4. CYTOCHEMICAL LOCALIZATION AND REGULATION OF ACID
4.3 RESULTS
4.3.7 Confocal microscopy
154 However, strong fluorescence was observed at 0.05 mM, mainly on the edge of the hyphae (Figure 4.7E). Because of the relatively low level of the resolution given by light microscopy, it was impossible to localize the reactions precisely in the mycobiont. Extending incubation time and increasing the ELF-97 did not enhance any staining pattern to allow any localization at the ultrastructural level. To gain a better understanding of the localization of the enzyme, the confocal microscope was used as an alternative.
155 Figure 4.10: (A-E) Lichen thallus visualized using confocal microscope. The thallus was visualized without any substrate, to ascertain the extent of autofluorescence in the sample. (A) Combined image, where algae can be seen as red round spots and lichen thallus as blue. (B) DIC image visualized as grey.
(C) Natural autofluorescence (blue) visualized under 488 laser excitation. (D) Algal cells as red spot (E) Unknown flourescing substance in the hyphae. Bar = 10 µM.
C
D E B A
10 µm algae
thallus
156 Figure 4.11: (A-I) Mycelium stained with Vector blue III substrate (VB), viewed with a confocal microscope. (A) In the control treatment (devoid of substrate), the cytoplasm appeared empty. (B) The distribution of VB could be seen in the cytoplasm, membrane and surface of the hyphae. (C) Strong fluorescence was visualized at 10 mM Pi. (D) Apase detected at 100 mM Pi treatment. (E) Control treatment devoid of substrate. (F) VB fluorescence was distributed all over the cells in older cultures with a treatment of 0.5 mM Pi. (G) DIC image of F. (H) Fluorescence detected from the cytoplasm as well as vacuole from 10 mM Pi treatment. (I) A detailed image of fluorescing organelles from the mycelium cultured under 100 mM, where apase could be seen in the vacuoles and small structures presumed to be endosomes (E) or prevacuoles (V).
A-Control B-0.05 mM C-10mM D-100mM
V
E I-100 mM G-0.05 mM
F-0.05 mM
E-control H-10 mM
157 To verify the nature of the organelles stained by vector blue substrate, and to rule out that this was not an artefact of fixation, the mycelium was stained only with a vital stain, FM4-64. The mycelia were analyzed by confocal microscopy after 15-30 min initial incubation with FM4-64.
To differentiate the autofluorescence from FM4-64, controls were measured first to establish their maximum excitation. The natural autofluorescence was allocated a green pseudo-colour and fluorescence by FM4-64 substrate was allocated red to make a contrast. Autofluorescence (AF) appeared as a cloud of green (Figure 4.12A). FM4-64 was excited by a laser 488 nm. At low Pi (0.05 mM), poor internalisation of FM4-64 dye was observed (Figure 4.12A). The hyphal cell did not incorporate the stain but it was scattered on the surface (Figure 4.12A). The mycelium was damaged and stunted.
Using 1.0 mM Pi, strong FM4-64 fluorescence was observed, which appeared like strong red dots (Figure 4.12B). The FM4-64 was noted to stain only round punctuate organelles; no staining of the cell membrane was observed (Figure 4.12B-C). FM4-64 was seen as a strong red fluorescent spot on the mycelium which were distinct from autofluorescence displayed by mycelium in general (Figure 4.12C-F). It was noted that, at 1.0 mM Pi treatment, the mycelium looks stunted, swollen and starting to form colloid growth (Figure 4.12C). The FM4-64 staining pattern identified several red punctuate structures (red fluorescence) of different sizes which were more numerous in the Pi-starved cultures (Figure 4.12C-F). At 100 mM Pi, few fluorescing organelles were observed compared to those found at 1.0 mM (Figure 4.12G-L).
Relatively large organelles were found in the higher treatment (100 mM Pi). For instance, the organelle in Figure 4.12G, identified as a mature vacuole, was located in the cytoplasm and separated by a distinct septum wall (Figure 4.12G).
