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Publisher Summary In higher plants, photosynthesis occurs mainly in the mesophyll, the chloroplast-rich tissue of leaves. The cellular contents are surrounded by a plasma membrane called the plasmalemma and are enclosed by a cell wall. The cell contains organelles, each with its own characteristic shape, which divide the cell into various compartments. Each compartment has specialized metabolic functions, which will be discussed in detail in the following chapters. The largest organelle, the vacuole, usually fills about 80% of the total cell volume. Chloroplasts represent the next largest compartment, and the rest of the cell volume is filled with mitochondria, peroxisomes, the nucleus, the endoplasmic reticulum, the Golgi bodies, and, outside these organelles, the cell plasma, called cytosol. In addition, there are oil bodies derived from the endoplasmic reticulum. These oil bodies, which occur in seeds and some other tissues, are storage organelles for triglycerides. The nucleus is surrounded by the nuclear envelope, which consists of the two membranes of the endoplasmic reticulum. The space between the two membranes is known as the perinuclear space. Nuclear pores with a diameter of about 50 nm interrupt the nuclear envelope. The nucleus contains chromatin, consisting of DNA double strands that are stabilized by being bound to basic proteins (histones). The genes of the nucleus are collectively referred to as the nuclear genome. Within the nucleus, usually off-center, lies the nucleolus, where ribosomal subunits are formed. These ribosomal subunits and the messenger RNA formed by transcription of the DNA in the nucleus migrate through the nuclear pores to the ribosomes in the cytosol, the site of protein biosynthesis. The synthesized proteins are distributed among the different cell compartments according to their final destination.

Before formally considering diffusion and related topics, this chapter outlines the structure of certain plant cells and tissues, thus introducing most of the anatomical terms. The chapter illustrates a representative leaf cell from a higher plant and illustrates the larger subcellular structures. Surrounding the protoplast is the cell wall, composed of cellulose and other polysaccharides, which helps provide rigidity to individual cells as well as to the whole plant. The cell wall contains numerous relatively large interstices, so it is not the main permeability barrier to the entry of water or small solutes into plant cells. The main barrier, a cell membrane known as the plasma membrane (or plasmalemma), is found inside the cell wall and surrounds the cytoplasm. The permeability of this membrane varies with the particular solute, so the plasma membrane can regulate what enters and leaves a plant cell. The cytoplasm contains organelles such as chloroplasts and mitochondria, which are membrane-surrounded compartments in which energy can be converted from one form to another. Also in the cytoplasm are microbodies such as peroxisomes, numerous ribosomes, and proteins, as well as many other macromolecules and structures that influence the thermodynamic properties of water. The term cytoplasm includes the organelles (but generally not the nucleus), whereas the term cytosol refers to the cytoplasmic solution delimited by the plasma membrane and the tonoplast but exterior to the organelles.

The introduction of different micromanipulation techniques into reproductive and developmental biology has helped us not only to answer many essential biological questions but it is now evident that these techniques also have wide practical applications. In human‐assisted reproduction, the most commonly used approach is the injection of a donor sperm into the oocyte cytoplasm—intracytoplasmic sperm injection. It is, however, speculated that with these techniques it would be also possible to improve the oocyte developmental potential especially in those cases when the quality of the cytoplasm is rather poor and thus its function is compromised. Another important application would be the elimination of mutated mitochondrial DNA (mtDNA) by transferring the nuclear material from an abnormal oocyte into a healthy donor oocyte cytoplast. Some of these techniques were already successfully tested in experimental animals, but it is evident that before their introduction into human medicine many questions must be answered, and we must be sure that these approaches are absolutely or almost absolutely safe. In our contribution, we will be specifically oriented to the nuclear (nuclear material) replacement approaches that could be potentially used to prevent the transmission of mutated mtDNA from mother to offspring. Because these techniques are very delicate, some training with oocytes from other species other than human is strongly recommended. © 2007, Elsevier Inc.

