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Results for “Cell Surface Extensions”

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PubMed2004

Ultrastructural study of echinocytes induced by poly (ethylene glycol)-cholesterol.

Poly (ethylene glycol)-cholesterol (PEG-Chol) consists of a hydrophilic PEG and hydrophobic cholesterol moiety. When PEG-Chol was applied to erythrocytes, the reagent quantitatively induced protrusions by exclusively distributing in the outer monolayer of the membrane. This kind of response has been regarded as a general response that reduces the stress of expansion of the outer monolayer. However, the relationship between the membrane architecture and the distribution of such molecules is unknown. In this study, we examined the distribution of tagged PEG-Chol along the shape change pathway. The echinocytic shape was initiated by the initial formation of bumps on the rim of the discoid, which subsequently elongated as protrusions. These protrusions contained aggregates of granular structures, which appeared to accommodate the increase in the outer monolayer area. At higher concentrations, PEG-Chol further induced sphero-echinocytosis that resulted in numerous branched protrusion processes. We found that PEG-Chol was exclusively distributed in these protrusions and, in particular, accumulated at the tips. These results suggested that externally intercalated PEG-Chol was sequestrated from erythrocytes as membrane protrusions through an as-yet-unknown mechanism.

Cell Surface Extensions↗

PubMed2002

Elucidation of the functions of genes central to diterpene metabolism in tobacco trichomes using posttranscriptional gene silencing.

The functions of two key, trichome-expressed genes were assessed using different posttranscriptional gene silencing strategies (PTGS). Efficient RNA interference (RNAi) revealed the function of a cembratriene-ol (CBT-ol) cyclase gene responsible for conversion of geranylgeranyl pyrophosphate to CBT-ols, and verified the function of a P450 gene responsible for conversion of CBT-ols to CBT-diols. CBT-diols are abundant diterpenes that comprise about 60% and 10% of trichome exudate weight and leaf dry weight, respectively, in Nicotiana tabacum, T.I. 1068. The relative efficiencies and levels of suppression using antisense (AS), sense co-suppression (S), and RNAi were compared for these two genes. With a partial cDNA of the P450 gene, the suppression efficiencies (percent of primary transformants with high CBT-ols/CBT-diols) were low, 3.3% for AS and 0% for S plants. In contrast, using RNAi with a partial gene sequence, a knockdown efficiency of about 45% was achieved. For the CBT-ol cyclase gene, no suppression was observed using partial cDNAs in AS or S orientations, while RNAi with a partial gene sequence yielded an efficiency of about 64%. The efficiencies of gene silencing using full-length coding regions of both genes in AS and S orientations were </=20%. Our results identify the function of a CBT-ol cyclase gene and demonstrate the efficacy and superiority of RNAi for assessing the functions of two trichome-specific genes that encode enzymes having widely different functions.

Cell Surface Extensions↗

PubMed2002

Gradual shifts in sites of free-auxin production during leaf-primordium development and their role in vascular differentiation and leaf morphogenesis in Arabidopsis.

The major regulatory shoot signal is auxin, whose synthesis in young leaves has been a mystery. To test the leaf-venation hypothesis [R. Aloni (2001) J Plant Growth Regul 20: 22-34], the patterns of free-auxin production, movement and accumulation in developing leaf primordia of DR5::GUS-transformed Arabidopsis thaliana (L.) Heynh. were visualized. DR5::GUS expression was regarded to reflect sites of free auxin, while immunolocalization with specific monoclonal antibodies indicated total auxin distribution. The mRNA expression of key enzymes involved in the synthesis, conjugate hydrolysis, accumulation and basipetal transport of auxin, namely indole-3-glycerol-phosphate-synthase, nitrilase, IAA-amino acid hydrolase, chalcone synthase and PIN1 as an essential component of the basipetal IAA carrier, was investigated by reverse transcription-polymerase chain reaction. Near the shoot apex, stipules were the earliest sites of high free-auxin production. During early stages of primordium development, leaf apical dominance was evident from strong beta-glucuronidase activity in the elongating tip, possibly suppressing the production of free auxin in the leaf tissues below it. Hydathodes, which develop in the tip and later in the lobes, were apparently primary sites of high free-auxin production, the latter supported by auxin-conjugate hydrolysis, auxin retention by the chalcone synthase-dependent action of flavonoids and also by the PIN1-component of the carrier-mediated basipetal transport. Trichomes and mesophyll cells were secondary sites of free-auxin production. During primordium development there are gradual shifts in sites and concentrations of free-auxin production occurring first in the tip of a leaf primordium, then progressing basipetally along the margins, and finally appearing also in the central regions of the lamina. This developmental pattern of free-auxin production is suggested to control the basipetal maturation sequence of leaf development and vascular differentiation in Arabidopsis leaves.

