Geoff Hyde | National Centre For Biological Sciences, Bangalore, India (original) (raw)

Papers by Geoff Hyde

Intrachromosomal somatic homologous recombination rates in transgenic Arabidopsis lines after inf... more Intrachromosomal somatic homologous recombination rates in transgenic Arabidopsis lines after infection with E. coli for various time periods. A. Line 651 B. Line R2L1. Bars indicate the standard error of the mean of three biological repeats, each consisting of about 140–160 plants. Numbers on top of the bars show the respective mean values, not significantly different (P > 0.05), as analysed by Duncan's multiple range test. Vertical axis shows the mutation rates. (PPT 128 kb)

C → T transition rates in transgenic Arabidopsis line M4, after infection with different Agrobact... more C → T transition rates in transgenic Arabidopsis line M4, after infection with different Agrobacterium strains for various time periods. A. After VOT infection. B. After XXX infection. Bars indicate the standard error of the mean of three biological repeats, each consisting of about 140-160 plants. Numbers on top of the bars show the respective mean values, not significantly different (P > 0.05), as analysed by Duncan's multiple range test. Vertical axis shows the mutation rates. (PPT 125 kb)

Intrachromosomal somatic homologous recombination rates in transgenic Arabidopsis lines (lines 65... more Intrachromosomal somatic homologous recombination rates in transgenic Arabidopsis lines (lines 651 and R2L1) after XXX infection for various time periods. A. Line 651 B. Line R2L1. Bars indicate the standard error of the mean of three biological repeats, each consisting of about 140–160 plants. Numbers on top of the bars show the respective mean values, not significantly different (P > 0.05), as analysed by Duncan's multiple range test. Vertical axis shows the mutation rates. (PPT 127 kb)

Background: Agrobacterium infection, which is widely used to generate transgenic plants, is often... more Background: Agrobacterium infection, which is widely used to generate transgenic plants, is often accompanied by T-DNA-linked mutations and transpositions in flowering plants. It is not known if Agrobacterium infection also affects the rates of point mutations, somatic homologous recombinations (SHR) and frame-shift mutations (FSM). We examined the effects of Agrobacterium infection on five types of somatic mutations using a set of mutation detector lines of Arabidopsis thaliana. To verify the effect of secreted factors, we exposed the plants to different Agrobacterium strains, including wild type (Ach5), its derivatives lacking vir genes, oncogenes or T-DNA, and the heat-killed form for 48 h post-infection; also, for a smaller set of strains, we examined the rates of three types of mutations at multiple time-points. The mutation detector lines carried a non-functional β-glucuronidase gene (GUS) and a reversion of mutated GUS to its functional form resulted in blue spots. Based on t...

Telomerase, particularly its main subunit, the reverse transcriptase, TERT, prevents DNA erosion ... more Telomerase, particularly its main subunit, the reverse transcriptase, TERT, prevents DNA erosion during eukaryotic chromosomal replication, but also has poorly understood non-canonical functions. Here, in the model social amoeba Dictyostelium discoideum, we show that the protein encoded by tert has telomerase-like motifs and regulates, non-canonically, important developmental processes. Expression levels of wild-type (WT) tert were biphasic, peaking at 8 and 12 h post-starvation, aligning with developmental events, such as the initiation of streaming (~7 h) and mound formation (~10 h). In tert KO mutants, however, aggregation was delayed until 16 h. Large, irregular streams formed, then broke up, leading to small mounds. The mound-size defect was not induced when a KO mutant of countin (a master size-regulating gene) was treated with TERT inhibitors but anti-countin antibodies did rescue size in the tert KO. Further, conditioned medium from countin mutants failed to rescue size in t...

BMC plant biology, 2015

Agrobacterium infection, which is widely used to generate transgenic plants, is often accompanied... more Agrobacterium infection, which is widely used to generate transgenic plants, is often accompanied by T-DNA-linked mutations and transpositions in flowering plants. It is not known if Agrobacterium infection also affects the rates of point mutations, somatic homologous recombinations (SHR) and frame-shift mutations (FSM). We examined the effects of Agrobacterium infection on five types of somatic mutations using a set of mutation detector lines of Arabidopsis thaliana. To verify the effect of secreted factors, we exposed the plants to different Agrobacterium strains, including wild type (Ach5), its derivatives lacking vir genes, oncogenes or T-DNA, and the heat-killed form for 48 h post-infection; also, for a smaller set of strains, we examined the rates of three types of mutations at multiple time-points. The mutation detector lines carried a non-functional β-glucuronidase gene (GUS) and a reversion of mutated GUS to its functional form resulted in blue spots. Based on the number of...

