Mechanisms of Organelle Inheritance in Dividing Plant Cells

Organelles form essential compartments of all eukaryotic cells. Mechanisms that ensure the unbiased inheritance of organelles during cell division are therefore necessary to maintain the viability of future cell generations. Although inheritance of organelles represents a fundamental component of the cell cycle, surprisingly little is known about the underlying mechanisms that facilitate unbiased organelle inheritance. Evidence from a select number of studies, however, indicates that ordered organelle inheritance strategies exist in dividing cells of higher plants. The basic requirement for unbiased organelle inheritance is the duplication of organelle volume and distribution of the resulting organelle populations in a manner that facilitates unbiased partitioning of the organelle population to each daughter cell. Often, partitioning strategies are specific to the organelle, being influenced by the functional requirements of the organelle and whether the cells are mitotically active or re-entering into the cell cycle. Organelle partitioning mechanisms frequently depend on interactions with either the actin or microtubule cytoskeleton. In this focused review, we attempt to summarize key findings regarding organelle partitioning strategies in dividing cells of higher plants. We particularly concentrate on the role of the cytoskeleton in mediating unbiased organelle partitioning.     

Orchestrating organelle inheritance in Saccharomyces cerevisiae

The biochemical functions of eukaryotic cells are often compartmentalized into membrane-bound organelles to increase their overall efficiency. Although some organelles can be formed anew, cells have evolved elaborate mechanisms to ensure the faithful inheritance of their organelles. In contrast to cells that divide by fission, the budding yeast Saccharomyces cerevisiae must actively and vectorially deliver half of its organelles to the growing bud. To achieve this, proteins called formins are strategically localized to the bud, where they assemble an array of actin cables that radiate deep into the mother cell. Class V myosin motors use these cables as tracks to transport various organelles, including peroxisomes, a portion of the vacuole and elements of the endoplasmic reticulum and Golgi complex. By contrast, mitochondria do not engage a myosin motor for their movement but instead use Arp2/3-nucleated actin polymerization for their bud-directed motility. The translocation machineries work cooperatively with molecular devices that retain organelles within both mother cell and bud to ensure an equitable division of organelles between them. While organelle inheritance requires specific proteins tailored for the inheritance of each type of organelle, it is becoming apparent that a set of fundamental rules underlies the inheritance of all organelles.     

Actin-filament-dependent remodeling of the vacuole in cultured mesophyll protoplasts

Summary. The ability of plant cells to dedifferentiate represents an important survival strategy invoked in a range of situations from repair mechanisms following wounding to apomixis. Dedifferentiation requires that somatic cells reprogram and enter the cell division cycle. This in turn necessitates the accurate partitioning of nuclear content and organelles, such as chloroplasts, to daughter cells, thereby ensuring continuity of cellular information systems. The distribution of cytoplasm and its organelle content in mature plant cells is governed by a large, central vacuole, with connections between distant cortical and perinuclear cytoplasmic domains mediated by transvacuolar strands. Here we examined the changes to vacuolar architecture in Arabidopsis thaliana protoplasts expressing a green-fluorescent protein fusion to a δ-tonoplast-intrinsic protein (δTIP). We found that vacuolar architecture became increasingly intricate during protoplast culture with the development of numerous transvacuolar strands. The development of an intricate vacuolar architecture was an actin filament- and not microtubule-dependent process, as is the case in interphase plant cells. Furthermore, we show that myosin is required for this increased complexity of vacuolar architecture and the formation of subcortical actin filament arrays. Despite the likelihood that increased vacuolar invagination would allow better redistribution of cytoplasmic organelles, we found that repositioning of chloroplasts from cortical to perinuclear cytoplasm was not dependent on transvacuolar strands. Our findings indicate that the vacuole is a dynamic entity that develops a complex architecture before dedifferentiating plant cells enter cell division. Supplementary material to this paper is available in electronic form at Correspondence and reprints: School of Environmental and Life Sciences, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.     

