Douglas J. Law

The Rim101p/PacC pathway and alkaline pH regulate pattern formation in yeast colonies.

Multicellular organisms utilize cell-to-cell signals to build patterns of cell types within embryos, but the ability of fungi to form organized communities has been largely unexplored. Here we report that colonies of the yeast Saccharomyces cerevisiae formed sharply divided layers of sporulating and nonsporulating cells. Sporulation initiated in the colonys interior, and this region expanded upward as the colony matured. Two key activators of sporulation, IME1 and IME2, were initially transcribed in overlapping regions of the colony, and this overlap corresponded to the initial sporulation region. The development of colony sporulation patterns depended on cell-to-cell signals, as demonstrated by chimeric colonies, which contain a mixture of two strains. One such signal is alkaline pH, mediated through the Rim101p/PacC pathway. Meiotic-arrest mutants that increased alkali production stimulated expression of an early meiotic gene in neighboring cells, whereas a mutant that decreased alkali production (cit1Δ) decreased this expression. Addition of alkali to colonies accelerated the expansion of the interior region of sporulation, whereas inactivation of the Rim101p pathway inhibited this expansion. Thus, the Rim101 pathway mediates colony patterning by responding to cell-to-cell pH signals. Cell-to-cell signals coupled with nutrient gradients may allow efficient spore formation and spore dispersal in natural environments.

Terminal regions of mouse nebulin: sequence analysis and complementary localization with N-RAP.

The regions of mouse nebulin extending from the ends of the super repeats to the C-terminus and N-terminus were cloned and sequenced. Comparison of the mouse sequence with the previously published human sequence shows that the terminal regions of nebulin are highly conserved. The four phosphorylation motifs and SH3 domain found at the C-terminus of mouse nebulin are identical to those found in human nebulin, with the exception of four conservative substitutions. The modules linking this C-terminal region to the super repeats have deletions relative to both fetal and adult human nebulins that correspond to integral numbers of modules, making the mouse C-terminal simple repeat region among the shortest observed to date. The N-terminal region and the C-terminal modules were expressed in Escherichia coli and used for antibody production. Immunofluorescent labeling of these regions of nebulin in isolated myofibrils demonstrates that they are located near the center of the sarcomere and near the Z-line, respectively. Immunogold labeling with antibodies raised against the N-terminal nebulin sequence localizes this region in the A-band near the tips of the thin filaments. Nebulin localization is complementary to that of N-RAP, another muscle-specific protein containing nebulin-like super repeats; nebulin is exclusively found in the sarcomeres, while N-RAP is confined to the terminal bundles of actin filaments at the myotendinous junction. Cell Motil. Cytoskeleton 3:211-222, 2000 Published 2000 Wiley-Liss, Inc.

Flo11p adhesin required for meiotic differentiation in Saccharomyces cerevisiae minicolonies grown on plastic surfaces.

Saccharomyces cerevisiae grown on plastic surfaces formed organized structures, termed minicolonies, that consisted of a core of round (yeast-like) cells surrounded by chains of filamentous cells (pseudohyphae). Minicolonies had a much higher affinity for plastic than unstructured yeast communities growing on the same surface. Pseudohyphae at the surface of these colonies developed further into chains of asci. These structures suggest that pseudohyphal differentiation and sporulation are sequential processes in minicolonies. Consistent with this idea, minicolonies grown under conditions that stimulated pseudohyphal differentiation contained higher frequencies of asci. Furthermore, a flo11Δ mutant, which fails to form pseudohyphae, yielded normal sporulation in cultures, but was defective for minicolony sporulation. When minicolonies were dispersed in water and cells were then allowed to settle on the plastic surface, these cells sporulated very efficiently. Taken together, our results suggest that sporulation in minicolonies is stimulated by pseudohyphal differentiation because these pseudohyphae are dispersed from the core of the colony.

Improvement in survival and muscle function in an mdx/utrn−/− double mutant mouse using a human retinal dystrophin transgene

Duchenne muscular dystrophy is a progressive muscle disease characterized by increasing muscle weakness and death by the third decade. mdx mice exhibit the underlying muscle disease but appear physically normal with ordinary lifespans, possibly due to compensatory expression of utrophin. In contrast, double mutant mice (mdx/utrn(-/-)), deficient for both dystrophin and utrophin die by approximately 3 months and suffer from severe muscle weakness, growth retardation, and severe spinal curvature. The capacity of human retinal dystrophin (Dp260) to compensate for the missing 427 kDa muscle dystrophin was tested in mdx/utrn(-/-) mice. Functional outcomes were assessed by histology, EMG, MRI, mobility, weight and longevity. MCK-driven transgenic expression of Dp260 in mdx/utrn(-/-) mice converts their disease course from a severe, lethal muscular dystrophy to a viable, mild myopathic phenotype. This finding is relevant to the design of exon-skipping therapeutic strategies since Dp260 lacks dystrophin exons 1-29.

Flo11p adhesin required for meiotic differentiation in S. cerevisiae minicolonies grown on plastic surfaces

Saccharomyces cerevisiae grown on plastic surfaces formed organized structures, termed minicolonies, that consisted of a core of round (yeast-like) cells surrounded by chains of filamentous cells (pseudohyphae). Minicolonies had a much higher affinity for plastic than unstructured yeast communities growing on the same surface. Pseudohyphae at the surface of these colonies developed further into chains of asci. These structures suggest that pseudohyphal differentiation and sporulation are sequential processes in minicolonies. Consistent with this idea, minicolonies grown under conditions that stimulated pseudohyphal differentiation contained higher frequencies of asci. Furthermore, a flo11Δ mutant, which fails to form pseudohyphae, yielded normal sporulation in cultures, but was defective for minicolony sporulation. When minicolonies were dispersed in water and cells were then allowed to settle on the plastic surface, these cells sporulated very efficiently. Taken together, our results suggest that sporulation in minicolonies is stimulated by pseudohyphal differentiation because these pseudohyphae are dispersed from the core of the colony.

Flo11p adhesin required for meiotic differentiation in Saccharomyces cerevisiae minicolonies grown on plastic surfaces: Yeast minicolony development

Saccharomyces cerevisiae grown on plastic surfaces formed organized structures, termed minicolonies, that consisted of a core of round (yeast-like) cells surrounded by chains of filamentous cells (pseudohyphae). Minicolonies had a much higher affinity for plastic than unstructured yeast communities growing on the same surface. Pseudohyphae at the surface of these colonies developed further into chains of asci. These structures suggest that pseudohyphal differentiation and sporulation are sequential processes in minicolonies. Consistent with this idea, minicolonies grown under conditions that stimulated pseudohyphal differentiation contained higher frequencies of asci. Furthermore, a flo11Δ mutant, which fails to form pseudohyphae, yielded normal sporulation in cultures, but was defective for minicolony sporulation. When minicolonies were dispersed in water and cells were then allowed to settle on the plastic surface, these cells sporulated very efficiently. Taken together, our results suggest that sporulation in minicolonies is stimulated by pseudohyphal differentiation because these pseudohyphae are dispersed from the core of the colony.