Methods in Cell Biology, 1995

Journal of cell science, 1995

Growing hyphae of the ascomycete fungus Neurospora crassa contained a tip-high gradient of cytopl... more Growing hyphae of the ascomycete fungus Neurospora crassa contained a tip-high gradient of cytoplasmic Ca2+, which was absent in non-growing hyphae and was insensitive to Gd3+ in the medium. Patch clamp recordings in the cell-attached mode, from the plasma membrane of these hyphae, showed two types of channel activities; spontaneous and stretch activated. The spontaneous channels were identified as inward K+ channels based on inhibition by tetraethylammonium. The stretch activated channels had increased amplitudes in response to elevated Ca2+ in the pipette solution, and thus are permeable to Ca2+ and mediate inward Ca2+ movement. Gd3+, which is an inhibitor of some stretch activated channels, incompletely inhibited stretch activated channel activity. Both tetraethylammonium and Gd3+ only transiently reduced the rates of tip growth without changing tip morphology, thus indicating that the channels are not absolutely essential for tip growth. Furthermore, in contrast to the hyphae of...

Methods in cell biology, 1995

... is opened with forceps under liquid nitrogen; if not, the cups are separated using forceps or... more ... is opened with forceps under liquid nitrogen; if not, the cups are separated using forceps or the sliding wedge device of Craig et al. (1987). The frozen tissue is then transferred to a vial for freeze substitution over 3 days at -79 in 2% 1. High-Pressure and Plunge-Freeze Fixation ...

European journal of cell biology, 1995

A gradient of the divalent cation ionophore A23187 polarized axis establishment in regenerating h... more A gradient of the divalent cation ionophore A23187 polarized axis establishment in regenerating hyphal protoplasts and germinating cysts of the tip-growing oomycete Saprolegnia ferax. An average of sixty-three percent of new hyphae emerging from initially spherical protoplasts were oriented towards the ionophore source. This polarization was dependent on the presence of Ca2+ and could not be elicited by the presence of either 1 mM Mn2+ or Mg2+. A similar but less marked (56%) orientation was shown by germinating cysts, either with or without pretreatment with the anti-microtubule drug, nocodazole. Further, cyst-derived hyphae which did not originally emerge facing the ionophore source later showed a tendency to reorient their growth towards it. Since A23187 is known to facilitate the entry of Ca2+ into cells, and since, in protoplasts at least, the response is Ca2+ dependent, these results imply that an ionophore-generated Ca2+ gradient within the cells may be responsible for the ob...

European journal of cell biology, 1993

Zoospores of Phytophthora cinnamomi are formed by cleavage of a multinucleate sporangium and cont... more Zoospores of Phytophthora cinnamomi are formed by cleavage of a multinucleate sporangium and contain nine different components that are distributed or oriented about a well-defined axis running through a pair of basal bodies near the nucleus. In this study, the importance of the cytoskeleton in establishing and maintaining cellular polarity was examined by using the anti-microtubule drug oryzalin and the anti-microfilament drug cytochalasin D (CD). The effects of the drugs on uncleaved and cleaving sporangia were determined, using fluorescence microscopy, for six of the components that are polarized in untreated cleaved cells: an astral microtubule (MT) array, the nucleus, mitochondria and three different types of vesicles, two of which are involved in directed exocytosis. CD had no effect upon the MT arrays, the positioning of nuclei or the polarized redistribution of mitochondria and vesicles to the cortical cytoplasm, although it did cause abnormal cleavage. The effects of oryzal...

Cell Motility and the Cytoskeleton, 2002

Tubule formation is a widespread feature of the endomembrane system of eukaryotic cells, serving ... more Tubule formation is a widespread feature of the endomembrane system of eukaryotic cells, serving as an alternative to the better-known transport process of vesicular shuttling. In filamentous fungi, tubule formation by vacuoles is particularly pronounced, but little is known of its regulation. Using the hyphae of the basidiomycete Pisolithus tinctorius as our test system, we have investigated the effects of four drugs whose modulation, in animal cells, of the tubule/vesicle equilibrium is believed to be due to the altered activity of a GTP-binding protein (GTP␥S, GDP␤S, aluminium fluoride, and Brefeldin A). In Pisolithus tinctorius, GTP␥S, a non-hydrolysable form of GTP, strongly promoted vacuolar tubule formation in the tip cell and next four cells. The effects of GTP␥S could be antagonised by pre-treatment of hyphae with GDP␤S, a non-phosphorylatable form of GDP. These results support the idea that a GTP-binding protein plays a regulatory role in vacuolar tubule formation. This could be a dynamin-like GTP-ase, since GTP␥S-stimulated tubule formation has only been reported previously in cases where a dynamin is involved. Treatment with aluminium fluoride stimulated vacuolar tubule formation at a distance from the tip cell, but NaF controls indicated that this was not a GTP-binding-protein specific effect. Brefeldin A antagonised GTP␥S, and inhibited tubule formation in the tip cell. Given that Brefeldin A also affects the ER and Golgi bodies of Pisolithus tinctorius, as shown previously, it is not clear yet whether the effects of Brefeldin A on the vacuole system are direct or indirect. Cell Motil.

