1,721,012 research outputs found
Inhibition of actin polymerization by LatB affects endocytosis and secretion both in the apex and in the subapical regions of tobacco pollen tubes
No full textThe pollen tube is a tip growing cell that plays a fundamental role in the fertilization process of higher plants. Pollen tube growth is based on transport and accumulation of secretory vesicles. The incorporation of new segments of PM largely exceeds the quantity of membrane required for the extension, so the excess of PM must be removed by endocytosis. Recent studies showed internalization of subapical PM domains that were mainly recycled to exocytosis through the Golgi apparatus and a second mainly degradative pathway. The movements of the endomembrane system during the pollen tube growth depends on the concerted action and integrity of the cytoskeleton. In this study we disturb the system of cortical actin filaments using Latrunculin B to verify modifications in endocytosis, using specific FM dyes in time-lapse experiments and by ultrastructural experiments using charged Nanogold. Our results demonstrate that the system of cortical microfilaments determines a strong reduction of endocytosis and consequently the alteration of recycling through the Golgi, the inhibition of degradative pathways, and changes in the secretion pattern at the tip PM
Endocytic pathways and membrane recycling involve the actin filaments in tobacco pollen tubes
Full text linkThe pollen tube is a tip growing cell that plays a fundamental role in the fertilization process of higher plants. Pollen tube growth is based on transport and accumulation of secretory vesicles. The incorporation of new segments of PM largely exceeds the quantity of membrane required for the extension, so the excess of PM must be removed by endocytosis. Recent studies showed internalization of subapical PM domains that were mainly recycled to exocytosis through the Golgi apparatus and a second mainly degradative pathway involving PM retrieval at the tip. The movements of the endomembrane system during the pollen tube growth depends on the concerted action and integrity of the cytoskeleton. In this study we disturb the system of cortical actin filaments using Latrunculin B to verify modifications in endocytosis, using specific FM dyes in time-lapse experiments and by ultrastructural experiments using charged Nanogold. Our results demonstrate that the system of cortical microfilaments determines a strong reduction of endocytosis in the subapical regions and consequently the alteration of recycling through the Golgi and the inhibition of the degradative pathways
Microtubules affect endocytosis and membrane trafficking in the apical region in Nicotiana tabacum pollen tube
Full text linkThe pollen tube is a highly polarized cell that plays a fundamental role in the fertilization process of higher plants. Pollen tube growth depends on transport and accumulation of secretory vesicles (SVs) in the apical region where they fuse in a small area of the plasma membrane (PM) supplying material for the construction of new cell wall and providing, at the same time, new surface of PM. The incorporation of new PM area largely exceeds the quantity of membrane surface required for tube elongation, so the excess of PM must be removed through an active internalization process, known as endocytosis (Steer and Steer 1989). In vivo time-lapse experiments using FM4-64 combined with quantitative analysis allowed the observation that different endocytic pathways occurs in distinct zones of the tube, and that endocytic vesicles might substantially contribute to the V-shaped vesicle accumulation (Moscatelli et al. 2007; Zonia and Munnick 2008;). Pollen tube growth depends on the integrity of the cytoskeletal apparatus; is was shown that the actin cytoskeleton regulates cytoplasmic streaming and secretion and influence endocytosis in the shank of the tube (Moscatelli et al. 2012). However, the identification of kinesin-related polypeptides (Cai et al. 1993; Romagnoli et al. 2003) led to hypothesize that MTs could also have a role in membrane trafficking. In this study we disturb the system of MTs using 5M Nocodazole, to verify its effect on endocytosis and endosome transport. Time lapse experiments by using FM4-64 demonstrated that Nocodazole affects the PM internalization in the tip. Moreover, the use of FM4-64 in pollen tubes that transiently express GFP-RabA4b under the control of Lat52 pollen specific promoter, allowed us to show that MTs are involved in membrane sorting in the apex and/or in the progression of endocytic vesicles from the tip regions to vacuoles. Endocytosis dissection by using charged nanogold confirmed that Nocodazole has an effect in the internalization pathway occurring in the tip. In vitro binding assays showing that MTs are able to bind Syp-21 positive compartments confirmed a role of MTs in degradative pathways. In addition, FRAP experiments of the apical PM suggested that MTs are involved in maintaining the differential pattern of exocytosis, in different domains of the apical PM
