). Due to the disperse localization of Vps1p, minor fractions always seemed to colocalize with peroxisomes. Coordinated movement of Vps1p with individual peroxisomes occurred and persisted for several minutes. We surmise that Vps1p only transiently interacts with peroxisomes and that fission events of peroxisomes can occur without accumulation of high concentrations of the Vps1 protein at the peroxisome membrane.
The orderly, track-like movements of peroxisomes in dividing wild-type cells suggested the involvement of cytoskeletal components. All our evidence indicates that peroxisomes are bound in the mother cell by the cortical actin skeleton and that they actively slide along actin filaments to the site of bud appearance and subsequently into the bud itself. Our time lapse analyses of Cap2-YFP, CFP-PTS1labeled cells revealed directed peroxisomal movement relative to the Cap2-labeled actin patches along the cell cortex strictly following the polarity of the actin cytoskeleton. In fixed cells stained for actin, all the GFP-PTS1 fluorescently labeled peroxisomes colocalize with actin polymers. The dynamic movements of peroxisomes in life cells were lost upon treatment of the cells with Lat-A, a poison that breaks down actin filaments. Finally, the protein responsible for tracking of peroxisomes through the cell is Myo2, a typical actin-based motor protein. This was inferred from the behavior of several thermosensitive alleles of Myo2. In all cases dynamic movements of peroxisomes stopped after bringing the cells to the nonpermissive temperature. In contrast, the movements were unaffected when wild-type or a Myo4p deletion mutant were inspected under the same conditions. Moreover, no colocalization of peroxisomes with the microtubule cytoskeleton was observed nor was any effect noted in a mutant (spc72) unable to form astral microtubules, which obviates the need for other motor proteins than Myo2p. Together, these results indicate that partitioning of peroxisomes between mother and bud is dependent on the actin cytoskeleton and an actin-specific motor protein. In analogy to mitochondria retention of a subset of peroxisomes in the mother cell may also require actin, suggesting a dual role for the actin cytoskeleton.
Myo2p has also been implicated in vectorial transport of secretory vesicles, late Golgi compartment, and the vacuole to the emerging bud (Johnston et al., 1991; Govindan et al., 1995; Hill et al., 1996; Catlett and Weisman, 1998; Schott et al., 1999; Karpova et al., 2000; Rossanese et al., 2001). Selectivity in recognizing the vacuolar cargo was ascribed to the myosin tail (Catlett and Weisman, 1998, 2000). For instance, a number of mutations defined a short region that was required for specific binding to the vacuole. It will be interesting to explore whether a similar tail region can be defined for binding to peroxisomes and to find out which peroxisomal membrane protein is the target for Myo2p interaction. This is also of clinical interest. There is a great latitude in loss of peroxisome function before organismal life is compromised. As a result, a number of diseases are known that are caused by diminished or even complete absence of peroxisome function. Considering the above mentioned specificity in Myo2p tail function and the predicted existence of a peroxisome-specific interaction partner, it is reasonable to expect that deficiencies of the corresponding homologs in humans can be the cause of disease. This prediction is supported by the occurrence of Griscelli disease (Pastural et al., 1997; Menasche et al., 2000; Wilson et al., 2000). Some forms of this syndrome are due to mutations in the human Myosin-Va protein, a homologue of yeast Myo2p. A mutation resulting in truncation of the protein after the motor domain and an amino acid substitution in front of the globular tail domain were reported. It will be interesting to study the behavior of peroxisomes in Griscelli patient cells and to test if similar diseases related to (partial) loss of peroxisome function exist. Further research in yeast can help to reveal the severity of the phenotypes resulting from mutations in proteins of the peroxisome segregation machinery and may help to predict how such diseases might present themselves in patients.
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Materials and methods |
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Plasmids pEW161, pEW171, and pEW213 were constructed as follows. pEW161: PCR amplification of the HIS3 promoter with 5'-GGGAATTCTATTACTCTTGGCCTCCTC-3' introducing an EcoR1 site at the 5' end and a SacI site at the 3' end with primer 5'-GCAAGATAAACGAAGGCAAAG-3'. The fragment was subsequently introduced into YIplac204 (Gretz and Sugino) (pEW156) and YIplac211 (pEW157). GFP-PTS1 (from pEW88; Hettema et al., 1998) was cloned into pEW156 and pEW157 cut with BamHI and HindIII resulting in pEW160 and pEW161, respectively. pEW171: the NcoI-BstBI fragment GFP-PTS1 derived from pEW160 was swapped with that of CFP or YFP. pEW213: genomic Vps1 was PCRed with primer 5'-GGATAGTATGATAGCTTCAGAG-3' and primer 5'-GGGCTGCAGAACAGAGGAGACGATTTGATAG-3' amplifying its promoter, the VPS1 gene without the stop codon and introducing a PstI site. This fragment has been ligated in frame with YFP in YIplac211 using EcoR1 and PstI.