Oxidative phosphorylation (OXPHOS) is the only mammalian biochemical pathway dependent on the coordinated assembly of protein subunits encoded by both nuclear and mitochondrial DNA (mtDNA) genes. Cytoplasmic hybrid cells, cybrids, are created by introducing mtDNAs of interest into cells depleted of endogenous mtDNAs, and have been a central tool in unraveling effects of disease-linked mtDNA mutations. In this way, the nuclear genetic complement is held constant so that observed effects on OXPHOS can be linked to the introduced mtDNA. Cybrid studies have confirmed such linkage for many defined, disease-associated mutations. In general, a threshold principle is evident where OXPHOS defects are expressed when the proportion of mutant mtDNA in a heteroplasmic cell is high. Cybrids have also been used where mtDNA mutations are not known, but are suspected, and have produced some support for mtDNA involvement in more common neurodegenerative diseases. Mouse modeling of mtDNA transmission and disease has recently taken advantage of cybrid approaches. By using cultured cells as intermediate carriers of mtDNAs, ES cell cybrids have been produced in several laboratories by pretreatment of the cells with rhodamine 6G before cytoplast fusion. Both homoplasmic and heteroplasmic mice have been produced, allowing modeling of mtDNA transmission through the mouse germ line. We also briefly review and compare other transgenic approaches to modeling mtDNA dynamics, including mitochondrial injection into oocytes or zygotes, and embryonic karyoplast transfer. When breakthrough technology for mtDNA transformation arrives, cybrids will remain valuable for allowing exchange of engineered mtDNAs between cells.

Publisher Summary This chapter focuses on the nuclear reprogramming process and how both the technological parameters of the cloning process and the biological limitation of nuclear reprogramming might relate to the inefficiencies and phenotypes observed during the development of cloned animals. The four key parameters of nuclear transfer technology that influence the development of cloned embryos are the state and nature of the recipient cytoplast; the cell cycle status of the donor cell; the identity, differentiation state, and developmental potency of the donor cell; and the genetic influences on the successful development of cloned embryos. Although a G0 cell cycle state is clearly not necessary for the successful development of cloned embryos, the somatic nucleus has to be in a conformation compatible with nuclear envelope breakdown and chromosome condensation. The most interesting issue in cloning by nuclear transfer is epigenetic reprogramming. For clones to complete development, genes normally expressed during embryogenesis but silent in the somatic donor cell must be reactivated. Nuclei isolated from embryonic stem (ES) cells and embryonic blastomeres generate viable cloned animals with a significantly higher efficiency compared to any somatic donor cell type. During cloning, reprogramming has to occur in the short interval between transfer of the donor nucleus into the egg and the onset of cellular differentiation, a context that is dramatically different from normal fertilization. Because of cloning's inherent inefficiencies, the nuclear transfer experiments using lymphocytes and other terminally differentiated cells as nuclear donors must be carefully designed and carried out.

Publisher Summary The cell cycle of a growing cell is the period between the formation of the cell by the division of its mother cell and the time when the cell itself divides to form two daughters. This chapter describes the relation between the cell cycle and the production of embryos by nuclear transfer, and it focuses on the interactions between the donor nucleus and components of the recipient cytoplasm and their impact on subsequent development. The cell cycle stages of the recipient oocyte and the donor nucleus interact to affect the subsequent development of the nuclear transfer embryos. The recipient cytoplast or the combination of the recipient cytoplast and the nuclear donor cell may have other effects on the subsequent development. These effects can be split into two categories: chromatin fate and nuclear reprogramming. The reported differences in the behavior of the transferred chromatin may be dependent on a number of factors. The first factor may be species differences, the second factor may be the level of maturation-promoting actor (MPF) activity, and the third factor may be the source of the donor cell and the method of reconstruction. The changes that are required to modify the donor chromatin to obtain development of the reconstructed embryo is termed as “nuclear reprogramming.” The recipient cytoplasm may have effects on the ploidy of the reconstructed embryo, and hence it may also affect development and therefore nuclear reprogramming.

Publisher Summary This chapter discusses reproduction of centrosomes in vitro . The structural complexity of the animal centrosome, particularly the presence of the centriole pair as a conspicuous feature, suggests that a specific study of the animal centrosome is needed. An alternative approach to decipher the centrosome duplication pathway has been to use marine or amphibian eggs in which early development occurs without cell growth by segmentation, following each doubling of chromosomes and of the sperm-inherited centrosome. Xenopus egg extracts are the most characterized cell-free systems to observe not only centrosome assembly but also cell cycle-dependent regulation of this pathway. The chapter discusses a new strategy for developing a centrosome duplication assay in a Xenopus egg extract. The main feature of the assay is to use in situ centrosomes instead of isolated centrosomes. Triton X-100-extracted cytoplasts prepared from mouse L929 cells are used in this assay. This has several advantages: in the absence of nuclei, cytoplasts are very flat and are attached stably to the cover-slip on which the cells are grown. Their cytoplasmic compartments are distributed about the centrosome, which site at the geometric center of the cytoplast. Thus cytoplasts from early G1 cells provide a favorable starting material, as their centrosome does not initiate the structurally identifiable steps of duplication.

