Localization of calcineurin in the mature and developing retina.

We studied the localization of calcineurin by immunoblotting analysis and immunohistochemistry as a first step in clarifying the role of calcineurin in the retina. Rat, bovine, and human retinal tissues were examined with subtype-nonspecific and subtype-specific antibodies for the A alpha and A beta isoforms of its catalytic subunit. In mature retinas of the three species, calcineurin was localized mainly in the cell bodies of ganglion cells and the cells in the inner nuclear layer, in which amacrine cells were distinctively positive. The calcineurin A alpha and A beta isoforms were differentially localized in the nucleus and the cytoplasm of the ganglion cell, respectively. Calcineurin was also present in developing rat retinas, in which the ganglion cells were consistently positive for it. The presence of calcineurin across mammalian species and regardless of age shown in the present study may reflect its importance in visual function and retinal development, although its function in the retina has not yet been clarified. (J Histochem Cytochem 49:187-195, 2001)     

Mechanisms of radioresistance in terminally differentiated cells of mature retina

Retinopathy of animals is induced by many DNA-damaging agents. This fact shows that DNA lesions may initiate retinal degeneration. The aim of our work was to study the effects of gamma and proton irradiation and single administration of methylnitrosourea (MNU) on mice retina. We assessed morphological changes, DNA damage and repair, as well as expression of proteins (p53, ATM, PARP, FasR, and caspase 3) participating in apoptosis in retina. 14 Gy was the equitoxic dose for induction of DNA single-strand breaks by both gamma- and proton irradiation. However, protons were twice as effective as γ rays in induction of DNA double-strand breaks. All breaks have been repaired for ≤10 h. Irradiation resulted in increased expression of p53 and ATM. Seven days after irradiation, no signs of cell death and retinal degeneration were observed. Proton irradiation with 25 Gy resulted in destructive changes in retina localized mainly in the photoreceptor layer. These changes were accompanied by enhanced expression of proapoptotic proteins. A single systemic administration of MNU (70 mg/kg) increased intracellular levels of p53, PARP, FasR, and Caspase 3 followed by destructive changes in retina with sings of apoptosis in photoreceptors. Similarly to irradiation, a halved MNU dose did not exhibit a cytotoxic effect on retina. A high level of spontaneous DNA damage at apurine and apyrimidine sites were observed in mouse retina. The results show that there is a genotoxic threshold in initiation of retinal cell death in vivo. It is suggested that topoisomerase 2 translates primary DNA damage into a cytotoxic effect in retina.     

Mechanisms of radioresistance in terminally differentiated cells of mature retina

Retinopathy of animals is induced by many agents damaging DNA. This fact shows that DNA lesions may initiate retinal degeneration. The aim of our work was to study the effects of gamma and proton irradiation, and methylnitrosourea (MNU) on mice retina. We evaluated morphological changes, DNA damage and repair in retina, and expression of 5 proteins participating in apoptosis: p53, ATM, FasR, PARP and caspase 3 active. Dose of 14 Gy is equitoxic in terms of induction of DNA single strand breaks by both gamma and proton irradiation. But protons were 2 fold more effective than gamma-rays in induction of DNA double strand breaks. All breaks were repaired within < or =10 h. Irradiation resulted in increased expression of p53 and ATM. But no sings of cell death and retinal degeneration were observed during 7 days after irradiation. Proton irradiation in dose of 25 Gy resulted in increasing over time destructive changes localized mainly in photoreceptor layer of retina. These changes were followed by increased expression of proapoptotic proteins. A single systemic administration of MNU (70 mg/kg) increased intracellular levels of p53, PARP, FasR, caspase 3 active, which was followed by destructive changes in retina with sings of apoptosis of photoreceptors. As in the case of irradiation, the 2-fold dose reduction of MNU abrogated cytotoxic effect of MNU on retina. High level of spontaneous DNA damage such as apurine and apyrimidine sites were observed in mouse retina. The results of our study demonstrate the occurrence of genotoxic threshold in the initiation of retinal cell death in vivo. Topoisomerase 2 of retina is suggested to translate primary DNA damage to cytotoxic effect.     