Cell Motility and the Cytoskeleton, 1999

While it is now recognised that transport within the endomembrane system may occur via membranous... more While it is now recognised that transport within the endomembrane system may occur via membranous tubules, spatial regulation of this process is poorly understood. We have investigated the role of the cytoskeleton in regulating the motility and morphology of the motile vacuole system in hyphae of the fungus Pisolithus tinctorius by studying (1) the effects of anti-microtubule (oryzalin, nocodazole) and anti-actin drugs (cytochalasins, latrunculin) on vacuolar activity, monitored by fluorescence microscopy of living cells; and (2) the ultrastructural relationship of microtubules, actin microfilaments, and vacuoles in hyphae prepared by rapid-freezing and freeze-substitution. Anti-microtubule drugs reduced the tubular component of the vacuole system in a dose-dependent and reversible manner, the extent of which correlated strongly with the degree of disruption of the microtubule network (monitored by immunofluorescence microscopy). The highest doses of anti-microtubule drugs completely eliminated tubular vacuoles, and only spherical vacuoles were observed. In contrast, anti-actin drugs did not reduce the frequency of tubular vacuoles or the motility of these vacuoles, even though immunofluorescence microscopy confirmed perturbation of microfilament organisation. Electron microscopy showed that vacuoles were always accompanied by microtubules. Bundles of microtubules were found running in parallel along the length of tubular vacuoles and individual microtubules were often within one microtubule diameter of a vacuole membrane. Our results strongly support a role for microtubules, but not actin microfilaments, in the spatial regulation of vacuole motility and morphology in fungal hyphae. Cell Motil.

The Protistan Cell Surface, 1994

The oomycetes are a class of protists that produce biflagellate asexual zoospores. Members of the... more The oomycetes are a class of protists that produce biflagellate asexual zoospores. Members of the oomycetes have close phylogenetic affinities with the chromophyte algae and are widely divergent from the higher fungi. This review focuses on two genera, Phytophthora and Pythium, which belong to the family Pythiaceae, and the order Peronosporales. These two genera contain many species that cause serious diseases in plants. Molecules on the surface of zoospores and cysts of these organisms are likely to play crucial roles in the infection of host plants. Knowledge of the properties of the surface of these cells should thus help increase our understanding of the infection process. Recent studies of Phytophthora cinnamomi and Pythium aphanidermatum have used lectins to analyse surface carbohydrates and have generated monoclonal antibodies (MAbs) directed towards a variety of zoospore and cysts surface components. Labelling studies with these probes have detected molecular differences between the surface of the cell body and of the fagella of the zoospores. They have been used to follow changes in surface components during encystment, including the secretion of an adhesive that bonds the spores to the host surface. Binding of lectin and antibody probes to the surface of living zoospores can induce encystment, giving evidence of cell receptors involved in this process. Freeze-substitution and immunolabelling studies have greatly augmented our understanding of the synthesis and assembly of the zoospore surface during zoosporogenesis. Synthesis of a variety of zoospore components begins when sporulation is induced. Cleavage of the multinucleate sporangium is achieved through the progressive extension of partitioning membranes, and a number of surface an-* Correspondence and reprints: Plant tigens are assembled onto the zoospore surface during cleavage. Comparisons of antibody binding to many isolates and species of Phytophthora and Pythium have revealed that surface components on zoospores and cysts exhibit a range of taxonomic specificities. Surface antigens or epitopes may occur on only a few isolates of a species; they may be species-specific, genus-specific or occur on the spores of both genera. Spore surface antigens thus promise to be of significant value for studies of the taxonomy and phylogeny of these protists, as well as for disease diagnosis.

Advances in Botanical Research, 1997

... However, regular microtubule-dependent nuclear spacing also exists in other plant, animal and... more ... However, regular microtubule-dependent nuclear spacing also exists in other plant, animal and fungal coenocytic structures, many of which do not become cleaved (eg McNaughton and Goff, 1990). In some situations, regular ...

Protoplasma, 1994

A small proportion of nucleate subprotoplasts (karyoplasts) and enucleate subprotoptasts (cytopla... more A small proportion of nucleate subprotoplasts (karyoplasts) and enucleate subprotoptasts (cytoplasts) are formed during the preparation ofprotoplasts from the filamentous green alga Mougeotia. Regeneration of Mougeotia protoplasts is an orderly process known to involve reorganisation of cortical microtubules into polar arrays centered upon two opposing foci, synthesis of new cell walls and elongation to reform cylindrical cells. The ability of cytoplasts to carry out microtubule reorganisation and cell wall synthesis was investigated by combining Hoechst staining, to distinguish cytoplasts from karyoplasts and protoplasts, with immunofluorescent staining of microtubules and Calcofiuor or Tinopal staining of cell walls. Cytoplasts survived at least 20 h in culture, but did not elongate. However, cytoplasts did participate in the first steps of protoplast regeneration. The majority of cytoplasts synthesized some cell wall material, while a small proportion was able to form ordered arrays of cortical microtubules indistinguishable from those in regenerating nucleate protoplasts. These results demonstrate the ability of plant microtubules to form new, orderly arrays in the absence of a nucleus, and suggest that the reestablishment of axiality in the protoplasts does not require a nucleus or nuclear DNA transcription.

