Membrane geometry of "open" prolamellar bodies.

The "open" type of prolamellar body in etiplasts was examined by electron microscopy to characterise its three-dimensional organisation. As in more compact forms of prolamellar body, its basic geometric unit is a tetrahedrally branched tubule. In the "open" type, these lie smoothly confluent with one another at the vertices of 5- and 6-membered rings which circumscribe the faces of three kinds of polyhedra: pentagonal dodecahedra (with 12 pentagonal faces), 14-hedra (2 opposite hexagonal faces joined by two circlets of six pentagonal faces), and 15-hedra (3 hexagonal and 12 pentagonal faces). These polyhedra join confluently in their turn, sharing faces with one another in at least one recognisable super-structure which accounts for the appearance of "open" prolamellar bodies in many ultrathin sections. In this organisation, columns of pentagonal dodecahedra are arranged at 120 degrees to one another in the x-y-plane of the lattice. They do not fill the plane but intersect so as to delimit voids in the form of hexagonally arranged 14-hedra (with hexagonal rings in the x-y-plane). Strata of this type alternate with strata made of face-sharing 15-hedra (with their hexagonal rings normal to x-y), which also delimit 14-hedra. The 14-hedra thus lie in register in the z-axis in hexagonally arranged columns, normal to the alternating strata. Other possible organisations cannot be excluded and local variations and dislocations certainly occur, but many micrographs that display elements of symmetry in "open" prolamellar bodies can be matched to thin slices through such a model. Its geometry is like that of the cages of water molecules in type IV (sensu Jeffrey = type III sensu O'Keeffe) clathrate-hydrates, point group P6/mmm, but about two orders of magnitude larger.

The cytoplast concept in dividing plant cells: cytoplasmic domains and the evolution of spatially organized cell.

The unique cytokinetic apparatus of higher plant cells comprises two cytoskeletal systems: a predictive preprophase band of microtubules (MTs), which defines the future division site, and the phragmoplast, which mediates crosswall formation after mitosis. We review features of plant cell division in an evolutionary context and from the viewpoint that the cell is a domain of cytoplasm (cytoplast) organized around the nucleus by a cytoskeleton consisting of a single "tensegral" unit. The term "tensegrity" is a contraction of "tensional integrity" and the concept proposes that the whole cell is organized by an integrated cytoskeleton of tension elements (e.g., actin fibers) extended over compression-resistant elements (e.g., MTs).During cell division, a primary role of the spindle is seen as generating two cytoplasts from one with separation of chromosomes a later, derived function. The telophase spindle separates the newly forming cytoplasts and the overlap between half spindles (the shared edge of two new domains) dictates the position at which cytokinesis occurs. Wall MTs of higher plant cells, like the MT cytoskeleton in animal and protistan cells, spatially define the interphase cytoplast. Redeployment of actin and MTs into the preprophase band (PPB) is the overt signal that the boundary between two nascent cytoplasts has been delineated. The "actin-depleted zone" that marks the site of the PPB throughout mitosis may be a more persistent manifestation of this delineation of two domains of cortical actin. The growth of the phragmoplast is controlled by these domains, not just by the spindle. These domains play a major role in controlling the path of phragmoplast expansion. Primitive land plants show different morphological changes that reveal that the plane of division, with or without the PPB, has been determined well in advance of mitosis.The green alga Spirogyra suggests how the phragmoplast system might have evolved: cytokinesis starts with cleavage and then actin-related determinants stimulate and positionally control cell-plate formation in a phragmoplast arising from interzonal MTs from the spindle. Actin in the PPB of higher plants may be assembling into a potential furrow, imprinting a cleavage site whose persistent determinants (perhaps actin) align the outgrowing edge of the phragmoplast, as in Spirogyra. Cytochalasin spatially disrupts polarized mitosis and positioning of the phragmoplast. Thus, the tensegral interaction of actin with MTs (at the spindle pole and in the phragmoplast) is critical to morphogenesis, just as they seem to be during division of animal cells. In advanced green plants, intercalary expansion driven by turgor is controlled by MTs, which in conjunction with actin, may act as stress detectors, thereby affecting the plane of division (a response clearly evident after wounding of tissue). The PPB might be one manifestation of this strain detection apparatus.