Mitosis

Within the last decade, the study of mitosis has evolved into a multidisciplinary science in which findings from fields as diverse as chromosome biology and cytoskeletal architecture have converged to present a more cohesive understanding of the complex events that occur when cells divide. The largest strides have been made in the identification and characterization of regulatory enzymes (kinases and phosphatases) that modulate mitotic activity, as well as a number of the proteins and structural components (spindle, chromosomes, nuclear envelope) which carry out the mitotic instructions. One emerging theme appears to be that molecular signalling, which can involve modification of components (such as phosphorylation) or even their specific destruction, monitors the state of the mitotic cell at all stages. One of the major challenges for the future will be the identification of addititonal targets of the signalling machinery, as well as new regulatory components and their targets on the chromosomes, on the spindle, and at the cleavage furrow.     

Molecular characterization of cell types in the developing,mature, and regenerating fish retina

The fish retina has become a powerful model system for the study of different aspects of development and regeneration. An important aspect in understanding retinal anatomy and function is to trace the development of various cell types during embryonic stages. Several markers that detect the cessation of proliferative activity have been used in studies of cellular birth days, in order to follow the temporal progression of retinogenesis. Moreover, by using cell type-specific markers, the onset of differentiation can be determined by identifying the earliest time points for which immunolabeling is observed. Additionally, fish retinal regeneration research holds the potential of providing new avenues for the treatment of degenerative diseases of the retina. Retinal markers constitute powerful tools in studies of retinal regeneration, because they allow characterization of the cell types involved in nerve tissue regeneration, providing insights into different aspects of this process. In this review, after presenting several structural and histological aspects of the mature and developing fish visual system, data on the use of various neurochemical markers specifically indicating cell types of the fish neural retina are summarized. This will be done through a review of the pertinent literature, as well as by drawing on our own experience gathered through recent studies on fish retinogenesis.     

Regulation of Mitosis in Living Cells. I. Mitosis under Controlled Conditions

A quantitative method has been devised to study mitosis in vitro by phase contrast and polarization microscopy. Mitosis in cell-wall-free endosperm cells of Haemanthus kathrinar Baker (the African blood lily) has been divided into 18 arbitrary stages or events. The time course for the various stapes, as well as the percentage of cells that proceed from one stage to another during a four hour observation period, are presented. Cells that were in prophase when selected for study proceeded from nuclear membrane breakdown to melaphase in 60 minutes and remained in melaphase for 30 minutes. Only 13 minutes was required to proceed from onset of anaphase to mid-anaphasc. Mid-anaphase provides a clear and precise baseline for determining the time required for succeeding stages to appear. The cell plate made its appearance 40 minutes after mid-anaphase and was completely formed 20 minutes later. The nuclear membranes also became evident at this latter time and nucleoli were visible 30 minutes later. Thus, the average time for a cell observed initially in prophase to proceed from nuclear membrane breakdown to formation of two daughter cells was just over three hours. A high percentage of cells that were in late prophase or later stages of mitosis at the time of initial observation completed mitosis during the observation period. The effect of the length of time a cell is subjected to experimental conditions upon its subsequent behaviour is assessed. These results form the basis for future studies of the effects of chemicals, particularly herbicides, upon cells in mitosis as observed in vitro by phase contrast and polarization microscopy.     

Air-breathing adaptation in a marine Devonian lungfish

Recent discoveries of tetrapod trackways in 395 Myr old tidal zone deposits of Poland (Niedźwiedzki et al. 2010 Nature 463, 43–48 (doi:10.1038/nature.08623)) indicate that vertebrates had already ventured out of the water and might already have developed some air-breathing capacity by the Middle Devonian. Air-breathing in lungfishes is not considered to be a shared specialization with tetrapods, but evolved independently. Air-breathing in lungfishes has been postulated as starting in Middle Devonian times (ca 385 Ma) in freshwater habitats, based on a set of skeletal characters involved in air-breathing in extant lungfishes. New discoveries described herein of the lungfish Rhinodipterus from marine limestones of Australia identifies the node in dipnoan phylogeny where air-breathing begins, and confirms that lungfishes living in marine habitats had also developed specializations to breathe air by the start of the Late Devonian (ca 375 Ma). While invasion of freshwater habitats from the marine realm was previously suggested to be the prime cause of aerial respiration developing in lungfishes, we believe that global decline in oxygen levels during the Middle Devonian combined with higher metabolic costs is a more likely driver of air-breathing ability, which developed in both marine and freshwater lungfishes and tetrapodomorph fishes such as Gogonasus.     