Protoplasma, 1990

Initially non-polar protoplasts of the green aIga Mougeotia will regenerate to re-establish their... more Initially non-polar protoplasts of the green aIga Mougeotia will regenerate to re-establish their original cylindrical cell shape. The orientation of the growth axis of regenerating protoplasts held in agarose was independent of both the direction of incident white light and gravity. Protoplasts elongated parallel to applied DC electric fields of approx. 0.2 V cm-i (1 mV/protoplast) and greater, with an increasing percentage oriented with increasing field strength. At the maximum field strength used (10mV/cell), 53% of protoplasts were oriented within • I 0 ~ of the 0/180 ~ axis of the field. In untreated controls, the orientation of elongation was random. Protoplast survival was unaffected by field treatment. Some protoplasts (up to 37% in 10mV/cell fields) formed outgrowths towards the cathode and occasionally towards the anode. Regenerating protoplasts in fields displayed the normal sequence of microtubule reorganization. This means that the positioning of the ordered symmetrical array of microtubules centred on two loci that appears within 3 to 4 h, and the subsequent organization of mierotubules by 8 to 12h into a band that intersects both foci and which is transverse to the axis of elongation (Galway and Hardham 1986), may be controlled by externally applied electric fields. In the region of this microtubule band, the applied field causes the plasma membrane to be stretched parallel to the field (Bryant and Wolfe 1987). We suggest that microtubules may become oriented perpendicular to the direction of field-induced membrane stretching, and that membrane stretching may be one of the orienting mechanisms for membrane-linked microtubules in elongating plant cells.

Protoplasma, 1997

Summary Membrane-impermeant fluorescent probes, such as Lucifer Yellow carbohydrazide, 6-carboxy... more Summary Membrane-impermeant fluorescent probes, such as Lucifer Yellow carbohydrazide, 6-carboxyfluorescein, and high-molecular-mass fluorescent dextrans (10 and 70 kDa) are not internalised by actively-growing hyphal tip-cells ofPisolithus tinctorius even after prolonged exposure to the probe. These findings suggest that fluid-phase endocytosis may not occur in these fully turgid tip-growing hyphae. In contrast, a number of membrane-permeant fluorescent probes, including 6-carboxfluorescein diacetate, the

Protoplasma, 1991

The effects of high pressure on the ultrastructure of sporangia of Phytophthora einnamomi and P. ... more The effects of high pressure on the ultrastructure of sporangia of Phytophthora einnamomi and P. paImivora have been examined by comparing sporangia frozen in a Balzers hyperbaric freezer or pressurized in a French pressure cell with sporangia plunge frozen at ambient pressure. Both freeze fixation methods provided excellent preservation of most cell structures, but one organelle type seen in plunge frozen material, the large peripheral vesicle (LPV), was not observed in high pressure frozen sporangia. Instead, these sporangia contained large irregularly shaped structures which exhibit the patterns of spatial distribution and, for P. cinnamomi, the monoclonal antibody binding characteristic of LPVs. These findings suggest that some factor of the hyperbaric freezing process causes LPVs to be degraded. Sporangia of P. cinnamomi that had been pressurized in a French pressure cell also exhibited large structures with the spatial distribution and monoclonal antibody binding characteristic of LPVs. The apparent expansion of LPVs that follows from both pressurizing treatments causes considerable passive disruption of sporangial structure. This is the first report of a major disturbance of cell structure from use of the Balzers hyperbaric freezer, and reflects the lability, noted in previous work, of LPVs in Phytophthora.

Protoplasma, 1997

Motile tubular vacuole systems have been visualised using DIC optics in living hyphae of Pisolith... more Motile tubular vacuole systems have been visualised using DIC optics in living hyphae of Pisolithus tinctorius without loading of any fluorescent tracer. Adding new medium, with or without the tracer CFDA, alters the motility of this system and increases the number of tubules. This response has been shown in individual hyphal tip cells and quantified in populations of tip cells. Vacuoles with motile tubules are also demonstrated in more basal cells of the hyphae, within 600 gm of the growing hyphal front. The vacuoles in these cells show more limited motility, but similarly respond to addition of new medium by increased motility and tubular activity. This demonstration that the vacuole system in more mature regions is both motile and interconnected as in the tips, and similarly responds to changes in external conditions, supports the hypothesis that the vacuole system may play a role in long-distance transport. Vacuoles in the most mature cells, more than 600 am behind the hyphal growth zone are not motile. They do not respond to these stimuli and remain spherical and isolated. There are many explanations for this and the present lack of response does not exclude the transport hypothesis. The findings further support the concept that tubular vacuole systems are equivalent to animal endosomal/lysosomal systems and have implications for their motility, especially their plasticity in response to external stimuli, such as fluorescent tracers.