A gamma-tubulin that associates specifically with centrioles in HeLa cells and the basal body complex in Chlamydomonas.

gamma-Tubulin is a putative component of microtubule initiating material. To further explore its subcellular distribution in plant and animal cells, we have raised a polyclonal antibody, Rb27, directed towards a conserved region (EEFATEGTDRKDVFFY) of the gamma-tubulin molecule. Immunoblotting of cell protein extracts with Rb27 reveals a polypeptide band of M(r) 49 kD in HeLa and a 58 kD band in Chlamydomonas. Although these polypeptides are comparable in size to forms of gamma-tubulin detected previously in mammalian and plant protein extracts by other antibodies to gamma-tubulin, by immunofluorescence microscopy Rb27 gives localization patterns not previously seen. It localizes specifically with the centrioles in HeLa cells and with the basal body complex in Chlamydomonas. Other gamma-tubulin antibodies label pericentriolar material. Because of the similarities in the size of the polypeptides recognized by our and other gamma-tubulin antibodies, and a restricted co-localization with known microtubule-organizing centres in evolutionarily distant organisms, we propose that Rb27 recognizes a novel conserved gamma-tubulin isotype.

The microtubular cytoskeleton during development of the zygote, proemhryo and free-nuclear endosperm in Arabidopsis thaliana (L.) Heynh.

The microtubular cytoskeleton has been studied during development of the zygote, proembryo and free-nuclear endosperm in A. thaliana using immunofluorescence localization of tubulin in enzymatically isolated material. Abundant micro tubules (MTs) are found throughout proembryogenesis. Microtubules in the coenocytic endosperm are mainly internal. By contrast, there is a re-orientation of MTs to a transverse cortical distribution during zygote development, predominantly in a subapical band which accompanies a phase of apical extension. The presence of these cortical arrays coincides with the elongation of the zygote. Cortical arrays also accompany elongation of the cylindrical suspensor. Extensive networks of MTs ramify throughout the cytoplasm of cells in the proembryo proper. Perinuclear arrays are detected in a number of cell types and MTs contribute to typical mitotic configurations during nuclear divisions. Preprophase bands of MTs are absent throughout megasporogenesis and embryo-sac development and do not occur in endosperm cell divisions. We have observed MTs throughout the first division cycle of the zygote. By placing the observed stages in a most probable sequence, we have identified this cell cycle as the point during embryogenesis at which a preprophase band is reinstated as a regular feature of cell division. Preprophase bands were observed to predict planes of cytokinesis in cell divisions up to the octant stage.

The microtubular cytoskeleton during development of the zygote, proembryo and free-nuclear endosperm inArabidopsis thaliana (L.) Heynh.

The microtubular cytoskeleton has been studied during development of the zygote, proembryo and free-nuclear endosperm inA. thaliana using immunofluorescence localization of tubulin in enzymatically isolated material. Abundant micro tubules (MTs) are found throughout proembryogenesis. Microtubules in the coenocytic endosperm are mainly internal. By contrast, there is a re-orientation of MTs to a transverse cortical distribution during zygote development, predominantly in a subapical band which accompanies a phase of apical extension. The presence of these cortical arrays coincides with the elongation of the zygote. Cortical arrays also accompany elongation of the cylindrical suspensor. Extensive networks of MTs ramify throughout the cytoplasm of cells in the proembryo proper. Perinuclear arrays are detected in a number of cell types and MTs contribute to typical mitotic configurations during nuclear divisions. Preprophase bands of MTs are absent throughout megasporogenesis and embryo-sac development and do not occur in endosperm cell divisions. We have observed MTs throughout the first division cycle of the zygote. By placing the observed stages in a most probable sequence, we have identified this cell cycle as the point during embryogenesis at which a preprophase band is reinstated as a regular feature of cell division. Preprophase bands were observed to predict planes of cytokinesis in cell divisions up to the octant stage.

DNA probes show genetic variation in cyanobacterial symbionts of the azolla fern and a closer relationship to free-living nostoc strains than to free-living anabaena strains.