Acyltransferases↗

PubMed2003

The distributional changes and role of microtubules in Nod factor-challenged Medicago sativa root hairs.

The normal tip-growing pattern exhibited by root hairs of legumes is disrupted when the hair is exposed to Nod factors generated by compatible bacteria capable of inducing nodule formation. Since microtubules (MTs) play an important role in regulating directionality and stability of apical growth in root hairs [T.N. Bibikova et al. (1999) Plant J 17:657-665], we examined the possibility that Nod factors might affect the MT distribution patterns in root hairs of Medicago sativa L. We observed that Nod factor application caused rapid changes in the pattern of MTs starting as early as 3 min after perfusion. Within 3 to 10 min after Nod factor application, first endoplasmic and then cortical MTs depolymerised, initially at the proximal ends of cells. Twenty minutes after exposure to Nod factors, a transverse band of microtubules was seen behind the tip, while almost all other MTs had depolymerised. By 30 min, very few MTs remained in the root hair and yet by 1 h the MT cytoskeleton re-formed. When Nod factors were applied in the presence of 10 microM oryzalin or 5 microM taxol, the MTs appeared disintegrated while the morphological effects, such as bulging and branching, became enhanced. Compared to the treatments with oryzalin or taxol alone, the combinatory treatments exhibited higher growth rates. Since microtubule reorganization is one of the earliest measurable events following Nod factor application we conclude that microtubules have an important role in the early phases of the signalling cascade. Microtubule involvement could be direct or a consequence of Nod factor-induced changes in ion levels.

Cell Polarity↗

PubMed2001

Detoxification of cadmium in tobacco plants: formation and active excretion of crystals containing cadmium and calcium through trichomes.

In tobacco (Nicotiana tabacum L.), long and short trichomes can be distinguished morphologically. The established function of long trichomes is to exude a sticky gum containing diterpenes, whereas that of short trichomes is not known. When tobacco seedlings were exposed to toxic levels of cadmium (Cd), growth was retarded, but trichome number was increased up to 2-fold in comparison with untreated samples. Observation by variable-pressure scanning electron microscopy (VP-SEM) indicated that large crystals of 150 microm in size were formed on head cells of both short and long trichomes. An energy-dispersive X-ray analysis system fitted with VP-SEM revealed the crystals to contain amounts of Cd and calcium (Ca) at much higher concentrations than in the head cells themselves. Transmission electron microscopy demonstrated crystal formation in amorphous osmiophilic deposits in vacuoles. When seedlings were treated with Cd in the presence of Ca, tolerance was increased in proportion to the increase in Ca concentration. These results indicate that tobacco plants actively exclude toxic Cd by forming and excreting Cd/Ca-containing crystals through the head cells of trichomes.

Biotransformation↗

PubMed2004

Projections and chemistry of Dogiel type II neurons in the mouse colon.

The physiological properties, shapes, projections and neurochemistries of Dogiel type II neurons have been thoroughly investigated in the guinea-pig intestine in which these neurons have been identified as intrinsic primary afferent neurons. Dogiel type II neurons in the myenteric ganglia of mice have similar physiological properties to those in guinea-pigs but whether other features of the neurons are similar is unknown. We have used intracellular dye-filling, retrograde tracing, immunohistochemistry and nerve lesions to determine salient features of Dogiel type II neurons of the mouse colon. Dye-filling showed that the neurons provide profuse terminal networks in the myenteric ganglia and also have axons that project towards the mucosa. Retrograde tracing and lesion studies showed that these axons provide direct innervation to the mucosa. High proportions of the neurons had immunoreactivity for calretinin, calbindin, choline acetyltransferase, the purine P2X2 receptor and calcitonin gene-related peptide (CGRP). CGRP was the most selective marker of the neurons. Following surgery to remove an area of myenteric plexus, the CGRP-immunoreactive nerve fibres in the mucosa degenerated. Thus, Dogiel type II neurons in mice have similar shapes and projections but some differences in chemistry from those in guinea-pigs. The close similarities between the two species in the shapes, projections and electrophysiology of these neurons suggest that they serve the same functions in both species.

Animals↗

PubMed2006

Morphological analysis of lamellar structures in mouse type II pneumocytes by quick-freezing and freeze-drying with osmium tetroxide vapor-fixation.

