Rapid whole cell imaging reveals a calcium-APPL1-dynein nexus that regulates cohort trafficking of stimulated EGF receptors.

The endosomal system provides rich signal processing capabilities for responses elicited by growth factor receptors and their ligands. At the single cell level, endosomal trafficking becomes a critical component of signal processing, as exemplified by the epidermal growth factor (EGF) receptors. Activated EGFRs are trafficked to the phosphatase-enriched peri-nuclear region (PNR), where they are dephosphorylated and degraded. The details of the mechanisms that govern the movements of stimulated EGFRs towards the PNR, are not completely known. Here, exploiting the advantages of lattice light-sheet microscopy, we show that EGFR activation by EGF triggers a transient calcium increase causing a whole-cell level redistribution of Adaptor Protein, Phosphotyrosine Interacting with PH Domain And Leucine Zipper 1 (APPL1) from pre-existing endosomes within one minute, the rebinding of liberated APPL1 directly to EGFR, and the dynein-dependent translocation of APPL1-EGF-bearing endosomes to the PNR within ten minutes. The cell spanning, fast acting network that we reveal integrates a cascade of events dedicated to the cohort movement of activated EGF receptors. Our findings support the intriguing proposal that certain endosomal pathways have shed some of the stochastic strategies of traditional trafficking and have evolved processes that provide the temporal predictability that typify canonical signaling.

Retention of fluorescent probes during aldehyde-free anhydrous freeze-substitution.

Fluorescent probes are widely used for microscopy of live-cell processes, but few such probes can also be used with classically fixed or otherwise immobilized material, and none has been used without aldehyde fixation, which can introduce artefacts of structure and probe localization. Here we show that the fluorescence patterns in fungal hyphae loaded with chloromethyl aminocoumarin (CMAC), and then anhydrously freeze-substituted, without any aldehyde fixation, are similar to those seen in living hyphae. Probe loss into the mounting medium (Spurr's resin) with CMAC and five other probes tested indicated that some unwanted solubilization of probe occurred during embedding, but nevertheless vacuoles could be imaged by their retention of probe.

Brefeldin A affects growth, endoplasmic reticulum, Golgi bodies, tubular vacuole system, and secretory pathway in Pisolithus tinctorius.

Brefeldin A (BFA) reduced radial growth in Pisolithus tinctorius at a concentration as low as 2 microM. Use of endoplasmic reticulum (ER)-Tracker dye, unconjugated BFA, and fluorescent BFA (BODIPY-BFA) allowed comparison of the effects of BFA on the endomembrane system of P. tinctorius at the light and electron microscope levels. Both ER-Tracker dye and BODIPY-BFA have been shown previously to label the ER. Unconjugated BFA and BODIPY-BFA modified the ER network and disrupted the tubular vacuole system in the tip region. The ultrastructure in freeze-substituted hyphae showed that BFA treatment resulted in (i) disruption of the Spitzenkörper, (ii) reduction in number of apical vesicles, (iii) redistribution and mild dilation of ER, and (iv) persistence and increased size and complexity of Golgi bodies. The effects of BFA on the ER were only partially reversible in the time period examined. We conclude that in P. tinctorius, BFA as the free metabolite or BODIPY-BFA affects the tubular vacuole system as well as anterograde membrane flow between the ER and the Golgi bodies and post-Golgi transport.

ER-Tracker dye and BODIPY-brefeldin A differentiate the endoplasmic reticulum and golgi bodies from the tubular-vacuole system in living hyphae of Pisolithus tinctorius.

