The Cytoplasmic pH Influences Hyphal Tip Growth and Cytoskeleton-Related Organization

The regulatory role of protons in hyphal tip growth was investigated by using membrane-permeant weak acids to acidify cytoplasm of the oomycete Saprolegnia ferax. Acetic acid decreased cytoplasmic pH from approximately pH 7.2 to 6.8, as shown by SNARF-1 measurements of cytoplasmic pH. Inhibition of growth in a dose-dependent manner by acetic, propionic, and isobutyric acid was accompanied by changes in positioning and morphology of mitochondria and nuclei, condensation of chromatin, disruptions in peripheral actin, and increases in hyphal diameter. These cellular alterations were fully reversible, and during recovery, major cytoplasmic movements and extensive apical vacuolations were observed. The results are consistent with proton regulation of the cytoskeleton, nuclear matrix, and/or chromosomes. However, a macroscopic cytoplasmic gradient of H+ in hyphae was not revealed by SNARF-1, indicating that if such a H+ gradient were required, it must occur at a finer level than we detected.     

Carbon dioxide induces increases in guard cell cytosolic free calcium

The hypothesis that increases in cytosolic free calcium ([Ca²⁺]_i) are a component of the CO₂ signal transduction pathway in stomatal guard cells of Commelina communis has been investigated. This hypothesis was tested using fura-2 fluorescence ratio photometry to measure changes in guard cell [Ca²⁺]_i in response to challenge with 700 µl l⁻¹ CO₂. Elevated CO₂ induced increases in guard cell [Ca²⁺]_i which were similar to those previously reported in response to abscisic acid. [Ca²⁺]_i returned to resting values following removal of the CO₂ and further application of CO₂ resulted in a second increase in [Ca²⁺]_i. This demonstrated that the CO₂-induced increases in [Ca²⁺]_i were stimulus dependent. Removal of extracellular calcium both prevented the CO₂-induced increase in [Ca²⁺]_i and inhibited the associated reduction in stomatal aperture. These data suggest that Ca²⁺ acts as a second messenger in the CO₂ signal transduction pathway and that an increase in [Ca²⁺]_i may be a requirement for the stomatal response to CO₂.     

A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation.     

Location of the gene involving the small eye mutation on mouse chromosome 2 suggests homology with human aniridia 2 (AN2)

Using an interspecific backcross, we have mapped the gene involved in the mouse Small eye mutation (SeyMH) relative to six cloned markers on chromosome 2 (Hox-5.1, Cas-1, Fshb, Bmp-2a, and ld) and the agouti locus. The results suggest that the Sey gene maps between Fshb and Cas-1. Human mapping studies have shown that the aniridia (AN2) gene, which is part of the Wilms tumor susceptibility, aniridia, genitourinary abnormalities, and mental retardation (WAGR) complex, is also between FSHB and CAT on human chromosome 11. The conserved linkage of the cloned markers and the similarity of the Sey/+ and AN2/+ phenotypes suggest that the gene involved in the Sey mutation is the mouse homolog of the human AN2 gene.     

Monitoring chilling injury: A comparison of chlorophyll fluorescence measurements, post-chilling growth and visible symptoms of injury in Zea mays

Changes in chlorophyll fluorescence emission from maize ( Zea mays L. cv. Northern Belle) seedlings chilled at 1.5°C in the dark for 3–30 h were compared with the ability of plants to resume growth in the immediate post-chilling period and with the development of visible symptoms of injury to the leaves. During chilling, the maximal rate of increase of the induced chlorophyll fluorescence rise. F_R, was measured on secondary leaf tissue. F_R decreased exponentially, at approximately the same rate in plants grown and chilled in hydroponic pots, in leaves detached from similar plants and in plants that were removed from the hydroponic pots and laid on wet filter paper adjacent to the detached leaves. The half-fall time for F_R in the 3 treatments was 7.8 ± 1.3 h, 8.6 ± 0.6 h and 8.8 ± 1.0 h, respectively. Following seedling removal from 1.5°C and return to 25/15°C, relative growth rates were determined from daily measurements of plant fresh weight gain. Compared with non-chilled seedlings, plants chilled for 3 h and longer showed depressed rates of growth. Inhibition of growth in the immediate post-chilling period (0–27 h) was linearly related to the duration of the chilling period and had a high positive correlation with the decrease in chlorophyll fluorescence (linearly related to log F_R) sustained during the chilling exposure. Visible symptoms of chilling injury developed during the post-chilling period on seedlings chilled for longer than 3 h. The decrease in log F_R during chilling was also linearly correlated with the severity of visual symptoms of chilling injury expressed in the post-chilling period. It is concluded that the extent of chilling injury in maize can be rapidly and non-destructively assessed from measurements of chlorophyll fluorescence.     

Elongation Factor 1A-1 Is a Mediator of Hepatocyte Lipotoxicity Partly through Its Canonical Function in Protein Synthesis

Elongation factor 1A-1 (eEF1A-1) has non-canonical functions in regulation of the actin cytoskeleton and apoptosis. It was previously identified through a promoter-trap screen as a mediator of fatty acid-induced cell death (lipotoxicity), and was found to participate in this process downstream of ER stress. Since ER stress is implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), we investigated the mechanism of action of eEF1A-1 in hepatocyte lipotoxicity. HepG2 cells were exposed to excess fatty acids, followed by assessments of ER stress, subcellular localization of eEF1A-1, and cell death. A specific inhibitor of eEF1A-1 elongation activity, didemnin B, was used to determine whether its function in protein synthesis is involved in lipotoxicity. Within 6 h, eEF1A-1 protein was modestly induced by high palmitate, and partially re-localized from its predominant location at the ER to polymerized actin at the cell periphery. This early induction and subcellular redistribution of eEF1A-1 coincided with the onset of ER stress, and was later followed by cell death. Didemnin B did not prevent the initiation of ER stress by high palmitate, as indicated by eIF2α phosphorylation. However, consistent with sustained inhibition of eEF1A-1-dependent elongation activity, didemnin B prevented the recovery of protein synthesis and increase in GRP78 protein that are normally associated with later phases of the response to ongoing ER stress. This resulted in decreased palmitate-induced cell death. Our data implicate eEF1A-1, and its function in protein synthesis, in hepatocyte lipotoxicity.