Nitric oxide modulates specific steps of auxin-induced adventitious rooting in sunflower

Present work on indole-3-acetic acid (IAA)-induced adventitious rooting in sunflower hypocotyl highlights a clear demarcation of nitric oxide (NO)-dependent and NO-independent roles of auxin in this developmental process. Of the three phases of adventitious rooting, induction is strictly auxin-dependent though initiation and extension are regulated by an interaction of IAA with NO. A vital role of auxin-efflux transporters (PIN) is also evident from 1-napthylphthalamic acid (NPA)-triggered suppression of adventitious roots (AR). Use of actin depolymerizing agent, latrunculin B (Lat B), has demonstrated the necessity of functional actin filaments in auxin-induced AR response, possibly through its effect on actin-mediated recycling of auxin transporter proteins. Thus, evidence for a linkage between IAA, NO and actin during AR formation has been established.Key words: adventitious roots, auxin, sunflowerAdventitious roots (AR) are post-embryonic roots known to originate from stem, leaf petiole and non-pericycle tissue of old roots. In young stem, AR commonly arise from the interfascicular parenchyma while they appear from vascular rays near the cambium in older stem. Formation of AR begins with re-differentiation of predetermined cells which switch from their morphogenetic path to act as mother cells for initiation of root primordia.¹ The process of AR formation consists of three physiologically interdependent phases: induction, initiation and extension.¹ Induction phase comprises of various molecular and biochemical events but no morphologically visible changes appear during this phase. Formation of multilayered cells and conception of root primordia occurs during initiation phase. During expression phase, root primordia exhibit intra-stem growth and their emergence through epidermis. Various environmental and endogenous factors, such as temperature, light, hormones (particularly auxin), sugars and mineral salts, act as cues for promoting redifferentiation of predetermined cells resulting in root induction.The three phases of AR formation are known to be regulated by alterations in the endogenous level of auxin.² A transient increase in auxin concentration has been reported during induction phase, which is followed by a decrease and again an increase during expression phase.³ Auxin transport to and from the responding region is essential for root organogenesis. Acropetal transport of auxin occurs through the vascular cylinder and the basipetal transport takes place through the epidermal and subtending cortical cells.⁴ Polar transport of auxin from the shoot apical meristem to the rooting region is primarily facilitated by auxin-influx (AUX1) and -efflux (PIN) transporters. Asymmetric localization of these transporter proteins in the vascular cambium cells is responsible for differential distribution of auxin in a particular zone of cells.⁵ Polar auxin transport is known to be inhibited by 1-napthylphthalamic acid (NPA, a phytotropin). This inhibition is mediated through a binding of NPA molecule to putative NPA-binding protein (NBP), which is functionally associated with PIN proteins.⁶ Efflux transporters exhibit rapid turnover in plasma membrane.⁷ High affinity of NBP for actin filaments,^8,9 suggests its involvement in the cycling and polar distribution of PIN proteins.¹⁰ Organization of actin filaments is known to be rapidly, reversibly and specifically disrupted by Latrunculin B (Lat B), a macrolide toxin isolated from Latrunculia magnificia, a red sea sponge.¹¹ Lat B associates with actin monomers in 1:1 ratio, thereby preventing their repolymerization into filaments, resulting in a complete shift from F-actin to G-actin.¹² Owing to its well-understood and simple mode of action and low effective dosage, Lat B has supplanted the classic actin-depolymerizing drug cytochalasin D¹³ in pharmacological investigations. In the past few years, significant work has been done on NO as a signaling molecule in a variety of plant developmental processes.¹⁴ Nitric oxide is known to play a crucial role in root development.¹⁵Using sunflower as a model system, present work has been undertaken to investigate the possible role of NO during IAA-induced adventitious rooting in hypocotyl explants. Since auxin action is principally based on PIN-regulated polar transport of IAA molecules, and PIN proteins are known to exhibit actin-asssisted rapid recycling in the target cells, attempts have been made in the present work to find a correlation between auxin transport, actin and NO, using specific pharmacological agents. Additionally, effect of Cyclosporin A (CsA), an inhibitor of nitric oxide synthase (NOS) in animal systems,¹⁶ has also been investigated. These specific agents have been used to monitor root initiation at the target sites in our attempts to decipher a signaling cascade for AR.Seeds of Sunflower (Helianthus annuus L. cv. Morden) were germinated on moist germination sheets at 25 ± 2°C under continuous illumination of 4.3 Wm⁻². Hypocotyls from 4 d old seedlings and with similar growth rate, were excised up to 6 cm below the cotyledonary node. Hypocotyl explants with apical meristem intact but cotyledons excised were selected for the present work with a view to provide a continuity of the endogenous auxin source. Similar explants were also recently used by Huang et al.¹⁷ to investigate indole-3-butyric acid (IBA)-induced AR formation in mung bean (Phaseolus radiatus L.). Using IAA instead of IBA for such investigations was preferred for the present work keeping in view that the two auxins seem to employ different transport proteins for their polar transport.¹⁸ Freshly harvested explants were put upright in glass vials with their proximal cut ends dipped in 1 ml of different concentrations (1, 5, 10 and 15 µM) of IAA, thus bathing the hypocotyls up to 5 mm of their lower ends. Explants were maintained in dark during the course of experiments. The number of AR visible on hypocotyl surface was recorded daily up to 4 days of incubation. Concentration of IAA thus observed optimal for rooting (10 µM) was used for all further experiments. Similarly, various concentrations of other test solutions, namely NPA (auxin efflux blocker; 1 and 10 µM), Lat B (an inducer of actin depolymerization; 25, 50 and 100 nM), CsA (an inhibitor of cyclophilins; 1, 5 and 10 µM), sodium nitroprusside (SNP; NO donor; 1, 5, 10 and 100 µM) and 2-phenyl-4,4,5,5-tetramethyllimidazoline-1-oxyl-3-oxide (PTIO; NO scavenger; 1 and 1.5 mM), were initially used to select their respective optimal concentrations. Based on these preliminary experiments NPA, Lat B, CsA, SNP and PTIO were used at 10 µM, 100 nM, 10 µM, 100 µM and 1.5 mM, respectively, for all subsequent experiments. Some other treatment combinations, namely NPA (10 µM) + IAA (10 µM); LatB (100 nM) + IAA (10 µM); CsA (10 µM) + IAA (10 µM); SNP (100 µM) + NPA (10 µM) and PTIO (1.5 mM) + IAA (10 µM) were also used to investigate their effects on adventitious rooting. Hypocotyl explants incubated in distilled water served as control. Morphological observations of rooting response were imaged after 7 days of incubation, using Nikon digital camera fitted on a stereomicroscope (Stemi 2000, Zeiss, Germany). Detailed evaluation of root initiation was observed after clearing by immersing the explants in a 3:1 solution of ethanol: acetic acid overnight. They were then transferred to 2 N NaOH solution, left overnight, washed once with distilled water and stained with safranin solution for 2–3 min. Excess stain was removed by repeated washing in distilled water. The lower 2 cm region of hypocotyl explants was then cut and mounted on a glass slide to examine endogenous root initials, using a stereomicroscope (Stemi 2000, Zeiss, Germany) fitted with a Nikon camera.Root initiation and extension in the basal region of hypocotyl explants maintained in distilled water indicates the expected basipetal transport of the inducing factor (endogenous IAA) from the intact meristem, as also reported earlier.¹⁹ Treatment with IAA (10 µM) elicited two effects on hypocotyl explants in comparison to those subjected to distilled water treatment: (1) Formation of greater number of root initials, (2) Greater extension of the initiated roots (Figs. 1 and ​and22). A response similar to that evoked by IAA is also evident in hypocotyl explants treated with 100 µM of SNP (Figs. 1 and ​and22). Recently SNP (NO donor) has been reported to evoke dose-dependent response on AR formation in marigold.²⁰ In the present work, treatment with variable concentrations of SNP, ranging from 1–100 µM, lead to a gradual increase in the number and extension growth of AR till 100 µM. Pagnussat et al.²¹ and Liao et al.²⁰ have used 10 µM and 50 µM as effective SNP concentration in cucumber and marigold, respectively. Thus, optimal concentration of SNP for AR formation is species-dependent. In presence of PTIO (1.5 mM; a specific NO scavenger), complete suppression of AR was evident in sunflower, as also reported earlier in mung bean.¹⁷ Combination of PTIO with IAA lead to root initiation only (no extension growth). NPA (10 µM) blocked AR initiation by endogenous (distilled water treatment) and exogenous IAA (Fig. 1). Application of NPA inhibits polar auxin transport, thus reducing the optimal concentration of IAA required for AR formation at the hypocotyl base (zone of AR formation). Thus, no evidence of root initials was evident in presence of NPA, which is also reported in cucumber²² and loblolly pine,²³ respectively. Though NO is expected to act downstream of IAA²⁴ but a treatment of SNP in combination with NPA (present work) lead to complete suppression of AR formation. Our unpublished observations have indicated the expression of NO in the interfascicular cells after induction phase (i.e., during AR iniation and extension). Thus, it can be proposed that IAA is involved in induction phase of adventitious rooting independent of NO, while initiation and extension phases appear to involve IAA-NO interaction. CsA-cyclophilin complex is known to inhibit calcineurin (a protein phosphatase) and NOS activity in animal systems.²⁵ Treatment of hypocotyl explants with CsA (10 µM) lead to formation of fewer number of roots which exhibited extension growth. Oh et al.²⁶ reported a significant reduction in the number of roots in the presence of CsA in hypocotyl explants from tomato. Subjecting hypocotyl explants with a combination of CsA and IAA lead to formation of fewer number of root initials, reaffirming the involvement of NO in auxin action in the developmental process under investigation (AR). However, further investigations on the role of cyclophilins and NOS in auxin-modulated AR formation are required to pinpoint their specific sites of action in this developmental process. Treatment with Lat B (+ and − IAA) lead to complete AR suppression in sunflower hypocotyl explants. Actin-mediated polar localization of PIN proteins is responsible for polar auxin transport and disruption of microfilaments by Lat-B would thus, directly affect IAA transport leading to the observed AR suppression. These observations indicate a convergence of the effect of IAA with that of NO and a role of a well organized actin in the responding cells.Open in a separate windowFigure 1Effect of IAA and various other pharmacological agents on adventitious rooting in hypocotyl explants. Morphological observations of rooting response (A and C). Evaluation of endogenous root initiation and elongation observed in cleared explants stained with safranin (B and D). Scale bar represents 3 mm.Open in a separate windowFigure 2Quantitative analysis of AR initiation in presence of distilled water, IAA and various pharmacological agents in hypocotyl explants of sunflower. Each datum presents a mean and standard error from at least three observations.To sum up, present investigations provide evidence for a linkage between auxin-induced AR response in seedling hypocotyls and NO (Fig. 3). Both endogenous and exogenous IAA-mediated AR induction seem to depend on actin. Significance of actin in this developmental response has become evident via its role in the cycling of auxin efflux proteins (PIN). The three phases of AR formation can be differentiated from each other in terms of their sensitivities to IAA and NO. AR induction phase seems to be governed by auxin alone, independent of NO. NO seems to become operative in this auxin-modulated response (AR) during initiation and extension phase only. Investigations are being undertaken in the author''s laboratory to visualize and quantitate the NO signal in the IAA-responding hypocotyl explants so that the phasing of the role of NO during AR formation can be precisely predicted.Open in a separate windowFigure 3Schematic presentation of the probable events associated with NO-mediated adventitious rooting.     