Different endocytic pathways require the coordinated action of actin filaments and microtubules in tobacco pollen tube
No full textPollen tubes are highly polarized cells in which both cytoplasmic constituents and proteins/lipids are distributed differentially in the cytoplasm and on the plasma membrane (PM). The equilibrium between exo- and endocytosis, defining PM composition, allows pollen tubes to successfully interact with molecules of the female tissues during guided migration through the style. It was shown that incorporation of new segments of PM exceeds the quantity of membrane required for the extension, so the excess of PM must be removed by endocytosis. Recent studies showed internalization of subapical PM domains that were mainly recycled to exocytosis through the Golgi apparatus and a second mainly degradative pathway. PM internalization and movements of the endomembrane system during the pollen tube growth depends on the concerted action and integrity of the cytoskeleton. The use of low concentration of Latrunculin B showed that both actin-dependent and actin-indepedent endocytosis work in tobacco pollen tube, internalizing tracts of PM destined to recycling and to degradation and evidenced the presence of distinct degradation pathways. Intriguingly, although most studies concentrate on exocytosis and distension in the tip region, this paper demonstrate that an actin-dependent, still uncharacterized secretory activity occurs in the flanks of tobacco pollen tubes. On the other hand, perturbation of microtubules by using Nocodazole showed that they are not involved in PM internalization. Colocalization experiments using dyes for Golgi, acidic compartments and FM4-64 in vivo assays suggested that Microtubule play a role in the transport of endosomes internalized in the tip region. These data have been also confirmed by ultrastructural observations of negatively charged naogold, whose transport to vacuoles appeared to be retarded in the presence of nocodazole
Pendrin overexpression affects cell volume recovery, intracellular pH and chloride concentration after hypotonicity-induced cell swelling
No full textThe pendrin (SLC26A4 or PDS) gene is responsible,
when mutated, for the Pendred syndrome, a recessive
disorder characterized by sensorineural hearing
loss often accompanied by thyroid dysfunctions.
Pendrin protein is an anion exchanger and
we focused on a still unexplored function that it
might play in view of its importance in the inner ear:
Cl- fluxes regulation during cellular volume control.
We challenged HEK-293 Phoenix cells
over-expressing wild type pendrin (PDS HEK cells)
together with the EYFP (Enhanced Yellow Fluorescent
Protein) or over-expressing the EYFP alone (control
HEK cells) with hypo-osmolar solutions. Taking
advantage of the confocal optical sectioning
we measured the cell volume. In addition, we
determined the intracellular pH and chloride
concentration with fluorescent probes (EYFP and
seminaphthorhodafluor-5F, SNARF-5F). Consequently,
we could estimate simultaneously Clfluxes,
cellular volume and intracellular pH
variations. Cl- movements markedly differed
between PDS and control HEK cells upon hypotonic
shock and are accompanied by an attenuation of
the swelling induced pH drop in PDS HEK cells. The
contemporary measurements of the three variables
not yet reported in living cells, allowed to assess a
possible influence of pendrin upregulation in volume
homeostasis and evidenced its participation to
Cl- fluxes
A megalin-like receptor is involved in protein endocytosis by Bombyx mori midgut cells in culture
No full textProtein absorption by the insect midgut currently attracts increasing research efforts, in part fostered by the need to develop efficient strategies for oral delivery of bioinsecticides targeting haemocoelic receptors. Gut absorption of undegraded proteins has been unequivocally demonstrated in a number of insect species in vivo but only recently the mechanism involved has been clarified: we have proved that the isolated midgut of Bombyx mori larvae performs the transepithelial translocation of the model protein albumin by transcytosis.
