It cuts two ways: microtubule loss during Alzheimer disease

EMBO J 32: 2920–2937 10.1038/emboj.2013.207; published online September242013Microtubule loss from axons and dendrites is a key contributor to nervous system degeneration during Alzheimer disease. Previous evidence suggested a simple pathway by which tau dissociation from microtubules in the axon allows excess severing of microtubules by katanin. Now, new evidence has emerged for a more complex pathway by which abnormal tau invasion into dendrites, triggered by Aβ oligomers, results in excess severing of microtubules by spastin.Alzheimer disease (AD) is a member of a category of neurodegenerative disorders called tauopathies (Wang and Liu, 2008). These are diseases of the nervous system in which tau becomes abnormally phosphorylated, and thereby detaches from microtubules. As the microtubules lose tau, they diminish in number and density, and this loss of microtubule mass negatively impacts the capacity of the neuron to maintain axonal transport and synaptic connections. Terms such as disintegrate or ‘fall apart'' are often used to describe the effect on the microtubules as they lose tau, but to date there has been very little information on how this happens. There is no mechanistic evidence to support the view that the microtubules become less stable and simply disassemble by their normal dynamic properties.One possibility is that tau normally protects microtubules from being destroyed by various proteins in the axon that would otherwise cut them into pieces or in some other way break them down. This presumably reflects a physiological mechanism wherein the regulation of tau dissociation from the microtubule via signalling pathways controls when and where microtubule breakage normally occurs. When a pathological condition causes tau to detach from microtubules, they become extremely sensitive to such factors. In addition, there is strong evidence that the abnormal tau, whether soluble or filamentous, can elicit toxic gain-of-function effects on the axon (Wang and Liu, 2008).To make matters even more complex, AD is not a pure tauopathy. Beta amyloid (Aβ) accumulates abnormally in the brain during AD, and this prompts tau to become hyperphosphorylated and lose association with microtubules. However, the Aβ can also elicit microtubule loss, independent of tau dissociation from the microtubules. In AD, there is also a loss of microtubules from dendrites, and this introduces an additional degree of complexity. Tau is normally less enriched in dendrites than axons. In AD, tau invades dendrites abnormally through deregulation of its normal sorting mechanism, and this somehow leads to microtubule loss from dendrites (Zempel et al, 2010).Microtubule loss is a common end point of multiple pathways, some involving loss of tau function, others involving gain-of-function effects of abnormal tau, and still others working through tau-dependent Aβ toxicity. All of this is not to say that the effects on microtubules are the only reason or even the principal explanation for axonal degeneration in AD, but the loss of microtubules is an important contributor to nervous system degeneration. Preventing or reversing the effects on microtubules could help stave off degeneration and hence provide patients with additional years of cognitive health and better quality of life.Microtubule assembly and disassembly occur from the ends of a microtubule, mainly (and often exclusively) at the plus end of the microtubule in living cells. Proteins that regulate microtubule stability affect the rate of these dynamics at microtubule ends. In recent years, a great deal of attention has focused on a category of proteins, termed microtubule-severing proteins. These proteins are enzymes that yank at the microtubule anywhere along its length to pull out a tubulin subunit, and thereby ‘cut'' the microtubule by causing it to break into pieces (Roll-Mecak and Vale, 2008). If the microtubule is sufficiently stable in the region of the break, the parent microtubule is cut into two shorter microtubules that persist, with minimal disassembly of either of the two pieces. If a microtubule is severed in its more labile region, the breakage could cause a great deal of disassembly. If the tubulin being yanked is situated at one of the ends of a microtubule, the result would be a shortening of the microtubule from that end; that is, disassembly. Thus, microtubule severing in the axon can certainly lead to microtubule loss, either by cutting the polymer all the way to subunits, inducing disassembly directly from microtubule ends or promoting disassembly as a secondary effect to the cutting.To date, the AAA enzymes katanin and spastin are the best studied of the microtubule-severing proteins (Yu et al, 2008). Spastin was originally identified as the product of the gene whose mutations are the chief cause of hereditary spastic paraplegia. Curiously, neurons express levels of spastin and katanin that are theoretically high enough to completely sever all of the microtubules in the neuron to subunits (Solowska et al, 2008), and yet this does not happen. Various regulatory mechanisms presumably keep the activities of the severing proteins in check. One of these mechanisms, in the case of katanin, is microtubule-bound tau, which protects the microtubule from being accessed by katanin (Qiang et al, 2006).Could microtubule loss in AD be due, at least in part, to excess microtubule severing due to deregulation of microtubule-severing proteins? We have posited that heightened severing of the microtubules by katanin, as the microtubules lose association with tau, is a contributing factor to the degradation of microtubules in the axons of AD patients (Sudo and Baas, 2011). A role for spastin in this pathway is questionable, because tau does not appear to protect microtubules against spastin as effectively as it does against katanin (Qiang et al, 2006). However, we now know that spastin is far from irrelevant to AD, as an exciting new article from the Mandelkow and Dawson laboratories implicates spastin in an entirely different pathway for microtubule loss in AD (Zempel et al, 2013). Whereas the katanin pathway is more relevant to axons, this new spastin pathway is more relevant to dendrites.In this new work, Zempel et al (2013) exposed mature primary neurons to oligomers of Aβ and observed microtubule breakdown in dendrites that had been invaded by tau. They found that the missorting of tau leads to an elevation of TTLL6 (Tubulin-Tyrosine-Ligase-Like-6) in dendrites, and this results in a marked increase in the polyglutamylation status of the microtubules. Because spastin has a strong preference for polyglutamylated microtubules, the microtubules become more sensitive to spastin-induced severing. Exactly why katanin is not a factor remains unclear, as polyglutamylation renders microtubules more sensitive to both of the severing proteins, not just spastin (Lacroix et al, 2010). Perhaps some of the tau that invades the dendrite is able to bind to microtubules and protect them from katanin, or perhaps katanin is less potent in dendrites because their microtubules are poorly acetylated, as katanin prefers acetylated microtubules to unacetylated ones (Sudo and Baas, 2011). Whatever the case, these new studies suggest that spastin, a protein whose mutations cause an entirely different neurodegenerative disease, is also a major factor in AD.What are the implications of these findings for AD treatment? In recent years, there have been encouraging results on rodent models for AD, in which behavioural improvement and enhanced neuronal vitality were observed when the animals were treated with drugs that stabilize microtubules against disassembly (Zhang et al, 2012). Such drugs are currently in clinical trials for AD (Barten et al, 2012). This strategy is based on the presumption that the microtubule loss that occurs in AD is due to destabilization of the microtubules. However, the results discussed here suggest that the primary cause of the microtubule loss could be something quite different, namely excess severing of microtubules. In this regard, it is relevant that both katanin and spastin seem to have a preference for severing stable microtubules (Lacroix et al, 2010; Sudo and Baas, 2011). Therefore, while treatment with a microtubule-stabilizing drug would mitigate disassembly that occurs as an aftereffect of microtubule severing, the severing events themselves would likely be increased (Figure 1). Heightened microtubule severing in axons and dendrites, even if the total levels of microtubule mass are preserved, could result in a gradual shift from a normal distribution of long and short microtubules to a predominance of microtubules too short to support sustained excursions of organelle transport. Long microtubules are also necessary as compression-bearing struts that prevent axons and dendrites from collapsing on themselves. We suspect that appropriate treatment regimes can be devised to prevent such dire consequences from happening, but we would advocate for the development of new drugs that inhibit microtubule-severing proteins. Such drugs may prove to be a better approach (on their own or in combination with a stabilizing drug) for preserving the fidelity of axonal and dendritic microtubules in AD patients. Given that the structure of the severing proteins is known, it may be straightforward to develop inhibitors, especially to their ATPase domains.Open in a separate windowFigure 1Microtubules in axons and dendrites consist of a stable region towards the minus end of the microtubule and a labile region towards the plus end, as well as a pool of free tubulin subunits. Microtubule severing is a normal event in the neuron, when tightly regulated. Abnormal (deregulated) microtubule severing is posited to account for microtubule loss in AD. Severing in the stable region of the microtubule would create two new microtubules, with fairly minimal disassembly of either one. Severing in the labile region of the microtubule would result in notably more disassembly. Severing at the end of the microtubule would result in disassembly. Because known microtubule-severing proteins favour the stable region of the microtubule, treatment of AD with a microtubule-stabilizing drug may mitigate disassembly that occurs as an aftereffect of the severing, but the severing events themselves would likely increase.     

