Putting the Cell in Order

Organization is one of the most conspicuous features of cells. Not only are cells highly ordered (in the sense of regularity and predictability), but also they are organized: their order has purpose, or function. How does biological organization arise, and how is it transmitted from one generation to the next? A key element is genetic information encoded in DNA. Many scientists hold that DNA is the master molecule of life that prescribes all that cells are and do, and the general public has swallowed that doctrine whole. There is truth in this view of biological organization, inasmuch as genes do specify the chemical structure (and thereby the function) of proteins, nucleic acids, and (indirectly) many other biomolecules. But that is only part of an increasingly complex story. The higher levels of cell organization are not spelled out in the genes; they arise by self-organization, and are commonly transmitted to the next generation because the mother cell is architecturally continuous with its daughter. DNA provides an indispensable database, but does not direct the show. Organisms are better understood as complex interactive systems composed of genetically specified elements.

A simple method for the application of exogenous phytohormones to the grass leaf base protodermal zone to improve grass leaf epidermis development research

Background The leaf epidermis functions to prevent the loss of water and reduce gas exchange. As an interface between the plant and its external environment, it helps prevent damage, making it an attractive system for studying cell fate and development. In monocotyledons, the leaf epidermis grows from the basal meristem that contains protodermal cells. Leaf protoderm zone is covered by the leaf sheath or coleoptile in maize and wheat, preventing traditional exogenous phytohormone application methods, such as directly spraying on the leaf surface or indirectly via culture media, from reaching the protoderm areas directly. The lack of a suitable application method limits research on the effect of phytohormone on the development of grass epidermis. Results Here, we describe a direct and straightforward method to apply exogenous phytohormones to the leaf protoderms of maize and wheat. We used the auxin analogs 2,4-D and cytokinin analogs 6-BA to test the system. After 2,4-D treatment, the asymmetrical division events and initial stomata development were decreased, and the subsidiary cells were induced in maize, the number of GMC (guard mother cell), SMC (subsidiary mother cell) and young stomata were increased in wheat, and the size of the epidermal cells increased after 6-BA treatment in maize. Thus, the method is suitable for the application of phytohormone to the grass leaf protodermal areas. Conclusions The method to apply hormones to the mesocotyls of maize and wheat seedlings is simple and direct. Only a small amount of externally applied substances are needed to complete the procedure in this method. The entire experimental process lasts for ten days generally, and it is easy to evaluate the phytohormones’ effect on the epidermis development.

Morpho-Histology, Endogenous Hormone Dynamics and Transcriptome Profiling in Dacrydium Pectinatum during Male Cone Development

Dacrydium pectinatum de Laubenfels is a perennial gymnosperm species dominant in tropical montane rain forests. Due to severe damages by excessive deforestation, typhoons, and other external forces, the population of the species has been significantly reduced. Furthermore, its natural regeneration is poor. To better understand the male cone development in D. pectinatum, we examined the morphological and anatomical changes, analyzed the endogenous hormone dynamics, and profiled gene expression. The morpho-histological observations suggest that the development of D. pectinatum male cone can be largely divided into four stages: microspore primordium formation (April to May), microspore sac and pollen mother cell formation (July to November), pollen mother cell division (January), and pollen grain formation (February). The levels of gibberellins (GA), auxin (IAA), abscisic Acid (ABA), cytokinin (CTK), and jasmonic acid (JA) fluctuated during the process of male cone development. The first transcriptome database for a Dacrydium species was generated, revealing >70,000 unigene sequences. Differential expression analyses revealed several floral and hormone biosynthesis and signal transduction genes that could be critical for male cone development. Our study provides new insights on the cone development in D. pectinatum and the foundation for male cone induction with hormones and studies of factors contributing to the species’ low rate of seed germination.

Septin Remodeling During Mammalian Cytokinesis

Cytokinesis mediates the final separation of a mother cell into two daughter cells. Septins are recruited to the cleavage furrow at an early stage. During cytokinetic progression the septin cytoskeleton is constantly rearranged, ultimately leading to a concentration of septins within the intercellular bridge (ICB), and to the formation of two rings adjacent to the midbody that aid ESCRT-dependent abscission. The molecular mechanisms underlying this behavior are poorly understood. Based on observations that septins can associate with actin, microtubules and associated motors, we review here established roles of septins in mammalian cytokinesis, and discuss, how septins may support cytokinetic progression by exerting their functions at particular sites. Finally, we discuss how this might be assisted by phosphoinositide-metabolizing enzymes.

