Experimental measurement of endocytosis in fungal hyphae.

The present study examines the notion that polarized exocytosis in the tips of growing hyphae creates an excess of plasma membrane and thus the need for its removal by endocytosis. To measure endocytosis experimentally, we developed a photobleaching (FRAP) procedure to count endocytic events in hyphae of Neurospora crassa carrying a fluorescent tag on the actin-binding protein fimbrin (FIM-1-GFP). Given 40 nm as the average diameter of endocytic vesicles, we calculated that about 12.5% of the plasma membrane discharged in the apex becomes endocytosed in the subapex. According to our calculations, the GFP-tagged hyphae of N. crassa, measured under the constrained conditions of confocal microscopic examination, needed about 8800 vesicles/min to extend their plasma membrane or about 9800/min, if we include predicted demands for cell wall growth and extracellular secretion. Our findings support the notion that exocytosis and endocytosis operate in tandem with the latter serving as a compensatory process to remove any excess of plasma membrane generated by the intense exocytosis in the hyphal tips. Presumably, this tandem arrangement evolved to support the hallmark features of fungi namely rapid cell extension and abundant secretion of hydrolytic enzymes

Invariant Optical Color Correlation for Recognition of Vibrio Cholerae

Abstrac

Coronin Is a Component of the Endocytic Collar of Hyphae of Neurospora crassa and Is Necessary for Normal Growth and Morphogenesis

Coronin plays a major role in the organization and dynamics of actin in yeast. To investigate the role of coronin in a filamentous fungus (Neurospora crassa), we examined its subcellular localization using fluorescent proteins and the phenotypic consequences of coronin gene (crn-1) deletion in hyphal morphogenesis, Spitzenkörper behavior and endocytosis. Coronin-GFP was localized in patches, forming a subapical collar near the hyphal apex; significantly, it was absent from the apex. The subapical patches of coronin colocalized with fimbrin, Arp2/3 complex, and actin, altogether comprising the endocytic collar. Deletion of crn-1 resulted in reduced hyphal growth rates, distorted hyphal morphology, uneven wall thickness, and delayed establishment of polarity during germination; it also affected growth directionality and increased branching. The Spitzenkörper of Δcrn-1 mutant was unstable; it appeared and disappeared intermittently giving rise to periods of hyphoid-like and isotropic growth respectively. Uptake of FM4-64 in Δcrn-1 mutant indicated a partial disruption in endocytosis. These observations underscore coronin as an important component of F-actin remodeling in N. crassa. Although coronin is not essential in this fungus, its deletion influenced negatively the operation of the actin cytoskeleton involved in the orderly deployment of the apical growth apparatus, thus preventing normal hyphal growth and morphogenesis

Invariant recognition of polychromatic images of Vibrio cholerae O1

7 pages, 5 figures.-- ©2002 Society of Photo-Optical Instrumentation Engineers.Cholera is an acute intestinal infectious disease. It has claimed many lives throughout history, and it continues to be a global health threat. Cholera is considered one of the most important emergence diseases due its relation with global climate changes. Automated methods such as optical systems represent a new trend to make more accurate measurements of the presence and quantity of this microorganism in its natural environment. Automatic systems eliminate observer bias and reduce the analysis time.We evaluate the utility of coherent optical systems with invariant correlation for the recognition of Vibrio cholerae O1. Images of scenes are recorded with a CCD camera and decomposed in three RGB channels. A numeric simulation is developed to identify the bacteria in the different samples through an invariant correlation technique. There is no variation when we repeat the correlation and the variation between images correlation is minimum. The position-, scale-, and rotation-invariant recognition is made with a scale transform through the Mellin transform. The algorithm to recognize Vibrio cholerae O1 is the presence of correlation peaks in the green channel output and their absence in red and blue channels. The discrimination criterion is the presence of correlation peaks in red, green, and blue channels.Peer reviewe