Splenogonadal Fusion: An Unusual Case of an Acute Scrotum

We highlight a case on a normal left testicle with a fibrovascular cord with three nodules consistent with splenic tissue. The torsed splenule demonstrated hemorrhage with neutrophilic infiltrate and thrombus consistent with chronic infarction and torsion. Splenogonadal fusion (SGF) is a rather rare entity, with approximately 184 cases reported in the literature. The most comprehensive review was that of 123 cases completed by Carragher in 1990. Since then, an additional 61 cases have been reported in the scientific literature. We have studied these 61 cases in detail and have included a summary of that information here.Key words: Splenogonadal fusion, Acute scrotumA 10-year-old boy presented with worsening left-sided scrotal pain of 12 hours’ duration. The patient reported similar previous episodes occurring intermittently over the past several months. His past medical history was significant for left hip dysplasia, requiring multiple hip surgeries. On examination, he was found to have an edematous left hemiscrotum with a left testicle that was rigid, tender, and noted to be in a transverse lie. The ultrasound revealed possible polyorchism, with two testicles on the left and one on the right (Figure 1), and left epididymitis. One of the left testicles demonstrated a loss of blood flow consistent with testicular torsion (Figure 2).Open in a separate windowFigure 1Ultrasound of the left hemiscrotum reveals two spherical structures; the one on the left is heterogeneous and hyperdense in comparison to the right.Open in a separate windowFigure 2Doppler ultrasound of left hemiscrotum. No evidence of blood flow to left spherical structure.The patient was taken to the operating room for immediate scrotal exploration. A normalappearing left testicle with a normal epididymis was noted. However, two accessory structures were noted, one of which was torsed 720°; (Figure 3). An inguinal incision was then made and a third accessory structure was noted. All three structures were connected with fibrous tissue, giving a “rosary bead” appearance. The left accessory structures were removed, a left testicular biopsy was taken, and bilateral scrotal orchipexies were performed.Open in a separate windowFigure 3Torsed accessory spleen with splenogonadal fusion.Pathology revealed a normal left testicle with a fibrovascular cord with three nodules consistent with splenic tissue. The torsed splenule demonstrated hemorrhage with neutrophillic infiltrate and thrombus consistent with chronic infarction and torsion (Figure 4).Open in a separate windowFigure 4Splenogonadal fusion, continuous type with three accessory structures.     