Characterization of germline development and identification of genes associated with germline specification in pineapple

Understanding germline specification in plants could be advantageous for agricultural applications. In recent decades, substantial efforts have been made to understand germline specification in several plant species, including Arabidopsis, rice, and maize. However, our knowledge of germline specification in many agronomically important plant species remains obscure. Here, we characterized the female germline specification and subsequent female gametophyte development in pineapple using callose staining, cytological, and whole-mount immunolocalization analyses. We also determined the male germline specification and gametophyte developmental timeline and observed male meiotic behavior using chromosome spreading assays. Furthermore, we identified 229 genes that are preferentially expressed at the megaspore mother cell (MMC) stage during ovule development and 478 genes that are preferentially expressed at the pollen mother cell (PMC) stage of anther development using comparative transcriptomic analysis. The biological functions, associated regulatory pathways and expression patterns of these genes were also analyzed. Our study provides a convenient cytological reference for exploring pineapple germline development and a molecular basis for the future functional analysis of germline specification in related plant species.

Membrane fission during bacterial spore development requires DNA-driven cellular inflation

Bacteria require membrane fission for cell division and endospore formation. FisB catalyzes membrane fission during sporulation, but the molecular basis is unclear as it cannot remodel membranes by itself. Sporulation initiates with an asymmetric division that generates a large mother cell and a smaller forespore that contains only 1/4 of its complete genome. As the mother cell membranes engulf the forespore, a DNA translocase pumps the rest of the chromosome into the small forespore compartment, inflating it due to increased turgor. When the engulfing membranes undergo fission, the forespore is released into the mother cell cytoplasm. Here we show that forespore inflation and FisB accumulation are both required for efficient membrane fission. We suggest that high membrane tension in the engulfment membrane caused by forespore inflation drives FisB-catalyzed membrane fission. Collectively our data indicate that DNA-translocation has a previously unappreciated second function in energizing FisB-mediated membrane fission under energy-limited conditions.

The fate of notch-1 transcript is linked to cell cycle dynamics by activity of a natural antisense transcript

A core imprint of metazoan life is that perturbations of cell cycle are offset by compensatory changes in successive cellular generations. This trait enhances robustness of multicellular growth and requires transmission of signaling cues within a cell lineage. Notably, the identity and mode of activity of transgenerational signals remain largely unknown. Here we report the discovery of a natural antisense transcript encoded in exon 25 of notch-1 locus (nAS25) by which mother cells control the fate of notch-1 transcript in daughter cells to buffer against perturbations of cell cycle. The antisense transcript is transcribed at G1 phase of cell cycle from a bi-directional E2F1-dependent promoter in the mother cell where the titer of nAS25 is calibrated to the length of G1. Transmission of the antisense transcript from mother to daughter cells stabilizes notch-1 sense transcript in G0 phase of daughter cells by masking it from RNA editing and resultant nonsense-mediated degradation. In consequence, nAS25-mediated amplification of notch-1 signaling reprograms G1 phase in daughter cells to compensate for the altered dynamics of the mother cell. The function of nAS25/notch-1 in integrating G1 phase history of the mother cell into that of daughter cells is compatible with the predicted activity of a molecular oscillator, slower than cyclins, that coordinates cell cycle within cell lineage.