Thigmomorphogenesis in Solanum lycopersicum: Morphological and biochemical responses in stem after mechanical stimulation

The activation of the phenylpropanoid pathway in plants by environmental stimuli is one of the most universal biochemical stress responses known. In tomato plant, rubbing applied to a young internode inhibit elongation of the rubbed internode and his neighboring one. These morphological changes were correlated with an increase in lignification enzyme activities, phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidases (POD), 24 hours after rubbing of the forth internode. Furthermore, a decrease in indole-3-acetic acid (IAA) content was detected in the rubbed internode and the upper one. Taken together, our results suggest that decrease in rubbed internode length is a consequence of IAA oxidation, increases in enzyme activities (PAL, CAD and POD), and cell wall rigidification associated with induction of lignification process.Key words: Mechanical stimulation, PAL, CAD, POD, IAAIn their environment, plants are constantly submitted to several stimuli such as wind, rain and wounding. The growth response of plants to such stimuli was termed thigmomorphogenesis and was observed in a wide range of plants.^1–3 The most common thigmomorphogenetic response is a retardation of tissue elongation accompanied by an increase in thickness.⁴ The plant response to mechanical perturbation is mainly restricted to the young developing internode, since no influence can be detected when the internode has reached its final length.^5,6 These plant growth modifications, which characterize thigmomorphogenesis, are related to biochemical events associated with lignification process⁷ and ethylene production.^8,9In tomato plant the length of internodes 4 (N4) and 5 (N5) was measured 14 days after rubbing of the fourth internode. Results reported in Figure 1 show that rubbing led to a significant reduction of elongation of the stressed internode (N4) (decrease of N4 length from 4.3 cm in the control plant to 2.9 in the rubbed one). This effect was not limited to the rubbed area but affected also the elongation of the neighboring internodes (N5) that were shorter in rubbed plants than in control ones.Open in a separate windowFigure 1Internode lengths of control and rubbed plants measured 14 day after mechanical stress applied to the fourth internode. Standard errors are indicated by vertical bars.Results reported in Figure 2 show an increase in PAL activity in both internodes N4 and N5, 24 hours after mechanical stress application as compared with corresponding controls. CAD activity was also investigated in N4 and N5, 24 h after rubbing of the fourth internode. Results presented in Figure 3 show that mechanical stress application induces a strong increase of CAD activity in the rubbed internode N4 (5.3 nkatal μg-1 protein) with an approximately two-fold increase when compared to control tomato internodes (2.3 nkatal μg-1 protein). Further, CAD activity in N5 was also increased in the rubbed internode (5.538 nkatal μg-1 protein) as compared with the control one (3.256 nkatal μg-1 protein).Open in a separate windowFigure 2PAL activity of internode 4, and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.Open in a separate windowFigure 3CAD activity of internode 4, and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.Syringaldazine (S-POD) and gaïacol (G-POD) peroxidase activities were measured in tomato N4 and N5. Results reported in Figure 4 show an increase in soluble peroxidase activity with both substrates in the rubbed internode N4 as compared with control plant. Enhancement in peroxidase activities in N4 was more pronounced with gaïacol (80.7 U) as an electron donor than syringaldazine (33.8 U). Similar results were observed in internode 5 as compared with control one (Fig. 4).Open in a separate windowFigure 4(A) Syringaldazine-POD (Syr-POD) activity of internode 4 and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars. (B) Gaiacol-POD (G-POD) activity of internode 4 and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.IAA was quantified in control and rubbed plant internodes 24 h after rubbing of the fourth internode. Results reported in figure 5 show that in control sample and as expected, the content of IAA was found to be higher in the younger internode (N5) as compared to the older one (N4). Rubbing led to a significant decrease in IAA levels in N4 (5.06 nmol g⁻¹ MF⁻¹) as compared with corresponding controls (7.27 nmol g⁻¹ MF⁻¹). Similar results were observed in internode 5, where IAA content was reduced from 16.52 nmol g⁻¹ MF⁻¹ in control internode to 12.35 nmol g⁻¹ MF⁻¹ in the rubbed internode (Fig. 5).Open in a separate windowFigure 5IAA Level of internode 4 and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.The results reported here establish an evident correlation between growth limitation of the rubbed internode and their degree of lignification, the increase in lignification enzymes activities and auxin degradation after mechanical stress application.Auxin seems to be involved in thigmomorphogenesis.¹⁰ It was proposed that MIS (Mechanically-induced stress) has opposite effects on auxin levels in the two species studied to date, Phaseolus vulgaris¹⁰ and Bryonia dioica.^11,12 Auxin level as measured by bioassay, increased in Phaseolus vulgaris following rubbing of the stem.¹⁰ It was proposed that a build up of auxin may result from the reduced polar transport of IAA at the rubbed internode, causing a build up of IAA in the stem tissue. Exogenous IAA did not reverse the MIS inhibition of growth in Phaseolus vulgaris and high levels of IAA retarded growth in non-stressed plants.¹⁰ Thus, retardation of extension growth in Phaseolus vulgaris may have been caused by high levels of endogenous auxin and the increase in stem diameter by increased ethylene production.⁴ However, ethylene increases radial growth only if auxin is present.¹³Boyer¹¹ reported a decrease in auxinlike activity in Bryonia dioica following MIS and this was confirmed in the same species by Hofinger et al.¹² who reported a decrease in IAA using gas chromatography-mass spectrometry. Auxin catabolism was accompanied with changes in both soluble and ionically bound cell wall basic peroxidases¹⁴ and the appearance of an additional peroxidase. This can suggest that in Bryonia, auxin catabolism is hastened by mechanical stimulated peroxidase. In addition, Boyer et al.¹⁵ reported that lithium pre-treatment prevents both thigmomorphogenesis and appearance of specific cathodic isoperoxidase in Bryonia plants subjected to MIS. This is give further credence to the possibility that the peroxidase-auxin system is involved in Bryonia thigmomorphogenesis. In addition, ethylene increases peroxidase activity which reduces the auxin content in the tissue to a level low enough not to support normal growth. We have evidence that decrease of auxin level contribute to mechanism leading to tomato internode inhibition subjected to mechanical stress.Growth inhibition has been suggested to be the result of tissues lignification.⁶ As the initial enzyme in the monolignol biosynthesis pathway, PAL has a direct influence on lignin accumulation.¹⁶ The characteristics of lignin differ among cell wall tissues and plant organs.¹⁷ It comprises polyphenolic polymers derived from the oxidative polymerization of different monolignols, including p-coumaryl, coniferyl and sinapyl alcohols via a side pathway of phenylalanine metabolism leading to lignin synthesis.¹⁸ The increase in lignin content in the rubbed tomato internode could be a response mechanism to mechanical damage caused by rubbing.³ It is known that plants create a natural barrier that includes lignin and suberin synthesis, components directly linked to support systems.^19,20The increase in lignin content of rubbed tomato internode³ is paralleled by a rise in CAD activity and whilst such direct proportionality between CAD activity and lignin accumulation does not always agree with the results in the literature, it clearly is responding in ways similar to those of the other enzymes in the pathway.²¹Mechanical stress-induced membrane depolarization would generate different species of free radicals and peroxides, which in turn initiate lipid peroxidation.²² The degradation of cell membranes is suggested to bring about rapid changes in ionic flux, especially release of K⁺ which would result in an enhanced endogenous Ca/K ratio and in leakage of solutes, among them electron donors such as ascorbic acid and phenolic substances. The increased intracellular relative calcium level activated secretion of basic peroxidases²³ into the free space where, in association with the electron donors and may be with the circulating IAA, they eliminate the peroxides, and facilitated binding of basic peroxidases to membrane structures allowing a role as 1-aminocyclopropane-1-carboxylic acid (ACC)-oxidases. The resulting IAA and ACC oxidase-mediated changes in ethylene production²⁴ would further induce (this time through the protein synthesis machinery) an increase in activity of phenylalanine ammonia-lyase and peroxidases. The resulting lignification and cell wall rigidification determines the growth response of tomato internode to the mechanical stress.     

Ethylene-Stimulated Nutations Do Not Require ETR1 Receptor Histidine Kinase Activity