Mitosis is swell

Cell volume and dry mass are typically correlated. However, in this issue, Zlotek-Zlotkiewicz et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201505056) and Son et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201505058) use new live-cell techniques to show that entry to mitosis coincides with rapid cell swelling, which is reversed before division.How growth is linked to division by the cell cycle regulatory network is an important open question in cell biology (Turner et al., 2012; Ginzberg et al., 2015). Yet, what is meant by cell growth? Different methods have been used to estimate either the total dry mass of the cell, total protein content, or cell volume. Although these parameters are often highly correlated, they are not the same. In budding yeast, growth parameters are nearly interchangeable as cell density changes only about 1% through the division cycle (Bryan et al., 2010). In contrast, cell density can drop by over 50% during a rapid growth phase in hypertrophic chondrocytes, which are responsible for determining bone length (Cooper et al., 2013). However, because of the current lack of similarly dramatic examples, it is assumed that chondrocytes are a special case and that most animal cells also exhibit little variation in cell density, as recently measured (Bryan et al., 2014). Yet this assumption has not been thoroughly tested because of the difficulty of measuring cell volume in animal cells, which are often irregularly shaped.Measuring cell volume is even more challenging in live cells. Whereas there is an accurate live-cell method for measuring dry mass in quantitative phase microscopy (Sung et al., 2013), live-cell volume measurements of adherent cells have been difficult because of their irregular geometry. Current methods are mostly based on 3D geometric reconstructions from confocal sections. However, confocal microscopy has poor resolution in the z-dimension, and increasing the number of z-sections to better estimate the cell membrane location and improve accuracy can be phototoxic.In this issue, Son et al. and Zlotek-Zlotkiewicz et al. applied two different methods to accurately measure cell volume changes in live cells. Son et al. (2015) used a variation of the suspended microchannel resonator pioneered by the Manalis laboratory (Fig. 1 A; Burg et al., 2007). In this method, the resonance frequency of the device shifts when a cell enters a part of a microchannel because the cell is of a different density than the surrounding media. The change in resonance frequency can therefore be used to calculate the buoyant mass of the cell. Changing the media of the microchannel to one of different density and then performing the same measurement for the same cell allows the accurate calculation of both cell dry mass and volume. One limitation of the microchannel resonator method is that the cells are required to be nonadherent so that they can be moved into and out of the resonator. To measure cell volume of adherent cells, Zlotek-Zlotkiewicz et al. (2015) used a microchamber culture device with a low 15–25-µm adjustable ceiling (Fig. 1 B). Cells were grown in a media containing fluorescent dye–labeled dextran. Cell volume could then be measured from epifluorescence images because the cells displaced the fluorescent dextran in proportion to their volume. This method was combined with quantitative phase microscopy to measure dry mass.Open in a separate windowFigure 1.Two new live-cell measurements of cell volume and mass reveal that cells swell in mitosis. (A) Schematic of microchannel resonator whose frequency is determined by the cells’ buoyant mass. Live-cell measurements in two media of different density allow calculation of cell volume and density (modified from Son et al., 2015). (B) Using epifluorescence microscopy, cell volume can be measured as the amount of dye-labeled dextran displaced in a low-ceiling culture chamber. (C) Cell density is constant through the cell cycle except in mitosis, when cells swell (modified from Son et al., 2015). (D) In the context of an animal tissue, mitotic swelling may generate a larger, rounder space to promote accurate and rapid chromosome segregation.Both Son et al. (2015) and Zlotek-Zlotkiewicz et al. (2015) applied their methods to precisely and noninvasively measure the volume and density dynamics in growing and dividing mammalian cells (Fig. 1 C). During most of the cell cycle, density is constant and dry mass is correlated with