scholarly journals Visualization of the Peroxisomal Compartment in Living Mammalian Cells: Dynamic Behavior and Association with Microtubules

1997 ◽  
Vol 136 (1) ◽  
pp. 71-80 ◽  
Author(s):  
Erik A.C. Wiemer ◽  
Thibaut Wenzel ◽  
Thomas J. Deerinck ◽  
Mark H. Ellisman ◽  
Suresh Subramani
Keyword(s):  
Mammalian Cells ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Small Subset ◽  
Subcellular Compartment ◽  
Directional Movement ◽  
Green Fluorescent

Peroxisomes in living CV1 cells were visualized by targeting the green fluorescent protein (GFP) to this subcellular compartment through the addition of a COOH-terminal peroxisomal targeting signal 1 (GFP–PTS1). The organelle dynamics were examined and analyzed using time-lapse confocal laser scanning microscopy. Two types of movement could be distinguished: a relatively slow, random, vibration-like movement displayed by the majority (∼95%) of the peroxisomes, and a saltatory, fast directional movement displayed by a small subset (∼5%) of the peroxisomes. In the latter instance, peak velocities up to 0.75 μm/s and sustained directional velocities up to 0.45 μm/s over 11.5 μm were recorded. Only the directional type of motion appeared to be energy dependent, whereas the vibrational movement continued even after the cells were depleted of energy. Treatment of cells, transiently expressing GFP–PTS1, with microtubule-destabilizing agents such as nocodazole, vinblastine, and demecolcine clearly altered peroxisome morphology and subcellular distribution and blocked the directional movement. In contrast, the microtubule-stabilizing compound paclitaxel, or the microfilament-destabilizing drugs cytochalasin B or D, did not exert these effects. High resolution confocal analysis of cells expressing GFP–PTS1 and stained with anti-tubulin antibodies revealed that many peroxisomes were associated with microtubules. The GFP–PTS1–labeled peroxisomes were found to distribute themselves in a stochastic, rather than ordered, manner to daughter cells at the time of mitosis.

Postharvest Handling Conditions Affect Internalization of Salmonella in Baby Spinach during Washing

2013 ◽  
Vol 76 (7) ◽  
pp. 1145-1151 ◽  
Author(s):  
VICENTE M. GÓMEZ-LÓPEZ ◽  
ALICIA MARÍN ◽  
ANA ALLENDE ◽  
LARRY R. BEUCHAT ◽  
MARÍA I. GIL
Keyword(s):  
Green Fluorescent Protein ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Negative Temperature ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Temperature Differential ◽  
Postharvest Handling ◽  
Green Fluorescent ◽  
Spinach Leaves

Internalization of foodborne pathogens in fruits and vegetables is an increasing safety concern. The aim of this research was to assess the potential for internalization of an enteric pathogen (Salmonella enterica serotype Typhimurium) in a leafy vegetable (baby spinach) during washing as influenced by three postharvest handling conditions: (i) illumination, (ii) negative temperature differential, and (iii) relative humidity (RH). To compare these potential postharvest handling conditions, leaves were exposed to different levels of illumination (0, 1,000, and 2,000 lx), temperature differential (5, 11, 14, 20, and 26uC), and RH (99, 85, and 74%) for a short time before or during washing. Washing of baby spinach was carried out in water containing green fluorescent protein–tagged Salmonella Typhimurium (6.5 log CFU/ml) at 5uC for 2 min, followed by surface disinfection with chlorine (10,000 μg/ml) for 1 min, two rinses in water for 10 s, and spin drying for 15 s. Internalization was assessed by enumerating the pathogen on Salmonella-Shigella agar and by confocal laser scanning microscopy. Illumination of spinach leaves before and during washing and a negative temperature differential during washing did not significantly (P > 0.05) increase the number of internalized bacteria. However, exposure of leaves to low-RH conditions before washing, which reduced the tissue water content, decreased internalization of Salmonella compared with internalization in baby spinach exposed to high RH (P ≤ 0.05). Green fluorescent protein–tagged Salmonella Typhimurium was visualized by confocal laser scanning microscopy at a depth of up to 30 μm beneath the surface of spinach leaves after exposure to a high inoculum level (8 log CFU/ml) for an extended time (2 h). Results show that internalization of Salmonella into baby spinach leaves can occur but can be minimized under specific postharvest handling conditions such as low RH.