Peroxisomal peripheral membrane protein YlInp1p is required for peroxisome inheritance and influences the dimorphic transition in the yeast Yarrowia lipolytica

Eukaryotic cells have evolved molecular mechanisms to ensure the faithful inheritance of organelles by daughter cells in order to maintain the benefits afforded by the compartmentalization of biochemical functions. Little is known about the inheritance of peroxisomes, organelles of lipid metabolism. We have analyzed peroxisome dynamics and inheritance in the dimorphic yeast Yarrowia lipolytica. Most peroxisomes are anchored at the periphery of cells of Y. lipolytica. In vivo video microscopy showed that at cell division, approximately half of the anchored peroxisomes in the mother cell are dislodged individually from their static positions and transported to the bud. Peroxisome motility is dependent on the actin cytoskeleton. YlInp1p is a peripheral peroxisomal membrane protein that affects the partitioning of peroxisomes between mother cell and bud in Y. lipolytica. In cells lacking YlInp1p, most peroxisomes were transferred to the bud, with only a few remaining in the mother cell, while in cells overexpressing YlInp1p, peroxisomes were preferentially retained in the mother cell, resulting in buds nearly devoid of peroxisomes. Our results are consistent with a role for YlInp1p in anchoring peroxisomes in cells. YlInp1p has a role in the dimorphic transition in Y. lipolytica, as cells lacking the YlINP1 gene more readily convert from the yeast to the mycelial form in oleic acid-containing medium, the metabolism of which requires peroxisomal activity, than does the wild-type strain. This study reports the first analysis of organelle inheritance in a true dimorphic yeast and identifies the first protein required for peroxisome inheritance in Y. lipolytica.     

Spatial structure of mitochondria and ER denotes changes in cell physiology of cultured tobacco protoplasts

The structure of mitochondria and of the endoplasmic reticulum (ER) in mesophyll protoplasts and regenerated cells was studied in vivo using the dye DiOC₆(3) (3,3'-dihexyloxacarbocyanine iodide) and confocal laser scanning microscopy (CLSM). The relation to the cell's physiology was investigated using a hormone-based model system for elongation and division. The structure of the mitochondria and of their population depends on the status of the cell. In freshly isolated protoplasts small spherical mitochondria are clustered around the nucleus and the chloroplasts. During the first 4 days of culture they are transformed into long vermiform organelles which distribute evenly throughout the cytoplasm. In a medium containing only auxins, cells then enter a period of expansion. Their mitochondria retain the same structure but increase in quantity. In a medium with auxins and cytokinins cells start dividing. Their mitochondria typically become numerous and very small, and are distributed throughout the cytoplasm. Both types of organization were observed during weeks of ongoing expansion or division. The ER is always present as a network close to the cell membrane. In freshly isolated protoplasts a considerable part of the ER is clustered around the chloroplasts, the remaining part of the network has a reduced complexity and is partly broken. During subsequent protoplast culture the network grows into a complex web with fine meshes incorporating lots of plate-like structures. This is the case in elongating cells as well as in dividing cells. Finally, the ER looks similar to the structure found in epidermal cells of the intact plant.     

The cytoskeleton maintains organelle partitioning required for single-cell C4 photosynthesis in Chenopodiaceae species

Recently, three Chenopodiaceae species, Bienertia cycloptera, Bienertia sinuspersici, and Suaeda aralocaspica, were shown to possess novel C(4) photosynthesis mechanisms through the compartmentalization of organelles and photosynthetic enzymes into two distinct regions within a single chlorenchyma cell. Bienertia has peripheral and central compartments, whereas S. aralocaspica has distal and proximal compartments. This compartmentalization achieves the equivalent of spatial separation of Kranz anatomy, including dimorphic chloroplasts, but within a single cell. To characterize the mechanisms of organelle compartmentalization, the distribution of the major organelles relative to the cytoskeleton was examined. Examination of the distribution of the cytoskeleton using immunofluorescence studies and transient expression of green fluorescent protein-tagged cytoskeleton markers revealed a highly organized network of actin filaments and microtubules associating with the chloroplasts and showed that the two compartments in each cell had different cytoskeletal arrangements. Experiments using cytoskeleton-disrupting drugs showed in Bienertia and S. aralocaspica that microtubules are critical for the polarized positioning of chloroplasts and other organelles. Compartmentalization of the organelles in these species represents a unique system in higher plants and illustrates the degree of control the plant cell has over the organization and integration of multiorganellar processes within its cytoplasm.     