Triple X Egyptian woman and a Down's syndrome offspring

The 47, XXX karyotype (triple X) has a frequency of 1 in 1000 female newborns. However, this karyotype is not usually suspected at birth or childhood. Female patients with a sex chromosome abnormality may be fertile. In patients with a 47, XXX cell line there appears to be an increased risk of a cytogenetically abnormal child but the extent of this risk cannot yet be determined; it is probably lower in the non-mosaic 47, XXX patient than the mosaic 46, XX/47, XXX one. We describe a new rare case of triple X woman and a Down''s syndrome offspring. The patient is 26 years of age. She is a housewife, her height is 160 cm and weight is 68 kg and her physical features and mentality are normal. She has had one pregnancy at the age of 25 years resulted in a girl with Down''s syndrome. The child had 47 chromosomes with trisomy 21 (47, XX, +21) Figure 1. The patient also has 47 chromosomes with a triple X karyotype (47, XX, +X) Figure 2. The patient''s husband (27 years old) is physically and mentally normal. He has 46 chromosomes with a normal XY karyotype (46, XY). There are neither Consanguinity between her parent''s nor she and her husband.Open in a separate windowFigure 1Karyotype 47, XX + 21 of the daughter of Triple X syndromeOpen in a separate windowFigure 2Karyptype 47, XX + X of the Down syndrome''s mother     