Growth of Vibrio cholerae O1 in Red Tide Waters off California

Vibrio cholerae serotype O1 is autochthonous to estuarine and coastal waters. However, its population dynamics in such environments are not well understood. We tested the proliferation of V. cholerae N16961 during a Lingulodinium polyedrum bloom, as well as other seawater conditions. Microcosms containing 100-kDa-filtered seawater were inoculated with V. cholerae or the 0.6-μm-pore-size filterable fraction of seawater assemblages. These cultures were diluted 10-fold with fresh 100-kDa-filtered seawater every 48 h for four cycles. Growth rates ranged from 0.3 to 14.3 day(−1) (4.2 day(−1) ± 3.9) for V. cholerae and 0.1 to 9.7 day(−1) (2.2 ± 2.8 day(−1)) for bacterial assemblage. Our results suggest that dissolved organic matter during intense phytoplankton blooms has the potential to support explosive growth of V. cholerae in seawater. Under the conditions tested, free-living V. cholerae was able to reach concentrations per milliliter that were up to 3 orders of magnitude higher than the known minimum infectious dose (10(4) cell ml(−1)) and remained viable under many conditions. If applicable to the complex conditions in marine ecosystems, our results suggest an important role of the growth of free-living V. cholerae in disease propagation and prevention during phytoplankton blooms

Localization and role of MYO-1, an endocytic protein in hyphae of Neurospora crassa.

The subapical endocytic collar is a prominent feature of hyphae of Neurospora crassa. It comprises a dynamic collection of actin patches associated with a number of proteins required for endocytosis, namely, ARP-2/3 complex, fimbrin, coronin, etc. We presently show that MYO-1 is another key component of this endocytic collar. A myo-1 sequence was identified in the genome of N. crassa and used it to generate a strain with a myo-1-sgfp allele under the ccg1 promoter. Examination of living hyphae by confocal microscopy, revealed MYO-1-GFP located mainly as a dynamic collection of small patches arranged in collar-like fashion in the hyphal subapex. Dual tagging showed MYO-1-GFP partially colocalized with two other endocytic proteins, fimbrin and coronin. MYO-1 was also present during septum formation. By recovering a viable strain, albeit severely inhibited, after deletion of myo-1, it was possible to investigate the phenotypic consequences of the elimination of MYO-1. Deletion of myo-1 caused a severe reduction in growth rate (95%), near absence of aerial mycelium and no conidiation. A reduced uptake of the lipophilic dye FM4-64 indicated a deficiency in endocytosis in the Δmyo-1 mutant. Hyphae were produced by the Δmyo-1 mutant but their morphogenesis was severely affected; hyphal morphology was distorted displaying irregular periods of isotropic and polarized growth. The morphological alterations were accompanied, and presumably caused, by a disruption in the organization and dynamics of a myosin-deprived actin cytoskeleton that, ultimately, compromised the stability and function of the Spitzenkörper as a vesicle supply center

Correlación digital del color para el reconocimiento de Vibrio cholerae 01 en muestras ambientales y de laboratorio

En general, el conteo directo refleja más la abundancia microbiológica que el conteo de placas. Se han desarrollado técnicas microbiológicas para reconocer y contar microorganismos en sistemas naturales pero han tenido problemas con la confiabilidad de sus resultados. Frecuentemente, métodos distintos dan diferentes resultados para la enumeración de un organismo específico. Para la examinación directa al microscopio de bacterias, los sistemas digitales automatizados representan un posible avance en la identificación y conteo, eliminando una predisposición del observador y reduciendo costos y tiempo de análisis. En este trabajo se desarrolló un programa computacional para evaluar la utilidad de sistemas ópticos coherentes para el reconocimiento de Vibrio cholerae 01, mediante correlación de color en cultivos de laboratorio y de muestras ambientales teñidas con anticuerpos monoclonales fluorescentes. De muestras de laboratorio probamos 94 muestras positivas y 115 negativas y 33 muestras positivas y 34 negativas de muestras ambientales y 613 muestras positivas y 546 negativas de muestras de mesocosmos. En muestras de laboratorio se llevó a cabo una correcta identificación, un conteo de células y una discriminación a un 100%. La sensibilidad del sistema digital en muestras ambientales como en muestras análogas al ambiente varió de 91% a 94% y tuvo un 99,5% de discriminación entre otras bacterias o partículas. Basados en los valores absolutos de correlación en los componentes rojo, verde y azul de las imágenes policromáticas de V. cholerae 01 (canales RGB), el algorítmo para contar e identificar correlacionó bien con los picos en la salida del canal verde y no hubo picos de salida en los canales rojo y azul. El criterio de discriminación correlacionó bien con los picos presentes en los canales rojo y azul. Concluímos que el sistema digital de correlación de color para identificar y contar V. cholerae 01 de muestras ambientales y de laboratorio es una herramienta útil con alta confiabilidad