Ethylene influences the growth and development of plants through the action of receptors that have homology to bacterial two-component receptors. In bacteria these receptors function via autophosphorylation of a His residue in the kinase domain followed by phosphotransfer to a conserved Asp residue in a response regulator protein. In Arabidopsis, two of the five receptor isoforms are capable of His kinase activity. However, the role of His kinase activity and phosphotransfer is unclear in ethylene signaling. A previous study showed that ethylene stimulates nutations of the hypocotyl in etiolated Arabidopsis seedlings that are dependent on the ETR1 receptor isoform. The ETR1 receptor is the only isoform in Arabidopsis that contains both a functional His kinase domain and a receiver domain for phosphotransfer. Therefore, we examined the role that ETR1 His kinase activity and phosphotransfer plays in ethylene-stimulated nutations.Key Words: ethylene, nutations, signal transduction, receptors, histidine kinase, phosphotransfer, two component signallingThe gaseous plant hormone ethylene has a role in a variety of physiological events in higher plants such as seed germination, abscission, senescence, fruit ripening, and growth regulation.¹ In etiolated Arabidopsis seedlings, ethylene causes reduced growth of the hypocotyl and root, increased radial expansion of the hypocotyl, and increased tightening of the apical hook.^2,3Previous studies have identified components in the ethylene signaling pathway and led to an inverse-agonist model for signal transduction.^4,5 According to this model, responses to ethylene are mediated by a family of five receptors (ETR1, ERS1, ETR2, EIN4, ERS2) in Arabidopsis that have homology to bacterial two-component receptors.^6–9 In bacterial systems, two-component receptors transduce signal via the autophosphorylation of a His residue in the kinase domain, followed by the transfer of phosphate to a conserved Asp residue in the receiver domain of a response regulator protein.¹⁰ The ethylene receptors of plants can be divided into two subfamilies based on sequence homology in the ethylene-binding domains.¹¹ ETR1 and ERS1 belong to subfamily I, contain all amino acid residues needed for His kinase activity,^6,12 and show His kinase activity in vitro.^13,14 ETR2, EIN4, and ERS2 belong to subfamily II, contain degenerate His kinase domains^7,9 and have Ser/Thr kinase activity in vitro.¹⁴ ERS1 shows both His and Ser/Thr kinase activities in vitro depending on the assay conditions used.¹⁴ While the kinase domain of ETR1 appears to be required for signaling,¹⁵ kinase activity is not.^15–17 It is unclear whether or not histidine kinase activity is involved in ethylene signaling, although, this activity might be involved in growth recovery after ethylene removal.¹⁷Recently, high-resolution, time-lapse imaging revealed that prolonged treatment with ethylene stimulates nutational bending of etiolated Arabidopsis hypocotyls.¹⁸ Nutations are oscillatory bending movements caused by localized differential growth¹⁹ that were originally termed “circumnutations”.²⁰ Nutations have been posited to be important for seedlings to penetrate through the soil²⁰ and thus could be critical for seedling survival. In support of this hypothesis, nutations of rice roots have been reported to increase soil penetration.²¹Mutational analysis revealed that many of the known ethylene signaling components including CTR1, EIN2, EIN3 and EIL1 are involved in signaling leading to ethylene-stimulated nutations.¹⁸ Surprisingly, loss-of-function mutations in ETR1 eliminated ethylene-stimulated nutations while combinatorial loss-of-function mutations in the other four receptor isoforms led to constitutive nutations in air.¹⁸ These results support a model where all the receptors are involved in ethylene-stimulated nutations but the ETR1 receptor is required for and has a contrasting role from the other receptor isoforms in this nutation phenotype. Since the ETR1 receptor is the only receptor isoform that contains both a functional His-kinase domain and a receiver domain,^6,13,14 the roles of His kinase activity and phosphorelay in the nutation phenotype were examined in the current study.Previous work showed that the nutation phenotype in etr1-7 loss-of-function mutants could be rescued with a wild-type, genomic ETR1 transgene.¹⁸ Etr1-7 mutants transformed with a kinase-inactivated genomic ETR1 transgene (gETR1 (G₂)) where the two conserved glycines in the G₂ box of the histidine kinase domain (G545, G547) were changed to alanines were examined to determine if ETR1 His kinase activity is required for ethylene-stimulated nutations. This construct lacks histidine autophosphorylation in vitro.²² Figure 1 shows that ethylene stimulates nutations in etr1-7 gETR1(G₂) seedlings. The period of these nutations was 4.7 ± 1.5 h which is similar to values obtained previously for wild-type seedlings (4.7 ± 1h) and somewhat longer than etr1-7 seedlings transformed with wild-type, genomic ETR1 (3.2 ± 0.6 h). However, the amplitude of these nutations (3.7 ± 1.0°) was approximately half that of nutations previously observed in wild-type seedlings (9.1 ± 6.0°) as well as etr1-7 seedlings transformed with wild-type, genomic ETR1 (8.2 ± 3.6°). This suggests that ETR1 histidine kinase activity is not required for ethylene-stimulated nutations but might have a role in modulating nutation amplitudes.Open in a separate windowFigure 1Ethylene stimulates nutations of etr1-7 seedlings transformed with a kinase-inactivated ETR1 transgene. The hypocotyl angles for four etr1-7 mutants transformed with a kinase-inactivated genomic ETR1 transgene (gETR1(G₂)) are shown. Transformants were obtained from Eric Schaller and have been described previously.²² In this and the following figure, etiolated Arabidopsis seedlings were imaged from the side at 15 min intervals while growing along a vertically orientated agar plate and the hypocotyl angle measured as described previously.¹⁸ Black and gray lines are used to help distinguish the movements of individual seedlings. All seedlings were grown in the presence of 5 µM AVG to block biosynthesis of ethylene by the seedlings. Seedlings were grown in air for 2 h prior to treatment with 10 µL L⁻¹ ethylene (Open in a separate window).To determine whether phosphotransfer through the receiver domain of ETR1 is required for the nutation phenotype, seedlings deficient in ethylene receptor isoforms containing a receiver domain (ETR1, ETR2, EIN4) were transformed with a mutant ETR1 transgene lacking the conserved Asp₆₅₉ required for phosphotransfer (getr1-[D]). Previous work showed that etr1-6 etr2-3 ein4-4 triple loss-of-function mutant seedlings failed to nutate and this nutation phenotype could be rescued when these mutants were transformed with wild-type, genomic ETR1 transgene.¹⁸ Similarly, transformation of the etr1-6 etr2-3 ein4-4 triple mutants with getr1-[D] rescued the nutation phenotype in most seedlings observed (Fig. 2). However, some seedlings (four of the eleven observed) failed to nutate. The reason for this variable rescue is unclear but could reflect differences in expression levels of the mutant transgene in individual plants. Alternatively, this variable rescue could reflect functional differences between the mutant and wild-type transgene suggesting a modulating role for phosphotransfer through the receiver domain of ETR1. Two independent lines were observed with similar results. Of those that did nutate, the period of nutations was 5.0 ± 1.2 h and the amplitude 7.6 ± 3.8° which is similar to values obtained previously for wild-type plants as well as plants transformed with a wild-type, genomic ETR1 transgene.¹⁸Open in a separate windowFigure 2Ethylene stimulates nutations of etr1-6 etr2-3 ein4-4 seedlings transformed with an ETR1 transgene mutated at Asp₆₅₉. The hypocotyl angles from seven etr1-6 etr2-3 ein4-4 triple mutants transformed with an ETR1 transgene mutated at Asp₆₅₉ (getr1[D]) are shown in two panels. One seedling in (A) (black) had no measurable nutations while one in (B) (black) had very small nutations.Conclusions from this and the previous study are that the ETR1 receptor has a unique role in ethylene-stimulated nutations. However, this role does not require either histidine kinase activity or phosphotransfer through the receiver domain of ETR1.     

Low temperature acclimation mediated by ethanol production is essential for chilling tolerance in rice roots

Rice seedlings (Oryza sativa L.) were subjected to low temperature pretreatment (LT-PT; 10°C) for various length of time followed by a 48-h chilling temperature stress (2°C). Chilling tolerance of rice roots was improved with increasing duration of LT-PT, but HT-PT longer than 12 h gave no additional improvement. LT-PT did not change in fatty acid composition in rice roots under the present experimental condition. Alcohol dehydrogenase (ADH) activity and ethanol concentration in the roots were increased with increasing duration of LT-PT up to 12 h, which indicates that LT-PT increased ethanol fermentation in the roots. 4-Methylpyrazole, a potent inhibitor of ADH, reduced the ethanol concentration and the chilling tolerance in the roots. This reduction of the chilling tolerance recovered with exogenously applied ethanol. Ethanol also induced 21- and 33-kD protein synthesis in the roots and these proteins may contribute the improvement of the tolerance. The present research suggests that LT-PT may increase chilling tolerance in rice roots owing to ethanol production, and ethanol may trigger a signal transduction cascade, which might lead to a decrease in membrane damage and injury.Key words: acclimation, alcohol dehydrogenase, chilling tolerance, ethanol, heat shock protein, low temperature, Oryza sativaAlcohol dehydrogenase (ADH; EC.1.1.1.1) gene and protein were induced by low temperature in Arabidopsis, maize and rice seedlings.^1,2,3 ADH is an enzyme involved in ethanolic fermentation and essential for plants to survive under anaerobic conditions.^4,5 However, it is unlikely that the induction of ADH by low temperture is due to a switch from aerobic respiration to anaerobic respiration as reported with anaerobic conditions.^2,6 Therefore, it is not clear that biological meanings of the induction of ADH in low temperature conditions.Rice seedlings (Oryza sativa L. cv. Nipponbare) were subjected to low temperature pretreatment (LT-PT; 10°C) for various length of time (1, 2, 4, 6, 12, 18, 24 h) followed by a 48-h chilling temperature stress (2°C). Chilling tolerance of rice roots was improved with increasing duration of LT-PT, but HT-PT longer than 12 h gave no additional improvement. LT-PT did not change in any fatty acid compositions in rice roots under the present experimental condition. Several plant species, such as oat, rye and spinach increased freezing tolerance due to the increasing unsaturation of fatty acids in plasma membranes, but this cold acclimation process required exposure of these plants to subzero temperature for 2–3 weeks.^7,8LT-PT increased ADH activity and ethanol concentration in rice roots, and the activity and the concentration were increased with increasing duration of LT-PT up to 12 h. Thus, LT-PT induced ethanolic fermentation system and stimulated ethanol production in the roots. 4-Methylpyrazole, which is a potent inhibitor of ADH and prevents ethanol production,⁹ reduced rice root growth to 40% of LP-PT root growth (Fig. 1), and the ethanol accumulation in the roots. This growth inhibition by 4-methylpyrazole recovered with exogenously applied ethanol. These results suggest that ethanol produced by LT-PT may contribute the chilling tolerance in the roots of the rice seedlings. In addition, an ADH deficient mutant of maize seedlings, which can not produce ethanol, was more sensitive to chilling temperature than their wild types.⁶Open in a separate windowFigure 1Effects of ethanol and 4-methylpyrazole on root growth of rice seedlings. Three-day-old rice seedlings were treated 12-h LT-PT (10°C) with or without 100 mM ethanol and/or 5 mM 4-methylpyrazole at 25°C for 24 h, and then subjected to chilling stress treatment (2°C, 48 h). Elongation of rice roots was determined over 48 h at 25°C after chilling stress treatment. Non-stressed seedlings and ethanol-treated seedlings were grown at 25°C. Chilling stressed seedlings were grown at 25°C for 24 h, and then subjected to chilling stress treatment. Means ± SE from five independent experiments with 20 plants for each determination are shown.When the seedlings were subjected to chilling temperature stress after ethanol treatment without LT-PT, the growth inhibition of rice roots by chilling temperature recovered from 22% to 71% of that of nonstressed roots (Fig. 1), which suggests that exogenously applied ethanol may improve chilling tolerance in the roots. It is also found that the ethanol treatment did not change in fatty acid composition in the roots at the temperature of this treatment (25°C).Chilling temperature induced lipid degradation in plant cells of cold-sensitive plants, such as cucumber, rice and soybean, as measured by an increase in malondialdehyde, which is a decomposition product of phospholipid peroxidation.¹⁰ Lipid peroxidation occurs when polyunsatured fatty acids are released from phospholipids by phospholipases and became substrates for lipoxygenases. Changes in the structural composition of the plasma membranes by lipid peroxidation cause the phase transition of the membrane from liquid to gel and the inactivation of membrane bound enzymes such as plasma membrane ATPase. Thus, the phase transition of the membranes was thought to be one of the primary causes of chilling injury.^11–13The addition of C₁ to C₆ alcohols including ethanol to model membranes increased fluidity of the membranes and lowered the phase transition temperature of the membranes.^14,15,16 Therefore, ethanol produced by LT-PT may prevent the phase transition of the membrane from liquid to gel, and lower the phase transition temperature of the membranes, which may contribute the acclimation to the chilling tolerance. In addition, ethanol induced an increase in ATPase activity in plasma membranes,⁶ and prevented chilling-induced ion leakage from plant tissues.¹⁷Ethanol is also known to stimulate the synthesis of heat shock protein (HSP) in yeast, bacteria and some other plants.^18,19 We thus determined the effect of ethanol on protein synthesis in rice roots by SDS-gel electrophoresis, and found that 21- and 33-kD protein synthesis were induced by ethanol. These proteins were also induced by heat shock treatment (45°C, 20 min). HSP was shown to be associated with the development of low temperature tolerance in spinach.^20,21 Thus, 21- and 33-kD proteins induced by ethanol may contribute the improvement of the chilling tolerance.The present research suggests that LT-PT-induced chilling tolerance may be owing to ethanol accumulation in rice roots. Accumulated ethanol may increase the fluidity of plasma membranes and lower the phase transition temperature of the membranes, and may also induce protein synthesis. This hypothesis is supported by exogenously applied ethanol which increased the chilling tolerance. Thus, ethanol might trigger a signal transduction cascade, which would lead to a decrease in membrane damage and injury. Further work needs to be done to test this possibility.     