Intrachromosomal somatic homologous recombination rates in transgenic Arabidopsis lines after inf... more Intrachromosomal somatic homologous recombination rates in transgenic Arabidopsis lines after infection with E. coli for various time periods. A. Line 651 B. Line R2L1. Bars indicate the standard error of the mean of three biological repeats, each consisting of about 140–160 plants. Numbers on top of the bars show the respective mean values, not significantly different (P > 0.05), as analysed by Duncan's multiple range test. Vertical axis shows the mutation rates. (PPT 128 kb)

C → T transition rates in transgenic Arabidopsis line M4, after infection with different Agrobact... more C → T transition rates in transgenic Arabidopsis line M4, after infection with different Agrobacterium strains for various time periods. A. After VOT infection. B. After XXX infection. Bars indicate the standard error of the mean of three biological repeats, each consisting of about 140-160 plants. Numbers on top of the bars show the respective mean values, not significantly different (P > 0.05), as analysed by Duncan's multiple range test. Vertical axis shows the mutation rates. (PPT 125 kb)

Intrachromosomal somatic homologous recombination rates in transgenic Arabidopsis lines (lines 65... more Intrachromosomal somatic homologous recombination rates in transgenic Arabidopsis lines (lines 651 and R2L1) after XXX infection for various time periods. A. Line 651 B. Line R2L1. Bars indicate the standard error of the mean of three biological repeats, each consisting of about 140–160 plants. Numbers on top of the bars show the respective mean values, not significantly different (P > 0.05), as analysed by Duncan's multiple range test. Vertical axis shows the mutation rates. (PPT 127 kb)

Background: Agrobacterium infection, which is widely used to generate transgenic plants, is often... more Background: Agrobacterium infection, which is widely used to generate transgenic plants, is often accompanied by T-DNA-linked mutations and transpositions in flowering plants. It is not known if Agrobacterium infection also affects the rates of point mutations, somatic homologous recombinations (SHR) and frame-shift mutations (FSM). We examined the effects of Agrobacterium infection on five types of somatic mutations using a set of mutation detector lines of Arabidopsis thaliana. To verify the effect of secreted factors, we exposed the plants to different Agrobacterium strains, including wild type (Ach5), its derivatives lacking vir genes, oncogenes or T-DNA, and the heat-killed form for 48 h post-infection; also, for a smaller set of strains, we examined the rates of three types of mutations at multiple time-points. The mutation detector lines carried a non-functional β-glucuronidase gene (GUS) and a reversion of mutated GUS to its functional form resulted in blue spots. Based on t...

Telomerase, particularly its main subunit, the reverse transcriptase, TERT, prevents DNA erosion ... more Telomerase, particularly its main subunit, the reverse transcriptase, TERT, prevents DNA erosion during eukaryotic chromosomal replication, but also has poorly understood non-canonical functions. Here, in the model social amoeba Dictyostelium discoideum, we show that the protein encoded by tert has telomerase-like motifs and regulates, non-canonically, important developmental processes. Expression levels of wild-type (WT) tert were biphasic, peaking at 8 and 12 h post-starvation, aligning with developmental events, such as the initiation of streaming (~7 h) and mound formation (~10 h). In tert KO mutants, however, aggregation was delayed until 16 h. Large, irregular streams formed, then broke up, leading to small mounds. The mound-size defect was not induced when a KO mutant of countin (a master size-regulating gene) was treated with TERT inhibitors but anti-countin antibodies did rescue size in the tert KO. Further, conditioned medium from countin mutants failed to rescue size in t...

BMC plant biology, 2015

Agrobacterium infection, which is widely used to generate transgenic plants, is often accompanied... more Agrobacterium infection, which is widely used to generate transgenic plants, is often accompanied by T-DNA-linked mutations and transpositions in flowering plants. It is not known if Agrobacterium infection also affects the rates of point mutations, somatic homologous recombinations (SHR) and frame-shift mutations (FSM). We examined the effects of Agrobacterium infection on five types of somatic mutations using a set of mutation detector lines of Arabidopsis thaliana. To verify the effect of secreted factors, we exposed the plants to different Agrobacterium strains, including wild type (Ach5), its derivatives lacking vir genes, oncogenes or T-DNA, and the heat-killed form for 48 h post-infection; also, for a smaller set of strains, we examined the rates of three types of mutations at multiple time-points. The mutation detector lines carried a non-functional β-glucuronidase gene (GUS) and a reversion of mutated GUS to its functional form resulted in blue spots. Based on the number of...

