Variations in mean spore projected area a and mean spore circularity b at different time intervals. Automatic image analysis was performed for different values of C s,min. The times used were 0 circles , 2 triangles , 4 diamonds and 6 squares h. Growth kinetics of A. Approximately elements were analysed for each time-point.
Images taken from the same hour old sample of A. Variations in mean total hyphal length a and mean number of tips b at different time intervals. Automatic image analysis was performed for different values of C h,max. The times used were 14 circles , Variations in mean number of tips as determined by automatic image analysis for different values of L b,min. The routine translation of morphological data into actionable bioprocess strategies remains a priority target in industrial fungal fermentation.
Computer-aided image analysis is an essential enabling tool in achieving this goal. Two complementary approaches may be discerned in the literature to reconcile the complexity of a typical vegetative mycelium with the practical limitations of imaging technology. The detailed analysis of a small number of individual hyphal elements in both submerged [ 33 , 43 ] and solid-state culture systems [ 44 , 45 ] has contributed much to our understanding of apical growth processes.
An alternative approach, aimed at process optimization within bioreactor systems, has been to derive average data from large cell populations [ 42 ], with extension of the analysis to a consideration of both micromorphological and macromorphological forms [ 18 ].
In this study a new system has been presented for quantitative analysis of spore and hyphal morphology. The method obviates the need for purchase of relatively expensive commercial image-analysis software by adding utility on to the publicly available ImageJ platform, a system of proven usefulness in this field [ 46 , 47 ]. Using A. In presenting complex fungal conformations in an essentially two-dimensional format, membrane immobilization confines cultures to a single focal plane, while being directly relevant to maintaining the natural spatial arrangement of vegetative mycelia encountered in solid state culture.
Archiving of membrane-bound material is also possible, as the culture is fixed, stained, and killed during specimen preparation. It has been found that samples stored for up to two years after preparation are still suitable for imaging. Work is currently underway to extend the system to submerged culture systems by filtering liquid phase mycelial samples through cellulose nitrate filters for subsequent analysis. In common with all image-analysis systems, the analysis routines described here depend on high-quality input images and are sensitive to elevated levels of artefact, which may result if large amounts of sediment are present in solutions used during filtration.
As such, these young hyphae and spore clusters are typically excluded from the analysis. In order to fully characterise this transition from spore to hypha, a means of distinguishing between these different objects is required.
Boundary shape descriptors [ 48 , 49 ] could be utilised in conjunction with the morphological thresholds used in the current study to this end. A means of breaking up clumps of spores in the inoculum, such as sonication, may also help to alleviate the problem. Membranes have previously been used to analyse the growth of fungi on solid substrates and have been combined with image analysis and light microscopy, but generally with low magnification, low resolution capability.
Cellulose acetate membranes have been used to study the growth of Trichoderma virens [ 50 ] in conjunction with a dissecting microscope. Image analysis has been used in the enumeration of the fractal dimension of Pycnoporus cinnabarinus [ 51 ] and Trichoderma viride [ 52 ] colonies immobilized on polycarbonate and cellophane membranes, respectively.
The quantification of septation in Streptomyces tendae was achieved using cellophane membranes in conjunction with image analysis [ 53 ], but this method involved the highly skilful transfer of mycelial matter from the membrane to a microscope slide for analysis.
An analysis of a large bank of images and the subsequent calculation of properties such as spore swelling rate, hyphal growth rate, and hyphal branching rate can be achieved in a matter of minutes, excluding the time necessary for image capture.
In the analysis of each hyphal element, the coordinates of each tip and branch-point are recorded. This information could be used to geometrically map the foraging strategies of fungi on solid substrates, which has been previously studied using the calculation of the local fractal dimension within a colony [ 52 ].
Given the demonstrated relationship between morphology and productivity in industrial fungal fermentation processes, there is a need for development of automated image-analysis techniques for accurate and rapid quantification of this fundamental bioprocess characteristic.