The lamellar body is a membranous structure periodically laminating in vesicles that is known as the most distinctive feature of type II pneumocytes by conventional preparation methods for transmission electron microscopy. The quick-freezing and freeze-drying method, followed by osmium tetroxide vapor-fixation (QF-FD-OsV), was performed to examine the in situ morphology of the lamellar body in type II pneumocytes of living mouse lungs. Typical lamellar structures were rarely seen in vesicles of the type II pneumocytes, but amorphous components and dispersed stripes were often detected in the vesicles, as revealed by the QF-FD-OsV method. To clarify how the lamellar body was formed during the conventional preparation steps, lung tissues of mice were treated with different fixation procedures, such as immersion-fixation with osmium tetroxide or perfusion-fixation with glutaraldehyde followed by osmium tetroxide, in combination with alcohol dehydration or QF-FD-OsV. In addition to lamellar bodies of type II pneumocytes in the specimens with alcohol dehydration, some lamellar structures were also formed even with the QF-FD-OsV method. These findings suggest that the labile lamellar body is easily modified and formed during both chemical fixation and alcohol dehydration steps.

Animals↗

PubMed2002

Epidermal and hypodermal characteristics in North American Cactoideae (Cactaceae).

Dermal and hypodermal anatomical features of 70 species representing 21 genera of North American Cactoideae were studied. Results show that all species examined have parallelocytic stomata and anticlinal wall surface varies from straight to undulate. Cuticle thickness is mostly narrow (1-10 microm) contrary to the general opinion that cuticle is thick in most cacti; however, few species such as Ariocarpus fissuratus and several species of Pachycereus show a distinctive thick cuticle. More than 80% of the species studied have a single-layered epidermis. Papillae occur in eight species belonging to four genera. Notable papillae are a feature shared by all members of Peniocereus subg. Peniocereus. Other species show a bullate surface produced by irregular patches of secondary epidermal cell divisions. Commonly, the hypodermis is composed of more than two cell layers with distinctive collenchymatous walls as reported in many South American species. Silica bodies, prismatic crystals, druses, sphaerocrystals, and tannins are the most common cellular inclusions that distinguish several genera and appear to have taxonomic value. However, a more thorough search in species of Cephalocereus, Coryphantha, Echinocereus, Mammillaria, Neobuxbaumia, Pilosocereus, and Turbinicarpus is needed to support the previous assertion.

Cactaceae↗

PubMed2006

"HAIRY CANOLA"--Arabidopsis GL3 induces a dense covering of trichomes on Brassica napus seedlings.

Transformation with the Arabidopsis bHLH gene 35S:GLABRA3 (GL3) produced novel B. napus plants with an extremely dense coverage of trichomes on seedling tissues (stems and young leaves). In contrast, trichomes were strongly induced in seedling stems and moderately induced in leaves of a hairy, purple phenotype transformed with a 2.2 kb allele of the maize anthocyanin regulator LEAF COLOUR (Lc), but only weakly induced by BOOSTER (B-Peru), the maize Lc 2.4 kb allele, or the Arabidopsis trichome MYB gene GLABRA1 (GL1). B. napus plants containing only the GL3 transgene had a greater proportion of trichomes on the adaxial leaf surface, whereas all other plant types had a greater proportion on the abaxial surface. Progeny of crosses between GL3+ and GL1+ plants resulted in trichome densities intermediate between a single-insertion GL3+ plant and a double-insertion GL3+ plant. None of the transformations stimulated trichomes on Brassica cotyledons or on non-seedling tissues. A small portion of bHLH gene-induced trichomes had a swollen terminal structure. The results suggest that trichome development in B. napus may be regulated differently from Arabidopsis. They also imply that insertion of GL3 into Brassica species under a tissue-specific promoter has strong potential for developing insect-resistant crop plants.

Amino Acid Sequence↗

PubMed1986

Role of mucus in the repair of gastric epithelial damage in the rat. Inhibition of epithelial recovery by mucolytic agents.