Migration of Salmonella Enteritidis Phage Type 30 through Almond Hulls and Shells

2008 ◽  
Vol 71 (2) ◽  
pp. 397-401 ◽  
Author(s):  
MICHELLE D. DANYLUK ◽  
MARIA T. BRANDL ◽  
LINDA J. HARRIS
Keyword(s):  
Green Fluorescent Protein ◽  
Laser Scanning ◽  
Salmonella Enteritidis ◽  
Fluorescent Protein ◽  
Phage Type ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Aqueous Environment ◽  
Serovar Typhimurium ◽  
Green Fluorescent

The ability of Salmonella to migrate from an external aqueous environment through the almond hull and shell, and to colonize the kernel, was evaluated in two ways. First, the outer surface of shell halves from five varieties of almonds that differed in shell hardness were placed in contact with a suspension of Salmonella enterica serovar Enteritidis phage type 30 for 24hat24°C. Salmonella Enteritidis was isolated from the inside of these almond shells in 46 and 100% of the samples, by direct swabbing of the inner surface of the shell and by enrichment from the swab, respectively. These findings suggested that hardness of the shell is not a significant factor in the migration of the pathogen through that tissue. In addition, both motile and nonmotile strains of S. enterica serovar Typhimurium migrated through the almond shells to the same extent under the conditions of this assay, indicating that bacterial migration through the wet shell may be a passive process. Second, whole almonds (intact hull, shell, and kernel) were soaked for 24 to 72 h at 24°C in a suspension of Salmonella Enteritidis phage type 30 labeled with the green fluorescent protein. Green fluorescent protein–labeled Salmonella cells were observed on the outer and inner surfaces of both the almond hull and shell, and on the kernel, by confocal laser scanning microscopy. Our data provide direct evidence that wet conditions allow for Salmonella migration through the hull and shell and onto the almond kernel, thus providing a means by which almond kernels may become contaminated in the field.

Alginate production affects Pseudomonas aeruginosa biofilm development and architecture, but is not essential for biofilm formation

2004 ◽  
Vol 53 (7) ◽  
pp. 679-690 ◽  
Author(s):  
Andres Plata Stapper ◽  
Giri Narasimhan ◽  
Dennis E. Ohman ◽  
Johnny Barakat ◽  
Morten Hentzer ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Cell System ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Alginate Production ◽  
Green Fluorescent ◽  
Biofilm Development

Extracellular polymers can facilitate the non-specific attachment of bacteria to surfaces and hold together developing biofilms. This study was undertaken to qualitatively and quantitatively compare the architecture of biofilms produced by Pseudomonas aeruginosa strain PAO1 and its alginate-overproducing (mucA22) and alginate-defective (algD) variants in order to discern the role of alginate in biofilm formation. These strains, PAO1, Alg+ PAOmucA22 and Alg− PAOalgD, tagged with green fluorescent protein, were grown in a continuous flow cell system to characterize the developmental cycles of their biofilm formation using confocal laser scanning microscopy. Biofilm Image Processing (bip) and Community Statistics (comstat) software programs were used to provide quantitative measurements of the two-dimensional biofilm images. All three strains formed distinguishable biofilm architectures, indicating that the production of alginate is not critical for biofilm formation. Observation over a period of 5 days indicated a three-stage development pattern consisting of initiation, establishment and maturation. Furthermore, this study showed that phenotypically distinguishable biofilms can be quantitatively differentiated.

scholarly journals Systemic Colonization of Potato Plants by a Soilborne, Green Fluorescent Protein-Tagged Strain of Dickeya sp. Biovar 3

2010 ◽  
Vol 100 (2) ◽  
pp. 134-142 ◽  
Author(s):  
Robert Czajkowski ◽  
Waldo J. de Boer ◽  
Henk Velvis ◽  
Jan M. van der Wolf
Keyword(s):  
Green Fluorescent Protein ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Agar Medium ◽  
Lateral Roots ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Potato Plants ◽  
Soil Inoculation ◽  
Green Fluorescent

Colonization of potato plants by soilborne, green fluorescent protein (GFP)-tagged Dickeya sp. IPO2254 was investigated by selective plating, epifluorescence stereo microscopy (ESM), and confocal laser scanning microscopy (CLSM). Replicated experiments were carried out in a greenhouse using plants with an intact root system and plants from which ca. 30% of the lateral roots was removed. One day after soil inoculation, adherence of the pathogen on the roots and the internal colonization of the plants were detected using ESM and CLSM of plant parts embedded in an agar medium. Fifteen days post-soil inoculation, Dickeya sp. was found on average inside 42% of the roots, 13% of the stems, and 13% of the stolons in plants with undamaged roots. At the same time-point, in plants with damaged roots, Dickeya sp. was found inside 50% of the roots, 25% of the stems, and 25% of the stolons. Thirty days postinoculation, some plants showed true blackleg symptoms. In roots, Dickeya sp. was detected in parenchyma cells of the cortex, both inter- and intracellularly. In stems, bacteria were found in xylem vessels and in protoxylem cells. Microscopical observations were confirmed by dilution spread-plating the plant extracts onto agar medium directly after harvest. The implications of infection from soilborne inoculum are discussed.

scholarly journals Phenological and Phytochemical Changes Correlate with Differential Interactions of Verticillium dahliae with Broccoli and Cauliflower