Publisher Summary This chapter provides a brief review of the expanding field of in vitro apoptosis studies. First, the chapter describes the cell-free systems reported thus far and provides some comments on the unique attributes of each system. Second, tentative criteria for validation of new apoptotic systems are proposed. These are based on the current understanding of the morphological and biochemical processes of apoptotic cell death. Finally, the methodologies that have been used for analyzing the systems are described together with a number of the more significant findings achieved from such analyses. The advantage of cell-free systems in the study of apoptosis is that they can overcome the stochastic nature of the onset of apoptotic events in cell populations. Several cell-free systems have been devised that recapitulate the morphological changes of apoptosis using components isolated from cells such as nuclei, cytoplasm, and organelles. These cell-free systems offer several advantages in the biochemical analysis of cell death pathways. The nuclei used as substrates for in vitro apoptosis reactions are typically isolated from healthy cells. Thus, the apoptosis-inducing signal comes from the extracts rather than the nuclei. These extracts seem to show no species or tissue specificity for substrate nuclei. The extracts can induce apoptotic changes in nuclei from a wide variety of sources such as human HeLa cervical carcinoma cells, GM3798 lymphoblastoid cells, Jurkat T cells, CEM T lymphoblastoid cells, mouse S49 cells, mouse liver, hamster liver, rat liver, rat thymocytes, and Xenopus sperm nuclei assembled in vitro. This apparently passive role of nuclei in the execution of apoptosis is consistent with experiments indicating that apoptotic changes can proceed even in cytoplasts, which lack nuclei.

Publisher Summary This chapter describes platelet-mediated transformation of human mitochondrial DNA-less ( ρ ˚)cells. The isolation of human cell lines completely devoid of mitochondrial DNA (mtDNA) and the demonstration that these cell lines can be repopulated with exogenous mtDNA by mitochondria-mediated transformation represents a major advance for human mitochondrial genetics studies. mtDNA-less ( ρ o ) cells have proved to be very useful for the study of mtDNA-linked diseases. The method for introducing mitochondria derived from cells of patients or normal human individuals into ρ ˚ cells involves fusion of enucleated cells (cytoplasts) with the ρ ˚ cells. An alternative method for introducing mitochondria into human ρ ˚ 0 cells has been developed, which involves the fusion with these cells of platelets derived from the individual's blood. The advantage of this method is that there is no need to establish cultures derived from the cells of the patient or the normal individual.

Publisher Summary This chapter describes methods for the transfer of mitochondria from one cellular environment to another, focusing on methods for the transfer of mitochondria into human cells lacking mtDNA ( ρ ˚ cells). The relatively large sizes of these organisms has facilitated the introduction into them of subcellular fractions containing mitochondria by microinjection. These investigations utilize mtDNA-encoded mutations specifying morphological or growth characteristics, or associate with specific drug resistance markers, for isolating mitochondrial transformants among the injected cells. The development of efficient, large-scale methods to obtain enucleated mammalian cells, termed “cytoplasts,” provides a basis for the transfer of mitochondria into cells by the fusion of cytoplasts with the cells of interest to form cytoplasmic hybrids, termed “cybrids.” The methods described in the chapter require donor mitochondria containing a selectable marker. The requirement for a selectable marker in donor mitochondria limits the utility of these systems for mitochondrial transfer, as there are only a small number of selectable markers associated with resistance to specific drugs available.

Publisher Summary This chapter discusses the progress of nuclear transplantation studies in mammals, the important biological factors influencing the development of eggs and embryos reconstituted by nuclear transplantation, and the prospects for future studies in this field. The transplantation of a nucleus to an appropriate cytoplast and the subsequent analysis of its embryonic development provide the most vigorous and direct tests of genomic totipotency of a cell nucleus. The most advanced studies are those carried out in amphibia. Totipotency, the capacity to direct the formation of fertile frogs, has been demonstrated for the nuclei of many kinds of embryonic cells; pluripotency, as judged by the development of heart-beating larvae, has been proved for all the cell nuclei tested to date.