A role for mucus in providing a microenvironment over sites of gastric damage, which is conducive to reepithelialization, has been proposed. We tested this hypothesis by examining the effects of disruption of such mucus on the recovery of epithelial integrity after damage induced by 50% ethanol. Exposure of an ex vivo chambered gastric mucosa to topically applied 50% ethanol resulted in copious release of mucus, cellular debris, and plasma, which formed a continuous cap over the mucosal surface. Ethanol-induced gastric damage was accompanied by extensive surface epithelial cell damage and a marked decrease in transmucosal potential difference. During the 30 min after ethanol was removed from the chamber, the epithelium became reestablished and the potential difference gradually recovered to 94% of the level before ethanol treatment. However, if the mucolytic agents N-acetylcysteine (5%) or pepsin (0.5%) were added to the bathing solutions, the "mucoid cap" disintegrated and the recovery of potential difference was significantly retarded (recovering to only 51% and 52% of levels before ethanol treatment). Histologic evaluation confirmed that mucosae treated with either agent had significantly less (p less than 0.005) intact epithelium at the end of the experiment. Removal of the mucoid cap with forceps caused a similar inhibition of the repair of the epithelium and the recovery of potential difference. Both mechanical and chemical (N-acetylcysteine) disruption of the mucoid cap resulted in a significant increase in the mucosal leakage of albumin and hemoglobin, supporting previous histologic evidence that the mucoid cap traps blood components over the damaged mucosa. These studies support the hypothesis that mucus released in response to topical application of an irritant plays an important role in the repair of epithelial damage through the process of restitution.

Acetylcysteine↗

PubMed2004

A requirement of MAPKAPK2 in the uropod localization of PTEN during FMLP-induced neutrophil chemotaxis.

The directionality control in chemotaxis is the result of a reciprocal regulation of PI3-kinase and PTEN subcellular localization. MK2(-/-) neutrophils have a directionality loss in fMLP-induced chemotaxis. We found that in polarized WT neutrophils PTEN was localized in the uropod region. However, MK2(-/-) neutrophils or p38 MAPK inhibitor-SB203580-pretreated WT neutrophils showed a disrupted PTEN subcellular localization. Some PTEN was localized at the leading edge of the polarized neutrophils, which may lower the concentration of PI3-kinase lipid product PtdIns(3,4,5)P3 required for directionality sensing. FMLP-stimulated MK2(-/-) neutrophils or SB203580-pretreated WT neutrophils also had disrupted F-actin polarization. F-actin polymerization inhibitor lantrunculin-B disrupted the polarization of PTEN, but not PtdIns(3,4,5)P3. The results suggest that PTEN uropod polarization is F-actin polymerization-dependent and may be through the effect of MK2 on F-actin polarization.

Actins↗

PubMed2006

Phosphorylation of serine 709 in GIT1 regulates protrusive activity in cells.

G protein-coupled receptor kinase-interacting protein (GIT)1 is a multidomain, adaptor protein that regulates cellular processes, such as migration and protrusive activity, by bringing together various signaling molecules, including PIX, PAK, and paxillin. Mutants of GIT1, which lack the C-terminal paxillin binding domain, fail to mediate its effects on migration and protrusions, suggesting that sites within this domain are critical to GIT1 function. In this study, we show that serine 709, which is located within the paxillin binding domain, regulates GIT1 function. Phosphorylation of serine 709 is necessary for GIT1-induced effects on protrusions. Phosphorylation of this site also regulates GIT1 interaction with paxillin, which could serve to target GIT1 to the leading edge of cells. As shown by an in vitro kinase assay, PAK phosphorylates GIT1 on serine 709. Taken together, our results indicate that GIT1 phosphorylation on serine 709 increases its binding to paxillin and regulates protrusive activity in cells.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Adhesions that mediate invasion.

Infiltration of new tissue areas requires that a mammalian cell overcomes the physical and biochemical barrier of the surrounding extracellular matrix. Cell migration during embryonic development, and growth, invasion and dispersal of metastatic tumor cells depend to a large extent on the controlled degradation of extracellular matrix components. Localized degradation of the surrounding matrix is seen at defined adhesive (podosomes) and/or protrusive (invadopodia) locations in a variety of normal cells and aggressive carcinoma cells, suggesting that these membrane-associated cellular devices have a central role in mediating polarized migration in cells that cross-tissue boundaries. Here, we will discuss the recent advances and developments in this field, and provide our provisional outlook into the future understanding of the principles of focal extracellular matrix degradation by podosomes and invadopodia.

Animals↗

PubMed2004

Spherocyte shape transformation and release of tubular nanovesicles in human erythrocytes.

We have studied dodecylmaltoside-induced echinocyte-spheroechincyte-spherocyte shape transformation and membrane vesiculation using transmission electron microscopy (TEM) on freeze-fracture replicas. It is indicated that spherical erythrocyte shape at higher dodecylmaltoside concentration is formed due to loss of membrane in the process where small, mostly tubular nanovesicles are released predominantly from the top of echinocyte and spheroechinocyte spicules.

Cell Shape↗

PubMed2004

Protein synthesis is necessary for dendritic spine proliferation in adult brain slices.