volume. However, the researchers found that cell volume, but not dry mass, increases rapidly as cells enter mitosis. This osmotic swelling occurs during prophase and prometaphase before being reversed in anaphase and telophase. Collectively, the work of both teams also determined that mitotic swelling is driven by osmotic water exchange and requires the activity of the Na/H ion exchanger but is not dependent on the actomyosin cortex, endocytosis, or cytokinesis. Whereas previous studies gave contradictory results, the two papers in this issue show that there is a reversible 10–30% volume increase during mitosis depending on the type of cell.The establishment of cell swelling during mitosis raises the question of its function. In laboratory conditions, mitotic animal cells lose surface adhesion and are spherical. This spherical geometry is accompanied by an increase in intracellular hydrostatic pressure (Stewart et al., 2011). In the in vivo context of an animal tissue, an increase in intracellular pressure accompanied by cell swelling would allow cells to push against their neighbors and open up additional space for mitosis (Fig. 1 D; Son et al., 2015; Zlotek-Zlotkiewicz et al., 2015). The mitotic acquisition of a larger, more spherical geometry may be important because physically preventing cells from rounding up retards mitosis and promotes inaccurate chromosome segregation (Lancaster et al., 2013). Alternatively, the dilution of the cytoplasm by swelling might change the physicochemical properties of the intracellular environment to facilitate chromosomal movement and segregation or change the kinetics of biochemical reactions (Son et al., 2015).Live-cell methods that accurately measure volume will most obviously be useful for studies of how cell growth is linked to cell cycle progression but are unlikely to be limited to this application. For example, it would be interesting to follow the dynamics of cell volume and density in other processes in which the surface area to volume ratio can change rapidly, such as cell migration (Traynor and Kay, 2007). Depending on the environment, cells can switch from actin-driven motility to hydrostatic pressure–driven bleb-based motility (Sahai and Marshall, 2003; Zatulovskiy et al., 2014). Because this motility switch strongly depends on the osmolarity of the environment (Fedier and Keller, 1997; Yoshida and Soldati, 2006), it is likely to be accompanied by and perhaps even require cell swelling.Although the swelling of animal cells has been mostly neglected, cell swelling is not unusual in other eukaryotic lineages. Unlike animal cells, which have a flexible cell geometry that can rapidly be remodeled, plant and fungal cells have a stiff cell wall and cannot easily change their geometry. Nevertheless, plants and fungi can use regulated swelling to move on time scales faster than that of growth (Skotheim and Mahadevan, 2005). For example, a stem bending to track the sun is caused by cells on one side swelling, whereas those on the other side shrink. This differential swelling allows the stem to bend because the plant tissue is connected by elastic cell walls. Using such differential swelling, plants and fungi can perform impressive coordinated movements to track the sun, compete for territory, disperse seeds or spores, and catch prey (Attenborough, 1995). Although swelling-based movements have long been appreciated in the context of plants, there is no a priori reason animal cells might not also harness such mechanisms to perform important functions. The further development and dissemination of technologies to accurately measure cell volume, density, and dry mass, such as those described in this issue, will be essential to determine the extent to which animal cells harness swelling.     

Mitosis: Introduction

Even before biochemical and genetic approaches were employed, many researchers were captivated and astounded by mitosis. Cell biologists have been enthralled by its sheer beauty for more than a century (Fig. 1). Yanagida, who probably contributed more to the genetic dissection of mitotic processes than any other scientist, described it as a “cellular festival”, dramatically thrust between the humdrum chores of daily life. True enough; if the cell cycle were a theatrical performance, mitosis would be the final climatic act. In this Spotlight, a few of the exciting aspects of mitosis are reviewed.

Key Words:

Mitosis, Chromosome, Segregation, Cytokinesis, Mitotic spindle, Skeletor