Edge Of Colony Research Articles

Automated cell colony counting and analysis using the circular Hough image transform algorithm (CHiTA)

We present an automated cell colony counting method that is flexible, robust and capable of providing more in-depth clonogenic analysis than existing manual and automated approaches. The full form of the Hough transform without approximation has been implemented, for the first time. Improvements in computing speed have facilitated this approach. Colony identification was achieved by pre-processing the raw images of the colonies in situ in the flask, including images of the flask edges, by erosion, dilation and Gaussian smoothing processes. Colony edges were then identified by intensity gradient field discrimination. Our technique eliminates the need for specialized hardware for image capture and enables the use of a standard desktop scanner for distortion-free image acquisition. Additional parameters evaluated included regional colony counts, average colony area, nearest neighbour distances and radial distribution. This spatial and qualitative information extends the utility of the clonogenic assay, allowing analysis of spatially-variant cytotoxic effects. To test the automated system, two flask types and three cell lines with different morphology, cell size and plating density were examined. A novel Monte Carlo method of simulating cell colony images, as well as manual counting, were used to quantify algorithm accuracy. The method was able to identify colonies with unusual morphology, to successfully resolve merged colonies and to correctly count colonies adjacent to flask edges.

Regulated Secretion of a Protease Activates Intercellular Signaling during Fruiting Body Formation in M. xanthus

In response to starvation Myxococcus xanthus initiates a developmental program that culminates in fruiting body formation. There are two morphogenetic events in this program, aggregation and sporulation, which are temporally and spatially coordinated by the contact-dependent intercellular C-signal protein (p17). p17 is generated by proteolytic cleavage of the p25 precursor protein, which accumulates in the outer membrane of vegetative and starving cells. However, p17 generation is restricted to starving cells. Here we identify the subtilisin-like protease PopC that is directly responsible for cleavage of p25. PopC accumulates in the cytoplasm of vegetative cells but is selectively secreted during starvation coinciding with the generation of p17. Consequently, p25 and PopC only encounter each other in starving cells. Thus, restriction of p25 cleavage to starving cells occurs by a compartmentalization mechanism that depends on selective secretion of PopC during starvation. Our results provide evidence for regulated proteolysis via regulated secretion.

Weathering the Storm: How Wind and Waves Impact Western Grebe Nest Placement and Success

Abstract Nests of many over-water nesting birds are frequently subjected to wave action from repeated windstorms. Colonial grebes often lose over 50% of nests in any particular breeding season to wave action, depending on factors such as storm activity and habitat quality. We examined nest structure survival in a colony of Western Grebes (Aechmophorus occidentalis) in relation to wave action due to wind. Initial nests were 66% successful in hatching at Lake Christina, Minnesota, in 2005 compared to only 48% in 2006. In 2005, initial nests that survived and hatched young were closer to the central tuber-mat of the colony and closer to individual bulrush island centers than were nests lost during storms. In 2006, a year of high storm activity, nests located near the colony edge most prone to wave attack were less likely to last the season and produce young than were nests in central or more protected areas of the colony. Buffer zones of vegetation surrounding the nest were the single most important factor i...

Swarming of Pseudomonas aeruginosa PAO1 without differentiation into elongated hyperflagellates on hard agar minimal medium

Polar flagellated Pseudomonas aeruginosa PAO1 demonstrated extensive spreading growth in 2 days on 1.5% agar medium. Such spreading growth of P. aeruginosa PAO1 strains was absent on Luria-Bertani 1.5% agar medium, but remarkable on Davis minimal synthetic agar medium (especially that containing 0.8% sodium citrate and 1.5% Eiken agar) under aerobic 37 degrees C conditions. Analyses using isogenic mutants and complementation transformants showed that bacterial flagella and rhamnolipid contributed to the surface-spreading behavior. On the other hand, a type IV pilus-deficient pilA mutant did not lose the spreading growth activity. Flagella staining of PAO1 T cells from the frontal edge of a spreading colony showed unipolar and normal-sized rods with one or two flagella. Thus, the polar flagellate P. aeruginosa PAO1 T appears to swarm on high-agar medium by producing biosurfactant rhamnolipid and without differentiation into an elongated peritrichous hyperflagellate.