Several organelles were identified in mycelium grown at 1.0 mM (Figure 4.13A-D). On closer observation, the apical region known as Spitzenköper (SPK), satellite Spitzenköper (SSPK) were observed at the edge of the hyphae (Figure 4.13D). A cluster of red punctuate structures (E), thought to be endosomes could also be seen. The bigger round clusters were identified as mature vacuoles (V) (Figure 4.13D). The staining of FM4-64 appeared to follow a hypothetical model of the organization of the vesicle trafficking network in a growing hypha based upon the pattern of FM4-64 staining (Figure 4.13E, adapted from FISCHER-PARTON et al., 2000).
158 Figure 4.12: (A-L) Confocal images of the mycelium of C. portentosa displaying the internalization of FM4-64. (A) 0.05 mM Pi after staining with FM4-64. (B) 1.0 mM Pi after staining with FM4-64. (C) A detailed image of 1.0 mM Pi. Note numerous fluorescing organelles. (D) FM4-64. (E) Autofluorescence.
(F) DIC image. (G) 100 mM Pi after staining with FM4-64. Note, the absent of small fluorescing organelles. A mature vacuole was easily distinguished (white). (H) DIC image. (J) Autofluorescence.
(K) FM4-64 staining. (L) A combined image.
A
F-DIC E-AF D-FM4-64
v
SPK
C-combined
A-0.05 mM FM4-64 B-1.0 mM FM4-64
L-combined J-AF H-DIC
K-FM4-64 G-combined
septum
Vacuole
10 µm
159 Figure 4.13: (A-D) Internalization of the FM4-64 by living hyphae grown under phosphorus starvation (1.0 mM Pi). (A) DIC represents an image under bright field light. (B) Autofluorescence (AF) seen in the background as green. (C) Fm4-64 fluorescent viewed as red dots. E = endosomes, V = vacuoles, SPK = satellite Spitzenköper and S = representing apical region known as Spitzenköper. (E) A hypothetical model of the organization of the vesicle trafficking network in a growing hypha based upon the pattern of FM4-64 staining (adapted from FISCHER-PARTON et al., 2000).
A- DIC
B- AF
C-FM4-64
SPK
SSPK E D-combined
V
E CW
Red line-vesicle trafficking Blue line- endocytosis Green line- exocytosis Black line- membrane Thick grey -wall cell wall E
160 In a second experiment, two fluorophores were used to label the mycelium. The mycelium was stained with Vector blue III first and then later co-labelled with FM4-64. The two stains were excited by two lasers, as described before and pseudo-colour green allocated to vector blue and red to FM4-64. At 100 mM Pi, colocalization of FM4-64 and vector blue was observed (Figure 4.14A-E).
Figure 4.14: (A-E) Interactive representation of apical branching of mycelium of C. portentosa, showing a colocalization of VB and FM4-64 after co-labelling. The mycelium was grown at 100 mM Pi, co-stained with Vector blue and FM-4-64. (A) Combined image of the fungus (gray). (B) Vector blue.
(C) FM4-64 (red). (D) DIC image. (E) Magnification of the zone in A, displaying colocalization of VB and FM4-64 (yellow). Note the enzyme is clearly represented by green pseudo colour, whereas FM4-64 is represented by red colour. Note the staining of different shaped organelles by both fluorophores. Bars
= 2 µm.
A
20 µm
A
E- combined C-FM4-64
D-DIC B-VB
161 At low concentration, there was a lot of background. It appeared that the fungus at this stage was dead, as no internalization was observed. Staining with vector blue revealed that the enzyme was all over the surface of the mycelium. However, the internalization of FM4-64 was not observed.
The colocalization (Figure 4.14E) was further confirmed by ImageJ Analysis where the Pearson coefficient was 0.88, indicating a very strong colocalization. Colocalization was first analyzed using isotropic structures using the VAN STEENSEL et al. (1996) method, which could distinguish between colocalization, exclusion and unrelated signals. A Pearson„s coefficient where r=0.804 was obtained. These results revealed that the enzyme was found in the same organelles that were stained by FM4-64.
162