2011 ◽  
Vol 101 (5) ◽  
pp. 523-534 ◽  
Author(s):  
S. M. C. Njoroge ◽  
G. E. Vallad ◽  
S.-Y. Park ◽  
S. Kang ◽  
S. T. Koike ◽  
...  
Keyword(s):  
Verticillium Dahliae ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Plant Phenology ◽  
Laser Scanning Microscopy ◽  
Plant Age ◽  
Confocal Laser ◽  
Glucosinolate Content ◽  
Developing Seed ◽  
Green Fluorescent

Cauliflower (Brassica oleracea var. botrytis subvar. cauliflora) is susceptible to wilt caused by Verticillium dahliae but broccoli (B. oleracea var. italica subvar. cyamosa) is not. Infection of broccoli and cauliflower by a green fluorescent protein-expressing isolate of V. dahliae was examined using epifluorescence and confocal laser-scanning microscopy to follow infection and colonization in relation to plant phenology. Plant glucosinolate, phenolic, and lignin contents were also assayed at 0, 4, 14, and 28 days postinoculation. V. dahliae consistently infected and colonized the vascular tissues of all cauliflower plants regardless of age at inoculation, with the pathogen ultimately appearing in the developing seed; however, colonization decreased with plant age. In broccoli, V. dahliae infected and colonized root and stem xylem tissues of plants inoculated at 1, 2, or 3 weeks postemergence. However, V. dahliae colonized only the root xylem and the epidermal and cortical tissues of broccoli plants inoculated at 4, 5, and 6 weeks postemergence. The frequency of reisolation of V. dahliae from the stems (4 to 22%) and roots (10 to 40%) of mature broccoli plants was lower than for cauliflower stems (25 to 64%) and roots (31 to 71%). The mean level of aliphatic glucosinolates in broccoli roots was 6.18 times higher than in the shoots and did not vary with age, whereas it was 3.65 times higher in cauliflower shoots than in the roots and there was a proportional increase with age. Indole glucosinolate content was identical in both cauliflower and broccoli, and both indole and aromatic glucosinolates did not vary with plant age in either crop. Qualitative differences in characterized glucosinolates were observed between broccoli and cauliflower but no differences were observed between inoculated and noninoculated plants for either broccoli or cauliflower. However, the phenolic and lignin contents were significantly higher in broccoli following inoculation than in noninoculated broccoli or inoculated cauliflower plants. The increased resistance of broccoli to V. dahliae infection was related to the increase in phenolic and lignin contents. Significant differential accumulation of glucosinolates associated with plant phenology may also contribute to the resistant and susceptible reactions of broccoli and cauliflower, respectively, against V. dahliae.

scholarly journals Translocation of Incoming Pseudorabies Virus Capsids to the Cell Nucleus Is Delayed in the Absence of Tegument Protein pUL37

2009 ◽  
Vol 83 (7) ◽  
pp. 3389-3396 ◽  
Author(s):  
Mirjam Krautwald ◽  
Walter Fuchs ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter
Keyword(s):  
Laser Scanning ◽  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Laser Scanning Microscopy ◽  
Rabbit Kidney ◽  
Confocal Laser ◽  
Tegument Proteins ◽  
Green Fluorescent ◽  
Virus Mutant

ABSTRACT After fusion of the envelope of herpesvirus particles with the host cell plasma membrane, incoming nucleocapsids are transported to nuclear pores. Inner tegument proteins pUL36, pUL37, and pUS3 remain attached to the nucleocapsid after entry and therefore might mediate interactions between the nucleocapsid and cellular microtubule-associated motor proteins during transport. To assay for the role of pUL37 in this process, we constructed a pUL37-deleted pseudorabies virus mutant, PrV-ΔUL37/UL35GFP, which expresses a fusion protein of green fluorescent protein (GFP) and the nonessential small capsid protein pUL35, resulting in the formation of fluorescently labeled capsids. Confocal laser-scanning microscopy of rabbit kidney cells infected with PrV-ΔUL37/UL35GFP revealed that, whereas penetration was not affected in the absence of pUL37, nuclear translocation of incoming particles was delayed by approximately 1 h compared to PrV-UL35GFP, but not abolished. In contrast, phenotypically complemented pUL37-containing virions of PrV-ΔUL37/UL35GFP exhibited wild type-like entry kinetics. Thus, the presence of pUL37 is required for rapid nuclear translocation of incoming nucleocapsids.

