Phytophthora capsici homologue of the cell cycle regulator SDA1 is required for sporangial morphology,mycelial growth and plant infection

SDA1 encodes a highly conserved protein that is widely distributed in eukaryotic organisms. SDA1 is essential for cell cycle progression and organization of the actin cytoskeleton in yeasts and humans. In this study, we identified a Phytophthora capsici orthologue of yeast SDA1, named PcSDA1. In P. capsici, PcSDA1 is strongly expressed in three asexual developmental states (mycelium, sporangia and germinating cysts), as well as late in infection. Silencing or overexpression of PcSDA1 in P. capsici transformants affected the growth of hyphae and sporangiophores, sporangial development, cyst germination and zoospore release. Phalloidin staining confirmed that PcSDA1 is required for organization of the actin cytoskeleton. Moreover, 4′,6‐diamidino‐2‐phenylindole (DAPI) staining and PcSDA1‐green fluorescent protein (GFP) fusions revealed that PcSDA1 is involved in the regulation of nuclear distribution in hyphae and sporangia. Both silenced and overexpression transformants showed severely diminished virulence. Thus, our results suggest that PcSDA1 plays a similar role in the regulation of the actin cytoskeleton and nuclear division in this filamentous organism as in non‐filamentous yeasts and human cells.     

Identification of cystatin as a component of carp chorion

An ovary-specific cystatin is immunocytochemically demonstrated to be localized in the chorions, cortical granules, and yolk granules of carp oocytes, as well as in the follicle cells surrounding oocytes. During cortical reaction, cystatin is exocytosed from cortical granules into the perivitelline space. In situ hybridization confirms that cystatin is synthesized by oocytes and follicle cells. Western blotting reveals that chorion cystatin appears in multiple bands of high molecular weight (from 65 kDa to larger than 200 kDa). No cystatin monomer of 14 kDa is found. These results indicate that chorion cystatin is conjugated with other chorion components. Two forms of conjugates are found. In one form, cystatin, ZP2, fibroin-like substance (FLS), and cathepsin-like substance (CLS) are conjugated, which is extracted by sodium dodecyl sulfate. In the other form, cystatin, FLS, and CLS are conjugated, which is extracted by guanidine thiocyanate (GTC). Most chorion cystatin of oocytes and ovulated eggs is solubilized by GTC, while a large amount of cystatin remains in the fertilization envelope of cortical reacted eggs after extraction by GTC. Mol. Reprod. Dev. 51:430–435, 1998. © 1998 Wiley-Liss, Inc.     

Role of N-WASP in Endothelial Monolayer Formation and Integrity

Endothelial cells (ECs) form a monolayer that serves as a barrier between the blood and the underlying tissue. ECs tightly regulate their cell-cell junctions, controlling the passage of soluble materials and immune cells across the monolayer barrier. We studied the role of N-WASP, a key regulator of Arp2/3 complex and actin assembly, in EC monolayers. We report that N-WASP regulates endothelial monolayer integrity by affecting the organization of cell junctions. Depletion of N-WASP resulted in an increase in transendothelial electrical resistance, a measure of monolayer integrity. N-WASP depletion increased the width of cell-cell junctions and altered the organization of F-actin and VE-cadherin at junctions. N-WASP was not present at cell-cell junctions in monolayers under resting conditions, but it was recruited following treatment with sphingosine-1-phosphate. Taken together, our results reveal a novel role for N-WASP in remodeling EC junctions, which is critical for monolayer integrity and function.     

Analysis of the growth of Chlorella cultures using electronically measured cell-volume distribution data

Cell-number density and cell-volume distribution data were obtained from cultures of Chlorella fusca var. vacuolata Shihira & Krauss growing under both continuous and periodic illumination. Mean, median and modal cell-volumes were calculated from the cell-volume distributions and a high correlation shown between mean and median values, whilst mean and modal values did not correlate well. It was concluded that where the computation necessary for deriving mean cell-volumes was not practicable, the median cell-volume was the next most useful statistic.

Synchrony indices of discontinuously illuminated cultures gave similar values when based on change in cell-number and change in mean cell-volume. Cultures under continuous illumination showed synchronous divisions at the beginning of growth. These divisions gave high values of a synchrony index based on change in cell-number, but low values of an index based on change in mean cell-volume. It was concluded that mean cell-volume data are more sensitive to the occurrence of unbalanced growth than analysis of cell-number data.