Two Fluorescent Markers Identify the Vacuolar System of Schizophyllum commune

Vacuole-mediated proteolysis is important to sustained growth of filamentous wood-decaying fungi such as Schizophyllum commune. Demonstrating that specific proteases are vacuole associated has been difficult in these organisms due to the lack of specific markers for vacuolar compartments. We used 5-(and 6-)-carboxy-2′, 7′-dichlorofluorescein diacetate (carboxy-DCFDA) and a proprietary vacuolar membrane marker for yeast (MDY-64; Molecular Probes) for in situ fluorescent labeling of the vacuoles of S. commune mycelia grown on microscope slides. MDY-64 labels numerous small vesicles in S. commune mycelia in addition to larger vacuolar structures. In contrast, carboxy-DCFDA apparently is taken up by a subset of the MDY-64-labeled vesicles, accumulating primarily in larger vacuoles. Staining of mycelia with carboxy-DCFDA shows a transition from mostly cytoplasmic fluorescence in apical cells with little vacuolar fluorescence to nearly complete sequestration of the stain in vacuoles of older cells. In penultimate cells, both cytoplasm and vacuolar structures fluoresce. Vacuoles stained with carboxy-DCFDA typically were spherical and ranged in size from 0.4 μm to 3.2 μm in diameter with a mean of 1.8 um. Occasionally, in penultimate cells, tubular structures which stained with carboxy-DCFDA were found. ScPrB, a principal enzyme of nitrogen-limitation induced autolysis in S. commune, copurified in sucrose density gradients with carboxy-DCFDA and acid phosphatase, demonstrating its vacuolar localization. Received: 23 December 1998 / Accepted: 11 January 1999     

Mitogen-activated protein kinase dynamics during the meiotic G2/MI transition of mouse spermatocytes

Cellular and genetic approaches were used to investigate the requirements for activation during spermatogenesis of the extracellular signal-regulated protein kinases (ERKs), more commonly known as the mitogen-activated protein kinases (MAPKs). The MAPKS and their activating kinases, the MEKs, are expressed in specific developmental patterns. The MAPKs and MEK2 are expressed in all premeiotic germ cells and spermatocytes, while MEK1 is not expressed abundantly in pachytene spermatocytes. Phosphorylated (active) variants of these kinases are diminished in pachytene spermatocytes. Treatment of pachytene spermatocytes with okadaic acid (OA), to induce transition from meiotic prophase to metaphase I (G2/MI), resulted in phosphorylation and enzymatic activation of ERK1/2. However, U0126, an inhibitor of the ERK-activating kinases, MEK1/2, did not inhibit OA-induced MAPK activation or chromosome condensation. Analysis of spermatocytes lacking MOS, a mitogen-activated protein kinase kinase kinase responsible for MEK and MAPK activation, revealed that MOS is not required for OA-induced activation of the MAPKs. OA-induced MAPK activation was inhibited by butyrolactone I, an inhibitor of cyclin-dependent kinases 1 and 2 (CDK1, CDK2); thus, these kinases may regulate MAPK activity. Additionally, spermatocytes lacking CDC25C condensed bivalent chromosomes and activated both MPF and MAPKs in response to OA treatment; therefore, there is a CDC25C-independent pathway for MPF and MAPK activation. These studies reveal that spermatocytes do not require either MOS or CDC25C for onset of the meiotic division phase or for activation of MPF and the MAPKs, thus implicating a novel pathway for activation of the ERK1/2 MAPKs in spermatocytes.     

Buteo Nesting Ecology: Evaluating Nesting of Swainson’s Hawks in the Northern Great Plains

Swainson’s hawks (Buteo swainsoni) are long-distance migratory raptors that nest primarily in isolated trees located in areas of high grassland density. In recent years, anthropogenic conversion of grassland habitat has raised concerns about the status of the breeding population in the northern Great Plains. In 2013, we initiated a study to investigate the influence of extrinsic factors influencing Swainson’s hawk nesting ecology in north-central South Dakota and south-central North Dakota. Using ground and aerial surveys, we located and monitored nesting Swainson’s hawk pairs: 73 in 2013 and 120 in 2014. We documented 98 successful breeding attempts that fledged 163 chicks; 1.52 and 1.72 fledglings per successful nest in 2013 and 2014, respectively. We used Program MARK to evaluate the influence of land cover on nest survival. The top model, S _{Dist2Farm+%Hay}, indicated that nest survival (fledging at least one chick) decreased as nests were located farther from farm sites and as the percent of hay cover increased within 1200-m of the nest site (34.4%; 95% CI = 27.6%–42.3%). We used logistic regression analysis to evaluate the influence of landscape variables on nest-site selection; Swainson’s hawks selected for nest sites located closer to roads. We suggest that tree belts associated with farm sites, whether occupied or not, provide critical breeding sites for Swainson’s hawks. Additionally, poor breeding success may be related to the late migratory behavior of this species which requires them to occupy marginal habitat due to other raptors occupying the most suitable habitat prior to Swainson’s hawks arriving to the breeding grounds.