Structural basis of transmembrane domain interactions in integrin signaling

Cell surface receptors of the integrin family are pivotal to cell adhesion and migration. The activation state of heterodimeric αβ integrins is correlated to the association state of the single-pass α and β transmembrane domains. The association of integrin αIIbβ3 transmembrane domains, resulting in an inactive receptor, is characterized by the asymmetric arrangement of a straight (αIIb) and tilted (β3) helix relative to the membrane in congruence to the dissociated structures. This allows for a continuous association interface centered on helix-helix glycine-packing and an unusual αIIb(GFF) structural motif that packs the conserved Phe-Phe residues against the β3 transmembrane helix, enabling αIIb(D723)β3(R995) electrostatic interactions. The transmembrane complex is further stabilized by the inactive ectodomain, thereby coupling its association state to the ectodomain conformation. In combination with recently determined structures of an inactive integrin ectodomain and an activating talin/β complex that overlap with the αβ transmembrane complex, a comprehensive picture of integrin bi-directional transmembrane signaling has emerged.Key words: cell adhesion, membrane protein, integrin, platelet, transmembrane complex, transmembrane signalingThe communication of biological signals across the plasma membrane is fundamental to cellular function. The ubiquitous family of integrin adhesion receptors exhibits the unusual ability to convey signals bi-directionally (outside-in and inside-out signaling), thereby controlling cell adhesion, migration and differentiation.^1–5 Integrins are Type I heterodimeric receptors that consist of large extracellular domains (>700 residues), single-pass transmembrane (TM) domains, and mostly short cytosolic tails (<70 residues). The activation state of heterodimeric integrins is correlated to the association state of the TM domains of their α and β subunits.^6–10 TM dissociation initiated from the outside results in the transmittal of a signal into the cell, whereas dissociation originating on the inside results in activation of the integrin to bind ligands such as extracellular matrix proteins. The elucidation of the role of the TM domains in integrin-mediated adhesion and signaling has been the subject of extensive research efforts, perhaps commencing with the demonstration that the highly conserved GFFKR sequence motif of α subunits (Fig. 1), which closely follows the first charged residue on the intracellular face, αIIb(K989), constrains the receptor to a default low affinity state.¹¹ Despite these efforts, an understanding of this sequence motif had not been reached until such time as the structure of the αIIb TM segment was determined.¹² In combination with the structure of the β3 TM segment¹³ and available mutagenesis data,^6,9,10,14,15 this has allowed the first correct prediction of the overall association of an integrin αβ TM complex.¹² The predicted association was subsequently confirmed by the αIIbβ3 complex structure determined in phospholipid bicelles,¹⁶ as well as by the report of a similar structure based on molecular modeling using disulfide-based structural constraints.¹⁷ In addition to the structures of the dissociated and associated αβ TM domains, their membrane embedding was defined^{12,13,16,18,19} and it was experimentally recognized that, in the context of the native receptor, the TM complex is stabilized by the inactive, resting ectodomain.¹⁶ These advances in integrin membrane structural biology are complemented by the recent structures of a resting integrin ectodomain and an activating talin/β cytosolic tail complex that overlap with the αβ TM complex,^20,21 allowing detailed insight into integrin bi-directional TM signaling.Open in a separate windowFigure 1Amino acid sequence of integrin αIIb and β3 transmembrane segments and flanking regions. Membrane-embedded residues^{12,13,16,18,19} are enclosed by a gray box. Residues 991–995 constitute the highly conserved GFFKR sequence motif of integrin α subunits.     

A new callose function: Involvement in differentiation and function of fern stomatal complexes

Callose in polypodiaceous ferns performs multiple roles during stomatal development and function. This highly dynamic (1→3)-β-D-glucan, in cooperation with the cytoskeleton, is involved in: (a) stomatal pore formation, (b) deposition of local GC wall thickenings and (c) the mechanism of stomatal pore opening and closure. This behavior of callose, among others, probably relies on the particular mechanical properties as well as on the ability to form and degrade rapidly, to create a scaffold or to serve as a matrix for deposition of other cell wall materials and to produce fibrillar deposits in the periclinal GC walls, radially arranged around the stomatal pore. The local callose deposition in closing stomata is an immediate response of the external periclinal GC walls experiencing strong mechanical forces induced by the neighboring cells. The radial callose fibrils transiently co-exist with radial cellulose microfibrils and, like the latter, seem to be oriented via cortical MTs.Key words: callose, cytoskeleton, fern stomata, guard cell wall thickening, stomatal function, stomatal pore formationCallose represents a hemicellulosic matrix cell wall component, usually of temporal appearance, which is synthesized by callose synthases, enzymes localized in the plasmalemma and degraded by (1→3)-β-glucanases.^1–4 It consists of triple helices of a linear homopolymer of (1→3)-β-glucose residues.^5–7 The plant cell is able to form and degrade callose in a short time. On the surface of the plasmolyzed protoplast a thin callose surface film may arise within seconds.⁸ Callose is the only cell wall component that is implicated in a great variety of developmental plant processes, like cell plate formation,^9–11 microspore development,^12–14 trafficking through plasmodesmata,^15,16 formation and closure of sieve pores,¹⁶ response of the plant cells to multiple biotic and abiotic stresses,^4,5 establishment of distinct “cell cortex domains”,¹⁷ etc.Despite the widespread occurrence of callose, its general function(s) is (are) not well understood (reviewed in refs. ⁴ and ⁵). It may serve as: a matrix for deposition of other cell wall materials, as in developing cell plates;⁹ a cell wall-strengthening material, as in cotton seed hairs and growing pollen tubes;¹⁸ a sealing or plugging material at the plasma membrane of pit fields, plasmodesmata and sieve plate pores;¹⁶ a mechanical obstruction to growth of fungal hyphae or a special permeability barrier, as in pollen mother cell walls and muskmelon endosperm envelopes.^4,19,20 The degree of polymerization, age and thickness of callose deposits may cause variation in its physical properties.⁵Evidence accumulated so far showed that a significant number of ferns belonging to Polypodiales and some other fern classes forms intense callose deposits in the developing GC wall thickenings.^21–28 This phenomenon has not been observed in angiosperm stomata, although callose is deposited along the whole surface of the young VW and in the VW ends of differentiating and mature stomata (our unpublished data; reviewed in refs ²⁹ and ³⁰).Stomata are specialized epidermal bicellular structures (Fig. 1A) regulating gas exchange between the aerial plant organs and the external environment. Their appearance in the first land plants was crucial for their adaptation and survival in the terrestrial environment. The constituent GCs have the ability to undergo reversible changes in shape, leading to opening and closure of the stomatal pore (stomatal movement). The mechanism by which GCs change shape is based on: (a) the particular mechanical properties of GC walls owed to their particular shape, thickening, fine structure and chemical composition and (b) the reversible changes in vacuole volume, in response to environmental factors, through fairly complicated biochemical pathways.^30–33Open in a separate windowFigure 1(A) Diagrammatic representation of an elliptical stoma. (B–E) Diagram to show the process of stomatal pore formation in angiosperms (B and C) and Polypodiales ferns (D and E). The arrows in (B) indicate the forming stomatal pore. DW, dorsal wall; EPW, external periclinal wall; GC, guard cell; IPW, internal periclinal wall; ISP, internal stomatal pore; PE polar ventral wall end; VW, ventral wall.The present review is focused on the multiple-role of callose in differentiating and functioning fern stomata, as they are substantiated by the available information, including some unpublished data, and in particular in: stomatal pore formation, deposition of GC wall thickenings and opening and closure of the stomatal pore. The mode of deposition of fibrillar callose deposits in GC walls and the mechanism of their alignment are also considered.     

Heme-independent soluble and membrane-associated peroxidase activity of a Zea mays annexin preparation