While tremendous progress has been made in this area in recent years, many of the reported techniques are specific to the submerged culture format. Those techniques that have been developed for the analysis of solid-state cultures have generally focussed on the macro-morphological form of fungal colonies, quantifying global properties such as fractal dimension and area coverage.
The method presented here enables high-resolution characterization of early fungal development from the time of inoculation. It is hoped that the use of this technique will lead to a greater understanding of fungal growth kinetics and their relationship to macro-morphological forms and productivity in industrial fermentation processes. We thank P. Taylor for her technical assistance in this study. Google Scholar.
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Application of neural network and cluster analysis for characterization of mycelial morphology Microb Cell Fact 5 3 Agger T , Spohr AB , Carlsen M , Nielsen J Growth and product formation of Aspergillus oryzae during submerged cultivations: verification of a morphologically structured model using fluorescent probes Biotechnol Bioeng 57 Papagianni M Quantification of the fractal nature of mycelial aggregation in Aspergillus niger submerged cultures Microb Cell Fact 5 5 Jones CL 2-D wavelet packet analysis of structural self-organisation and morphogenic regulation in filamentous fungal colonies Complex Int 3 1.
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Sign In or Create an Account. Sign In. Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. Materials and methods. Morphological quantification of filamentous fungal development using membrane immobilization and automatic image analysis.
Oxford Academic. Cecilia Chan. Gwilym A Williams. Select Format Select format. Permissions Icon Permissions. Abstract Mycelial morphology is a critically important process property in industrial fermentations of filamentous micro-organisms, as particular phenotypes are associated with maximum productivity.
Image analysis plug-ins were developed for the public domain, Java-based image processing program, ImageJ v1. The software begins by requesting a set of data for the analysis from the user, together with the directory in which the bank of images to be analysed is contained. The analysis then proceeds automatically until all the images in the specified directory have been analysed.
The process for each individual image is outlined as follows Fig. Open in new tab Download slide. Algorithm used for characterization of fungal micro-morphology. Artefactual points sites where the skeleton is greater than one pixel in width can lead to the incorrect classification of branch-points, while artefactual branches can lead to an over-estimation of hyphal length and the number of hyphal tips. The second stage begins by scanning the image in a raster fashion until a hyphal tip is located.
A tip is defined as a black pixel with just one other black pixel in the immediate neighbourhood Fig. The location of the tip is recorded and the skeleton is then traced from this tip along the hyphal length until either another tip or branch-point is reached. A branch-point is defined as a black pixel with three or more black pixels in the immediate neighbourhood, with no two of these neighbours joined.
The length of the branch is then calculated on the basis of the number of pixels traversed. If the length of the branch is less than the specified minimum L b,min it is removed. The scan of the image then proceeds from the point where the initial tip was located and the process continues until the end of the image has been reached. Once pruned, the skeletonised hyphae are suitable for analysis Fig.
The routine begins by scanning the image in a raster fashion until a hyphal tip is located. Part I: detailed morphological model based on the description of individual hyphae. Momamy M, Taylor I Landmarks in the early duplication cycles of Aspergillus fumigatus and Aspergillus nidulans : polarity, germ tube emergence and septation.
Microbiology — Biosci Biotechnol Biochem 58 4 — Appl Environ Microbiol 68 4 — Nielsen J A simple morphologically structured model describing the growth of filamentous microorganisms. Biotechnol Bioeng 41 7 — Nielsen J, Krabben P Hyphal growth and fragmentation of Penicillium chrysogenum in submerged cultures. Biotech Bioeng 46 6 — Biotechnol Adv — Papagianni M Fungal morphology and metabolite production in submerged mycelial processes. Papagianni M, Mattey M, Kristiansen B Citric acid production and morphology of Aspergillus niger as functions of the mixing intensity in a stirred tank and a tubular loop bioreactor.
Biochem Eng J 2 3 — Papagianni M, Mattey M, Kristiansen B The influence of glucose concentration on citric acid production and morphology of Aspergillus niger in batch and glucostat culture.