Publisher Summary Cell enucleation and fusion provide a technology to determine if the cell cytoplasm contains stable regulatory substances that might modulate nuclear gene expression. It has been reported that added cytoplasm can have either a permanent, long-lived (2–8 weeks), short-lived (1–2 days), or no effect on the phenotype of the nuclear recipient. The protocols described in this chapter may be useful for helping to dissect the nature of these cytoplasmic regulatory substances. In addition, by fusing a cytoplast with a karyoplast, a viable “reconstituted” cell can be produced. Alternatively, by fusing a cytoplast from one cell to another whole cell, a cytoplasmic hybrid or cybrid is formed. Finally, by fusing a karyoplast to another whole ceil it is possible to form a nuclear hybrid. The various protocols for the production and isolation of these cellular constructs are described in this chapter.

Publisher Summary This chapter describes the methods for enucleation and reconstruction of interferon-producing cells. The extent of enucleation can be increased by centrifugation in the presence of the drug, probably because the nucleus is forced into a cytoplasmic stalk, which subsequently severs spontaneously. The percentage of efficiency of enucleation is readily estimated by staining the cytoplasts with crystal violet after recovery; any nucleated cells are then obvious, and both nucleated and enucleated cells can be counted. Cell reconstruction can be separated into two stages—namely, the agglutination of cytoplasts and karyoplasts by the Sendai virus and the actual fusion. Mouse L cells produce high titers of interferon (up to 10 5 international research units) when induced with NDV strain F. However, enucleated cells do not produce any interferon, whereas reconstructed cells produce as much interferon per cell as do normal L cells. Thus the interferon system, requiring a nuclear gene and cytoplasmic protein synthesis, has also been reconstructed.

Publisher Summary This chapter discusses the preparation and cryopreservation of cytoplasts from human phagocytes. Intact phagocytes can be activated by soluble or particulate stimuli to generate reactive oxygen products and arachidonic acid metabolites. Neutrophil cytoplasts, in contrast to intact neutrophils, can be cryopreserved with the complete maintenance of functional properties. In a density gradient, cells will move until they reach a layer with a specific gravity higher than that of their own specific gravity. At temperatures above the transition temperature of the lipids in the neutrophil plasma membrane (27° C) and in the presence of the microfilament-destroying agent cytochalasin B, a high centrifugal force will cause partitioning of each neutrophil into a cytoplast and a karyoplast. This process involves fusion of the plasma membrane to form a cytoplast and a karyoplast, without spilling of cell contents into the gradient. Neutrophil cytoplasts consist of a closed vesicle of plasma membrane, filled with cytoplasm and some endoplasmic reticulum. The volume of the cytoplasts is about one-fourth, the surface area is about one-third, and the amount of cytoplasm is about one-half of that of the original neutrophils.

Publisher Summary This chapter describes the techniques required to construct large, viable, homogeneous populations of true cytoplasmic-nuclear hybrids. These techniques are being applied to the construction of novel systems for investigating gene expression and its control in mammalian cells. Thus, the cytoplasts and karyoplasts prepared from various differentiated and transformed cell lines that exhibit distinct morphological and/or biochemical traits are fused to form true cytoplasmic-nuclear hybrid cells. The hybrids and their progeny are analyzed for the maintenance, loss, or acquisition of the parental cell traits. The chapter outlines the methods for the Preparation and Characterization of Karyoplasts and of Cytoplasts. The procedure for nuclear transplantation involves the preparation of Sendai virus, virus-mediated fusion, characterization of hybrids, and genetic selection techniques. The selection procedure should be applicable for the selection of cytoplasmic-nuclear hybrids. Thus, before fusing cytoplasts and karyoplasts derived from cell lines of interest, the mutants resistant to appropriate drugs, and toxins can first be selected.