Twenty-two isolates of Anabaena azollae derived from seven Azolla species from various geographic and ecological sources were characterized by DNA-DNA hybridization. Cloned DNA fragments derived from the genomic sequences of three different A. azollae isolates were used to detect restriction fragment length polymorphism among all symbiotic anabaenas. DNA clones were radiolabeled and hybridized against southern blot transfers of genomic DNAs of different isolates of A. azollae digested with restriction endonucleases. Eight DNA probes were selected to identify the Anabaena strains tested. Two were strain specific and hybridized only to A. azollae strains isolated from Azolla microphylla or Azolla caroliniana. One DNA probe was section specific (hybridized only to anabaenas isolated from Azolla ferns representing the section Euazolla), and five other probes gave finer discrimination among anabaenas representing various ecotypes of Azolla species. These cloned genomic DNA probes identified 11 different genotypes of A. azollae isolates. These included three endosymbiotic genotypes within Azolla filiculoides species and two genotypes within both A. caroliniana and Azolla pinnata endosymbionts. Although we were not able to discriminate among anabaenas extracted from different ecotypes of Azolla nilotica, Azolla mexicina, Azolla rubra and Azolla microphylla species, each of the endosymbionts was easily identified as a unique genotype. When total DNA isolated from free-living Anabaena sp. strain PCC7120 was screened, none of the genomic DNA probes gave detectable positive hybridization. Total DNA of Nostoc cycas PCC7422 hybridized with six of eight genomic DNA fragments. These data imply that the dominant symbiotic organism in association with Azolla spp. is more closely related to Nostoc spp. than to free-living Anabaena spp.

Preprophase bands of microtubules and the cell cycle: Kinetics and experimental uncoupling of their formation from the nuclear cycle in onion root-tip cells.

We have studied the timing of preprophase band (PPB) development in the division cycle of onion (Allium cepa L.) root-tip cells by combinations of immunofluorescence microscopy of microtubules, microspectrophotometry of nuclear DNA, and autoradiography of [(3)H]thymidine incorporation during pulse-chase experiments. In normally grown onion root tips, every cell with a PPB had the G2 level of nuclear DNA. Some were in interphase, prior to chromatin condensation, and some had varying degrees of chromatin condensation, up to the stage of prophase at which the PPB-prophase spindle transition occurs. In addition, autoradiography showed that PPBs can be formed in cells which have just finished their S phase, and microspectrophotometry enabled us to detect a population of cells in G2 which had no PPBs, these presumably including cells which had left the division cycle. The effects of inhibitors of DNA synthesis showed that the formation of PPBs is not fully coupled to events of the nuclear cycle. Although the mitotic index decreased 6-10-fold to less than 0.5% when roots were kept in 20 µg·ml(-1) aphidicolin for more than 8 h, the percentage of cells containing PPBs did not decrease in proportion: the number of cells in interphase with PPBs increased while the number in prophase decreased. Almost the same phenomena were observed in the presence of 100 µg·ml(-1) 5-aminouracil and 40 µg·ml(-1) hydroxyurea. In controls, all cells with PPBs were in G2 or prophase, but in the presence of aphidicolin, 5-aminouracil or hydroxyurea, some of the interphase cells with PPBs were in the S phase or even in the G1 phase. We conclude that PPB formation normally occurs in G2 (in at least some cases very early in G2) and that this timing can be experimentally uncoupled from the timing of DNA duplication in the cell-division cycle. The result accords with other evidence indicating that the cytoplasmic events of cytokinesis are controlled in parallel to the nuclear cycle, rather than in an obligatorily coupled sequence.

Preprophase bands, phragmoplasts, and spatial control of cytokinesis.

Features of preprophase bands (PPBs) of microtubules (MTs), and the spatial relationship between phragmosomes, PPB sites, and developing phragmoplasts during cytokinesis, are reviewed, setting new observations in the context of current knowledge. PPBs in onion root tip cells are present by the beginning of the G2 period of the cell cycle. They are at first wide, but later become more compact, narrower bands. MTs traverse the cytoplasm between the band at the cell cortex and the nuclear envelope. This whole assemblage of nucleus, PPB and intervening MTs remains together when the cell is ruptured during preparation for examination by immunofluorescence microscopy. Double bands are occasionally seen in early stages of PPB development, perhaps as a consequence of double induction from neighbouring cells. Calmodulin is not present in PPBs at a higher concentration than in the general cytoplasm, but it is more abundant in parts of the spindle and in the phragmoplast. The PPB MTs disappear at prophase, but nevertheless the new cell plate fuses with the parental cell walls at the PPB site. This spatial relationship can be disrupted by treatment with CIPC. Another experimental disruption of the relationship, accomplished by making minute wounds in the PPB site of mitotic cells in Tradescantia stamen hairs, is described. In other experiments on these cells the phragmoplast is shown to become tethered to the PPB site when the cell plate is half to three-quarters developed, although the telophase nuclei are free to move. Rhodamine-labelled phalloidin reveals putative F-actin in the phragmoplast of Tradescantia, but not in the gap between the extending phragmoplast and the PPB site. Rhodamine-labelled phalloidin also stains cytoplasmic strands that exist when cytoplasmic streaming occurs before and after (but not during) mitosis. Cytochalasin B treatment blocks incorporation of actin into the phragmoplast, which, however, can still develop, though usually abnormally. The F-actin of the phragmoplast may function in consolidation of the cell plate, rather than in spatial guidance of its growth toward the PPB site at the cell surface.

Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells.

Cells were prepared for indirect immunofluorescence microscopy after paraformaldehyde fixation of multicellular root apices and brief incubation in cell wall-digesting enzymes. This allowed subsequent separation of the tissue into individual cells or short files of cells which were put onto coverslips coated with polylysine. Unlike spherical protoplasts made from living tissues, these preparations retain the same polyhedral shape as the cells from which they are derived. Cellular contents, including organized arrays of microtubules, are likewise structurally stabilized. Antibodies to porcine brain tubulin react with all types of microtubule array known to occur in plant meristematic cells, namely, interphase cortical microtubules, pre-prophase bands, the mitotic spindle, and phragmoplast microtubules. The retention of antigenicity in permeabilized, isolated, stabilized cells from typical, wall-enclosed plant cells has much potential for plant immunocytochemistry, and in particular should facilitate work on the role of microtubules in the morphogenesis of organized plant tissues.

Interpolation of microtubules into cortical arrays during cell elongation and differentiation in roots of Azolla pinnata.

Longitudinal sections of roots of Azolla pinnata R. Br. were prepared for electron microscopy so that cortical microtubules could be counted along the longitudinal walls in cell files in the endodermis, pericycle, and inner and outer cortex, and in sieve and xylem elements. With the exception of the xylem, where there are no transverse cell divisions, each file of cells commences with its initial cell and then possesses a zone of concomitant cell expansion and transverse cell division, followed, after completion of the divisions, by a zone of terminal cell differentiation. The cells augment their population of cortical microtubules as they elongate and divide, showing a net increase of up to 0.6 micron of polymerized microtubule length per min. Two main sub-processes were found: (i) When a longitudinal wall is first formed it is supplied with a higher number of microtubules per unit length of wall than it will have later, when it is being expanded. This initial quota becomes diluted as the second sub-process commences. (ii) The cells interpolate new microtubules at a rate which is characteristic of the cell, and, in the endodermis, of the face of the cell, while the cell elongates. Most cell types thus maintain a set density of cortical microtubules while they elongate and divide. Comparisons of endodermal cells in untreated controls, and roots that had been treated with colchicine, low temperature, or high pressure indicate that the initial quota of microtubules, and the later interpolations, and differentially sensitive to microtuble perturbations. Three types of behaviour, all related to changes in the cell walls, were noted as cortex, xylem and sieve element cells entered their respective phases of cell differentiation. The cortical cells expanded in all dimensions, and the interpolation of microtubules diminished or ceased. The sieve elements continued to elongate, and interpolated at a high rate, reaching unusually high densities of microtubules when the cell walls were being thickened. During this period a net increase of 2.0 micron of polymerized microtubule length per min was calculated. Thereafter interpolation ceased and the density of microtubules declined. The sample applied to developing xylem except that, because wall-thickening is localized rather than widespread, the rise and subsequent fall in the density of microtubules was less marked. The data are discussed in relation to the participation of microtubules in wall deposition and to the hypothesis that cortical microtubules arise in discrete zones along the edges of cells.

Structure of cortical microtubule arrays in plant cells.

Serial sectioning was used to track the position and measure the lengths of cortical microtubules in glutaraldehyde-osmium tetroxide-fixed root tip cells. Microtubules lying against the longitudinal walls during interphase, those overlying developing xylem thickenings, and those in pre-prophase bands are oriented circumferentially but on average are only about one-eighth of the cell circumference in length, i.e., 2-4 micrometer. The arrays consist of overlapping component microtubules, interconnected by cross bridges where they are grouped and also connected to the plasma membrane. Microtubule lengths vary greatly in any given array, but the probability that any pass right around the cell is extremely low. The majority of the microtubule terminations lie in statistically random positions in the arrays, but nonrandomness in the form of groups of terminations and terminations in short lines parallel to the axis of cell elongation has been observed. Low temperature induces microtubule shortening and increases the frequency of C-shaped terminations over the 1.7% found under normal conditions; colchicine and high pressures produce abnormally large proportions of very short microtubules amongst those that survive the treatments. Deuterium oxide (D2O) treatment probably induces the formation of additional microtubules as distinct from increasing the length of those already present. The distribution of C-shaped terminations provides evidence for at least local polarity in the arrays. The validity of the findings is discussed, along with implications for the development, maintenance, and orientation of the arrays and their possible relationship to the orientation of cellulose deposition.