Enzyme Microb Technol — Papagianni M, Moo-Young M Protease secretion in glucoamylase producer Aspergillus niger cultures: fungal morphology and inoculum effects. Biotechnol Bioeng 42 1 — In: Scheper T ed Advances in biochemical engineering biotechnology.
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Grimm, S. Kelly, R. You can also search for this author in PubMed Google Scholar. Correspondence to D. The action of these forces can be clearly visualized as the squeezing of flexible targets like RBCs captured in phagocytic cups Araki et al. Inhibiting actin polymerization and remodelling prevented the formation of actin jackets and consequently, the formation of these constrictions. However, myosin II activity was dispensable for these processes Fig.
Actin jackets constrict the Salmonella -FBTs. Cells were fixed, external sections of the S-FBTs were immunolabeled blue , and F-actin was stained green. Right: higher magnification of the S-FBT inverted image , showing its constriction by the actin jacket brackets. Solid line indicates the cell boundary.
B Constriction of the S-FBT shown in A, as measured by the fluorescence intensity of the indicated regions along the filament. Cells were fixed 10 min after the initial attachment, and cell membrane labeled with CD11b antibodies red. F-actin is shown in green. Bottom: higher magnifications of framed regions. All micrographs shown are merged confocal planes not deconvolved. Collectively, these results suggest that the diffusion barriers are mediated by the actin jackets closing the aperture of the T-PCs.
To investigate this possibility, we stabilized actin jackets with jasplakinolide Fig. As suggested by the trends in Fig. Altogether, these results indicate that the diffusion barriers depended on molecular sieves, probably formed by receptor-ligand binding. Because both T-PC formation and diffusional barriers occurred in the absence of opsonins Fig. S5 , this suggests that nonopsoninc receptors could mediate this process. However, the receptors involved in the nonopsonic uptake of Lp are unknown Khelef et al.
Because T-PCs are long-lasting, nonhydrolytic compartments, we speculated that filamentous bacteria held inside them could have longer time to inject effectors and interfere with phagocytosis before being exposed to a lysosomal environment. To investigate this, we followed the fate of intracellular viable Lp in RAW macrophages. Effectors secreted through the type IV secretion system allow Lp to avoid phagosomal trafficking, converting its intracellular compartment into an ER-derived vacuole, the Legionella -containing vacuole LCV; Isberg et al.
In agreement with our hypothesis, the trend in Fig. Filamentous morphology contributes to L. Data shown are percentages from a representative experiment from two repeats. C Length of intracellular bacteria and the associated compartment from A. Red lines indicate medians.
D—G Live-cell video microscopy analysis. D Elongation of intracellular Lp02 filaments followed over time by time-lapse microscopy. Bacterial length was measured at indicated times using the software Volocity.
Data shown are lengths from one representative experiment out of 3 repeats where 11 Lp02 filaments were analyzed. E Snapshots from time-lapse imaging of RAW cell infected with Lp02 showing the replication of filamentous Lp02 and production of bacillary progeny. Relative time is indicated on the top right corner and infected cell is delineated by dashed line.
Insets show DIC images. F Infected cells were identified and followed over time to assess the fate of individual bacteria destroyed, replicating, or nonreplicating. Data shown are percentages from a representative experiment out of two independent experiments. G Length of intracellular Lp02 measured at the time the time-lapse acquisitions were started and their fate.
Red lines indicate medians from three independent experiments. At least 15 intracellular Lp02 were analyzed in each case. We next investigated whether length could favor intracellular replication of Lp. Live-cell imaging of individual intracellular bacterial filaments over time revealed that not only did filamentous Lp02 form an LCV, they were also able to replicate Video 7. In the course of 15 h, the filaments first elongated Fig. This is illustrated in Video 7 and in the time-lapse video microscopy sequence from Fig.
After analyzing Lp02 filaments over several movies from independent experiments, we found that Assesment of the fate of intracellular Lp02 as a function of bacterial length showed a trend suggesting that the longest filaments were the ones that replicated more frequently Fig. This trend was in agreement with that from Fig. Collectively, these results indicate that in concert with effectors, filamentous morphology can aid L.