Significance of microtubule catastrophes at focal adhesion sites

Directional cell migration requires cell polarization and asymmetric distribution of cell signaling. Focal adhesions and microtubules are two systems which are essential for these. It was shown that these two systems closely interact with each other. It is known that microtubule targeting stimulates focal adhesion dissociation. Our recent study shows that focal adhesions, in turn, specifically induce microtubule catastrophe via a biochemical mechanism. We were able to track down one of the focal adhesion proteins paxillin which is involved in this process. Paxillin phosphorylation was previously shown to be the key component in the regulation of focal adhesion assembly or disassembly. Since microtubule catastrophe dynamic differs at the leading edge and cell rear, similar to paxillin phosphorylation levels, we suggest a model connecting asymmetric distribution of focal adhesions and asymmetric distribution of microtubule catastrophes at adhesion sites as a feedback loop.Key words: microtubule catastrophe, focal adhesion, microtubules, paxillin, cell motilityCell migration is important for many biological processes. It requires organized asymmetric dynamics of focal adhesions (FAs), sites where cells interact with extra cellular matrix. FAs appear at the leading edge as small transient dot-like structures termed focal complexes (FXs).^1,2 FX assembly and disassembly is regulated by phosphorilation status of paxillin a major FX protein.^3,4 Most of FXs form and disassemble rapidly. However, some adhesions mature in a force-dependent manner, into larger late adhesions. This process, involves both an increase in size and change in molecular composition^3,5 and is accompanied by a reduction in local paxillin phosphorylation.⁴ Late adhesions are more stable, immobile and undergo forced disassembly by multiple microtubule targeting events⁶ only underneath the approaching cell body or transform into fibrillar adhesions by a Src-dependent mechanism.⁷Similarly to the leading edge, proper adhesion patterns at the cell rear are also essential. Most trailing adhesions are initiated in protrusions at the rear and flanks of the cell as FX rapidly mature in response to tension and transform into sliding trailing adhesions.⁸ The process of sliding is complex. While adhesion proteins coupled with the actin cytoskeleton can be translocated relative to substratum, those that are associated with the membrane are thought to undergo treadmilling within the adhesion site.^9,10 Treadmilling, which includes disassembly of adhesion proteins at the distal end and reassembly at the proximal end,¹⁰ is accompanied by fusion with new adhesions formed in front of the sliding one.⁶ Thus, despite a protein composition similar to late adhesions, sliding adhesions are more dynamic. Not surprisingly, sliding adhesions have high paxillin phosphorylation at the distal end of the adhesion site, indicating very dynamic assembly/disassembly rates.⁴Several mechanisms have been proposed for the regulation of adhesion turnover (reviewed in ref. ¹¹). However, these have not accounted for the observed asymmetry of adhesion turnover. Understanding this requires examining the connection with another asymmetric intracellular system, the microtubule network. This dynamic network closely interacts with FAs. Microtubules play an essential role in cell migration and polarized distribution of signals within the cell. Multiple microtubule targeting to FA leads to their disassembly both at the leading edge and at the cell rear.⁶Unlike microtubule growth in other cell regions, growth at its leading edge is persistent, characterized by short periods of shrinkage.⁸ Simultaneous observation of microtubules and FAs show that microtubules specifically target adhesion sites.¹² More detailed analysis of microtubule dynamics reveals that FAs are preferable sites for microtubule catastrophes.¹³ Although FAs cover only about 5% of cell area more than 40% of catastrophes occur at these sites. The likelihood of microtubule catastrophe is seven times higher when a microtubule grows through a FA rather than through an adhesion-free area¹³ and about 90% of microtubules approaching adhesion sites undergo catastrophe. Although most of the catastrophes occur at late adhesions, due to their increased stability and lifespan, there is no difference in efficiency of catastrophe induction between small focal complexes and large rigid late adhesions.¹³ As FX do not have dense adhesion or actin plaque, it is likely that microtubule catastrophe is triggered by a biochemical mechanism rather than mechanical rigidity. This is also supported by the fact that mechanical obstacles in a cell do not necessarily cause microtubule catastrophe.¹³At the cell rear, microtubule dynamics differ from those at the leading edge. Microtubules spend less time in a growing phase and more time in pauses and shrinkage.⁸ Polymerization and depolymerization occur within a very limited area close to the cell edge.⁸ Live-cell imaging of cells expressing both microtubule and focal adhesion markers show that this complex dynamic sequence often happens within a single sliding adhesion. Microtubules that are captured at the proximal end of adhesion undergo multiple repetitive catastrophes at the distal end (Fig. 1) accompanied by rescue at the capture site. Thus, the capture mechanism significantly increases the lifetime of a microtubule and ensures that repetitive catastrophes occur at the single adhesion. This scenario leads to high catastrophe frequency at the cell rear, resulting in intensive catastrophe-dependent regulation in this cell region.Open in a separate windowFigure 1Multiple microtubule catastrophes at the sliding adhesion. (A) Frame from TIRF video sequence of a fish fibroblast cell (CAR) co-transfected with GFP-tubulin (green) to visualize microtubules and Cherry-Zyxin (red) to mark focal adhesions. The boxed region is presented in the kymograph in (B). Bar, 10 µm. (B) Kymograph of microtubule dynamics at a trailing end focal adhesion. Top panel shows microtubule (MT) only. Bottom panel shows life history plot of MT (green line shows movement of MT end) in relation to focal adhesions (red). Arrows show catastrophes at the distal end of adhesion, arrowheads show capture at the proximal end of adhesion.Detailed analysis of microtubule catastrophe localization shows that they occur at the areas of FAs where paxillin is enriched and highly phosphorylated.^4,13 Paxillin was shown to interact with microtubules through its Lim2/Lim3 domain.¹⁴ Purified GST-Lim2/Lim3 fragment injected into the cell localizes to FAs, displacing endogenous paxillin.¹³ This leads to a 40% decrease in the number of microtubule catastrophe events at adhesion sites,¹³ indicating that paxillin is needed for catastrophe initiation.In summary, we conclude that microtubule catastrophes at focal adhesions are specific events that are triggered by a biochemical mechanism. This process involves the focal adhesion protein paxillin, which may serve as a docking site for microtubules and/or microtubule catastrophe factors. The nature of catastrophe factors remains to be clarified. Possible mechanisms include molecules which induce microtubule catastrophe directly, such as stathmin,¹⁵ or molecules which regulate catastrophe-inducing factors activity. Alternatively, catastrophe factors at adhesion sites could act by removing stabilizing factors from microtubule tips. Thus, allowing already active catastrophe-inducing molecules such as kinesin-13 family member MCAK^16,17 to complete their function. Furthermore, microtubule catastrophe at paxillin-enriched areas, followed by release of microtubule-associated factors, may be involved in paxillin phosphorylation. This local regulation of adhesion disassembly would close the feed-back loop to microtubule regulation of FA turnover.In this model, asymmetric distribution of microtubule catastrophes is tightly linked to asymmetric regulation of FA. Since asymmetric FA dynamics in a cell are critical for organization of the actin cytoskeleton, tensile force distribution and directional cell migration, we conclude that microtubule catastrophes serve as important regulatory events for asymmetric signaling and dynamics of the whole cell (Fig. 2).Open in a separate windowFigure 2Model for asymmetric focal adhesion and microtubule dynamics. Focal complexes at the leading edge either disassemble or mature in response to tension. Microtubules undergo catastrophe both at focal complexes and late adhesions. Late adhesions disassemble in response to multiple microtubule targeting. At the cell rear a microtubule is captured at the proximal end of sliding adhesion and undergoes multiple catastrophes at its distal end, supporting disassembly of this region.     

ERECTA controls low light intensity-induced differential petiole growth independent of phytochrome B and cryptochrome 2 action in Arabidopsis thaliana