Dendritic spines, small protrusions from dendritic shafts, receive most of the excitatory synapses in cortical regions. Spines are highly plastic structures that can be rapidly produced or lost in response to a wide array of internal and external stimuli, and they proliferate in acute slice preparations [J. Neurosci. 19 (1999) 2876]. The goal of the present study was to determine if protein synthesis is necessary for this spine proliferation. We found that the addition of protein synthesis inhibitors to acute slices (in which spines otherwise proliferate) blocked new spine growth. Furthermore, a population of longer spines was observed after 2 h but these did not develop during protein synthesis blockade. These data suggest that protein synthesis is necessary for new spine growth in acute brain slice preparations and support literature suggesting that newly produced spines develop from filopodia-like protrusions.

Analysis of Variance↗

PubMed2004

Ultrastructure of the rostral ventral respiratory group neurons in the ventrolateral medulla of the rat.

The neurons in the ventrolateral medulla that project to the spinal cord are called the rostral ventral respiratory group (rVRG) because they activate spinal respiratory motor neurons. We retrogradely labeled rVRG neurons with Fluoro-Gold (FG) injections into the fourth cervical spinal cord segment to determine their distribution. The rostral half of the rVRG was located in the area ventral to the semicompact formation of the nucleus ambiguus (AmS). A cluster of the neurons moved dorsally and intermingled with the palatopharyngeal motor neurons at the caudal end of the AmS. The caudal half of the rVRG was located in the area including the loose formation of the nucleus ambiguus caudal to the AmS. We also labeled the rVRG neurons retrogradely with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) to determine their ultrastructural characteristics. The neurons of the rVRG were medium to large (38.1 x 22.1 microm), oval or ellipsoid in shape, and had a dark cytoplasm containing numerous free ribosomes, rough endoplasmic reticulum (rER), mitochondria, Golgi apparatuses, lipofuscin granules and a round nucleus with an invaginated nuclear membrane. The average number of axosomatic terminals in a profile was 33.2. The number of axosomatic terminals containing round vesicles and making asymmetric synaptic contacts (Gray's type I) was almost equal to those containing pleomorphic vesicles and making symmetric synaptic contacts (Gray's type II). The axodendritic terminals were large (1.55 microm), and about 60% of them were Gray's type I. The rVRG neurons have ultrastructural characteristics, which are different from the palatopharyngeal motor neurons or the prorpiobulbar neurons.

Animals↗

PubMed2005

Blockade of chloride conductance antagonizes PMA-induced ramification in the murine microglial cell line, BV-2.

In microglial cells, activation of ion channels and ion transporters is associated with the transformation from an amoeboid to a ramified phenotype and vice versa. In the present study, we evaluated the contributions of protein kinase C (PKC) activity and ion conductance to the phorbol 12-myristate 13-acetate (PMA)-dependent ramification in the murine microglial cell line, BV-2. In a first set of experiments, we showed that PMA, a commonly used activator of PKC, but not the bioinactive analog 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), induces ramification in BV-2 cells. Surprisingly, the PKC inhibitors calphostin C, chelerythrine, or bisindolylmaleimide II did not antagonize PMA-induced ramification. In a further set of experiments, we found that 4,4'-diisocyanatostilbene-2,2' disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), which block chloride channels and K-Cl cotransporters, and SKF 96365, a non-selective ion channel blocker, consistently suppressed PMA-induced ramification in BV-2 cells. Additional ion channel blockers, including lanthanides, amiloride, Ba2+, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), and flufenamic acid did not affect PMA-induced ramification in BV-2 cells. Cs+ accentuated the PMA-dependent ramification in BV-2 cells. Thus, our results indicate (1) that a PMA-binding protein, excluding PKC isoforms, is critical in structural remodeling of microglial cells and (2) that chloride conductance plays a pivotal role in induction of ramification in microglial cells.

Animals↗

PubMed2004

The WASP-Arp2/3 pathway: genetic insights.

Arp2/3 complex nucleates the formation of dendritic actin filament arrays, which are especially prominent at the leading edges of motile cells. Recent genetic and other loss-of-function studies have highlighted the importance of the Arp2/3 complex for normal cell functions, and especially for cell motility. WASP/Scar family proteins regulate the activity of the Arp2/3 complex, and also link it to several signaling pathways. Recent studies suggest that Scar is a more important regulator of Arp2/3 activity in actin-dependent morphological processes than WASP, which may have a more restricted role in specialized cellular events. It has also become clear that precise regulation of both Scar and WASP activity is of the utmost importance for their physiological functions.

Actin Cytoskeleton↗