Experimentally induced helper dispersal in colonially breeding cooperative cichlids

The ‘benefits of philopatry’ hypothesis states that helpers in cooperatively breeding species derive higher benefits from remaining home, instead of dispersing and attempting to breed independently. We tested experimentally whether dispersal options influence dispersal propensity in the cooperatively breeding Lake Tanganyika cichlids Neolamprologus pulcher and N. savoryi. Cooperative groups of these fishes breed in densely packed colonies, surrounded by unoccupied, but apparently suitable breeding habitat. Breeding inside colonies and living in groups seems to benefit individuals, for example by early detection and deterrence of predators. We show that despite a slight preference of both species for habitat with a higher stone cover, 40% of the preferred habitat remained unoccupied. On average, the colonies contained a higher number of (1) predators of adults, juveniles and eggs, (2) shelter competitors, and (3) other species including potential food competitors, compared to the outside colony habitat. Apparently, habitat differences cannot explain why these cichlids breed in colonies. Accordingly, dispersal may not be limited by a lack of suitable breeding shelters, but by the relatively higher risk of establishing an outside- compared to a within-colony breeding territory. To test whether cichlids prefer within- to outside-colony breeding territories, we provided breeding shelters inside the colony and at the colony edge and studied helper dispersal. As expected, significantly more shelters were occupied within the colony compared to the edge. New breeding pairs with several helpers occupied these shelters. We conclude that although breeding habitat is plentiful outside the colonies, helpers delay dispersal to obtain a higher quality breeding position within the group or colony eventually, or they disperse in groups. Our results suggest that (1) group augmentation and Allee effects are generally important for dispersal decisions in cooperatively breeding cichlids, consistent with the ‘benefits of philopatry hypothesis’, and (2) habitat saturation cannot fully explain delayed dispersal in these species.

Salmonella enterica phage-resistant mutant colonies display an unusual phenotype in the presence of phage Felix 01

To investigate irregular colony morphology formation in Salmonella enterica serovar Typhimurium DPC6046 in the presence of a lytic phage, Felix 01. Phage-resistant derivatives of the parent strain DPC6046 were isolated which exhibited an irregular colony morphology. These were subjected to viability studies by using confocal scanning laser microscopy and live/dead BacLight stain to evaluate the cell viability within the colony. The phenomenon was also observed with other S. enterica serotypes tested which were normally sensitive to phage Felix. In the case of strain DPC6046, dead cells were clearly evident at the irregular edges of the phage-resistant colonies in locations where the cell density was lower. This colony morphology was not apparent with two other Salmonella phages tested. These findings support the hypothesis that the unusual morphology is due to reversion to phage sensitivity and consequent cell death within the colony as it forms. The irregular colony morphology observed is peculiar to phage Felix. The confocal scanning laser microscopy methodology allowed the basis for the irregular morphology to be elucidated.

Identification of Fluorescent U(V) and U(VI) Microparticles in a Multispecies Biofilm by Confocal Laser Scanning Microscopy and Fluorescence Spectroscopy

Fluorescent uranium(V) and uranium(VI) particles were observed for the first time in vivo by a combined laser fluorescence spectroscopy and confocal laser scanning microscopy approach in a living multispecies biofilm grown on biotite plates. These particles ranged between 1 and 7 um in width and up to 20 microm in length and were located at the bottom and at the edges of biofilms colonies. Analysis of amplified 16S rRNA fragments and fluorescence in situ hybridization were used to characterize the biofilm communities. Laser fluorescence spectroscopy was used to identify these particles. The particles showed either a characteristic fluorescence spectrum in the wavelength range of 415-475 nm, indicative for uranium(V), or in the range of 480-560 nm, which is typical for uranium(VI). Particles of uranium(V) as well as uranium(VI) were simultaneously observed in the biofilms. These uranium particles were attributed for uranium(VI) to biologically mediated precipitation and for uranium(V) to redox processes taking place within the biofilm. The detection of uranium(V) in a multispecies biofilm was interpreted as a short-lived intermediate of the uranium(VI) to uranium(IV) redox reaction. Its presence clearly documents that the uranium(VI) reduction is not a two electron step but that only one electron is involved.