Here we present evidence that the phagocytosis of filamentous bacteria deviates from the canonical pathway delineated using spheroidal targets.
This variation includes spatiotemporal alterations that affect the morphogenesis of the phagocytic cup and the maturation of the phagosome containing FBTs. Importantly, these changes along with effectors allowed L. The key aspect of the above-described phenomena is that in comparison with the uptake of spheroidal particles, phagocytosis of FBTs required a long-lasting phagocytic cup. This distinguishing feature is determined by the way in which the FBT is captured and ingested.
As previously described for E. Similar to the extension of pseudopodia in the phagocytosis of spheroidal targets Swanson and Baer, , the elongation of the protrusion required actin turnover and thus could be inhibited by jasplakinolide.
Although myosin II was dispensable for this process Fig. S4 B , the involvement of other myosins, like myosin X Cox et al. Despite the extensive actin remodeling and internalization of large amounts of plasma membrane to ingest FBTs, this process occurred independently of PI3K activity, resembling the uptake of small targets Cox et al.
This is likely because FBTs are internalized through their poles and hence, the aperture of their phagocytic cups will be determined by the short axis of the filament, which is comparable to the size of small targets, including bacillary bacteria. Therefore, our results suggest that the aperture of the phagocytic cup could play a decisive role in how cells detect particle size. F-actin strongly accumulated, forming an elongated jacket around FBTs at the most distal portion of the phagocytic protrusion.
Formation of these jackets required actin treadmilling; however, the structure, once formed, was resistant to jasplakinolide treatment. We present evidence indicating that these actin jackets provided constriction forces, as was evident by the squeezing of the bacteria enclosed by these structures. Actin jackets are homologous to the actin furrow that constricts erythrocytes Araki et al. Nonetheless, other myosin motors, like myosin I, could be responsible for providing such contractile forces Araki et al.
These actin jackets likely mediate the formation of a diffusion barrier at the T-PC. Our data suggest that protease-sensitive molecular sieves, probably formed by nonopsonic receptors cross-linking the FBTs with the T-PCs, could be responsible for these barriers. Presence of fences at the phagocytic cups for spherical targets has been suggested Golebiewska et al.
Nevertheless, further work is required to elucidate the mechanisms involved in the formation of diffusional barriers. In the canonical pathway of phagocytosis, the remodeling of the phagocytic cup precedes and facilitates the scission of the nascent phagosome from the plasma membrane Flannagan et al. The latter event is considered a sine qua non condition for the initiation of the maturation of the newly formed phagosome.
However, notably, our results demonstrate that this scission from the membrane is not necessary for maturation, as T-PCs can undergo the same maturation steps as described for phagosomes in the canonical pathway.
This crucial difference between the phagocytic cup of spheroidal and filamentous targets is most likely a consequence of the longer time required for the sealing of the T-PCs. The duration of the phagocytic cup stage during phagocytosis of FBTs could simply provide enough time for maturation to occur before the sealing of the cup.
Indeed, the timings of the acquisition of phagosomal maturation markers by the T-PCs correspond well to those reported elsewhere for spheroid targets Vieira et al.
This was the consequence of the large molecular weight cut-off of the diffusion barriers at the phagocytic protrusion that allowed the leaking of small molecules into the milieu. Consequently, acidification and proteolytic activity was not detected in the T-PCs but was observed only after phagosomes were formed. According to the established phagocytic pathway, acidification of the phagosome is necessary for its fusion with the endocytic compartments needed for phagosomal maturation.
This concept is based on results from treatments where both the endosomes and the phagosomes were neutralized, making it difficult to distinguish if one or both of the two compartments need to be acidified for fusion to occur Flannagan et al. Our results show that the T-PCs, although neutral, can fuse with late endosomes and lysosomes, indicating that an acidic pH of endosomal compartments is likely the one required for this fusion and not necessarily that of the phagosomes.