Methods in Cell Biology, 1995

Journal of cell science, 1995

Growing hyphae of the ascomycete fungus Neurospora crassa contained a tip-high gradient of cytopl... more Growing hyphae of the ascomycete fungus Neurospora crassa contained a tip-high gradient of cytoplasmic Ca2+, which was absent in non-growing hyphae and was insensitive to Gd3+ in the medium. Patch clamp recordings in the cell-attached mode, from the plasma membrane of these hyphae, showed two types of channel activities; spontaneous and stretch activated. The spontaneous channels were identified as inward K+ channels based on inhibition by tetraethylammonium. The stretch activated channels had increased amplitudes in response to elevated Ca2+ in the pipette solution, and thus are permeable to Ca2+ and mediate inward Ca2+ movement. Gd3+, which is an inhibitor of some stretch activated channels, incompletely inhibited stretch activated channel activity. Both tetraethylammonium and Gd3+ only transiently reduced the rates of tip growth without changing tip morphology, thus indicating that the channels are not absolutely essential for tip growth. Furthermore, in contrast to the hyphae of...

Methods in cell biology, 1995

... is opened with forceps under liquid nitrogen; if not, the cups are separated using forceps or... more ... is opened with forceps under liquid nitrogen; if not, the cups are separated using forceps or the sliding wedge device of Craig et al. (1987). The frozen tissue is then transferred to a vial for freeze substitution over 3 days at -79 in 2% 1. High-Pressure and Plunge-Freeze Fixation ...

European journal of cell biology, 1995

A gradient of the divalent cation ionophore A23187 polarized axis establishment in regenerating h... more A gradient of the divalent cation ionophore A23187 polarized axis establishment in regenerating hyphal protoplasts and germinating cysts of the tip-growing oomycete Saprolegnia ferax. An average of sixty-three percent of new hyphae emerging from initially spherical protoplasts were oriented towards the ionophore source. This polarization was dependent on the presence of Ca2+ and could not be elicited by the presence of either 1 mM Mn2+ or Mg2+. A similar but less marked (56%) orientation was shown by germinating cysts, either with or without pretreatment with the anti-microtubule drug, nocodazole. Further, cyst-derived hyphae which did not originally emerge facing the ionophore source later showed a tendency to reorient their growth towards it. Since A23187 is known to facilitate the entry of Ca2+ into cells, and since, in protoplasts at least, the response is Ca2+ dependent, these results imply that an ionophore-generated Ca2+ gradient within the cells may be responsible for the ob...

European journal of cell biology, 1993

Zoospores of Phytophthora cinnamomi are formed by cleavage of a multinucleate sporangium and cont... more Zoospores of Phytophthora cinnamomi are formed by cleavage of a multinucleate sporangium and contain nine different components that are distributed or oriented about a well-defined axis running through a pair of basal bodies near the nucleus. In this study, the importance of the cytoskeleton in establishing and maintaining cellular polarity was examined by using the anti-microtubule drug oryzalin and the anti-microfilament drug cytochalasin D (CD). The effects of the drugs on uncleaved and cleaving sporangia were determined, using fluorescence microscopy, for six of the components that are polarized in untreated cleaved cells: an astral microtubule (MT) array, the nucleus, mitochondria and three different types of vesicles, two of which are involved in directed exocytosis. CD had no effect upon the MT arrays, the positioning of nuclei or the polarized redistribution of mitochondria and vesicles to the cortical cytoplasm, although it did cause abnormal cleavage. The effects of oryzal...

Cell Motility and the Cytoskeleton, 2002

Tubule formation is a widespread feature of the endomembrane system of eukaryotic cells, serving ... more Tubule formation is a widespread feature of the endomembrane system of eukaryotic cells, serving as an alternative to the better-known transport process of vesicular shuttling. In filamentous fungi, tubule formation by vacuoles is particularly pronounced, but little is known of its regulation. Using the hyphae of the basidiomycete Pisolithus tinctorius as our test system, we have investigated the effects of four drugs whose modulation, in animal cells, of the tubule/vesicle equilibrium is believed to be due to the altered activity of a GTP-binding protein (GTP␥S, GDP␤S, aluminium fluoride, and Brefeldin A). In Pisolithus tinctorius, GTP␥S, a non-hydrolysable form of GTP, strongly promoted vacuolar tubule formation in the tip cell and next four cells. The effects of GTP␥S could be antagonised by pre-treatment of hyphae with GDP␤S, a non-phosphorylatable form of GDP. These results support the idea that a GTP-binding protein plays a regulatory role in vacuolar tubule formation. This could be a dynamin-like GTP-ase, since GTP␥S-stimulated tubule formation has only been reported previously in cases where a dynamin is involved. Treatment with aluminium fluoride stimulated vacuolar tubule formation at a distance from the tip cell, but NaF controls indicated that this was not a GTP-binding-protein specific effect. Brefeldin A antagonised GTP␥S, and inhibited tubule formation in the tip cell. Given that Brefeldin A also affects the ER and Golgi bodies of Pisolithus tinctorius, as shown previously, it is not clear yet whether the effects of Brefeldin A on the vacuole system are direct or indirect. Cell Motil.