Annexins are cytosolic proteins capable of reversible, Ca²⁺-dependent membrane binding or insertion. Animal annexins form and regulate Ca²⁺-permeable ion channels and may therefore participate in signaling. Zea mays (maize) annexins (ZmANN33 and ZmANN35) have recently been shown to form a Ca²⁺-permeable conductance in planar lipid bilayers and also exhibit in vitro peroxidase activity. Peroxidases form a superfamily of intra- or extracellular heme-containing enzymes that use H₂O₂ as the electron acceptor in a number of oxidative reactions. Maize annexin peroxidase activity appears independent of heme and persists after membrane association, the latter suggesting a role in reactive oxygen species signaling.Key words: annexin, calcium, C2, lipoxygenase, maize, peroxidasePeroxidases may guard cells against the toxicity of reactive oxygen species (ROS), and are thought to be involved in stress signaling, auxin regulation and wall remodelling.¹ Recombinant Arabidopsis thaliana annexin 1 (AtANN1) has peroxidase activity in vitro,^2,3 as does an annexin4 from Brassica juncea BjANN1 and Capsicum annum⁵ CaANN24. Annexin peroxidase activity^2,3,6,7 appears to rely on a region (including a conserved His residue; His40) which is similar to the ∼30 amino acid heme-binding domain of plant peroxidases such as horse radish peroxidase (HRP). Mutagenesis of His40 in AtANN1 abolishes peroxidase activity.³In vitro peroxidase activity was also evident in a maize annexin preparation in which ZmANN33/35 were the predominant components but traces of a lipoxygenase and a novel C2-domain-containing protein were also evident.⁸ Peroxidase activity of this maize annexin preparation has Michaelis-Menten kinetics (Fig. 1A). K_m values (mean ± se estimated from Lineweaver-Burk plots) were 15 ± 5 µM and 31 ± 8 µM (n = 3) for maize annexin preparation and HRP respectively (Fig. 1B–D). The mean ± se maximum velocity (V_max) was 76 ± 14 µmole s⁻¹ µg⁻¹ protein compared with 2424 ± 115 µmole s⁻¹ µg⁻¹ for HRP. The K_m of the annexin preparation (15 µM) is within range of the soluble cytosolic heme-containing ascorbate peroxidases (APXs; pea⁹ 20 µM; tea¹⁰ APX1, 30 µM and APX2, 80 µM; rice¹¹ APX1, 33 µM and APX2, 76 µM). APXs are usually associated with tightly regulating the redox status of the cell.¹²Open in a separate windowFigure 1Peroxidase activity of maize annexin preparation measured using Amplex Red. (A) Effect of substrate concentration. Initial rate of Amplex Red oxidation to resorufin by annexin preparation (25 µg/ml) in the presence of a range of [H₂O₂] shows standard Michaelis-Menten behaviour (mean ± se from 3 independent trials). (B) Lineweaver-Burk analysis of the data in (A). (C) Comparison with HRP. Initial rate of Amplex Red oxidation by HRP (0.095 µg/ml) (mean ± se from 3 independent trials). (D) Lineweaver-Burk analysis of HRP reaction rates. Maize annexin preparation was isolated and assayed as described previously.⁸The previous study on the maize annexin preparation failed to detect heme.⁸ The putative heme-binding motif is present in maize annexins but the maize annexin preparation supporting peroxidase activity did not exhibit the Soret peak (in spectral analysis) that is characteristic of the presence of heme.⁸ As it was feasible that a heme moiety could have dissociated, protein was recovered from the Amplex Red peroxidase assay (pH 7.4 and 1 mM H₂O²) and re-tested for peroxidase activity using luminol-based detection. The annexin preparation still displayed peroxidase activity (Fig. 2A) therefore the null result for heme association was unlikely to be the consequence of experimental procedures. In separate tests, staining of native PAGE gels with 3,3′,5,5′ tetramethylbenzidine (TMB) revealed the presence of a heme moiety for as little as 0.095 ng (HRP) but yielded a null result for 10 µg annexin preparation (Fig. 2B). Thus it appears that heme is not essential for peroxidase activity of the annexin preparation.Open in a separate windowFigure 2Heme detection. (A) Peroxidase activity of annexin preparation recovered from spectral analysis with bovine serum albumen (BSA) as a negative control. Peroxidase activity was analysed using an ECL dot blot. Annexin, HRP (“proteins”; 5 µg and 2 ng respectively) and BSA (5 µg; “control”) were dotted onto nitrocellulose. The dot blot was treated with ECL solutions (Amersham) and used to expose photographic film for 5 minutes. (B) Staining of native PAGE gels reveals the presence of heme in 0.095 ng HRP but not in 10 µg annexin preparation. Native PAGE gels were stained for heme.²⁰ After incubation for 45 min in 1.25 mM 3,3′,5,5′tetramethylbenzidine (TMB); 30% (v/v) methanol, 175 mM sodium acetate (pH 5.0), gels were rinsed and incubated for up to 1 hour in 30% (v/v) isopropanol, 175 mM sodium acetate (pH 5.0). Results are representative of 3 determinations.The physiological significance of annexin peroxidase activity could depend on whether the protein is cytosolic or membrane-bound. However, the effects of Ca²⁺ and lipid binding have not been quantified to date. Addition of 10 mM Ca²⁺ caused a significant increase (29%, p = 5.52 × 10⁻⁵; n= 3, Student''s t-test) in peroxidase activity of the maize annexin preparation (1 mM H₂O₂) but had no significant effect on HRP (13 Addition of PS:PC liposomes depressed activity of both annexin preparation and HRP (with no Ca²⁺, by 65% for annexin and 22% for HRP). However, in the presence of liposomes, activity of the annexin preparation was still stimulated by addition of 10 mM Ca²⁺ (79%, p = 5.52 × 10⁻⁵; n = 3) while HRP was unresponsive.

Table 1

Peroxidase activity supported by maize annexin preparation or HRP

Co-localization of mitochondria with chloroplasts is a light-dependent reversible response

Co-localization of mitochondria with chloroplasts in plant cells has long been noticed as beneficial interactions of the organelles to active photosynthesis. Recently, we have found that mitochondria in mesophyll cells of Arabidopsis thaliana expressing mitochondrion-targeted green fluorescent protein (GFP) change their distribution in a light-dependent manner. Mitochondria occupy the periclinal and anticlinal regions of palisade cells under weak and strong blue light, respectively. Redistributed mitochondria seem to be rendered static through co-localization with chloroplasts. Here we further demonstrated that distribution patterns of mitochondria, together with chloroplasts, returned back to those of dark-adapted state during dark incubation after blue-light illumination. Reversible association of the two organelles may underlie flexible adaptation of plants to environmental fluctuations.Key words: Arabidopsis thaliana, blue light, chloroplast, green fluorescent protein, mesophyll cell, mitochondrion, organelle positioningHighly dynamic cell organelles, mitochondria, are responsible not only for energy production, but also for cellular metabolism, cell growth and survival as well as gene regulations.^1,2 Appropriate intracellular positioning and distribution of mitochondria contribute to proper organelle functions and are essential for cell signaling.^3,4 In plant cells operating photosynthesis, the co-localization of mitochondria with chloroplasts has been a well known phenomenon for a long period of time.^5,6,7 Physical contact of mitochondria with chloroplasts may provide a means to transfer genetic information from the organelle genome,⁸ as well as to exchange metabolite components; a process required for the maintenance of efficient photosynthesis.^9,10,11Using Arabidopsis thaliana stably expressing mitochondrion-targeted GFP,¹² we have recently examined a different aspect of mitochondria positioning. Although mitochondria in leaf mesophyll cells are highly motile under dark condition, mitochondria change their intracellular positions in response to light illumination.¹³ The pattern of light-dependent positioning of mitochondria seems to be essentially identical to that of chloroplasts.¹⁴ Mitochondria occupy the periclinal regions under weak blue light (wBL; 470 nm, 4 µmol m⁻²s⁻¹) and the anticlinal regions under strong blue light (sBL; 100 µmol m⁻²s⁻¹), respectively. A gradual increase in the number of static mitochondria located in the vicinity of chloroplasts in the periclinal regions with time period of wBL illumination clearly demonstrates that the co-localization of these two organelles is a light-induced phenomenon.¹³In the present study, to ask whether the light-dependent positioning of mitochondria is reversible or not, a time course of mitochondria redistribution was examined transferring the sample leaves from light to dark conditions. The representative results (Fig. 1) clearly show that mitochondria re-changed their positions within several hours of dark treatment. Immediately after dark adaptation, mitochondria in the palisade mesophyll cells were distributed randomly throughout the cytoplasm (Fig. 1A and ^{ref. 13}). Chloroplasts were distributed along the inner periclinal walls and the lower half of the anticlinal walls. On the contrary, mitochondria accumulated along the outer (Fig. 1B) and inner periclinal walls when illuminated with wBL. Chloroplast position was also along the outer and inner periclinal walls. Many of the mitochondria located near the chloroplasts lost their motility. When wBL-illuminated leaves were transferred back to dark condition, the numbers of mitochondria and chloroplasts present on the periclinal regions began to decrease within several hours (Fig. 1C). After 10 h dark treatment, distribution patterns of mitochondria as well as chloroplasts almost recovered to those of dark-adapted cells (Fig. 1D).Open in a separate windowFigure 1Distribution of mitochondria and chloroplasts on the outer periclinal regions of palisade mesophyll cells of A. thaliana under different light conditions. Mitochondria (green; GFP) and chloroplasts (red; chlorophyll autofluorescence) were visualized with confocal microscopy after dark adaptation (A), immediately after wBL (470 nm, 4 µmol m⁻²s⁻¹) illumination for 4 h (B), after dark treatment for 6 h (C) and 10 h (D) following the 4-h wBL illumination, respectively. Bar = 50 µm.To our knowledge, this may be the first report that directly demonstrates that wBL regulates mitochondria and chloroplast positioning in a reversible manner, though the nuclei in A. thaliana leaf cells were also found to reverse their positions when transferred from sBL to dark conditions.¹⁵ Reversible regulation of organelle positioning in leaf cells should play critical roles in adaptation of plants to highly fluctuating light conditions in the nature. Since distribution patterns of mitochondria under wBL and sBL are identical to those of chloroplasts, we can assume that phototropins, the BL receptors for chloroplast photo-relocation movement,¹⁶ may have some role in the redistribution of mitochondria. On the other hand, we also found that red light exhibited a significant effect on mitochondria positioning (Islam et al. 2009), suggesting an involvement of photosynthesis. These possibilities are now under investigation.     

Understanding the effect of mean pore size on cell activity in collagen-glycosaminoglycan scaffolds