Divide and multiply: organelle partitioning in yeast

The mechanisms ensuring accurate partitioning of yeast vacuoles and mitochondria are distinct, yet they share common elements. Both organelles move along actin filaments, and both organelles require fusion and fission to maintain normal morphology. Recent studies have revealed that while vacuolar inheritance requires a processive myosin motor, mitochondrial inheritance requires controlled actin polymerization. Distinct sets of proteins required for the fusion and fission of each organelle have also been identified.     

Myosin VI is required for asymmetric segregation of cellular components during C. elegans spermatogenesis

BACKGROUND: The asymmetric division of cells and unequal allocation of cell contents is essential for correct development. This process of active segregation is poorly understood but in many instances has been shown to depend on the cytoskeleton. Motor proteins moving along actin filaments and microtubules are logical candidates to provide the motive force for asymmetric sorting of cell contents. The role of myosins in such processes has been suggested, but few examples of their involvement are known. RESULTS: Analysis of a Caenorhabditis elegans class VI myosin deletion mutant reveals a role for this motor protein in the segregation of cell components during spermatogenesis. Mutant spermatocytes cannot efficiently deliver mitochondria and endoplasmic reticulum/Golgi-derived fibrous-body membranous organelle complexes to budding spermatids, and fail to remove actin filaments and microtubules from the spermatids. The segregation defects are not due to a global sorting failure as nuclear inheritance is unaffected. CONCLUSIONS: C. elegans myosin VI has an important role in the unequal partitioning of both organelles and cytoskeletal components, a novel role for this class of motor protein.     

Mitotic inheritance of endoplasmic reticulum in the primitive red alga Cyanidioschyzon merolae

Endoplasmic reticulum (ER) is a major site for secretory protein folding and lipid synthesis. Since ER cannot be synthesized de novo, it must be inherited during the cell cycle. Studying ER inheritance can however be difficult because the ER of typical plant and animal cells is morphologically complex. Therefore, our study used Cyanidioschyzon merolae, a species that has a simple ER structure, to investigate the inheritance of this organelle. Using immunofluorescence microscopy, we demonstrated that C. merolae contains a nuclear ER (nuclear envelope) and a small amount of peripheral ER extending from the nuclear ER. During mitosis, the nuclear ER became dumbbell-shaped and underwent division. Peripheral ER formed ring-like structures during the G1 and S phases, and extended toward the mitochondria and cell division planes during the M phase. These observations indicated that C. merolae undergoes closed mitosis, whereby the nuclear ER does not diffuse, and the peripheral ER contains cell cycle-specific structures.     

Emergent complexity in Myosin V-based organelle inheritance

How is adaptability generated in a system composed of interacting cellular machineries, each with a separate and functionally critical job to perform? The machinery for organelle inheritance is precisely one such system, requiring coordination between robust and ancient cellular modules, including the cell cycle, cytoskeleton, and organelle biogenesis/identity. Budding yeasts have emerged as powerful models to study these processes, which are critical for cellular survival, propagation, and differentiation, as organelles must compete for access to myosin V motors that travel along polarized actin cables to vectorially deliver bound cargo to the bud. Under the direction of the cell cycle, myosin V motors are recruited to organelles by specific interactions between their carboxyl-terminal globular tail domains and organelle-specific receptors. We used comparative genomics, phylogenetics, and secondary structure modeling to characterize the evolutionary history of these organelle-specific receptors. We find that while some receptors are retained widely across the animals and fungi, others are limited primarily to the Saccharomycetaceae family of budding yeast, with the emergent pattern of a conserved biogenic and inheritance factor often paired with an evolutionarily novel inheritance adaptor. We propose an evolutionary model whereby the emergence of myosin V-based organelle inheritance has utilized mechanisms of paralogy, mutation, and the appearance of pliable evolutionarily novel adaptor proteins. Our findings suggest an overarching evolutionary mechanism for how diverse cargoes compete for a single myosin V motor in organelle transport and detail one system's solution to obtaining evolutionary adaptability amongst constrained cellular modules.     