Food habits of the American mink Mustela vison in the Mazurian Lakeland, Northeastern Poland

Food habits of the American mink Mustela vison were studied based on the analysis of 2364 scat samples, collected at three lakes in Northeastern Poland. The mink preyed on a wide range of prey, but two types of prey, amphibians and fish, dominated in the diet of the mink during all the seasons. Frogs, and first and foremost, the common frog Rana temporaria, were hunted by the mink, mainly from the late autumn until the early spring, and comprised up to 83.9% of the prey biomass (the multiannual average for November–December at the Majcz Wielki Lake). The most frequently eaten fish were cyprinids and percids. Seasonality of fish consumption by mink was not as well pronounced as in the case of amphibians. At the first two lakes, fish were hunted mainly in the winter and in the early spring, whereas at the third lake in the summer. The highest multiannual average share of fish in the diet of the mink was recorded in March–April at Lake Tuchlin (69.2% of the prey biomass). Crayfish, which were recorded in the diet mainly in the late spring and in the summer, comprised up to 59.6% of the prey biomass for May–June at the Majcz Wielki Lake. Birds, mammals and insects were supplementary food for the mink. During the breeding season, mink predation on waterfowl and their broods was correlated with the abundance of crested grebe Podiceps cristatus and coot Fulica atra nests in the area. The diet of individual mink varied considerably and the share of birds in the diet of the mink was related to the distance from individual mink dens to the colonies of waterfowl. In May–June, adult birds, chicks and eggs comprised up to 73.6% of the prey biomass of a female mink that inhabited a den located 100 m from the colony's edge. At all three lakes, the diet of the mink was the most diverse in the late spring and in the summer. In May–August, the values of the mink food niche breadths were about twice those noted in winter months.

Calmodulin-like Protein Increases Filopodia-dependent Cell Motility via Up-regulation of Myosin-10

Human calmodulin-like protein (CLP) is an epithelial-specific protein that is expressed during cell differentiation but down-regulated in primary cancers and transformed cell lines. Using stably transfected and inducible HeLa cell lines, we found that CLP expression did not alter the proliferation rate and colony-forming potential of these cells. However, remarkable phenotypic changes were observed in CLP-expressing compared with control cells. Soft agar colonies of CLP-expressing cells had rough boundaries, with peripheral cells migrating away from the colony. Cells expressing CLP displayed a striking increase in the number and length of myosin-10-positive filopodia and showed increased mobility in a wound healing assay. This increase in wound healing capacity was prevented by small interference RNA-mediated down-regulation of myosin-10. Fluorescence microscopy and Western blotting revealed that CLP expression results in up-regulation of its target protein, myosin-10. This up-regulation occurs at the protein level by stabilization of myosin-10. Thus, CLP functions by increasing the stability of myosin-10, leading to enhanced myosin-10 function and a subsequent increase in filopodial dynamics and cell migration. In stratified epithelia, CLP may be required during terminal differentiation to increase myosin-10 function as cells migrate toward the upper layers and establish new adhesive contacts.

