Developments in nanotechnology are leading to a rapid proliferation of new materials that are likely to become a source of engineered nanoparticles (ENPs) to the environment, where their possible ecotoxicological impacts remain unknown. The surface properties of ENPs are of essential importance for their aggregation behavior, and thus for their mobility in aquatic and terrestrial systems and for their interactions with algae, plants and, fungi. Interactions of ENPs with natural organic matter have to be considered as well, as those will alter the ENPs aggregation behavior in surface waters or in soils. Cells of plants, algae, and fungi possess cell walls that constitute a primary site for interaction and a barrier for the entrance of ENPs. Mechanisms allowing ENPs to pass through cell walls and membranes are as yet poorly understood. Inside cells, ENPs might directly provoke alterations of membranes and other cell structures and molecules, as well as protective mechanisms. Indirect effects of ENPs depend on their chemical and physical properties and may include physical restraints (clogging effects), solubilization of toxic ENP compounds, or production of reactive oxygen species. Many questions regarding the bioavailability of ENPs, their uptake by algae, plants, and fungi and the toxicity mechanisms remain to be elucidated.

/pdf/environmental-behavior-and-ecotoxicity-of-engineered-3ivmobaxr6.pdf

Environmental behavior and ecotoxicity of engineered nanoparticles to algae, plants, and fungi

https://www.cell.com/cell/pdf/S0092-8674(03)00003-5.pdf

The Arabidopsis GNOM ARF-GEF Mediates Endosomal Recycling, Auxin Transport, and Auxin-Dependent Plant Growth

mRNA Localization: Gene Expression in the Spatial Dimension

In eukaryotic cells, compartments of the highly dynamic endomembrane system are acidified to varying degrees by the activity of vacuolar H+-ATPases (V-ATPases). In the Arabidopsis thaliana genome, most V-ATPase subunits are encoded by small gene families, thus offering potential for a multitude of enzyme complexes with different kinetic properties and localizations. We have determined the subcellular localization of the three Arabidopsis isoforms of the membrane-integral V-ATPase subunit VHA-a. Colocalization experiments as well as immunogold labeling showed that VHA-a1 is preferentially found in the trans-Golgi network (TGN), the main sorting compartment of the secretory pathway. Uptake experiments with the endocytic tracer FM4-64 revealed rapid colocalization with VHA-a1, indicating that the TGN may act as an early endosomal compartment. Concanamycin A, a specific V-ATPase inhibitor, blocks the endocytic transport of FM4-64 to the tonoplast, causes the accumulation of FM4-64 together with newly synthesized plasma membrane proteins, and interferes with the formation of brefeldin A compartments. Furthermore, nascent cell plates are rapidly stained by FM4-64, indicating that endocytosed material is redirected into the secretory flow after reaching the TGN. Together, our results suggest the convergence of the early endocytic and secretory trafficking pathways in the TGN.

Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis.

Fungal morphology and metabolite production in submerged mycelial processes.

Confocal microscopy of amphiphilic styryl dyes has been used to investigate endocytosis and vesicle trafficking in living fungal hyphae. Hyphae were treated with FM4-64, FM1-43 or TMA-DPH, three of the most commonly used membrane-selective dyes reported as markers of endocytosis. All three dyes were rapidly internalized within hyphae. FM4-64 was found best for imaging the dynamic changes in size, morphology and position of the apical vesicle cluster within growing hyphal tips because of its staining pattern, greater photostability and low cytotoxicity. FM4-64 was taken up into both the apical and subapical compartments of living hyphae in a time-dependent manner. The pattern of stain distribution was broadly similar in a range of fungal species tested (Aspergillus nidulans, Botrytis cinerea, Magnaporthe grisea, Neurospora crassa, Phycomyces blakesleeanus, Puccinia graminis, Rhizoctonia solani, Sclerotinia sclerotiorum and Trichoderma viride). With time, FM4-64 was internalized from the plasma membrane appearing in structures corresponding to putative endosomes, the apical vesicle cluster, the vacuolar membrane and mitochondria. These observations are consistent with dye internalization by endocytosis. A speculative model of the vesicle trafficking network within growing hyphae is presented.

Confocal microscopy of FM4-64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae

Regulated secretory vesicle delivery, vesicle fusion and rapid membrane recycling are all contentious issues with respect to tip growth in plant, fungal and animal cells. To examine the organisation and dynamics of membrane movements at the growing pollen tube apex and address the question of their relationship to growth, we have used the membrane stain FM4-64 both as a structural marker and as a quantitative assay. Labelling of living Lilium Longiflorum pollen tubes by FM4-64 resulted in a distinct staining pattern in the tube apex, which corresponds spatially to the previously identified cone-shaped 'apical clear zone' containing secretory vesicles. Dye uptake could be inhibited by sodium azide and followed a strict temporal sequence from the plasma membrane to a population of small (1-2 microm diameter) discrete internal structures, with subsequent appearance of dye in the apical region and ultimately in vacuolar membranes. Washout of the dye rapidly removed the plasma membrane staining, which was followed by a gradual decline in the apical fluorescence over more than an hour. Injected aqueous FM4-64 solution showed a relatively even distribution within the pollen tube. Association of FM4-64 with apical secretory vesicles was supported by the effects of the inhibitors Brefeldin-A and Cytochalasin-D, which are known to affect the localisation and number of such vesicles, on the FM4-64 staining pattern. Examination of the dynamics of FM4-64 labelling in the pollen tube tip by time-lapse observation, supported by fluorescence-recovery-after-photobleaching (FRAP) analysis, suggested the possibility of distinct pathways of bulk membrane movement both towards and, significantly, away from the apex. Quantitative analysis of FM4-64 distribution in the apex revealed that fluctuations in fluorescence 5 to 10 microm subapically, and to a lesser extent the apical 3 microm, could be related to the periodic oscillation in pollen tube growth rate. This data reveals a quantitative relationship between FM4-64 staining and growth rate within an individual tube.