Single columnar cells in culture, isolated from the larval midgut, represent the best tool to identify the mechanism involved in protein endocytosis, the fist step of the transcytotic process. Mature columnar cells were obtained from the proliferation and differentiation of stem cells detached from B. mori midgut epithelium and maintained in culture. We investigated FITC-albumin internalisation by confocal laser scanning microscopy. The protein uptake was time-dependent and strongly reduced by low temperatures and by metabolic inhibitors. Labelled albumin uptake as a function of increasing protein concentration showed a saturation kinetics and was inhibited by native albumin in a concentration-dependent manner. These data prove that albumin uptake is an active process and indicate that a receptor mediates the internalisation of the protein. The internalisation takes place by clathrin-mediated endocytosis, since two specific inhibitors of this process caused a significant reduction of the uptake, and clathrin and albumin colocalised in the intermicrovillar areas of the apical plasma membrane. The integrity of the cytoskeletal organisation is essential for the correct functioning of the endocytic machinery because preincubations with nocodazole or cytochalasin D induced a significant reduction of FITC-albumin uptake. RT-PCR analysis and colocalisation experiments indicated that the receptor involved is a putative homologue of megalin, the multiligand endocytic receptor belonging to the low-density lipoprotein (LDL)-receptor family, responsible for the uptake of a variety of molecules, albumin included, in many mammalian epithelial cells. Although the specific role of the putative megalin homologue in protein absorption by the midgut of lepidopteran larvae needs to be further clarified, our study indicates that this receptor is highly conserved during evolution
Inhibition of actin polymerisation by low concentration Latrunculin B affects endocytosis and alters exocytosis in shank and tip of tobacco pollen tubes
No full textPollen tube growth depends on the integrity of the actin cytoskeleton that regulates cytoplasmic streaming and secretion. To clarify whether actin also plays a role in pollen tube endocytosis, Latrunculin B (LatB) was employed in internalisation experiments with tobacco pollen tubes, using the lipophilic dye FM4-64 and charged nanogold. Time-lapse analysis and dissection of endocytosis allowed us to identify internalisation pathways with different sensitivity to LatB. Co-localisation experiments and ultrastructural observations using positively charged nanogold revealed that LatB significantly inhibited endocytosis in the pollen tube shank, affecting internalisation of the plasma membrane (PM) recycled for secretion, as well as that conveyed to vacuoles. In contrast, endocytosis of negatively charged nanogold in the tip, which is also conveyed to vacuoles, was not influenced. Experiments of fluorescence recovery after photobleaching (FRAP) of the apical and subapical PM revealed domains with different rates of fluorescence recovery and showed that these differences depend on the actin cytoskeleton integrity. These results show the presence of distinct degradation pathways by demonstrating that actin-dependent and actin-indepedent endocytosis both operate in pollen tubes, internalising tracts of PM to be recycled and broken down. Intriguingly, although most studies concentrate on exocytosis and distension in the apex, the present paper shows that uncharacterised, actin-dependent secretory activity occurs in the shank of pollen tubes
A megalin-like receptor is involved in protein endocytosis in the midgut of an insect (Bombyx mori, Lepidoptera)
No full textThe mechanism responsible for fluorescein isothiocyanate (FITC)-albumin internalization
by columnar cells in culture obtained from the midgut of Bombyx mori larvae was examined by confocal laser scanning microscopy. Protein uptake changed over time, and it appeared to be energy dependent,
since it was strongly reduced by both low temperatures and metabolic inhibitors. Labeled albumin uptake as a function of increasing protein concentration showed a saturation kinetics with a Michaelis constant
value of 2.0 +/- 0.6 microM. These data are compatible with the occurrence of receptor-mediated endocytosis. RT-PCR analysis and colocalization experiments with an anti-megalin primary antibody indicated that
the receptor involved was a putative homolog of megalin, the multiligand endocytic receptor belonging to the low-density lipoprotein receptor family, responsible for the uptake of various molecules,albumin included, in many epithelial cells of mammals. This insect receptor, like the mammalian counterpart, required Ca2+ for albumin internalization and was inhibited by gentamicin. FITC-albumin internalization
was clathrin mediated, since two inhibitors of this process caused a significant reduction of the uptake, and clathrin and albumin colocalized in the intermicrovillar areas of the apical plasma membrane.
The integrity of actin and microtubule organization was essential for the correct functioning of the endocytic machinery
Effects of the microtubulo inhibitor Nocodazole on endocytosis in pollen tubes of Nicotianan tabacum
No full textThe pollen tube is a highly polarized cell, it was generally accepted that secretory vesicles (SVs) are transported by the acto-myosin system to the tip region where they are accumulated in the clear zone. Recently, use of lypophilic dyes and analysis of vesicle dynamic suggest a new model of growth that the apical PM is not only the site of exocytosis but the endocytic vesicles significantly contribute to the V-shaped vesicle accumulation in addition to SVs.
In this study we disturb the system of Mts using low concentration of Nocodazole, to verify modifications in endocytosis and endosome trafficking.
Our results demonstrated that the perturbation of Mts had not influence on the distribution of the Afs and in the internalization of PM both in subapical and in the apical regions. Colocalization experiments using dyes for Golgi, acidic compartments and FM4-64 in vivo assays revealed that Mts play a role in the transport of endosomes internalized in the tip region. These data have been also confirmed by ultrastructural observations of negatively charged naogold, whose transport to vacuoles appeared to be retarded with nocodazol
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