For gene deletions we followed the EUROFAN guidelines (guidelines for EUROFAN B0 program ORF deletants, plasmid tools, and basic functional analyses are available at www.mips.biochem.mpg.de/proj/eurofan/index.html) for gene replacement in S. cerevisiae. We used the oligonucleotide pairs depicted in Table II for generation of the KanMX4, deletion cassettes, or YFP fusion constructs. To label microtubules we integrated plasmid pAFS125 into the ura31 locus (Straight et al., 1997). To label peroxisomes we integrated pEW161 or pEW177 into the ura3 or pEW171 into the trp1 locus. Microtubules or peroxisomes were clearly observable under the fluorescence microscope upon successful transformation. Labeling of Vps1 was performed by integrating pEW213 into the ura3 locus. Correct integration rescued the peroxisomal morphology defect and the temperature sensitivity of vps1
strains.
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Acquisition of still images
YFP-PTS1, CFP-PTS1, Cap2-YFP, and GFP-Tub1labeled wild-type and spc72 strains were grown in YPD medium to early log phase at 30°C, 3 µl of the culture were spread on a poly-L-lysinetreated slide overlaid with a coverslip and immediately used for microscopy. To simultaneously visualize peroxisomes and the actin cytoskeleton, GFP-PTS1 cells were grown in 25 ml of YPD at 30°C to early log phase and then fixed and stained (Amberg, 1998) using modifications to preserve GFP fluorescence: the first formaldehyde fixation was reduced to 5 min, the second to 30 min, and the rhodamine-phalloidin staining time to 30 min. It was essential to keep the pH above 8.0. Subsequently, the cells were immediately used for analysis. We acquired five z-axis planes spaced by 0.8 µm. In each z-axis plane we acquired one GFP and one rhodamine image using a Plan-Neofluar 100x, 1.3 oil, PH3 objective and a 2x Optovar (ZEISS) with 5 s exposure times, 100% fluorescence transmission. Each individual plane was then processed using the two-dimensional deconvolution algorithm of the AutoDeblur software (AutoQuant Imaging). Separately, the GFP and rhodamine images where then averaged into one plane, scaled, and converted to 8-bit images. We used different scaling parameters for the mother cell bodies and buds of rhodamine channelacquired images due to the much weaker signal of the actin cables in the mother cell compared with the patches in the bud. Finally, the phase-contrast, GFP, and rhodamine images were overlaid using Metamorphs "Overlay" function with default color balance settings using false color settings as depicted in the figure legend.
In vivo microscopy procedures and techniques
The video microscopy setup and in vivo time lapse procedures using GFP-PTS1, CFP-PTS1, Cap2-YFP, and GFP-Tub1labeled strains were described by Hoepfner et al. (2000). Imaging of the YFP/CFP variant of GFP was performed using filter sets 41028 and 31044v2 (Chroma Technology Corp.). For long term in vivo time-lapses we used diploid cells because spreading of the cells was better due to bipolar budding. Acquisition settings like interval time, exposure time, excitation light transmission, the number, and spacing of the z-axis planes are indicated in the video legends. To analyze the temperature-sensitive mutants at the nonpermissive temperature we used a temperature-adjustable stage (Biowerk). Acquisition and processing of images was performed using the Metamorph 4.1 program (Universal Imaging Corp.). Acquired z-axis stacks were merged into one plane using the "stack arithmetic: maximum" command of Metamorph. Stored images were then scaled and converted to 8-bit files. False color look-up tables were assigned to the individual channels as indicated in the figure legends. The phase-contrast and fluorescence 8-bit planes were then overlaid using the built-in "Overlay" command with default color balance.
Online supplemental material
For time-lapse analysis we assembled the picture files to videos in QuickTime format (Apple Computer) with a frame rate of 10 frames per second using the Premiere 4.2 program (Adobe Systems Europe). The videos are platform-independent and can be viewed using QuickTime movie player that can be downloaded at www.apple.com. Explanatory remarks and detailed information about acquisition settings are shown in the individual video legends associated with the figures showing representative still images of the time-lapse sequences. Videos are available at http://www.jcb.org/cgi/content/full/jcb.200107028/DC1.
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Footnotes |
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Ewald H. Hettema's present address is Cell Biology Division, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
* Abbreviations used in this paper: CFP, cyan fluorescent protein; GFP, green fluorescent protein; Lat-A, Latrunculin A; PBD, peroxisomal biogenesis disorder; PTS1, peroxisomal targeting signal type I; SPB, spindle pole body; YFP, yellow fluorescent protein.
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Acknowledgments |
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This work was supported by grants from the University of Basel and the Swiss Federal Office for Education and Science as well by the Netherlands Foundation for Scientific Research (NWO).
Submitted: 9 July 2001
Revised: 17 September 2001
Accepted: 16 October 2001
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References |
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