Dynamics of the apical vesicle accumulation and the rate of growth are related in individual pollen tubes.

The growing pollen tube provides an excellent single cell model system in which to study the mechanisms determining growth regulation, polarity and periodic behaviour. Previously, using FM4-64, we identified periodic movements within the apical vesicle accumulation that were related to the period of oscillatory growth. This suggested a more complex interdependence between membrane traffic, apical extension and periodicity than previously thought. To investigate this a comparison was made between normally growing and Brefeldin-A-treated, non-growing, tubes. Brefeldin-A treatment established an intriguing, stable yet dynamic system of membrane aggregations in the pollen tube tip that exhibited regular movements of material with a 5-7 second period compared with the normal ∼30 second periodicity observed in growing tubes. Heat treatment was found to reduce period length in both cases. After BFA treatment membrane was demonstrated to flow from the extreme pollen tube apex back through a distinct subapical Brefeldin-A-induced membrane accumulation. The effects of Brefeldin-A on the distribution of ER- and Golgi-targeted fluorescent proteins revealed that ER did not contribute directly to the system of membrane aggregations while only certain compartments of the Golgi might be involved. The involvement of membrane derived from the apical vesicle accumulation was strongly implicated. Calcium measurements revealed that Brefeldin-A abolished the typical tip-focused calcium gradient associated with growth and there were no obvious periodic fluctuations in apical calcium associated with the continued periodic Brefeldin-A membrane aggregation associated movements. Our experiments reveal an underlying periodicity in the pollen tube that is independent of secretion, apical extension and the oscillating tip-focused calcium gradient normally associated with growth, but requires an active actin cytoskeleton.

/pdf/pollen-tubes-exhibit-regular-periodic-membrane-trafficking-1b4k10jc0m.pdf

Pollen tubes exhibit regular periodic membrane trafficking events in the absence of apical extension

The existence of pronounced cytoplasmic pH gradients within the apices of tip-growing cells, and the role of cytoplasmic pH in regulating tip growth, were investigated in three different cell types: vegetative hyphae of Neurospora crassa; pollen tubes of Agapanthus umbellatus; and rhizoids of Dryopteris affinis gametophytes. Examination of cytoplasmic pH in growing cells was performed by simultaneous, dual emission confocal ratio imaging of the pH-sensitive probe carboxy SNARF-1. Considerable attention was paid to the fine tuning of dye loading and imaging parameters to minimise cellular perturbation and assess the extent of dye partitioning into organelles. With optimal conditions, cytoplasmic pH was measured routinely with a precision of between +/-0.03 and +/-0.06 of a pH unit and a spatial resolution of 2.3 microm2. Based on in vitro calibration, estimated values of mean cytoplasmic pH for cells loaded with dye-ester were between 7.15 and 7.25 for the three cell types. After pressure injecting Neurospora hyphae with dextran-conjugated dye, however, the mean cytoplasmic pH was estimated to be 7.57. Dextran dyes are believed to give a better estimate of cytoplasmic pH because of their superior localisation and retention within the cytosol. No significant cytoplasmic pH gradient (delta pH of >0.1 unit) was observed within the apical 50 microm in growing cells of any of the three cell types. Acidification or alkalinisation of the cytoplasm in Neurospora hyphae, using a cell permeant weak acid (propionic acid at pH 7.0) or weak base (trimethylamine at pH 8.0), slowed down but did not abolish growth. However, similar manipulation of the cytoplasmic pH of Agapanthus pollen tubes and Dryopteris rhizoids completely inhibited growth. Modification of external pH affected the growth pattern of all cell types. In hyphae and pollen tubes, changes in external pH were found to have a small transient effect on cytoplasmic pH but the cells rapidly readjusted towards their original pH. Our results suggest that pronounced longitudinal gradients in cytoplasmic pH are not essential for the regulation of tip growth.

Pronounced cytoplasmic pH gradients are not required for tip growth in plant and fungal cells

https://www.cell.com/current-biology/pdf/S0960-9822(08)00803-8.pdf

Richard M. Parton

Papers

Confocal microscopy of FM4-64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae

Dynamics of the apical vesicle accumulation and the rate of growth are related in individual pollen tubes.

Pollen tubes exhibit regular periodic membrane trafficking events in the absence of apical extension

Pronounced cytoplasmic pH gradients are not required for tip growth in plant and fungal cells

Changes in bicoid mRNA Anchoring Highlight Conserved Mechanisms during the Oocyte-to-Embryo Transition