Plants can respond quickly and profoundly to changes in their environment. Several species, including Arabidopsis thaliana, are capable of differential petiole growth driven upward leaf movement (hyponastic growth) to escape from detrimental environmental conditions. Recently, we demonstrated that the leucine-rich repeat receptor-like Ser/Thr kinase gene ERECTA, explains a major effect Quantitative Trait Locus (QTL) for ethylene-induced hyponastic growth in Arabidopsis. Here, we demonstrate that ERECTA controls the hyponastic growth response to low light intensity treatment in a genetic background dependent manner. Moreover, we show that ERECTA affects low light-induced hyponastic growth independent of Phytochrome B and Cryptochrome 2 signaling, despite that these photoreceptors are positive regulators of low light-induced hyponastic growth.Key words: hyponastic growth, petiole, Arabidopsis, low light, ERECTA, differential growth, phytochrome B, cryptochrome 2Plants must adjust growth and reproduction to adverse environmental conditions. Among the strategies that plants employ to escape from unfavorable conditions is differential petiole growth-driven upward leaf movement, called hyponastic growth. Arabidopsis thaliana is able to exhibit a marked hyponastic response upon flooding, which is triggered by endogenous accumulation of the gaseous phytohormone ethylene.¹ Moreover, a similar response is triggered upon low light intensity perception and in response to supra-optimal temperatures.^2–5 By tilting the leaves to a more vertical position during submergence and shading, the plants restore contact with the atmosphere and light, respectively. The kinetics of the hyponastic growth response induced by the various stimuli is remarkably similar. This led to the hypothesis that shared functional genetic components may be employed to control hyponastic growth. Yet, at least part of the signaling cascades is parallel, as the hormonal control of the response differs between the stimuli. Low light-induced hyponastic growth for example does not require ethylene action.² Whereas the response to heat is antagonized by this hormone.⁵ The abiotic stress hormone abscisic acid (ABA) antagonizes ethylene-induced hyponastic growth and stimulates heat-induced hyponastic growth.^5,6 Moreover, ethylene-induced hyponasty does not involve auxin action⁷ whereas both heat- and low light-induced hyponasty require functional auxin signaling and transport components.^2,5In our recent paper, published in The Plant Journal,⁸ we employed Quantitative Trait Locus (QTL) analysis to identify loci involved in the control of ethylene-induced hyponastic petiole growth. By analyzing induced mutants and by complementation analysis of naturally occurring mutant accessions, we found that the leucine-rich repeat receptor-like Ser/Thr kinase gene ERECTA (ER) is a positive regulator of ethylene-induced hyponastic growth and most likely is causal to one of the identified QTLs. In addition, we demonstrated that the ER dependency is not via ER mediated control of ethylene production or sensitivity.Since low light-induced hyponasty does not require ethylene action,² ER may be part of the proposed shared signaling cascade leading to hyponastic growth where ethylene and low light signals meet. Therefore, we studied low light intensity-induced hyponasty in various erecta mutants. Moreover, natural occurring er mutant accessions complemented with a functional, Col-0 derived, ER allele were tested. The response of Lan-0 (Lan-0; with functional ER) to low light was indistinguishable from the response of Landsberg erecta (Ler) (Fig. 1A). However, complemented Ler (ER-Ler) showed an enhanced response compared to Ler (Fig. 1B). The response of mutant er105 was slightly attenuated compared to the wild type Columbia-0 (Fig. 1C). Mutant er104, however, showed an indistinguishable hyponastic growth phenotype to low light compared to the wild type Wassilewskija-2 (Ws-2) (Fig. 1D). Complementation of the natural occurring erecta mutant accession Vancouver-0 (Van-0) resulted in an enhanced hyponastic growth response to low light (Fig. 1E), whereas this was not the case for Hiroshima-1 (Hir-1) (Fig. 1F). Together, these data suggest that ER acts as positive regulator of low light-induced hyponastic growth and therefore may be part of the shared signaling cascade towards differential petiole growth. Yet, the effect is strongly dependent on the genetic background since the effects were not observed in every accession tested.Open in a separate windowFigure 1ERECTA involvement in low light-induced hyponasty. Effect of exposure to low light (spectral neutral reduction in light intensity from 200 to 20 µmol m⁻² s⁻¹) on the kinetics of hyponastic petiole growth in Arabidopsis thaliana. (A) mutant (circles) Ler and wild type (dashed line) Lan-0, (B) Ler and Ler complemented (ER-; squares) with the Col-0 ERECTA allele (ER-Ler), (C) er105 and Col-0 wild type, (D) er104 and Ws-2 wild type, (E) natural mutant Van-0 and Van-0 complemented with the Col-0 ER allele (ER-Van-0), (F) natural mutant Hir-1 and Hir-1 complemented with the Col-0 ER allele (ER-Hir-1). Petiole angles were measured using time-lapse photography and subsequent image analysis. Data is pairwise subtracted, which corrects for diurnal petiole movement in control conditions. For details on this procedure, growth conditions and materials, transformation protocol, treatments, data acquirement and all analyses see.^1,8 Error bars represent standard errors; n ≥ 12.Phytochrome B (PhyB) and Cryptochrome 2 (Cry2) photoreceptor proteins are required for a full induction of low light-induced hyponastic growth.² We transformed the phyb5 cry2 mutant9 (Ler genetic background) with Col-0 derived ER. This complementation did not restore the ability of phyb5 cry2 to induce hyponastic growth to neither ethylene (data not shown) nor low light conditions (Fig. 2A). Mutant phyb5 cry2 plants have a typical constitutive shade avoidance phenotype, reflected by severely elongated organs. This includes enhanced inflorescence and silique length and thin inflorescences (Fig. 2B-D). Complementation with ER resulted in a significant additional effect on these parameters (Fig. 2B-D). Together, this suggests that ER is not an integral part of PhyB nor Cry2 signaling with respect to (hyponastic) growth. Moreover, PhyB and Cry2 control of plant architecture does not require ER action. Rather, ER seems to mediate growth via genetic interaction with light-reliant growth mechanisms, instead of being downstream of photoreceptor action. Studies on the effects of ER on shade avoidance responses and various hormone responses, including cytokinin and auxin, led to the similar conclusion, suggesting a possible role for ER as a molecular hub coordinating light- and hormone-mediated plant growth.^10,11 One could speculate that ER fine-tunes other (than light) environmental clues with light signaling components. A comparable conclusion was drawn previously for gibberellin (GA) reliant growth mechanisms, as er enhanced the negative effect on plant size of the short internode (shi) mutation12 and er represses the positive effect of the spindly mutation in a GA independent manner.¹³Open in a separate windowFigure 2Effects of ERECTA on light signaling. (A) Effect of exposure to low light (spectral neutral reduction in light intensity from 200 to 20 µmol m⁻² s⁻¹) on the kinetics of hyponastic petiole growth of Ler (dashed lines), the photoreceptor double mutant phyb5 cry2 (circles) and this mutant complemented with the Col-0 ERECTA (ER-phyb cry2; squares). For details see legend Figure 1. (B) Plant height, (C) silique length and (D) inflorescence stem thickness of the above mentioned lines. These parameters were measured when the last flower on the plant developed a silique. Plant height was measured from root/shoot junction to inflorescence top. Stem thickness was measured ∼1 cm above the root/shoot junction with a caliper and silique lengths were measured from representative pedicels in the top ∼10 cm of the main inflorescence stem. Error bars represent standard errors; n ≥ 12. Significance levels; *p < 0.05; **p < 0.01; ***p < 0.001; ns = non significant, by Students t-test.     