Mean pore size is an essential aspect of scaffolds for tissue-engineering. If pores are too small cells cannot migrate in towards the center of the construct limiting the diffusion of nutrients and removal of waste products. Conversely, if pores are too large there is a decrease in specific surface area available limiting cell attachment. However the relationship between scaffold pore size and cell activity is poorly understood and as a result there are conflicting reports within the literature on the optimal pore size required for successful tissue-engineering. Previous studies in bone tissue-engineering have indicated a range of mean pore sizes (96–150 µm) to facilitate optimal attachment. Other studies have shown a need for large pores (300–800 µm) for successful bone growth in scaffolds. These conflicting results indicate that a balance must be established between obtaining optimal cell attachment and facilitating bone growth. In this commentary we discuss our recent investigations into the effect of mean pore size in collagen-glycosaminoglycan (CG) scaffolds with pore sizes ranging from 85–325 µm and how it has provided an insight into the divergence within the literature.Key words: bone tissue engineering, cell adhesion, collagen, extracellular matrix, pore size, scaffoldThe goal of tissue engineering is to develop cell, construct and living system technologies to restore the structure and functional mechanical properties of damaged or degenerated tissue. While the field of tissue engineering may be relatively new, the idea of replacing tissue with another goes as far back as the 16^th century when an Italian, Gasparo Tagliacozzi (1546–99), Professor of Surgery and Anatomy at the Bologna University, described a nose replacement that he had constructed from a forearm flap in his work “De Custorum Chirurigia per Insitionem” (The Surgery of Defects by Implantation) which was published in 1597. In modern times, the techniques of transplanting tissue from one site to another in the same patient (an autograft) or from one individual to another (transplant or allograft) have been revolutionary and lifesaving. However major problems exist with both techniques. Harvesting autografts is expensive, painful, constrained by anatomical limitations and associated with donor-site morbidity due to infection and hemorrhage. Transplants have serious constraints. The major problem is accessing enough tissue and organs for all of the patients who require them. Transplants are strongly associated with rejection by the patient''s immune system and they are also limited by the potential risks of introducing infection or disease.Tissue engineering was born from the belief that primary cells could be isolated from a patient, expanded in vitro and seeded onto a substrate that could be grafted back into the patient.¹ It provides a biological alternative to transplantations and prosthesis. One of the first scaffolds pioneered for tissue regeneration was synthesized as a graft co-polymer of type I collagen and chondroitin 6-sulphate, a glycosaminoglycan. The development of these scaffolds, which are capable of supporting tissue synthesis when seeded with cells, marks the beginning of the field of tissue engineering.^2,3 Since this early work, there have been rapid advances in bone tissue engineering with the development of porous, biocompatible, three-dimensional scaffolds. Regardless of the application, the scaffold should be biocompatible and imitate both the physical and biological function of the native extracellular matrix (ECM), as the ECM provides a substrate with specific ligands for cell adhesion as well as physical support for cells.⁴ When designing scaffolds for any tissue engineering application, a major consideration is the mean pore size. Scaffolds must be permeable with interconnecting pores to facilitate cell growth, migration and nutrient flow. A previous study demonstrated that permeability increases with increasing pore size due to a reduction in specific surface area.⁵ If pores are too small, cell migration is limited, resulting in the formation of a cellular capsule around the edges of the scaffold. This in turn can limit the distribution of nutrients and removal of waste products resulting in necrotic regions within the construct. Conversely if pores are too large there is a decrease in specific surface area.³ It has been proposed that a reduction in specific surface area reduces the ligand density available for cells to bind to.⁶ Cellular activity is influenced by specific integrin-ligand interactions between cells and surrounding ECM. Initial cell adhesion mediates all subsequent events such as proliferation, migration and differentiation within the scaffold. As a result the mean pore size within a scaffold affects cell adhesion and ensuing proliferation, migration and infiltration. Therefore maintaining a balance between the optimal pore size for cell migration and specific surface area for cell attachment is essential.^4,7In our laboratory we use a composite scaffold fabricated from collagen and a glycosaminoglycan (GAG) for bone tissue engineering applications produced by a lyophilisation (freeze-drying) fabrication process. The first generation of this collagen-GAG (CG) scaffold was originally developed for skin regeneration but has since been applied to a number of other tissue engineering applications, due to its high biological activity and resultant ability to promote cell growth and tissue development.^2,8–12 Originally CG scaffolds were fabricated using a rapid uncontrolled quench process during lyophilisation which resulted in heterogeneous porous scaffolds with a large variation of pore size within certain areas of the scaffold.² When these scaffolds were used in previous studies they were visually examined so that the areas of variation could be avoided resulting in subjective selection of scaffold samples for analysis.⁸ However, an improved lyophilisation technique was later developed which incorporated a constant cooling rate which controlled the formation and growth of ice-crystals thus resulting in CG scaffolds with homogenous pore structures.¹³ The traditional final temperature of freezing used to produce these scaffolds is −40°C; however, further modifications to the lyophilisation process demonstrated that by changing the final temperature of freezing, it is possible to tailor the mean pore size in the scaffolds. This study showed that by varying the temperature of freezing from −40 to −10°C it was possible to produce homogenous CG scaffolds with mean pore sizes ranging from 96–151 µm.⁶A cellular solid is one made up of an interconnecting porous network and cellular solids modeling techniques can be used to describe both mechanical and microstructural (i.e., specific surface area) properties of scaffolds. A cellular solids model utilizing a tetrakaidecahedral unit cell (a 14-sided polyhedron that packs to fill space) was used to determine the effect of mean pore size on specific surface area. Specific surface area can be related to the relative density of a scaffold and using a tetrakaidecahedral unit cell it was possible to model the geometry of the CG scaffolds.^5,6,14 As a result the specific surface area (SA) per unit volume (V) available for cell adhesion in each of the scaffolds with different mean pore sizes (d) was estimated as: SA/V = 0.718/d(1)This relationship demonstrates that the specific surface area is inversely proportional to the mean pore size. The authors then carried out a simple experiment and seeded the scaffold range with osteoblasts and monitored initial cell adhesion up to 48 h post-seeding. Cell adhesion is the binding of cells to their extracellular environment via specific ligand-integrin interactions. The results demonstrated that cell adhesion decreased with increasing pore size and that the highest levels of cell attachment were found on the scaffolds with the smallest pore size (96 µm). The rationale for this result, as suggested by the authors, was the effect of specific surface area on cell adhesion due to the scaffolds with larger pores having less available specific surface area and thus a lower ligand density for initial cell attachment.^5,6The results of this study conflicted with other studies within the literature which demonstrate a need for larger pores. The relationship between scaffold pore size and cell activity is not fully understood and as a result, over the years there have been conflicting reports on the optimal pore size required for bone tissue engineering. Pores ranging from 20–1,500 µm have been used in bone tissue engineering applications.^15–18 Initial studies demonstrated that the minimum pore size for significant bone growth is 75–100 µm with an optimal range of 100–135 µm.^15,19 Since this early work it has been reported that pores greater than ∼300 µm are essential for vascularisation of constructs and bone ingrowth, while pores smaller than ∼300 µm can encourage osteochondral ossification.^20–22In a very recent study in our laboratory, which utilized improved technical capability of our freeze-drying system and introduced a novel annealing step during lyophilisation, we have been able to further expand the range of mean pore sizes produced in the CG scaffolds from 96–151 µm up to 85–325 µm.²³ We then investigated the effect of this new expanded range of scaffolds on initial cell attachment followed by migration and proliferation by monitoring cellular activity up to 7 days post-seeding (as opposed to 48 h in the earlier study⁶) to see whether the pattern of specific surface area affecting initial cell adhesion as seen in the previous studies would continue as cells proliferated.²⁴The results provide a possible insight into why there are conflicting reports in the literature on the optimal scaffold pore size for bone tissue engineering. A non-linear effect of pore size was seen on cell proliferation over the 7 day incubation period. Scaffolds with the largest pore size of 325 µm facilitated higher cell number at all time points in comparison to the other scaffold types. However, within the lower range of pore sizes there was a small peak in cell number at 24 h and 48 h post-seeding in scaffolds with a mean pore size of 120 µm. This peak disappeared by day 7 (Fig. 1). This peak is consistent with that seen in the earlier study⁶ and can therefore be explained by the effect of pore surface area on cell attachment. Collagen, a natural component of bone ECM, contains binding sites (ligands) that are recognized by specific cell surface receptors (integrins), the main collagen integrins being α₁β₁ and α₂β₁. Based on the interactions between integrins and their corresponding ligands, cells can detect subtle changes in ECM that can influence cell attachment and consequently determine cell proliferation, speed and migration. Our results reflected this within the smaller pore range (85–190 µm) when cell number was presented as a percentage of the cells seeded onto the scaffolds,²⁴ indicating that high specific surface area in scaffolds is important for optimal cell attachment. However, when this range of pore sizes was expanded (85–325 µm) the linear relationship between mean pore size and specific surface area was no longer applicable (Fig. 1) and scaffolds with the largest pores showed the highest cell numbers even though the surface area is lower than that for the other scaffold variants. We propose that the effect of specific surface area is overcome in larger pores by the improved potential for cell migration and proliferation as was seen histologically in scaffolds with 325 µm.Open in a separate windowFigure 1Effect of mean pore size on cell number at each time point. Cell number increases to a small peak 24 h post seeding in scaffolds with a pore size of 120 µm. This peak declines at later time points. Cell number significantly peaks in scaffolds with a mean pore size of 325 µm. *p < 0.001 (reviewed in ref. ²⁴).When seeding three-dimensional scaffolds it is desirable that the cells infiltrate and colonize the scaffold laying down their own ECM. The CG scaffolds are highly porous (∼99%)⁵ and it has previously been shown that cell migration behavior decreases with increasing pore size.²⁶ However, similarly to other studies,⁶ these results were based on limited range of mean pore sizes incubated for less than 48 h. In this study, migration of cells was assessed histologically after 7 days incubation. Cells were observed lining the pores in all scaffolds. However, cell aggregations were seen along the edges of the scaffolds with smaller pore sizes of 85 µm–120 µm limiting the number of cells infiltrating the scaffold (Fig. 2A). Cell aggregations form a “skin” around the outer surface of the scaffold which restricts the diffusion of nutrients and removal of waste from the cells colonizing the center of the scaffold. As the mean pore size increased, cells migrated further away from the edges and in towards the center of the scaffold until cells were seen colonizing the center of the scaffolds with the largest mean pore size of 325 µm (Fig. 2B). An increase in cell number was seen in 120 µm pore size, but the aggregations seen on the surface of these scaffolds compound the hypothesis that this peak was related to initial cell adhesion and the advantages of this pore size were lost with subsequent cell proliferation and migration.Open in a separate windowFigure 2Effect of mean pore size on cell infiltration and distribution CG scaffolds after 7 days. Scaffolds were stained with H&E: (A) 85 µm pore size at x40 magnification, (B) 325 µm pore size at ×40 magnification. Collagen scaffold is stained pink and cell nuclei a deep purple. The arrow indicates cell aggregations along the edges of the scaffold. Aggregations disappeared and cell migration increased with increasing pore size (reviewed in ref. ²⁴).The study²⁴ had a number of limitations. It was not possible to determine the upper pore size limit for cell activity within a CG scaffold. If the pores become too large the mechanical properties of the scaffold will be compromised due to void volume⁷ and as pore size increases further, the specific surface area will eventually reduce to a level that will limit cell adhesion. Furthermore, this study has determined the optimal pore size for MC3T3-E1 pre-osteoblast activity. It has been hypothesised that the optimal pore size will vary with different cell types⁶ and another recent study from our laboratory has demonstrated that mesenchymal stem cells seeded on the smaller range of CG scaffolds and maintained in osteogenic culture for 3 weeks showed improved osteogenesis on the scaffolds with bigger pores²⁵. For this reason it is important to repeat this study with different cell types. However, regardless of these limitations, this paper has demonstrated that mean pore size does affect cell behavior within a scaffold and that subtle changes in pore size can have a significant effect on cell behavior. We also provide an insight into why the literature reports conflicting results on the optimal pore size required for bone tissue engineering, whereby increased specific surface area provided by scaffolds with small pores has a benefi- cial effect on initial cell attachment, but this is overcome by the improved cellular infiltration provided by scaffolds with larger pores suggesting that these scaffolds might be optimal for longer term in vitro culture with the aim of facilitating bone tissue repair.     