Deletion of bem46 retards spore germination and may be related to the polar growth of Aspergillus fumigatus

2019 ◽  
Vol 58 (5) ◽  
pp. 690-697
Author(s):  
Yan Ma ◽  
Ying Ji ◽  
Jing Yang ◽  
Wen Li ◽  
Jiajuan Li ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Morphological Changes ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Polarized Growth ◽  
Bud Emergence ◽  
Green Fluorescent

Abstract Bud emergence 46 (BEM46), a member of the α/β hydrolase superfamily, has been reported to be essential for polarized growth in Neurospora crassa. However, the role of BEM46 in aspergillus fumigatus (A. fumigatus) remains unclear. In this study, we constructed an A. fumigatus strain expressing BEM46 fused with enhanced green fluorescent protein, and a Δbem46 mutant, to explore the localization and the role of growth of BEM46 in A. fumigatus, respectively. Confocal laser scanning microscopy revealed that BEM46 was dominantly expressed in the sites where hyphae germinated from conidia in A. fumigatus. When compared with the control strain, the Δbem46 mutant exhibited insignificant morphological changes but delayed germination. No significant changes were found regarding the radial growth of both strains in response to various antifungal agents. These results suggest that BEM46 plays an essential role in timely germination in A. fumigatus. From the observation of fluorescence localization, we infer that that BEM46 might be involved in polarized growth in A. fumigatus.

scholarly journals Fitness of Salmonella enterica serovar Thompson in the Cilantro Phyllosphere

2002 ◽  
Vol 68 (7) ◽  
pp. 3614-3621 ◽  
Author(s):  
Maria T. Brandl ◽  
Robert E. Mandrell
Keyword(s):  
Salmonella Enterica ◽  
Laser Scanning ◽  
Fruits And Vegetables ◽  
Fluorescent Protein ◽  
Bacterial Flora ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Human Pathogen ◽  
Dry Conditions ◽  
Green Fluorescent

ABSTRACT The epiphytic fitness of Salmonella enterica was assessed on cilantro plants by using a strain of S. enterica serovar Thompson that was linked to an outbreak resulting from cilantro. Salmonella serovar Thompson had the ability to colonize the surface of cilantro leaves, where it was detected by confocal laser scanning microscopy (CLSM) at high densities on the veins and in natural lesions. The population sizes of two common colonizers of plant surfaces, Pantoea agglomerans and Pseudomonas chlororaphis, were 10-fold higher than that of the human pathogen on cilantro incubated at 22°C. However, Salmonella serovar Thompson achieved significantly higher population levels and accounted for a higher proportion of the total culturable bacterial flora on cilantro leaves when the plants were incubated at warm temperatures, such as 30°C, after inoculation, indicating that the higher growth rates exhibited by Salmonella serovar Thompson at warm temperatures may increase the competitiveness of this organism in the phyllosphere. The tolerance of Salmonella serovar Thompson to dry conditions on plants at 60% relative humidity was at least equal to that of P. agglomerans and P. chlororaphis. Moreover, after exposure to low humidity on cilantro, Salmonella serovar Thompson recovered under high humidity to achieve its maximum population size in the cilantro phyllosphere. Visualization by CLSM of green fluorescent protein-tagged Salmonella serovar Thompson and dsRed-tagged P. agglomerans inoculated onto cilantro revealed that the human pathogen and the bacterial epiphyte formed large heterogeneous aggregates on the leaf surface. Our studies support the hypothesis that preharvest contamination of crops by S. enterica plays a role in outbreaks linked to fresh fruits and vegetables.

scholarly journals Green Fluorescent Protein-Marked Pseudomonas fluorescens: Localization, Viability, and Activity in the Natural Barley Rhizosphere

1999 ◽  
Vol 65 (10) ◽  
pp. 4646-4651 ◽  
Author(s):  
Bo Normander ◽  
Niels B. Hendriksen ◽  
Ole Nybroe
Keyword(s):  
Green Fluorescent Protein ◽  
Pseudomonas Fluorescens ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Fluorescence ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Protein Fluorescence ◽  
Dividing Cells ◽  
Green Fluorescent

ABSTRACT The gfp-tagged Pseudomonas fluorescensbiocontrol strain DR54-BN14 was introduced into the barley rhizosphere. Confocal laser scanning microscopy revealed that the rhizoplane populations of DR54-BN14 on 3- to 14-day-old roots were able to form microcolonies closely associated with the indigenous bacteria and that a majority of DR54-BN14 cells appeared small and almost coccoid. Information on the viability of the inoculant was provided by a microcolony assay, while measurements of cell volume, the intensity of green fluorescent protein fluorescence, and the ratio of dividing cells to total cells were used as indicators of cellular activity. At a soil moisture close to the water-holding capacity of the soil, the activity parameters suggested that the majority of DR54-BN14 cells were starving in the rhizosphere. Nevertheless, approximately 80% of the population was either culturable or viable but nonculturable during the 3-week incubation period. No impact of root decay on viability was observed, and differences in viability or activity among DR54-BN14 cells located in different regions of the root were not apparent. In dry soil, however, the nonviable state of DR54-BN14 was predominant, suggesting that desiccation is an important abiotic regulator of cell viability.

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