Creative destruction of the microtubule array

Comment on: Mukherjee S, et al. Cell Cycle 2012; 11:2359-66.Typical cells contain a dense array of microtubules that serves as a structural backbone and also provides a substrate against which molecular motor proteins generate force. Cells transitioning through the cell cycle or undergoing significant morphological changes must be able to tear apart the microtubule array and reconstruct it into new configurations, either partially or completely. The microtubule field was revolutionized in the 1980s with the introduction of the dynamic instability model,¹ now broadly recognized as a fundamental mechanism by which microtubule populations are reconfigured.² Dynamic instability involves the catastrophic disassembly of microtubules, generally from their plus ends, as well as the rapid reassembly of microtubules and selective stabilization of particular ones. Microtubules can be stabilized along their length by binding to various proteins and can be attached at their minus ends to structures such as the centrosome and “captured” at their plus ends by proteins in the cell’s cortex.² Given the contribution of these stabilizing and anchoring factors, additional mechanisms beyond dynamic instability are required to tear down previous microtubule structures so that new ones can be constructed. Borrowing from the field of economics, we refer to this as creative destruction.Various proteins such as stathmin³ and kinesin-13⁴ contribute to creative destruction by promoting loss of tubulin subunits from the ends of the microtubules. We find especially interesting a category of AAA enzymes called microtubule-severing proteins that use the energy of ATP hydrolysis to yank at tubulin subunits within the microtubule, thereby causing the lattice to break.⁵ If this occurs along the length of the microtubule, the microtubule will be severed into pieces. If this occurs at either of the two ends of the microtubule, the microtubule will lose subunits from that end. The first discovered and best-studied microtubule-severing proteins are katanin and spastin.Thanks to David Sharp and his colleagues at Albert Einstein College of Medicine, as well as other workers in the field, we now know that cells express at least five other AAA proteins with potential microtubule-severing properties, on the basis of sequence similarity to katanin and spastin in the AAA region.⁵ Two of these, called katanin-like-1 and katanin-like-2, are very similar to katanin. The three others are similar to one another, collectively termed fidgetins (fidgetin, fidgetin-like-1 and fidgetin-like-2). One possibility is that all seven of the microtubule-severing proteins are regulated similarly and are functionally redundant with one another. A more compelling possibility is that, while there is some functional redundancy, there is also a division of labor, with each severing protein displaying distinct properties and carrying out its own duties. Thus far, Sharp’s studies on mitosis support the latter scenario, with katanin, fidgetin and spastin having characteristic distributions within the spindle, resulting in unique phenotypes when depleted.⁶In a new article, Sharp’s group has confirmed that fidgetin has microtubule-severing properties. Interestingly, fidgetin depolymerizes microtubules preferentially from the minus end.⁷ In addition, the new work shows that in human U2OS cells, fidgetin targets to the centrosome, where most minus ends of microtubules are clustered, suggesting a scenario by which fidgetin suppresses microtubule growth from the centrosome as well as attachment to it. Consistent with this scenario, the authors show that experimental depletion of fidgetin reduces that speed of poleward tubulin flux as well as the speed of anaphase A chromatid-to-pole motion and also results in an increase in both the number and length of astral microtubules. Notably, this contrasts with katanin, which favors the plus ends of microtubules, for example, at the chromosome during cell division⁶ and at the leading edge of motile cells.⁸The authors close their article by pointing out that microtubule-severing is important beyond mitosis, for example, in the restructuring of the microtubule array in neurons and migrating cells, and we would point to plants as well.⁹ We previously described a mechanism called “cut and run,” wherein the severing of microtubules is important for motility within the microtubule array, as short microtubules are more mobile than long ones.⁹ Now, inspired by the work of Sharp and colleagues, we envision “creative destruction” as another way of understanding the crucial roles played by a diversity of microtubule-severing proteins in cells.     