Auxin perception and polar auxin transport are not always a prerequisite for differential growth

Using time-lapse photography, we studied the response kinetics of low light intensity-induced upward leaf-movement, called hyponastic growth, in Arabidopsis thaliana. This response is one of the traits of shade avoidance and directs plant organs to more favorable light conditions. Based on mutant- and pharmacological data we demonstrated that among other factors, functional auxin perception and polar auxin transport (PAT) are required for the amplitude of hyponastic growth and for maintenance of the high leaf angle, upon low light treatment. Here, we present additional data suggesting that auxin and PAT antagonize the hyponastic growth response induced by ethylene treatment. We conclude that ethylene- and low light-induced hyponastic growth occurs at least partly via separate signaling routes, despite their strong similarities in response kinetics.Key words: hyponastic growth, petiole, Arabidopsis, ethylene, low light, auxin, polar auxin transport, differential growthUpward leaf movement (hyponastic growth) is a trait of several plant species to escape from growth-limiting conditions.^1,2 Interestingly, Arabidopsis thaliana induces a marked hyponastic growth response triggered by various environmental stimuli, including complete submergence, high temperature, canopy shade and spectral neutral low light intensities (Fig. 1).^3–6 The paper of Millenaar et al. in the New Phytologist 2009,⁷ provides a detailed analysis of low light intensity-induced hyponastic growth and components of the signal transduction are characterized using time-lapse photography. Low light intensity-induced hyponastic growth is a component of the so-called shade avoidance syndrome. Light-spectrum manipulations and mutant analyses indicated that predominantly the blue light wavelength region affects petiole movement and fast induction of hyponastic growth to low light conditions involves the photoreceptor proteins Cryptochrome 1 (Cry1), Cry2, Phytochrome-A (PhyA) and PhyB. Moreover, we show that also photosynthesis-derived signals can induce differential growth.Open in a separate windowFigure 1Typical hyponastic growth phenotype of Arabidopsis thaliana. Side view of Columbia-0 plants treated 10 h with ethylene (5 µl l⁻¹) or low light (20 µmol m⁻² s⁻¹). Plants in control light conditions were in 200 µmol m⁻² s⁻¹. Both stimuli induce a clear leaf inclination (hyponasty) relative to the horizontal by differential growth of the petioles. Plants kept in control conditions only show modest diurnal leaf movement and leaf angles gradually decline over time due to maturation of the leaves. Note that the paint droplets were applied to facilitate quantitative measurement of leaf angle kinetics in a time-lapse camera setup.⁷The hyponastic growth response to low light intensity was not affected in several ethylene-insensitive mutant lines. Moreover, low light did not affect expression of ethylene inducible marker genes nor differences in ethylene release were noted. Therefore, we concluded that low light-induced hyponastic growth is independent of ethylene signaling. This is perhaps surprising, because ethylene is the main trigger of hyponastic growth induced by complete submergence in several species. Interestingly, both ethylene and low light can induce hyponastic growth in Arabidopsis with similar kinetics.³We showed that plants mutant in auxin perception components (transport inhibitor response1 (tir1) and tir1 afb1 afb2 afb3 quadruple, containing additional mutant alleles of TIR1 homologous F-box proteins) and plants mutant in (polar) auxin transport (tir3-1, pin-formed3 (pin3) and pin7) components had a lower hyponastic growth amplitude in low light conditions.⁷ Moreover, these mutants were less able to maintain the high leaf angles after the response maximum. Both characteristics were also noted in plants pre-treated with the polar auxin transport (PAT) inhibitor 2,3,5-triiodobenzoic acid (TIBA). We therefore concluded that auxin perception and PAT are involved in the regulation of low light-induced hyponastic growth.⁷ Interestingly, we observed that TIBA pretreatment did not inhibit ethylene-induced hyponastic growth. In fact, the response upon ethylene treatment was even modestly enhanced. In agreement with this observation, we show here that the above mentioned auxin perception and PAT mutants also showed a slightly enhanced hyponastic growth response upon ethylene treatment (Fig. 2).Open in a separate windowFigure 2Auxin involvement in ethylene induced hyponasty. Effect of exposure to ethylene (5 µl l⁻¹) on the kinetics of hyponastic petiole growth (A) in Arabidopsis thaliana Columbia-0 plants treated with 50 µm TIBa (open circles) or a mock treatment (line) adapted from Supporting Information Figure S3 of Millenaar et al. (2009)⁷ and (B–F) in Arabidopsis auxin signaling and polar auxin transport mutants (closed circles), compared to the wild type response to low light (lines). Petiole angles are pair wise subtracted, which corrects for diurnal petiole movement in control conditions. For details on this procedure, growth conditions, treatments, data acquirement and analysis see.^7,13 Error bars represent standard errors; n ≥ 12. mutants were obtained from the Nottingham Arabidopsis Stock Center (accession numbers are shown between brackets) or from the authors describing the lines. tir1-1 (n3798,¹⁴), tir1-1 afb1-1 afb2-1 (in a mixed Columbia/Wassilewskija background),¹⁵ tir3-1,¹⁴ pin3-4 (n9363,¹⁶) and pin7-1 (n9365,¹⁰).Despite that auxin and PAT are required for many differential growth responses such as phototropism and gravitropism,^8,11 these data indicate that auxin perception and PAT are not obligatory for ethylene-induced hyponasty in Arabidopsis per se. In fact, one might even conclude that auxin and PAT antagonizes ethylene-induced hyponasty. These results are partly in agreement with observations on the wetland species Rumex palustris, were pretreatment with the auxin-efflux carrier 1-naphthylphthalamic acid (NPA) resulted in doubling of the lag-phase for hyponastic growth under water, but hardly affected the amplitude of the response.¹²Together, this indicates that auxin is not always a prerequisite for differential growth responses. Based on the apparent contrasting effects of auxin perception and PAT in low light- and ethylene-induced hyponastic growth, we conclude that ethylene and low light induce hyponastic growth, at least partly, via separate signaling routes.     

Interplay between adhesion turnover and cytoskeleton dynamics in the control of growth cone migration