Publisher Summary This chapter discusses that cell enucleation and immunocytochemistry have provided direct evidence that the estrogen receptor is located exclusively in the nucleus of the cell and that little unoccupied receptor is found in the cytoplasm. It presents experiments in which cell enucleation techniques are used to separate cytoplasm from the nucleus of the cell without breaking open the cell and diluting its contents. Both the cytoplast and the remainder of the cell that contains the nucleus (nucleoplast) are alive and are surrounded by an intact plasma membrane. If the unoccupied receptor is a cytoplasmic protein, one would expect to find it in the cytoplasts and to find less receptor in the nucleoplasts, from which cytoplasm has been removed. However, little receptor was found in cytoplasts; instead, the unoccupied receptor was found in nucleus-containing fractions.

Mammalian cells can be separated into enucleated (cytoplast) and nucleated (karyoplast) fragments by subjecting cells to a centrifugal force in the presence of the fungal metabolite cytochalasin B. Cytoplasts and karyoplasts have been partially characterized regarding their ultrastrueture, enzymatic constitution, and their physiological behavior. The isolated cellular components can be fused to whole cells or to each other. The fusion of cytoplasts to whole cells provided the first demonstration of cytoplasmic inheritance in mammalian cells. Viable cells are formed by fusing the components to each other.

Publisher Summary This chapter draws attention to the most significant recent advances in research on Acetabularia and related Dasycladales. Progress in research on Acetabularia and related Dasycladales has been particularly rapid in the past few years. The foundation of an “International Research Group on Acetabularia” and the organization of three international symposia have greatly stimulated investigations on these giant unicellular marine algae. New ideas have arisen from these meetings and what is equally important, a sincere wish for international collaboration. Acetabularia is certainly one of the most useful organisms for studies on cellular growth and differentiation in the absence of the nucleus. It is particularly suitable for investigating the subtle relationships, which exist between nucleus and cytoplasm and for examining the intergenomic cooperation inside the eukaryotic cell. The possibility of obtaining anucleate cells or cell fragments, regenerating nucleate basal parts, cytoplasts, isolated nuclei and organelles permits various types of in vivo and in vitro biological and biochemical experiments. Moreover, elegant intra- and interspecific grafts are possible with different species of Acetabularia.

Publisher Summary Prescott et al . reported an efficient, relatively simple technique for the enucleation of large numbers of cultured mammalian cells. The method entails plating cells on a glass coverslip or some other surface and centrifuging the surface, cell-side down in medium containing the mold metabolite cytochalasin B. Prescott's method or variations of it have found widespread application. The use of enucleated cells, or cytoplasts, thus prepared is justified by the findings that cytoplasts retain many of the morphological and behavioral characteristics of whole cells. The enucleation method of Prescott nevertheless has certain drawbacks. These include the fact that the yield is limited by the surface area that can be exposed to centrifugal forces, and the method cannot be used on cells that adhere poorly or not at all to solid surfaces. Authors have therefore explored the possibility that cells can be enucleated while in suspension by isopycnic centrifugation in the presence of cytochalasin B, and have developed a procedure of high efficiency and yield that can theoretically be applied to almost any eukaryotic cell lacking a cell wall.

Cytoplasmic hybrids (cybrids) were made by fusing the chloramphenicol resistant (CAP-r) cytoplast of one mouse species with the chloramphenicol sensitive (CAP-s) whole cell of another mouse species or subspecies. The mitochondrial DNA (mt-DNA) of parents and cybrids was analysed by restriction endonuclease digestion and electrophoresis. Two types of cybrids were found: 1) those that maintained a balanced combination of endogenous, CAP-s and alien, CAP-r mt-DNA, and 2) those that showed repopulation by the alien CAP-r mt-DNA. When cybrids of the first type were returned to medium without CAP, the CAP-r phenotype segregated concordantly with the mt-DNA of the CAP-r subspecies, indicating that they are linked. In cybrids that showed repopulation, no segregation was observed, indicating that substitution was probably complete. No evidence of recombination was found. The repopulated heterospecific cybrids may be useful in determining which polypeptides are encoded by the mt-DNA. When CAP-s cells were treated with purified mt-DNA from CAP-r strains, CAP-s cells were efficiently “transformed” to CAP-r. Only the endogenous CAP-s mt-DNA was detected in these transformants. Fine structure restriction analysis suggests that small regions of the CAP-s mt-DNA may be altered, perhaps by incorporation of some CAP-r mt-DNA.