136 research outputs found
Ca2+ signalling and homeostasis during colony initiation in Neurospora crassa
No full textCalcium is a highly versatile intracellular signal molecule that can regulate numerous
different cellular functions. In filamentous fungi there is evidence for it being
involved in regulating various processes, including spore germination, hyphal tip
growth, hyphal branching and conidiation. During colony initiation in the
filamentous fungus Neurospora crassa, conidia form germ tubes which are involved
in colony establishment, and conidial anastomosis tubes (CATs) which are involved
in generating fused networks of conidial germlings. The primary research aim of this
thesis was to analyze the role of Ca2+-signalling and homeostasis during colony
initiation in N. crassa.
Removal of Ca2+ from the growth medium showed that external Ca2+ was necessary
for CAT fusion and, more specifically, was required for CAT chemoattraction. Two
L-type Ca2+ channel blockers (verapamil and diltiazem) with different modes of
action were found to inhibit both conidial germination and CAT fusion in wild type
strains and CAT fusion was shown to be more sensitive to these two drugs. These
channel blockers were additionally found to inhibit Ca2+ uptake by conidial
germlings of the wild type expressing the aequorin Ca2+ reporter. However, the
channel blockers also, unexpectedly, raised the cytosolic free Ca2+ ([Ca2+]c) resting
level in these germlings suggesting that they did not just inhibit L-type Ca2+ activity.
The morphological phenotypes (conidial germination, hyphal extension rate,
conidiation and hyphal branching) of 22 mutants defective in different components
of their Ca2+-signalling and homeostasis machinery were characterized in order to
identify their possible roles of Ca2+ during colony initiation and development. The
∆cch-1 mutant lacking the CCH-1 L-type Ca2+ channel gene exhibited a reduction in
CAT fusion. CAT fusion was decreased even further in a double mutant (∆cch-1∆mid-1) suggesting that that the CCH-1 and MID-1 proteins operate in
combination during this process. Increased extracellular Ca2+ partially restored the
phenotypes of the ∆cch-1, ∆mid-1 smco-1 and ∆cch-1∆mid-1 mutants which is
consistent with CCH-1 and MID-1 being involved in Ca2+ uptake from the external
medium.
Calcium signatures following mechanical perturbation were successfully measured in
populations of conidial germlings using aequorin expressed in the wild type and in
deletion mutants (∆cch-1, ∆yvc-1, ∆fig-1) lacking different Ca2+ channels. The
removal of external Ca2+ completely abolished the [Ca2+]c increase in response to
mechanical perturbation and CCH-1 was found to partly contribute to this increase in
[Ca2+]c. Various Ca2+-sensitive dyes (Oregon green 488, Fluo-4 and Calcium
Green-1) were also tested to determine if they can be used to image [Ca2+]c at the
single cell and subcellular levels. Only Fluo-4 allowed the measurement of [Ca2+]c in
individual cells but the changes in dye fluorescence in response to changes in [Ca2+]c
were too small to be useful for imaging [Ca2+]c dynamics at the subcellular level. The
other two dyes underwent rapid compartmentalization in organelles when loaded into
germlings.
The plant antifungal proteins (defensins), MsDef1, MtDef4 and PAF were all found
to disrupt Ca2+ signaling/homeostasis in conidial germlings of N. crassa. They all
inhibited the [Ca2+]c increase and raised the resting level of [Ca2+]c in response to
mechanical perturbation. Analysis of an aequorin expressing mutant that was
defective in glucosylceramide synthase (∆gcs) showed that the effects of MsDef1
(but not MtDef4) on [Ca2+]c were mediated by the sphingolipid glucosylceramide.
All of the defensins tested were found to exhibit different potencies with regard to
their inhibitory effects on conidial germination and CAT fusion
Phylogenetic relationships and estimation of divergence times among Sisoridae catfishes
No full textNineteen taxa representing 10 genera of Sisoridae were subjected to phylogenetic analyses of sequence data for the nuclear genes Plagl2 and ADNP and the mitochondrial gene cytochrome . The three data sets were analyzed separately and combined into a single data set to reconstruct phylogenetic relationships among Chinese sisorids. Both Chinese Sisoridae as a whole and the glyptosternoid taxa formed monophyletic groups. The genus is likely to be the earliest diverging extant genus among the Chinese Sisoridae. The four species included in the study formed a monophyletic group. was indicated to be earliest diverging glyptosternoid, followed by and . Our data supported the conclusion that and both formed a monophyletic group. On the basis of the fossil record and the results of a molecular dating analysis, we estimated that the Sisoridae diverged in the late Miocene about 12.2 Mya. The glyptosternoid clade was indicated to have diverged, also in the late Miocene, about 10.7 Mya, and the more specialized glyptosternoid genera, such as , originated in the Pleistocene (within 1.9 Mya). The speciation of glyptosternoid fishes is hypothesized to be closely related with the uplift of the Qinghai-Tibet Plateau.Nineteen taxa representing 10 genera of Sisoridae were subjected to phylogenetic analyses of sequence data for the nuclear genes Plagl2 and ADNP and the mitochondrial gene cytochrome . The three data sets were analyzed separately and combined into a single data set to reconstruct phylogenetic relationships among Chinese sisorids. Both Chinese Sisoridae as a whole and the glyptosternoid taxa formed monophyletic groups. The genus is likely to be the earliest diverging extant genus among the Chinese Sisoridae. The four species included in the study formed a monophyletic group. was indicated to be earliest diverging glyptosternoid, followed by and . Our data supported the conclusion that and both formed a monophyletic group. On the basis of the fossil record and the results of a molecular dating analysis, we estimated that the Sisoridae diverged in the late Miocene about 12.2 Mya. The glyptosternoid clade was indicated to have diverged, also in the late Miocene, about 10.7 Mya, and the more specialized glyptosternoid genera, such as , originated in the Pleistocene (within 1.9 Mya). The speciation of glyptosternoid fishes is hypothesized to be closely related with the uplift of the Qinghai-Tibet Plateau
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