Redistribution of Golgi stacks and other organelles during mitosis and cytokinesis in plant cells

We have followed the redistribution of Golgi stacks during mitosis and cytokinesis in living tobacco BY-2 suspension culture cells by means of a green fluorescent protein-tagged soybean alpha-1,2 mannosidase, and correlated the findings to cytoskeletal rearrangements and to the redistribution of endoplasmic reticulum, mitochondria, and plastids. In preparation for cell division, when the general streaming of Golgi stacks stops, about one-third of the peripheral Golgi stacks redistributes to the perinuclear cytoplasm, the phragmosome, thereby reversing the ratio of interior to cortical Golgi from 2:3 to 3:2. During metaphase, approximately 20% of all Golgi stacks aggregate in the immediate vicinity of the mitotic spindle and a similar number becomes concentrated in an equatorial region under the plasma membrane. This latter localization, the "Golgi belt," accurately predicts the future site of cell division, and thus forms a novel marker for this region after the disassembly of the preprophase band. During telophase and cytokinesis, many Golgi stacks redistribute around the phragmoplast where the cell plate is formed. At the end of cytokinesis, the daughter cells have very similar Golgi stack densities. The sites of preferential Golgi stack localization are specific for this organelle and largely exclude mitochondria and plastids, although some mitochondria can approach the phragmoplast. This segregation of organelles is first observed in metaphase and persists until completion of cytokinesis. Maintenance of the distinct localizations does not depend on intact actin filaments or microtubules, although the mitotic spindle appears to play a major role in organizing the organelle distribution patterns. The redistribution of Golgi stacks during mitosis and cytokinesis is consistent with the hypothesis that Golgi stacks are repositioned to ensure equal partitioning between daughter cells as well as rapid cell plate assembly.     

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

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.     

Mitochondrial inheritance in yeast

Mitochondria are the site of oxidative phosphorylation, play a key role in cellular energy metabolism, and are critical for cell survival and proliferation. The propagation of mitochondria during cell division depends on replication and partitioning of mitochondrial DNA, cytoskeleton-dependent mitochondrial transport, intracellular positioning of the organelle, and activities coordinating these processes. Budding yeast Saccharomyces cerevisiae has proven to be a valuable model organism to study the mechanisms that drive segregation of the mitochondrial genome and determine mitochondrial partitioning and behavior in an asymmetrically dividing cell. Here, I review past and recent advances that identified key components and cellular pathways contributing to mitochondrial inheritance in yeast. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.     

Maternal inheritance, sexual conflict and the maladapted male

Females differ from males in transmitting not only nuclear genes but also cytoplasmic genetic elements (CGEs), including DNA in mitochondria, chloroplasts and microorganisms that are present in the cell. Until recently, evolutionary research has adopted a nucleocentric approach in which organelles have been viewed as subservient energy suppliers. In this article, we propose that a more equitable view of nuclear genes and organelle genomes will lead to a better understanding of the dynamics of sexual selection and the constraints on male adaptation. Maternal inheritance of CGEs intensifies sexually-antagonistic coevolution and provides a parsimonious explanation for the relatively high frequency in males of such apparently maladaptive traits as infertility, homosexuality and baldness.     

Sharing the wealth: peroxisome inheritance in budding yeast

Eukaryotic cells have evolved molecular mechanisms to ensure the faithful partitioning of cellular components during cell division. The budding yeast Saccharomyces cerevisiae has to actively deliver about half of its organelles to the growing bud, while retaining the remaining organelles in the mother cell. Until lately, little was known about the inheritance of peroxisomes. Recent studies have identified the peroxisomal proteins Inp1p and Inp2p as two key regulators of peroxisome inheritance that perform antagonistic functions. Inp1p is required for the retention of peroxisomes in mother cells, whereas Inp2p promotes the bud-directed movement of these organelles. Inp1p anchors peroxisomes to the cell cortex by interacting with specific structures lining the cell periphery. On the other hand, Inp2p functions as the peroxisome-specific receptor for the class V myosin, Myo2p, thereby linking peroxisomes to the translocation machinery that propels peroxisome movement. Tight coordination between Inp1p and Inp2p ensures a fair and harmonious spatial segregation of peroxisomes upon cell division.     