Our data show that the aforementioned phenomena, along with the extended time required for phagosome sealing, has profound implications for the microbicidal capacity of macrophages. Filamentous Lp can escape phagosomal killing and replicate inside macrophages in a length-dependent manner. This suggests that the long residence time in the T-PCs could facilitate filamentous Lp survival by allowing the bacteria to maximize the delivery of effectors into the host cells before phagosomal sealing.
This would allow them to modify their intracellular compartment to favor survival and replication. Of note, a similar mechanism could potentially account for the inhibition of phagocytosis reported for filamentous uropathogenic E.
The uptake of pathogens by macrophages and their subsequent degradation in the phago-lysosomes constitutes an important component of the innate immune system Flannagan et al. Filamentation has been observed for other pathogens in response to antibiotics Chen et al. In summary, our results demonstrate that key aspects regarding the identity and remodeling of the phagocytic cup and the timing of phagosomal maturation are in fact conditioned by the morphology of the target. Alexa Fluor—conjugated secondary antibodies and phalloidin, LysoSensor green, tetramethylrhodamine dextrans, carbodiimide cross-linker, and DQ-ovalbumin were from Life Technologies.
Reagents for electron microscopy, latrunculin B and blebbistatin, were from Sigma-Aldrich. Cultures enriched in filamentous L. These precultures were subcultured at OD of 0. All fluorescent strains and mutants were made in the Lp02 background. Brassinga University of Manitoba, Winnipeg, Canada. As described in Brassinga et al. To obtain Salmonella typhimurium filaments, RFP-expressing bacteria were used.
Bacteria were cultured overnight in LB and then sub-cultured for an additional 4 h. Excess carbodiimide was removed by three washes in 1 ml of 0. FBTs were opsonized with 0. For assays using pharmacological inhibitors, phagocytosis assays were performed using FBTs as described above. Phagocytosis assays were performed as described above and particle internalization was measured after 50 min. For assays using proteases, phagocytic assays were performed as described above.
After this, cells were either left untreated, treated with 1. Endosomes and lysosomes were labeled using fluid-phase uptake of tetramethylrhodamine dextran 3 kD, 10 kD, and 70 kD, and DQ-ova. Phagocytosis assays were performed as described above. For fluorescence labeling of acidic compartments, 20 min after the onset of phagocytosis external sections of the FBTs were immunolabeled in the cold Alexa Fluor —conjugated secondary antibody. After this, the macrophages were incubated with LysoSensor green for 2 min, washed, and moved to precooled microscope stage for imaging.
S-FBTs were opsonized with 1. Cells were fixed 10 min after initial attachment. Live-cell video microscopy was performed to assess bacterial surival as described previously Goclaw-Binder et al. From previous experiments using FBTs and differential immunostaining, 5 h was sufficient time to allow for complete filament internalization. Images were acquired at indicated intervals in the presence of gentamycin. Lengths of intracellular bacteria were measured at indicated intervals to quantify elongation.
Cells were fixed 20 min after initial attachment and processed as described elsewhere Silver and Harrison, In brief, cells were fixed 20 min after initial FTB attachment using 2. Cells were dehydrated in ethanol and sputter-coated with gold.
The equipment was controlled by MetaMorph acquisition software Molecular Devices. Unless indicated otherwise, images shown for fixed cell analysis are deconvolved, merged z-planes. S2 shows FBT containing phagosomes positive for fluorescent dextrans of different molecular weights.
S3 shows that the phagocytic protuberance persists after cells are treated with latrunculin B. Video 4 shows the fluorescence produced as a result of the proteolytic destruction of an intracellular FBT cross-linked with DQ-ova.
Video 5 shows the destruction of an intracellular FBT. Video 7 shows the intracellular replication of a viable Lp filament. Video 8 shows an example of the destruction of intracellular Lp filament. We thank A. Brassinga for sharing the KB plasmid. We would like to thank S.
Voa3-GFP construct was provided by S. We thank R. Harrison, R. Botelho, A. Brahmendra, R. Philpott, I. Tattoli, S. Brunt, and D.
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