113 CHARACTERIZATION OF THE BOVINE EMBRYO-DERIVED CT-1 CELL LINE FOR STUDYING REGULATION OF TRANSCRIPTION FACTORS EXPRESSED WITHIN THE BOVINE TROPHECTODERM

Cloned bovine embryos have been shown to display deviations in gene expression of trophectodermal (TE) lineage-specific transcription factors, including Cdx2, Errβ, Eomes, Mash2, and Hand1. Misregulation of these genes in the early TE has been implicated to play a causal role in abnormal placental formation and fetal loss. Because suboptimal in vitro culture conditions fail to support development beyond hatching, very little is known about regulation of peri-attachment bovine embryo development. Trophectodermal-derived bovine cell lines, such as the CT-1 cell line produced by Talbot and colleagues (Talbot et al. 2000 Biol. Reprod. 62, 235–247) could prove beneficial for understanding how regulatory factors govern early bovine TE development. The CT-1 cell line has been cultured continuously for over 2 years and appears morphologically similar to primary bovine TE outgrowths. Furthermore, these cells produce interferon τ, a cytokine unique to the early bovine TE. The objective of this study was to develop a gene expression profile of selected developmental genes that play a potential role in commitment to the TE fate, maintenance of multipotency, or differentiation into a binucleate phenotype. The CT-1 colonies were initially expanded on gamma-irradiated STO cells at 37°C and 5% CO2 and passaged weekly. Cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1 mm sodium pyruvate and were passaged whenever colony edges touched. Cultures designated for RNA extraction and RT-PCR were then passaged into collagen-coated flasks or Cellbind® flasks (Corning, Lowell, MA, USA) at an average dilution of 1:4 for at least 6 feeder-free passages. Feeder-free culture at 37°C and 8.5% CO2 was optimal for adherence and growth. There were no observable differences in morphology, adherence, or growth rate between CT-1 cells grown on collagen film or a Cellbind® surface. The RT-PCR results indicate that the transcription factor Cdx2, which is homologous to the mouse master TE regulator, is abundantly expressed, whereas pluripotency specific Oct4 is weakly expressed. However, pluripotency specific transcription factors Nanog and Sox2 were undetectable. Transcription factors Eomes, Ets2, Errβ, and Id2, which may be required for proper bovine trophoblast elongation and developmental potential, were also expressed. Hand1 mRNA, a basic helix-loop-helix transcription factor that directs mouse trophoblast cells to differentiate into a giant cell phenotype, was also present and may play a role in binucleate cell formation. Together the data indicate that the CT-1 cell line can be utilized as a permissive cell line for studying transcriptional control of key TE regulatory factors that define the bovine TE lineage and allow better interpretation of gene expression data collected from assisted reproductive technology-derived embryos.

Cyclopamine treatment of human embryonic stem cells followed by culture in human astrocyte medium promotes differentiation into nestin- and GFAP-expressing astrocytic lineage

Human embryonic stem cells (hESCs) are able to differentiate into various cell types, including neuronal cells and glial cells. However, little information is available regarding astrocyte differentiation. This report describes the differentiation of hESCs into nestin- and GFAP-expressing astrocytes following treatment with cyclopamine, which is an inhibitor of Hedgehog (Hh) signaling, and culturing in human astrocyte medium (HAM). In hESCs, cyclopamine treatment suppressed the expression of Hh signaling molecules, the Hh signaling target gene, and ESC-specific markers. Clyclopamine also induced the differentiation of the cells at the edges of the hESC colonies, and these cells stained positively for the early neural marker nestin. Subsequent culturing in HAM promoted the expression of the astrocyte-specific marker GFAP, and these cells were also nestin-positive. These findings indicate that treatment with cyclopamine followed by culturing in HAM leads to the differentiation of hESCs into nestin- and GFAP-expressing astrocytic lineage.