Two fluorochromes, ER-TrackerTM Blue-White DPX dye and the fluorescent brefeldin A (BFA) derivative, BODIPY-BFA, label the endoplasmic reticulum (ER) in hyphal tips of Pisolithus tinctorius and allow its differentiation from the tubular-vacuole system at the light microscope level in living cells. The ER-Tracker dye labels a reticulate network similar in distribution to ER as seen in electron micrographs of freeze-substituted hyphae. BODIPY-BFA stains a thicker axially aligned structure with an expanded region at the apex, which is similar to that seen when hyphae are stained with ER-Tracker dye in the presence of unconjugated BFA. This structure is considered to be ER modified by BFA, a view supported by ultrastructural observations of the effect of BFA on the fungal ER. Both fluorescent probes also stain punctate structures, which are most likely to be Golgi bodies. Neither probe labels the tubular-vacuole system.

Microtubules, but not actin microfilaments, regulate vacuole motility and morphology in hyphae of Pisolithus tinctorius.

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.

The roles of Ca2+ and plasma membrane ion channels in hyphal tip growth of Neurospora crassa.

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 another tip growing organism, Saprolegnia ferax, tip-high gradients of neither spontaneous nor stretch activated channels were found. Voltage clamping of the apical plasma membrane potential in the range from -300 to +150 mV did not affect the rates of hyphal elongation. Collectively, these data suggest that ion transport across the plasma membrane at the growing tip in Neurospora is not obligatory for the maintenance of tip growth, but that a gradient of Ca2+, possibly generated from internal stores in an unknown way, is required.

Ca(2+)-dependent polarization of axis establishment in the tip-growing organism, Saprolegnia ferax, by gradients of the ionophore A23187.

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 observed polarizing influence on protoplasts and cysts. It is likely that the role of Ca2+ is essentially the same in both systems: a primary common factor in the establishment of the growth axis. The results provide evidence that Ca2+ polarizes hyphal growth and support the idea that Ca2+ has a ubiquitous, primary role in the initiation of polarity in tip-growing cells.

Microtubules regulate the generation of polarity in zoospores of Phytophthora cinnamomi.

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 oryzalin, however, indicate that the asymmetric disposition of the MT array is fundamental to zoospore polarities: when the array is itself eliminated with this drug, none of the other five elements show any signs of polar positioning within the cleaved sporangium. Oryzalin also caused abnormal cleavage similar to that seen in CD-treated cells. Most intriguing, however, was the finding that although the three vesicle types in cleaved, oryzalin-treated sporangia did not exhibit the polarized distribution seen in control and CD-treated cells, in many cases the vesicles had, nevertheless, lost their initially random distributions and had become concentrated in the cytoplasm adjacent to the abnormal cleavage planes. Thus although an intact MT array is required for segregation of the vesicles within the cortex, their redistribution to the cortex can somehow occur in the absence of MTs and actin microfilaments.

Freeze substitution reveals a new model for sporangial cleavage in Phytophthora, a result with implications for cytokinesis in other eukaryotes.

Rapid freezing and freeze substitution (RF-FS) have been used to re-examine the process by which the multinucleate sporangium of the Oomycetes, Phytophthora cinnamomi and P. palmivora, is subdivided into uninucleate zoospores. The results indicate a new model for sporangial cleavage in Phytophthora and suggest that the currently accepted model is based on interpretation of artefacts caused by chemical fixation. The previous model describes cleavage as a two-stage process in which specialized cleavage vesicles first become positioned at the boundaries of each future subdivision and later fuse to compartmentalize the sporangium. RF-FS, however, indicates that cleavage results from the progressive extension of paired sheets of membrane along the future subdivision boundaries. These sheets finally interconnect and subdivide the sporangium. Cleavage vesicles are only evident in preliminary stages of this process and are never aligned along the future boundaries, contrary to the observations of studies based on chemical fixation. Chemical fixation apparently causes the membranous sheets to vesiculate, even at relatively advanced stages of cleavage, thus giving the misleading impression that the resulting network of lined-up vesicles is an intermediate stage in the cleavage process. This finding has wide-ranging implications for the understanding of eukaryotic cytokinesis, because all previous studies that describe vesicle alignment and fusion have relied upon chemical fixation. Other novel features revealed by RF-FS include an extensive extracellular matrix within the sporangium that could be involved in zoospore release, and a trans-Golgi network.