FisB relies on homo-oligomerization and lipid binding to catalyze membrane fission in bacteria

Little is known about mechanisms of membrane fission in bacteria despite their requirement for cytokinesis. The only known dedicated membrane fission machinery in bacteria, fission protein B (FisB), is expressed during sporulation in Bacillus subtilis and is required to release the developing spore into the mother cell cytoplasm. Here, we characterized the requirements for FisB-mediated membrane fission. FisB forms mobile clusters of approximately 12 molecules that give way to an immobile cluster at the engulfment pole containing approximately 40 proteins at the time of membrane fission. Analysis of FisB mutants revealed that binding to acidic lipids and homo-oligomerization are both critical for targeting FisB to the engulfment pole and membrane fission. Experiments using artificial membranes and filamentous cells suggest that FisB does not have an intrinsic ability to sense or induce membrane curvature but can bridge membranes. Finally, modeling suggests that homo-oligomerization and trans-interactions with membranes are sufficient to explain FisB accumulation at the membrane neck that connects the engulfment membrane to the rest of the mother cell membrane during late stages of engulfment. Together, our results show that FisB is a robust and unusual membrane fission protein that relies on homo-oligomerization, lipid binding, and the unique membrane topology generated during engulfment for localization and membrane scission, but surprisingly, not on lipid microdomains, negative-curvature lipids, or curvature sensing.

Asymmetric localization of the cell division machinery during Bacillus subtilis sporulation

The Gram-positive bacterium Bacillus subtilis can divide via two modes. During vegetative growth, the division septum is formed at the midcell to produce two equal daughter cells. However, during sporulation, the division septum is formed closer to one pole to yield a smaller forespore and a larger mother cell. Using cryo-electron tomography, genetics and fluorescence microscopy, we found that the organization of the division machinery is different in the two septa. While FtsAZ filaments, the major orchestrators of bacterial cell division, are present uniformly around the leading edge of the invaginating vegetative septa, they are only present on the mother cell side of the invaginating sporulation septa. We provide evidence suggesting that the different distribution and number of FtsAZ filaments impact septal thickness, causing vegetative septa to be thicker than sporulation septa already during constriction. Finally, we show that a sporulation-specific protein, SpoIIE, regulates asymmetric divisome localization and septal thickness during sporulation.

NanH Is Produced by Sporulating Cultures of Clostridium perfringens Type F Food Poisoning Strains and Enhances the Cytotoxicity of C. perfringens Enterotoxin

ABSTRACT Clostridium perfringens type F food poisoning (FP) strains cause one of the most common foodborne illnesses. This FP develops when type F FP strains sporulate in the intestines and produce C. perfringens enterotoxin (CPE), which is responsible for the diarrhea and abdominal cramps of this disease. While C. perfringens can produce up to three different sialidases, the current study surveyed FP strains, which confirmed the results of a previous study that they consistently carry the nanH sialidase gene, often as their only sialidase gene. NanH production was found to be associated with sporulating cultures of the surveyed type F FP strains, including SM101 (a transformable derivative of a FP strain). The sporulation-associated regulation of NanH production by strain SM101 growing in modified Duncan-Strong medium (MDS) was shown to involve Spo0A, but it did not require the completion of sporulation. NanH production was not necessary for either the growth or sporulation of SM101 when cultured in MDS. In those MDS cultures, NanH accumulated in the sporulating mother cell until it was released coincidently with CPE. Since CPE becomes extracellular when mother cells lyse to release their mature spores, this indicates that mother cell lysis is also important for NanH release. The copresence of NanH and CPE in supernatants from lysed sporulating cultures was shown to enhance CPE cytotoxicity for Caco-2 cells. This enhancement was attributable to NanH increasing CPE binding and could be replicated with purified recombinant NanH. These in vitro findings suggest that NanH may be an accessory virulence factor during type F FP. IMPORTANCE Clostridium perfringens type F strains cause the second most common bacterial foodborne illness in the United States. C. perfringens enterotoxin (CPE) is responsible for the diarrhea and cramping symptoms of this food poisoning (FP). Previous studies showed that NanI sialidase can enhance CPE activity in vitro. While many type F FP strains do not produce NanI, they do consistently make NanH sialidase. This study shows that, like CPE, NanH is produced by sporulating type F FP strains and then released extracellularly when their sporulating cells lyse to release their mature spore. NanH was shown to enhance CPE cytotoxicity in vitro by increasing CPE binding to cultured Caco-2 cells. This enhancement could be important because many type F FP strains produce less CPE than necessary (in a purified form) to cause intestinal pathology in animal models. Therefore, NanH represents a potential accessory virulence factor for type F FP.