Frederick Jackson Who? The Spanish American Frontier in the Late Eighteenth Century

David J. Weber, pre-eminent historian of Spanish Borderlands, has focused his remarkable energy and prodigious erudition produce nothing less than a massive survey of Spanish in North and South America under Bourbon rule from roughly 1750 1800. His previous big book The Spanish Frontier in North America (1992) considered only one hemisphere. In Bdrbaros, Weber tackles both hemispheres and revisits earlier piece with new observations and arguments so that this work can be read alone although reader interested in this volume will benefit from studying earlier one. If The Spanish Frontier in North America established Weber's status ahead of his predecessors in Borderlands history, Herbert Eugene Bolton and John Francis Bannon, then Bdrbaros distinguishes him as the scholar of American in its broadest sense, moving far beyond more regional concerns of earlier writers who concentrated only on United States.1 While fully cognizant of Frederick Jackson Turner's frontier thesis, Turner is not cited here.2 Weber notes that Bourbon Reforms extended to very edges of Spain's colonies, affecting Indians who lived beyond Spanish political control. In late eighteenth century, of Spanish Empire acquired heightened strategic importance as they gained attention of Spain's European rivals or, in case of North America after 1783, young United States. Bourbon officials hoped consolidate political control over some of those strategic frontiers, secure them from Indian raiders and foreign interlopers, and make them more productive (p. 5). Weber's conceptual ideas are partly from work of Jeremy Adelman and Stephen Aron who write, By we understand a meeting place of peoples in which geographic and cultural borders were not clearly defined, and they designate borderlands for contested boundaries between colonial domains. They continue, [E]qually important history of and frontiers were ways in which Indians exploited these differences and compelled these shifts, partly resist

About the Cover.

Next article FreeAbout the CoverPDFPDF PLUSFull Text Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinked InRedditEmailQR Code SectionsMoreCoverMany invertebrates form colonies of genetically identical individuals in which some individuals become specialized for different functions. One such animal is Membranipora membranacea, a sheet-like bryozoan commonly found encrusting kelp blades in Puget Sound and many other temperate coastal areas.The main cover image shows two colonies of M. membranacea, the larger of which is 2 cm in diameter. Each of the rectangular, skeletal boxes in the colony houses an individual zooid that bears a crown of ciliated tentacles. The tentacles generate water currents that bring suspended food particles to the colony. Groups of zooids form openings (chimneys) that allow water to flow out of the colony after it has been filtered through the colony's feeding tentacles to capture food. Zooids in the center of chimneys lack feeding tentacles, but the tentacles of individuals surrounding chimneys are raised above those of their neighbors. Chimneys enhance feeding by preventing fluid pressure build-up within the colony. In the cover picture, chimneys appear as dark patches within the canopy formed by the tentacles.Because of its simple geometry and rapid growth, M. membranacea provides an excellent opportunity to investigate how fluid flow affects the development of fluid transport systems. Previous authors suggested that phenotypic plasticity in chimney spacing could be explained if water flow through the colony controls where chimneys form. To test whether water currents affect chimney formation, Michelangelo von Dassow, in a study described on pages 76-82 in this issue, injected water under the canopy of tentacles to enhance flow out the edge of the colony. In the colony shown in the upper portion of the cover image, a sheet of red light was positioned so that only the raised tentacles around chimneys were lit. As the colony grew outward, a new chimney-like opening formed distal to the polyethylene tube through which water was injected (toward the top of the image). The new opening distal to the site of water injection (right-hand tube) formed sooner than the new opening distal to the control site without water injection (left-hand tube). The ends of the tubes in the upper image were 0.8 cm apart. The observation that enhancing excurrent flow at the colony edge induced the formation of new chimneys supports the hypothesis that fluid flow controls the development of this fluid transport system. These results provide insight into the role of fluid flow in patterning animal colonies and in the development of fluid transport systems.Credits: Photos, Michelangelo von Dassow; Cover layout, Beth Liles (Marine Biological Laboratory). Next article DetailsFiguresReferencesCited by The Biological Bulletin Volume 211, Number 1August 2008 Published in association with the Marine Biological Laboratory Article DOIhttps://doi.org/10.1086/BBLv211n1cover Views: 39 © 2006 by Marine Biological Laboratory. All rights reserved.PDF download Crossref reports no articles citing this article.