The migration of neuronal growth cones, driving axon extension, is a fascinating process which has been subject of intense investigation over several decades. Many of the key underlying molecules, in particular adhesion proteins at the cell membrane which allow for target recognition and binding, and cytoskeleton filaments and motors which power locomotion have been identified. However, the precise mechanisms by which growth cones coordinate, in time and space, the transmission of forces generated by the cytoskeleton to the turnover of adhesion proteins are still partly unresolved. To get a better grasp at these processes, we put here in relation the turnover rate of ligand/receptor adhesions and the degree of mechanical coupling between cell adhesion receptors and the actin rearward flow. These parameters were obtained recently for N-cadherin and IgCAM based adhesions using ligand-coated microspheres in combination with optical tweezers and photo-bleaching experiments. We show that the speed of growth cone migration requires both a fairly rapid adhesion dynamics and a strong physical connection between adhesive sites and the cytoskeleton.Key words: actin retrograde flow, molecular clutch, myosin, N-cadherin, IgCAMGrowth cones are motile structures at the distal extremity of axons responsible for pathfinding and neurite extension during nervous system development and repair (Fig. 1A). Growth cone advance relies on two coupled processes. First, an internal dynamics of the cytoskeletal network, with actin polymerization occurring at the leading edge, depolymerization in the central region, and myosin activity pulling on lamellipodial actin filaments.¹ These integrated mechanisms altogether result in a continuous retrograde flow of actin (Fig. 1B). This flow provides the mechanical tension that drives axonal extension, through a connection to the dynamic array of microtubules that fills the axon and invades the growth cone central domain.² Second, there is repeated formation and dissociation of transient contacts between growth cones and the extracellular matrix or adjacent cells. These contacts are mediated by trans-membrane cell adhesion molecules (CAMs), e.g. integrins,³ immunoglobulin CAMs (IgCAMs)^4,5 and cadherins,^6,7 which form specific ligand/receptor bonds with variable lifetimes. A still open question is how these two processes, i.e. actin flow and adhesion dynamic, are coordinated at the growth cone level and contribute to set migration speed. A thorough understanding of these mechanisms is important both from a fundamental perspective and for the design of new compounds to foster axon regeneration after injury.Open in a separate windowFigure 1Growth cone advance and actin flow. (A) Growth cone from a 2 DIV rat hippocampal neuron plated on N-cadherin coated glass. This growth cone moved forward at a speed of about 1 µm/min (B) Raw fluorescence image of transfected actin-GFP. (C) Sequential actin-GFP images were subtracted, giving rise to intensity variations that display the movement of newly assembled actin (black). Note the rapid retrograde movement of actin spots (arrowheads), at a velocity of several µm/min.The coupling between actin-based motility and substrate adhesion has been shown for certain adhesion molecules such as NCAM and N-cadherin to involve a “molecular clutch” (Fig. 2). This mechanism implies a direct transmission of traction forces from the cytoskeleton to the substrate through a strong physical connection between the actin flow and ligand-bound adhesion receptors.^8,9 The connection is likely provided by adaptor proteins that can make transient bridges between actin filaments and the cytoplasmic domain of adhesion molecules, i.e. α-and β-catenin in the case of N-cadherin,¹⁰ ankyrin and ezrin in the case of IgCAMs such as L1.^11–14 These purely mechanical connections can also be accompanied by signalling events such as Rac-1 activation by N-cadherin liganding¹⁵ and phosphorylation of the L1 intracellular tail that regulates binding to ankyrin.^11,12 When only few molecular bonds are formed, e.g. at low ligand density, coupling to the actin flow is not strong enough, resulting in “slippage.” In this process, transient bonds can be formed and broken repeatedly between ligand-occupied adhesion receptors and the actin network. This is how the speed of growth cone translocation usually reaches at most 1 µm/min, whereas the internal actin flow rate proceeds at a rate of several µm/min (Fig. 1A and B). Such slippage is best demonstrated by the use of optical tweezers to impose low forces on ligand-coated microspheres presented to the growth cone dorsal surface (Fig. 3A). Beads tend to move rearward as they couple to the actin flow, and then suddenly snap back into the trap center, when receptor-cytoskeleton bonds break¹⁶ (the force of optical tweezers is usually not enough to rupture ligand-receptor bonds, which remain intact at the cell surface). Thus, a step in which a nucleating cluster of adhesion receptors recruits a minimal number of intracellular partners allowing coupling to the actin flow, can be a rate-limiting factor in growth cone progression.Open in a separate windowFigure 2Molecular components involved in growth cone migration. (A) Top view diagram showing filopodia which sense the environment, a flat lamellipodium which is the site of actin dynamics and the thicker central domain and axon which contain dynamic microtubules. The plus signs are sites of actin polymerization and the minus signs indicate actin depolymerization. (B) Side view showing the life cycle of ligand/receptor adhesions.Open in a separate windowFigure 3Optical tweezers and FRAP experiments to measure ligand/receptor and receptor/cytoskeleton dynamics. (A) Optical tweezers experiments performed on ligand-coated beads placed on the growth cone dorsal surface.¹⁶ (B) The distance traveled rearward with respect to the trap center is measured. A 2 min trajectory is indicated in red. A pooled parameter called coupling index taking into account the latency for bead escape, as well as the mean velocity and lateral diffusion of the bead, measures the strength of receptor/cytoskeleton interactions. (C) FRAP experiments on membrane GFP-tagged molecules accumulated at ligand-coated microspheres having sedimented on growth cones. The fluorescence intensity is normalized to represent the receptor enrichment level at the bead contact. (D) The recovered intensity is fit by a diffusion/reaction model, which yields a collective equilibrium turnover rate of ligand/receptor bonds. In red is the average of a series of individual curves (grey).In contrast, when strong connection is formed and if the substrate is resistant enough, then the molecular clutch engages and the cell reacts. In growth cones from Aplysia bag cells, forces were imposed on microspheres coated with ApCAM (the homolog of vertebrate NCAM) using a microneedle to locally block the retrograde actin flow. This was systematically followed by a protrusion of the microtubule-rich central domain towards those stiff contacts and forward expansion of the actin-rich lamellipodium.⁹ These phenomena were later shown to be controlled by a src protein kinase.¹⁷ In the case of rat hippocampal neurons, a dramatic accumulation of actin at N-cadherin coated microspheres is observed when the latter are restrained from moving rearward by a microneedle.¹⁶ This phenomenon is mediated by a connection between N-cadherin and α-catenin, likely triggering local actin polymerization. By careful analysis of the bead trajectories at varying ligand densities and computation of the latency for bead escape when the optical trap is applied continuously, one can extract a quantitative index of receptor-cytoskeleton coupling (Fig. 3B). Overall, a strong correlation was observed between such coupling index and the velocity of growth cone migration on N-cadherin substrates, both by varying N-cadherin ligand density and by expressing mutated N-cadherin molecules, supporting the clutch concept.¹⁶ This mechanism is consistent with in vivo experiments showing that overexpression of the N-cadherin intracellular tail in retinal ganglion cells results in severely impaired axon outgrowth.¹⁸ As a negative example of the clutch model, beads coated with fibronectin (our unpublished data) or anti-α1 integrin antibodies³ couple weakly to the actin flow in growth cones while, in parallel, the migration of growth cones on fibronectin- or collagen-coated substrates is rather limited.^6,19Molecular mechanisms parallel to the “clutch” can also be involved in growth cone migration. For example, IgCAM adhesions can not only couple to the rearward actin flow but also to static components of the cytoskeleton. Indeed, a 30% fraction of TAG-1 or anti-L1 coated beads can stay immobile on the growth cone surface.^12,20 These contrasting behaviors are likely mediated by interactions between the IgCAM intracellular domain and different binding partners (ankyrin vs. ERM),¹³ and may be responsible for the pauses which alternate with phases of growth cone advance. Also, homophilic adhesions between molecules of cadherin-11 couple very weakly to the actin flow, but promote substantial growth cone migration when cadherin-11 is presented as a substrate. This effect seems to be mediated by an independent interaction with the FGF receptor, which triggers actin dynamic through a signaling cascade.^21,22As growth cones migrate, adhesion sites must be recycled at a rate that somehow matches the speed of migration. Adhesion turnover can be schematically decomposed in several sequential phases (Fig. 2B). (1) Initiation of a first single ligand/receptor bond powered by membrane diffusion²³ and followed by trapping through a key/lock interaction; (2) Formation of small adhesion clusters through the recruitment of more ligand/receptor pairs, and possibly stabilized by cis-oligomerization (cadherins through the same interface as the trans-dimer, IgCAMs through FnIII domains). These clusters might form very transiently and serve as sites of actin recruitment, as demonstrated for N-cadherin;¹⁶ (3) contact maturation and possible reinforcement by connection to the cytoskeleton (as demonstrated for integrins in fibroblasts²⁴); (4) Adhesion rupture, which can proceed through ligand/receptor dissociation triggered by cytoskeleton tension. Indeed, the intrinsic lifetime of ligand/receptor bonds such as cadherins, is sensitive to the mechanical force applied on them.²⁵ Furthermore, the loosening of receptor/cytoskeketon connections can cause inside-out rupture of ligand/receptor bonds. This was demonstrated for fibronectin/integrin interactions by the fact that when fibronectin coated-beads reach the base of a fibroblast lamellipodium, they spontaneously detach from the cell surface.²⁶ In the case of very sticky ligand/receptor interactions such as SynCAM homophilic adhesions,²⁷ this process can actually be a limiting step that slows down growth cone advance. Indeed, SynCAM couples very well to the actin flow, but is unable to support growth cone migration.¹⁶ Finally, adhesion rupture might also proceed through membrane rupture, the adhesion receptors being extracted from the cell membrane and left behind on the substrate (demonstrated for integrins at the tail of fibroblasts²⁸).These basic processes can be accompanied by more complex and active phenomena, e.g. involving forward surface transport as shown for NCAM²⁹ or internal trafficking in the case of L1.³⁰ By interacting with the clathrin adaptor AP-2 through a specific RSLE motif in its intracellular tail, L1 can undergo endocytosis in the central domain and exocytosis at the periphery of the growth cone.³⁰ This mechanism generates a density gradient of L1 molecules which accelerates the formation of bonds with a variety of ligands, including L1 itself. The use of an L1-GFP construct in which the N-terminal GFP could be rapidly cleaved off by thrombin, together with L1-Fc microspheres manipulated by optical tweezers showed that local exocytosis of L1-rich vesicles at the growth cone periphery indeed participates in enhancing the formation of L1 homophilic contacts.³¹ We did not observe such internal traffic for N-cadherin within the growth cone, partly because of a difficulty to introduce a fluorescent protein tag in the ectodomain, which otherwise perturbs the adhesive function. However, the use of an N-cadherin molecule with triple mutation in the juxta-membrane domain that abolishes binding to p120 catenin, involved in the export of N-cadherin to the cell surface, suggested that recycling events might also play a role.³²By measuring the fluorescence recovery after photobleaching (FRAP) of GFP-tagged receptors transiently trapped at ligand-coated microspheres and analyzing the curves using a diffusion/reaction model, we were able to compute the equilibrium turnover rates of ligand/receptor pairs in controlled adhesive contacts involving many simultaneous bonds (Fig. 3C and D). We found that mature L1 homophilic adhesions recycle fast compared to other IgCAMs such as TAG-1/NrCAM adhesions,²⁰ likely owing to the specific internalization motif present in L1. Indeed, the recycling rate was reduced by a factor of 3 after truncation of the L1 intracellular tail, which prevented endocytosis.³¹ N-cadherin homophilic adhesions have an intermediate turnover rate, which is sensitive to the binding to catenin partners.³² Using these measurements as well as data from the literature, we plotted the impact of both receptor-cytoskeleton coupling and adhesion turnover rate on neurite outgrowth (Fig. 4A and B), which is strongly proportional to growth cone velocity.¹⁶ The graphs show that a strong coupling between ligand-occupied receptors and the actin flow is necessary, but not sufficient for neurite extension (Fig. 4C). Another requirement is that the turnover of ligand/receptor adhesions lies in an optimal range: not too high, otherwise bonds detach before coupling can occur, and not too slow either, since sticky bonds which do not rupture paralyze growth cone progression (Fig. 4D). A similar bell-shape curve between the strength of cell-substrate adhesion and cell migration speed was demonstrated for fibroblasts³³ and keratocytes,³⁴ indicating that these coupled mechanisms are fundamental to cell migration. To fully understand the quantitative relationship between adhesion turnover and the clutch process, it would be helpful to add data to this preliminary graph. For example, the extracellular matrix molecule laminin is known to support axon growth very efficiently but, to our knowledge, neither the coupling to the actin flow in growth cones or the adhesive turnover rate of integrins has been evaluated yet. Conversely, NCAM was shown to couple well to the actin flow³⁵ and induce neurite outgrowth,^4,36 but measurements of the lifetime of NCAM homophilic adhesions within growth cones are still lacking.Open in a separate windowFigure 4Impact of ligand/receptor turnover rate and receptor/cytoskeletal coupling on neurite outgrowth. (A and B) Example of 2 DIV rat hippocampal neurons plated on N-cadherin-Fc coated substrate and transfected at 1 DIV with N-cadherin-GFP. (A) DIC image. (B) Fluorescence image. The longest neurite, most likely the axon, is outlined by arrowheads. (C and D) In both graphs, the y-axis represents the longest neurite length after two days plating on ligand-coated glass. (Red) Rat hippocampal neurons transfected with either wild type or mutated N-cadherin molecules, interacting with purified N-cadherin ligands.^16,32 The scale in red intensity represents from dark to light: wild type N-cadherin, N-cadherin deleted of the whole ectodomain, N-cadherin truncated in the C-terminal region binding to β-catenin, N-cadherin with triple mutation in the juxta-membrane domain interacting with p120, and wild type N-cadherin in the presence of cytochalasin D. (Blue) Neurons transfected with either wild type L1 (dark) or L1 truncated in the intracellular tail (light), interacting with purified L1.³¹ Neurite growth on L1 was estimated from references.^7,14,30 (Grey) Interaction between endogenous SynCAM1 molecules expressed on growth cones and SynCAM-Fc ligands coated on microspheres or flat glass.¹⁶ The turnover of SynCAM homophilic interactions was estimated from SynCAM-coated Quantum dots detaching from neurons transfected with SynCAM1.⁴² (Green) Neuroblastoma cells expressing NrCAM-GFP in contact with TAG-1 coated microspheres.²⁰ DRG neurite growth on TAG-1 was taken from reference.⁴³ (Orange) The coupling index was taken from optical tweezers experiments using anti-β1 integrin coated beads interacting with DRG neurons,³ the turnover rate was inferred from FRAP experiments on fibroblast focal contacts⁴⁴ and neurite growth on fibronectin was estimated from reference.¹⁹ We omitted statistics for clarity. The SEM are usually in the order of 5–15% of the mean, for sample sizes of typically 20–30 beads (coupling index and turnover rate) and 40–100 transfected cells (neurite length).One important question is how these observations obtained from simplified in vitro systems using stiff substrates of well-defined geometry coated with specific purified proteins at controlled density, translate to the in vivo situation. There, the 3D substrate is comprised of extracellular matrix and multiple cell types, co-expressing many different CAMs that can bind simultaneously in various stoechiometries and also generating local gradients of chemo-attractant and chemo-repulsive signals.³⁷ Substrate flexibility is probably an important factor, since axons grow more slowly when neurons are plated on a layer of fibroblasts expressing CAMs²¹ than when molecules are immobilized on a substrate.^16,30 This preference for cells to move on stiff substrates, called durotaxis, has been well described for fibroblasts.³⁸ Another specific feature of the in vivo situation is the existence of decision points, often correlated with the presence of guidepost cells where growth cones make a pause and often change shape and reorient before turning to another direction.³⁹ This type of behavior has been successfully mimicked in vitro using artificial guideposts made of fibronectin or laminin coated microspheres.⁴⁰ Whereas growth cones display a fairly continuous displacement on a homogeneous substrate,¹⁶ the presence of these guideposts make growth cones either slow down, pause or even collapse, or conversely accelerate, depending on the CAM grafted on the bead.^40,41 Finally, the shape itself of the growth cone can be an indicator of its motile state:³⁹ this is also true in vitro where small growth cones are often the most rapid whereas large and flat growth cones stay rather immobile. Thus, although the in vivo situation seems at first sight awfully complex, some general trends can be explained given a small number of interacting molecular species and rather simple bio-chemical and mechanical models.In conclusion, the dynamic regulation of growth cone advance can take place at several levels: (1) the actin-associated proteins controlling actin dynamics (nucleation, polymerization, sequestering, branching); (2) the activity of motors pulling on the actin network, generating the retrograde flow; (3) the intracellular adaptor proteins that link actin to the CAMs; (4) the membrane delivery and retrieval of CAMs; (5) the ligand/receptor interaction properties themselves; and (6) the processes regulating microtubule assembly and microtubule/actin interactions at the base of the growth cone. The orchestration in time and space of all these processes generates the movement and reactivity of growth cones necessary to lead axons to their target cells.