Cell Motility and the Cytoskeleton, 1999

While it is now recognised that transport within the endomembrane system may occur via membranous... more While it is now recognised that transport within the endomembrane system may occur via membranous tubules, spatial regulation of this process is poorly understood. We have investigated the role of the cytoskeleton in regulating the motility and morphology of the motile vacuole system in hyphae of the fungus Pisolithus tinctorius by studying (1) the effects of anti-microtubule (oryzalin, nocodazole) and anti-actin drugs (cytochalasins, latrunculin) on vacuolar activity, monitored by fluorescence microscopy of living cells; and (2) the ultrastructural relationship of microtubules, actin microfilaments, and vacuoles in hyphae prepared by rapid-freezing and freeze-substitution. Anti-microtubule drugs reduced the tubular component of the vacuole system in a dose-dependent and reversible manner, the extent of which correlated strongly with the degree of disruption of the microtubule network (monitored by immunofluorescence microscopy). The highest doses of anti-microtubule drugs completely eliminated tubular vacuoles, and only spherical vacuoles were observed. In contrast, anti-actin drugs did not reduce the frequency of tubular vacuoles or the motility of these vacuoles, even though immunofluorescence microscopy confirmed perturbation of microfilament organisation. Electron microscopy showed that vacuoles were always accompanied by microtubules. Bundles of microtubules were found running in parallel along the length of tubular vacuoles and individual microtubules were often within one microtubule diameter of a vacuole membrane. Our results strongly support a role for microtubules, but not actin microfilaments, in the spatial regulation of vacuole motility and morphology in fungal hyphae. Cell Motil.

The Protistan Cell Surface, 1994

The oomycetes are a class of protists that produce biflagellate asexual zoospores. Members of the... more The oomycetes are a class of protists that produce biflagellate asexual zoospores. Members of the oomycetes have close phylogenetic affinities with the chromophyte algae and are widely divergent from the higher fungi. This review focuses on two genera, Phytophthora and Pythium, which belong to the family Pythiaceae, and the order Peronosporales. These two genera contain many species that cause serious diseases in plants. Molecules on the surface of zoospores and cysts of these organisms are likely to play crucial roles in the infection of host plants. Knowledge of the properties of the surface of these cells should thus help increase our understanding of the infection process. Recent studies of Phytophthora cinnamomi and Pythium aphanidermatum have used lectins to analyse surface carbohydrates and have generated monoclonal antibodies (MAbs) directed towards a variety of zoospore and cysts surface components. Labelling studies with these probes have detected molecular differences between the surface of the cell body and of the fagella of the zoospores. They have been used to follow changes in surface components during encystment, including the secretion of an adhesive that bonds the spores to the host surface. Binding of lectin and antibody probes to the surface of living zoospores can induce encystment, giving evidence of cell receptors involved in this process. Freeze-substitution and immunolabelling studies have greatly augmented our understanding of the synthesis and assembly of the zoospore surface during zoosporogenesis. Synthesis of a variety of zoospore components begins when sporulation is induced. Cleavage of the multinucleate sporangium is achieved through the progressive extension of partitioning membranes, and a number of surface an-* Correspondence and reprints: Plant tigens are assembled onto the zoospore surface during cleavage. Comparisons of antibody binding to many isolates and species of Phytophthora and Pythium have revealed that surface components on zoospores and cysts exhibit a range of taxonomic specificities. Surface antigens or epitopes may occur on only a few isolates of a species; they may be species-specific, genus-specific or occur on the spores of both genera. Spore surface antigens thus promise to be of significant value for studies of the taxonomy and phylogeny of these protists, as well as for disease diagnosis.

Advances in Botanical Research, 1997

... However, regular microtubule-dependent nuclear spacing also exists in other plant, animal and... more ... However, regular microtubule-dependent nuclear spacing also exists in other plant, animal and fungal coenocytic structures, many of which do not become cleaved (eg McNaughton and Goff, 1990). In some situations, regular ...

Protoplasma, 1994

A small proportion of nucleate subprotoplasts (karyoplasts) and enucleate subprotoptasts (cytopla... more A small proportion of nucleate subprotoplasts (karyoplasts) and enucleate subprotoptasts (cytoplasts) are formed during the preparation ofprotoplasts from the filamentous green alga Mougeotia. Regeneration of Mougeotia protoplasts is an orderly process known to involve reorganisation of cortical microtubules into polar arrays centered upon two opposing foci, synthesis of new cell walls and elongation to reform cylindrical cells. The ability of cytoplasts to carry out microtubule reorganisation and cell wall synthesis was investigated by combining Hoechst staining, to distinguish cytoplasts from karyoplasts and protoplasts, with immunofluorescent staining of microtubules and Calcofiuor or Tinopal staining of cell walls. Cytoplasts survived at least 20 h in culture, but did not elongate. However, cytoplasts did participate in the first steps of protoplast regeneration. The majority of cytoplasts synthesized some cell wall material, while a small proportion was able to form ordered arrays of cortical microtubules indistinguishable from those in regenerating nucleate protoplasts. These results demonstrate the ability of plant microtubules to form new, orderly arrays in the absence of a nucleus, and suggest that the reestablishment of axiality in the protoplasts does not require a nucleus or nuclear DNA transcription.