Opposing Roles of Zyxin/LPP ACTA Repeats and the LIM Domain Region in Cell-Cell Adhesion

Cadherins mediate cell-cell adhesion by linking cell junctions to actin networks. Although several actin regulatory systems have been implicated in cell-cell adhesion, it remains unclear how such systems drive cadherin-actin network formation and how they are regulated to coincide with initiation of adhesion. Previous work implicated VASP in assembly of cell-cell junctions in keratinocytes and the VASP-binding protein zyxin colocalizes with VASP at cell-cell junctions. Here we examine how domains in zyxin and its relative LPP contribute to cell-cell junction assembly. Using a quantitative assay for cell-cell adhesion, we demonstrate that zyxin and LPP function to increase the rate of early cell-cell junction assembly through the VASP-binding ActA repeat region. We also identify the LIM region of zyxin and LPP to be a regulatory domain that blocks function of these proteins. Deletion of the LIM domains drives adhesion and increases VASP level in detergent insoluble cadherin-actin. Dominant-negative zyxin/LPP mutants reduce the rate of adhesion, lower VASP levels in detergent-insoluble cadherin-actin networks, and allow for the accumulation of capping protein at cell-cell contacts. These data implicate the LIM domains of zyxin and LPP in regulating cell-cell junction assembly through VASP.

Mapping Bacterial Surface Population Physiology in Real-Time: Infrared Spectroscopy of Proteus Mirabilis Swarm Colonies

We mapped the space-time distribution of stationary and swarmer cells within a growing Proteus mirabilis colony by infrared (IR) microspectroscopy. Colony mapping was performed at different positions between the inoculum and the periphery with a discrete microscope-mounted IR sensor, while continuous monitoring at a fixed location over time used an optical fiber based IR-attenuated total reflection (ATR) sensor, or "optrode." Phenotypes within a single P. mirabilis population relied on identification of functional determinants (producing unique spectral signals) that reflect differences in macromolecular composition associated with cell differentiation. Inner swarm colony domains are spectrally homogeneous, having patterns similar to those produced by the inoculum. Outer domains composed of active swarmer cells exhibit spectra distinguishable at multiple wavelengths dominated by polysaccharides. Our real-time observations agree with and extend earlier reports indicating that motile swarmer cells are restricted to a narrow (approximately 3 mm) annulus at the colony edge. This study thus validates the use of an IR optrode for real-time and noninvasive monitoring of biofilms and other bacterial surface populations.

Two continuum models for the spreading of myxobacteria swarms

We analyze the phenomenon of spreading of a Myxococcus xanthus bacterial colony on plates coated with nutrient. The bacteria spread by gliding on the surface. In the first few hours, cell growth is irrelevant to colony spread. In this case, bacteria spread through peninsular protrusions from the edge of the initial colony. We analyze the diffusion through the narrowing reticulum of cells on the surface mathematically and derive formulae for the spreading rates. On the time scale of tens of hours, effective diffusion of the bacteria, combined with cell division and growth, causes a constant linear increase in the colony's radius. Mathematical analysis and numerical solution of reaction-diffusion equations describing the bacterial and nutrient dynamics demonstrate that, in this regime, the spreading rate is proportional to the square root of both the effective diffusion coefficient and the nutrient concentration. The model predictions agree with the data on spreading rate dependence on the type of gliding motility.

Effects of Nest Density, Location, and Timing on Breeding Success of Caspian Terns

Abstract One of the proposed benefits of colonial nesting in birds is the protection afforded against avian predators. This advantage may be counter-balanced by the negative effects of intraspecific aggression on breeding success. Effects of nest density, nest location within the colony, and timing of nest initiation on productivity of Caspian Terns (Sterna caspia) were investigated on Crescent Island in the mid-Columbia River, Washington, USA. In the absence of intense nest predation at the Crescent Island tern colony, it was hypothesized that nest density would be negatively associated with productivity. A rangefinder was used to determine spatial distribution of Caspian Tern nests, and these data used to calculate nest characteristics (nest density, nearest neighbor distance, and distance to colony edge) for a randomly-selected subset of nests monitored for nest chronology and productivity. Productivity did not differ between nests in high- and low-density areas of the colony, and was positively associ...