UV radiation reduces epidermal cell expansion in Arabidopsis thaliana leaves without altering cellular microtubule organization

Upon chronic UV treatment pavement cell expansion in Arabidopsis leaves is reduced, implying alterations in symplastic and apoplastic properties of the epidermal cells. In this study, the effect of UV radiation on microtubule patterning is analysed, as microtubules are thought to serve as guiding rails for the cellulose synthase complexes depositing cellulose microfibrils. Together with hemicelluloses, these microfibrils are regarded as the load-bearing components of the cell wall. Leaves of transgenic plants with fluorescently tagged microtubules (GFP-TUA6) were as responsive to UV as wild type plants. Despite the UV-induced reduction in cell elongation, confocal microscopy revealed that cellular microtubule arrangements were seemingly not affected by the UV treatments. This indicates an unaltered deposition of cellulose microfibrils in the presence of UV radiation. Therefore, we surmise that the reduction in cell expansion in UV-treated leaves is most probably due to changes in cell wall loosening and/or turgor pressure.Key words: arabidopsis, cell expansion, GFP-TUA6, leaf development, microtubule cytoskeleton, UV radiationPhotosynthetic functions such as solar light capture and carbon fixation are highly evolved features of plant leaves. To fulfil these functions in an optimal way, leaf development needs to be tuned to environmental conditions. Leaves are continuously exposed and subjected to environmental influences, which serve as co-regulators of leaf and plant development.¹ This ability of plants to adapt, secures the plant''s survival, even under non-optimal conditions. An example of a regulatory environmental parameter is solar light, indispensable for photosynthesis but potentially causing photoinhibition and/or UV-radiation stress. The highly energetic ultraviolet B (UV-B) rays of short wavelengths (280–315 nm) can both cause damage, as well as induce a range of specific metabolic and morphogenic plant responses. It was reported before that exposure to low dose UV radiation reduces Arabidopsis leaf size due to a decreased cell size.² Expansion of leaf epidermal cells of Arabidopsis thaliana is the combined action of promotion and restriction of growth, resulting in the typical irregular sinuous pavement cells. It has been postulated that cellulose microfibrils are responsible for generating a force opposing isotropic expansion by creating neck regions in between outgrowing lobes.³ As the microtubule cytoskeleton is believed to serve as guiding rails for the cellulose synthase complexes (CESAs),⁴ the deposition of the cellulose fibrils is intimately linked to the cortical microtubule arrangement. We have studied the UV-effect on microtubule organisation in leaf epidermal cells whose expansion had decreased upon this UV radiation. Microtubules in the adaxial pavement cells of the fourth leaf were monitored on several successive days in a transgenic line containing GFP fused to tubulin A6.⁵ The chronic UV treatment was started on day 0 when the plants were 2 weeks old, using UV exposure conditions as described in reference ². First the responsiveness of the GFP-TUA6 plants to UV radiation was evaluated. Similar to wild type (WT) plants,² the GFP-TUA6 plants had smaller leaves following 8 days of UV treatment (t-test, p < 0.01) (Fig. 1). This was caused by a significant reduction in the generalized cell area average of all measured cells, irrespective of the location within the leaf (Fig. 1; t-test, p < 0.01). In more detail, the average cell area within the base, middle and top zones of the GFP-TUA6 leaf was systematically lower in UV-treated leaves from 8 days after the treatment started onwards (data not shown).Open in a separate windowFigure 1Effect of UV radiation on leaf and cell area after different days of UV radiation. Open asterisks indicate a statistically significant difference in leaf area between UV-treated and control plants, black asterisks indicate statistically significant difference in cell area (t-test, *p < 0.05, **p < 0.01, ***p < 0.001). Error bars indicate the standard error for five different leaves at all measured time-points and 600, 170 and 180 cells at day 0, 8 and 12 respectively.As GFP-TUA6 leaves were as responsive to UV radiation as wild type leaves, confocal microscopy was used to visualize the organisation of the cortical microtubules facing the outer periclinal wall of the adaxial epidermis. No clear difference in microtubule (re)organization could be detected during the development of pavement cells, and throughout the UV treatment period. As shown in Figure 2 at day 2, pavement cells with comparable areas are similarly shaped in control and UV-irradiated plants and contain similar microtubule arrangements (Fig. 2 and marked cells). This means that microtubule organization is not directly affected by the UV exposure and that shape development proceeds in an analoguous manner as under control conditions. This lack of alteration in the microtubule arrangement can be observed for cells at the leaf tip, which were already in the process of lobe formation at the start of the exposure period, as well as for cells at the leaf base. Under our growth conditions, and in the monitored leaf number 4, cell proliferation still took place in this part of the leaf and lobes only started to appear on the cell surface. As microtubules are linked to the deposition of cellulose microfibrils, it can be assumed that no alterations in cellulose deposition occur upon UV treatment either. We can therefore conclude that the process of lobe formation and microtubule patterning is not impeded and that only the extent of cell expansion is restricted upon UV exposure.Open in a separate windowFigure 2Microtubule pattern in control and UV-exposed leaves visualized using GFP-TUA6 and confocal microscopy. Both images are from cells at the mid zone of the fourth leaf at day 2. Microtubules are similarly arranged in equally shaped and sized cells of control and UV-exposed leaves. The marked cells show a pattern whereby the tubules are centred in the neck regions between two outgrowing lobes.According to the Lockhart equation,⁶ cell (wall) growth is modulated by wall biomechanics and turgor pressure. Concerning turgor pressure, no clear differences in this factor between UV-exposed and control plants of Lactuca sativa L.⁷ and Pisum sativum⁸ could be observed, reinforcing the idea that especially the modulation of cell wall properties is the main factor causing the observed UV-induced reduction in cell expansion. Some reports indicate differential expression of wall loosening enzymes like expansins or xyloglucan endotransglycosylase/hydrolases (XTHs),^9,10 or cell wall strengthening enzymes as particular peroxidases⁷ after UV exposure. Another key event could involve UV-mediated changes in the phenylpropanoid pathway, which may cause changes in the lignin biosynthesis. As shown by the literature^11–14 lignin may well be an important modulator of cell wall architecture in Arabidopsis and therefore alterations in lignin synthesis could form the basis for morphological modifications. Further research on the cell wall properties of UV-treated plants may resolve this uncertainty.As a general conclusion we can state that the patterning of microtubules is not altered, but that alterations in cell wall composition or arrangements are the most plausible candidates for the observed reduction in pavement cell expansion upon chronic UV treatment.     