Protoplasma, 1990

Initially non-polar protoplasts of the green aIga Mougeotia will regenerate to re-establish their... more Initially non-polar protoplasts of the green aIga Mougeotia will regenerate to re-establish their original cylindrical cell shape. The orientation of the growth axis of regenerating protoplasts held in agarose was independent of both the direction of incident white light and gravity. Protoplasts elongated parallel to applied DC electric fields of approx. 0.2 V cm-i (1 mV/protoplast) and greater, with an increasing percentage oriented with increasing field strength. At the maximum field strength used (10mV/cell), 53% of protoplasts were oriented within • I 0 ~ of the 0/180 ~ axis of the field. In untreated controls, the orientation of elongation was random. Protoplast survival was unaffected by field treatment. Some protoplasts (up to 37% in 10mV/cell fields) formed outgrowths towards the cathode and occasionally towards the anode. Regenerating protoplasts in fields displayed the normal sequence of microtubule reorganization. This means that the positioning of the ordered symmetrical array of microtubules centred on two loci that appears within 3 to 4 h, and the subsequent organization of mierotubules by 8 to 12h into a band that intersects both foci and which is transverse to the axis of elongation (Galway and Hardham 1986), may be controlled by externally applied electric fields. In the region of this microtubule band, the applied field causes the plasma membrane to be stretched parallel to the field (Bryant and Wolfe 1987). We suggest that microtubules may become oriented perpendicular to the direction of field-induced membrane stretching, and that membrane stretching may be one of the orienting mechanisms for membrane-linked microtubules in elongating plant cells.

Protoplasma, 1997

Summary Membrane-impermeant fluorescent probes, such as Lucifer Yellow carbohydrazide, 6-carboxy... more Summary Membrane-impermeant fluorescent probes, such as Lucifer Yellow carbohydrazide, 6-carboxyfluorescein, and high-molecular-mass fluorescent dextrans (10 and 70 kDa) are not internalised by actively-growing hyphal tip-cells ofPisolithus tinctorius even after prolonged exposure to the probe. These findings suggest that fluid-phase endocytosis may not occur in these fully turgid tip-growing hyphae. In contrast, a number of membrane-permeant fluorescent probes, including 6-carboxfluorescein diacetate, the

Protoplasma, 1991

The effects of high pressure on the ultrastructure of sporangia of Phytophthora einnamomi and P. ... more The effects of high pressure on the ultrastructure of sporangia of Phytophthora einnamomi and P. paImivora have been examined by comparing sporangia frozen in a Balzers hyperbaric freezer or pressurized in a French pressure cell with sporangia plunge frozen at ambient pressure. Both freeze fixation methods provided excellent preservation of most cell structures, but one organelle type seen in plunge frozen material, the large peripheral vesicle (LPV), was not observed in high pressure frozen sporangia. Instead, these sporangia contained large irregularly shaped structures which exhibit the patterns of spatial distribution and, for P. cinnamomi, the monoclonal antibody binding characteristic of LPVs. These findings suggest that some factor of the hyperbaric freezing process causes LPVs to be degraded. Sporangia of P. cinnamomi that had been pressurized in a French pressure cell also exhibited large structures with the spatial distribution and monoclonal antibody binding characteristic of LPVs. The apparent expansion of LPVs that follows from both pressurizing treatments causes considerable passive disruption of sporangial structure. This is the first report of a major disturbance of cell structure from use of the Balzers hyperbaric freezer, and reflects the lability, noted in previous work, of LPVs in Phytophthora.

Protoplasma, 1997

Motile tubular vacuole systems have been visualised using DIC optics in living hyphae of Pisolith... more Motile tubular vacuole systems have been visualised using DIC optics in living hyphae of Pisolithus tinctorius without loading of any fluorescent tracer. Adding new medium, with or without the tracer CFDA, alters the motility of this system and increases the number of tubules. This response has been shown in individual hyphal tip cells and quantified in populations of tip cells. Vacuoles with motile tubules are also demonstrated in more basal cells of the hyphae, within 600 gm of the growing hyphal front. The vacuoles in these cells show more limited motility, but similarly respond to addition of new medium by increased motility and tubular activity. This demonstration that the vacuole system in more mature regions is both motile and interconnected as in the tips, and similarly responds to changes in external conditions, supports the hypothesis that the vacuole system may play a role in long-distance transport. Vacuoles in the most mature cells, more than 600 am behind the hyphal growth zone are not motile. They do not respond to these stimuli and remain spherical and isolated. There are many explanations for this and the present lack of response does not exclude the transport hypothesis. The findings further support the concept that tubular vacuole systems are equivalent to animal endosomal/lysosomal systems and have implications for their motility, especially their plasticity in response to external stimuli, such as fluorescent tracers.