Mass flow and pressure-driven hyphal extension in Neurospora crassa

Mass flow of cytoplasm in Neurospora crassa trunk hyphae was directly confirmed by injecting oil droplets into the hyphae. The droplets move in a manner similar to cytoplasmic particles and vacuoles within the hyphae. The direction of mass flow is towards the growing hyphal tips at the colony edge. Based on flow velocities (about 5 microm s(-1)), hyphal radius and estimates of cytoplasm viscosity, the Reynolds number is about 10(-4), indicating that mass flow is laminar. Therefore, the Poiseulle equation can be used to calculate the pressure gradient required for mass flow: 0.0005-0.1 bar cm(-1) (depending on the values used for septal pore radius and cytoplasmic viscosity). These values are very small compared to the normal hydrostatic pressure of the hyphae (4-5 bar). Mass flow stops after respiratory inhibition with cyanide, or creation of an extracellular osmotic gradient. The flow is probably caused by internal osmotic gradients created by differential ion transport along the hyphae. Apical cytoplasm migrates at the same rate as tip extension, as do oil droplets injected near the tip. Thus, in addition to organelle positioning mediated by molecular motors, pressure-driven mass flow may be an integral part of hyphal extension.

Genetic Evidence that GTP Is Required for Transposition of IS 903 and Tn 552 in Escherichia coli

Surprisingly little is known about the role of host factors in regulating transposition, despite the potentially deleterious rearrangements caused by the movement of transposons. An extensive mutant screen was therefore conducted to identify Escherichia coli host factors that regulate transposition. An E. coli mutant library was screened using a papillation assay that allows detection of IS903 transposition events by the formation of blue papillae on a colony. Several host mutants were identified that exhibited a unique papillation pattern: a predominant ring of papillae just inside the edge of the colony, implying that transposition was triggered within these cells based on their spatial location within the colony. These mutants were found to be in pur genes, whose products are involved in the purine biosynthetic pathway. The transposition ring phenotype was also observed with Tn552, but not Tn10, establishing that this was not unique to IS903 and that it was not an artifact of the assay. Further genetic analyses of purine biosynthetic mutants indicated that the ring of transposition was consistent with a GTP requirement for IS903 and Tn552 transposition. Together, our observations suggest that transposition occurs during late stages of colony growth and that transposition occurs inside the colony edge in response to both a gradient of exogenous purines across the colony and the developmental stage of the cells.

Polarized Gene Expression Determines Woronin Body Formation at the Leading Edge of the Fungal Colony

The Woronin body (WB) is a peroxisome-related organelle that is centered on a crystalline core of the HEX-1 protein, which functions to seal septal pores of filamentous ascomycetes in response to cellular damage. Here, we investigate the cellular and genetic control of WB-formation and show that polarized hex-1 gene expression determines WB-biogenesis at the growing hyphal apex. We find that intron splicing is coupled to efficient hex-1 gene expression and strikingly, when the yellow fluorescent protein was expressed from hex-1 regulatory sequences, we observed a fluorescent gradient that was maximal in apical cells. Moreover, endogenous hex-1 transcripts were specifically enriched at the leading edge of the fungal colony, whereas other transcripts accumulated in basal regions. Time-lapse confocal microscopy showed that HEX-1 crystals normally formed in the vicinity of the hyphal apex in large peroxisomes, which matured and were immobilized at the cell periphery as cells underwent septation. When the hex-1 structural gene was expressed from regulatory sequences of an abundant, basally localized transcript, WB-core formation was redetermined to basal regions of the colony, and these strains displayed loss-of-function phenotypes specifically in apical hyphal compartments. These results show that apically localized gene expression is a key determinant of spatially restricted WB-assembly. We suggest that this type of regulation may be widely used to determine cellular activity in apical regions of the fungal hypha.