Formative and proliferative cell divisions, cell differentiation, and developmental changes in the meristem of Azolla roots.

The root of the water fern Azolla is a compact higher-plant organ, advantageous for studies of cell division, cell differentiation, and morphogenesis. The cell complement of A. filiculoides Lam. and A. pinnata R.Br. roots is described, and the lineages of the cell types, all derived ultimately from a tetrahedral apical cell, are characterised in terms of sites and planes of cell division within the formative zone, where the initial cells of the cell files are generated. Subsequent proliferation of the initial cells is highly specific, each cell type having its own programme of divisions prior to terminal differentiation. Both formative and proliferative divisions (but especially the former) occur in regular sequences. Two enantiomorphic forms of root develop, with the dispositions of certain types of cell correlating with the direction, dextrorse or sinistrorse, of the cell-division sequence in the apical cells. Root growth is determinate, the apical cell dividing about 55 times, and its cell-cycle duration decreasing from an initial 10 h to about 4 h during the major phase of root development. Sites of proliferation progress acropetally during aging, but do not penetrate into the zone of formative divisions. The detailed portrait of root development that was obtained is discussed with respect to genetic and epigenetic influences; quantal and non-quantal cell cycles; variation in cell-cycle durations; relationships between cell expansion and cell division: the role of the apical cell; and the limitation of the total number of mitotic cycles during root formation.

Pre-prophase bands of microtubules in all categories of formative and proliferative cell division in Azolla roots.

Pre-prophase bands of microtubules were found in every category of cell division, symmetrical and asymmetrical, in the cell lineages of the root apex of Azolla pinnata R.Br. and A. filiculoides Lam., and in the transverse divisions in the cell files of the roots. They are also found in the asymmetrical cell division that gives rise to trichoblasts in roots of Hydrocharis dubia (B1). Backer. It is possible, in a variety of cell types in roots of Azolla, to predict within a fraction of a micrometre where a new cell wall will be located. In every such case the midline of the 1.5-3-µm-wide pre-prophase band anticipates this location. Each of the daughter cells thus inherits approximately half of the former pre-prophase band site. Images interpreted as stages of formation of the band were obtained, its microtubules replacing the interphase cortical arrays. In one highly asymmetrical division, band formation precedes migration of the nucleus to the site of mitosis. The asymmetrical division that gives rise to root hairs passes acropetally along every cell in the dermatogen layer, and preprophase bands were seen up to 8 cells in advance of the last completed division. Here, and in the zone of formative divisions, the band is present for much longer than the duration of mitosis. The ubiquity of the band in the Azolla root tip is discussed in relation to the literature, and a working hypothesis is presented that takes into account current knowledge of occurrence, development and function of the band.

Evidence for initiation of microtubules in discrete regions of the cell cortex in Azolla root-tip cells, and an hypothesis on the development of cortical arrays of microtubules.

Complexes of microtubules, vesicles, and (to varying degrees) dense matrix material around the microtubules were seen along the edges of cells in root apices of Azolla pinnata R.Br. (viewing the cells as polyhedra with faces, vertices and edges). They are best developed after cytokinesis has been completed, when the daughter cells are reinstating their interphase arrays of microtubules. They are not confined to edges made by the junction of new cell plates with parental walls, but occur also along older edges. Similar matrices and vesicles are seen amongst phragmoplast microtubules and where pre-prophase bands intersect the edges of cells. It is suggested that the complexes participate in the development of cortical arrays of microtubules. The observations are combined with others, made on pre-prophase bands and on the substructure of cortical arrays lying against the faces of cells, to develop an hypothesis on the development of cortical microtubules, summarised below: Microtubules are nucleated along the edges of cells, at first growing in unspecified orientations and then becoming bridged to the plasma membrane. Parallelism of microtubules in the arrays arises by inter-tubule cross-bridging. Lengths of microtubule are released from, or break off, the nucleating centres and are moved out onto the face of the cell by intertubule and tubule-membrane sliding, thus accounting for the presence there of short tubules with randomly placed terminations. The nucleating zones along cell edges might have vectorial properties, and thus be able to control the orientation of the microtubules on the different faces of the cell. Also, localised activation could generate localised arrays, especially pre-prophase bands in specified sites and planes. Two possible reasons for the spatial restriction of nucleation to cell edges are considered. One is that the geometry of an edge is itself important; the other is that along most cell edges there is a persistent specialised zone, inherited at cytokinesis by the daughter cells when the cell plate bisects the former pre-prophase-band zone.