Direct Targeting of Proteins from the Cytosol to Organelles: The ER versus Endosymbiotic Organelles

In eukaryotic cells consisting of many different types of organelles, targeting of organellar proteins is one of the most fundamental cellular processes. Proteins belonging to the endoplasmic reticulum (ER), chloroplasts and mitochondria are targeted individually from the cytosol to their cognate organelles. As the targeting to these organelles occurs in the cytosol during or after translation, the most crucial aspect is how specific targeting to these three organelles can be achieved without interfering with other targeting pathways. For these organelles, multiple mechanisms are used for targeting proteins, but the exact mechanism used depends on the type of protein and organelle, the location of targeting signals in the protein and the location of the protein in the organelle. In this review, we discuss the various mechanisms involved in protein targeting to the ER, chloroplasts and mitochondria, and how the targeting specificity is determined for these organelles in plant cells .     

Organelle inheritance in the yeast cell cycle

Cell proliferation requires the inheritance of subcellular organelles, yet little is known of the molecular basis of this essential process. Recent microscopy studies of the yeast Saccharomyces cerevisiae have characterized the cellular distribution of mitochondria, vacuoles and elements of the endoplasmic reticulum and Golgi complex. In addition, genetic and microscopical approaches have allowed the isolation and analysis of mutants defective in the inheritance of mitochondria and vacuoles. These investigations are leading to the identification of molecular components mediating the movement of organelles into daughter cells and have revealed that the inheritance of organelles is coordinated with other events of the cell division cycle.     

Mitochondrial manoeuvres: latest insights and hypotheses on mitochondrial partitioning during mitosis in Saccharomyces cerevisiae

Movement and positional control of mitochondria and other organelles are coordinated with cell cycle progression in the budding yeast, Saccharomyces cerevisiae. Recent studies have revealed a checkpoint that inhibits cytokinesis when there are severe defects in mitochondrial inheritance. An established checkpoint signaling pathway, the mitotic exit network (MEN), participates in this process. Here, we describe mitochondrial motility during inheritance in budding yeast, emerging evidence for mitochondrial quality control during inheritance, and organelle inheritance checkpoints for mitochondria and other organelles.     

Kinase-dead ATM protein causes genomic instability and early embryonic lethality in mice

Studies on cell division traditionally focus on the mechanisms of chromosome segregation and cytokinesis, yet we know comparatively little about how organelles segregate. Analysis of organelle partitioning in asymmetrically dividing cells has provided insights into the mechanisms through which cells control organelle distribution. Interestingly, these studies have revealed that segregation mechanisms frequently link organelle distribution to organelle growth and formation. Furthermore, in many cases, cells use organelles, such as the endoplasmic reticulum and P granules, as vectors for the segregation of information. Together, these emerging data suggest that the coordination between organelle growth, division, and segregation plays an important role in the control of cell fate inheritance, cellular aging, and rejuvenation, i.e., the resetting of age in immortal lineages.     

Actin and myosin function in directed vacuole movement during cell division in Saccharomyces cerevisiae

During cell division, cytoplasmic organelles are not synthesized de novo, rather they are replicated and partitioned between daughter cells. Partitioning of the vacuole in the budding yeast Saccharomyces cerevisiae is coordinated with the cell cycle and involves a dramatic translocation of a portion of the parental organelle from the mother cell into the bud. While the molecular mechanisms that mediate this event are unknown, the vacuole''s rapid and directed movements suggest cytoskeleton involvement. To identify cytoskeletal components that function in this process, vacuole inheritance was examined in a collection of actin mutants. Six strains were identified as being defective in vacuole inheritance. Tetrad analysis verified that the defect cosegregates with the mutant actin gene. One strain with a deletion in a myosin-binding region was analyzed further. The vacuole inheritance defect in this strain appears to result from the loss of a specific actin function; the actin cytoskeleton is intact and protein targeting to the vacuole is normal. Consistent with these findings, a mutation in the actin-binding domain of Myo2p, a class V unconventional myosin, abolishes vacuole inheritance. This suggests that Myo2p serves as a molecular motor for vacuole transport along actin filaments. The location of actin and Myo2p relative to the vacuole membrane is consistent with this model. Additional studies suggest that the actin filaments used for vacuole transport are dynamic, and that profilin plays a critical role in regulating their assembly. These results present the first demonstration that specific cytoskeletal proteins function in vacuole inheritance.