Age-related and origin-related control of the numbers of plasmodesmata in cell walls of developing Azolla roots.

Plasmodesmata were counted in the longitudinal and transverse walls in developmental sequences of merophytes in roots of Azolla pinnata R.Br. The differences between certain categories of longitudinal wall were traced to factors that govern the surface area of the cell plates, the density of plasmodesmata (number per unit area of cell plate), and the amount by which each type of plate expands. No evidence for secondary augmentation of plasmodesmatal numbers after the cell-plate stage of development was found, but plasmodesmata are lost from the walls of sieve and xylem elements during their differentiation. Losses caused by cell separation occur in other tissues. The relatively high density of plasmodesmata in transverse walls is based not so much on a high density in the cell plates as on the relatively low expansion that these walls undergo. There appears to be a compensatory mechanism that relates initial plasmodesmatal density to the future expansion of the cell plate. The root shows determinate growth, the apical cell dividing about 55 times. Beginning at about the 35th division there is a progressive failure to maintain the plasmodesmatal frequencies that were developed in earlier cell divisions in the apical cell. The divisions that occur within the later-produced merophytes also show progressive diminution of plasmodesmatal numbers. The result is that the apex of the root, and particularly the apical cell, becomes more and more isolated symplastically, a phenomenon which could account for its limited lifespan and the determinate growth pattern of the root.

Examination of ribosome-like particles in isolated prolamellar bodies.

Potential methods for the preparation of fractions enriched in prolamellar bodies (PLBs) were examined in detail. Sucrose density gradient centrifugation methods gave fractions consisting almost exclusively of PLBs whilst those methods employing differential centrifugation were quite successful but contained greater quantities of lamellar membranes. Greater difficulty was experienced in obtaining detached PLBs which retained their "ribosome-like" lattice particles. No modification to density gradient procedures was found which retained these particles but the omission of ethylene diaminetetraacetic acid (EDTA) from all media including that of lysis gave a hint that this was possible with differential centrifugal methods. This was developed to produce a successful method for the preparation of PLBs which retain the "ribosome-like" particles of the lattice. Such fractions from Avena sativa L. and Hordeum vulgare L. were treated with ribonuclease which completely removed these particles from the lattice structures implying that they may be "ribosomal" in nature. EDTA apparently has a critical effect on PLB structure at concentration lower than those that effect the chloroplast coupling factor particles but it is not known if it is a direct effort of PLB membranes, on the lattice particles or both.

The length and disposition of cortical microtubules in plant cells fixed in glutaraldehyde-Osmium tetroxide.

Serial sectioning has been used to show that the majority of circumferential microtubules lying in the cortex of root tip cells are much shorter than the cell circumference. The significance of this observation is briefly discussed.

Synthesis of the inner mitochondrial membrane and the intercalation of respiratory enzymes during the cell cycle of Chlorella.

The ratio of inner to outer mitochondrial membrane area remains close to 1-8 throughout the cell cycle in synchronized cells of Chlorella fusca var, vacuolata 211-8p. Using estimates of this ratio, together with our previous estimates of mitochondrial surface area, to calculate the absolute area of inner mitochondrial membrane, it is demonstrated that growth of the inner mitochondrial membrane during the cell cycle occupies an extended period and parallels the growth of the whole cell. In contrast, the synthesis of succinate dehydrogenase and cytochrome oxidase is restricted to the last third of the cell cycle. It is concluded that mitochondrial growth involves the intercalation of periodically synthesized respiratory enzymes into membranes made earlier in the cycle, with consequent 5-fold changes in the density of active enzyme molecules in the membrane. These observations are discussed in relation to the control of mitochondiral membrane synthesis, membrane assembly and respiration rate during the cell cycle.