Diversity in spindle morphology in Arabidopsis root tip

A primary function of the spindle apparatus is to segregate chromosomes into two equal sets in a dividing cell. It is unclear whether spindles in different cell types play additional roles in cellular regulation. As a first step in revealing new functions of spindles, we investigated spindle morphology in different cell types in Arabidopsis roots in the wild-type and the cytokinesis defective1 (cyd1) mutant backgrounds. cyd1 provides cells larger than those of the wild type for testing the cell size effect on spindle morphology. Our observations indicate that cell type (shape), not cell size, is likely a factor affecting spindle morphology. At least three spindle types were observed, including small spindles with pointed poles in narrow cells, large barrel-shaped spindles (without pointed poles) in wide cells, and spindles intermediate in pole focus and size in other cells. We hypothesize that the cell-type-associated spindle diversity may be an integral part of the cell differentiation processes.Key words: spindle pole, microtubule, morphogenesis, cell type, metaphaseThe cellular apparatus for chromosome segregation during mitosis is typically described as a spindle composed of microtubules and microtubule-associated proteins. Research on the structure and function of the spindle is usually conducted under the assumption that spindles are structurally the same or alike in different cell types in an organism. If the assumption is true, it would indicate that either the intracellular conditions in different dividing cells are very similar or the assembly and maintenance of the spindle are insensitive to otherwise variable intracellular conditions. But experimental evidence related to this assumption is relatively sparse.The root tip in Arabidopsis, as in other higher plants, contains dividing cells of different shapes and sizes. These cells include both meristem initial and derivative cells, with the former and latter being proximal and distal to the quiescent center, respectively.¹ The diversity in dividing cells in the root tip provides an opportunity for testing whether the spindles also exhibit diversity in morphology. To visualize the spindles at the metaphase stage in the root tip cells, we conducted indirect immunofluorescence labeling of the β-tubulin in single cells prepared from wild-type Arabidopsis (in Col-0 background) root tips as previously described in references ² and ³. The spindles in cells of different morphologies were then observed under a confocal laser scanning microscope.³ Three types of spindle were detected. The first type (Fig. 1A) was the smallest in width and length and had the most-pointed poles among the three types. The second type (Fig. 1B) was wider and longer than the first type but with less-pointed poles than the first type. The third type (Fig. 1C) was similar in height to the second type but lacked the pointed poles. In fact, the third type is shaped more like a barrel than a spindle. The first type was found in cells narrow in the direction parallel to the equatorial plane of the spindle, a situation opposite to that of the third type whose cells were wide in the equatorial direction. The wide cells containing the barrel-shaped spindles likely belonged to the epidermal layer in the root tip.¹ The second type was found in cells intermediate in width. Examples of metaphase spindles morphologically resembling the three types of spindles in Arabidopsis root can also be found in a previous report by Xu et al. even although spindle diversity was not the subject of the report.⁴ In Xu et al.''s report, type 1- or 2-like metaphase spindles can be identified in Figures 2B and 3A, and type 3-like metaphase spindles can be identified in Figures 1A and 3B. These observations indicate that at least three types of spindles exist in the root cells.Open in a separate windowFigure 1Spindles in wild-type root cells. (A) Type-1 spindle. (B) Type-2 spindle. (C) Type-3 spindle. The spots without fluorescence signals in the middle of the spindles are where the chromosomes were located. Scale bar for all the figures = 20 µm.Open in a separate windowFigure 2Spindles in cyd1 root cells. (A) Type-1 spindle. Arrows indicate the upper and lower boundaries of the cell. (B and C) Two type-2 spindles. (D and E) Two type-3 spindles. (F) DAPI-staining image corresponding to (E), showing chromosomes at the equatorial plane. Scale bar for the images = 20 µm.The above observations suggest that either the cell size or the cell type (shape) might be a factor in the type of spindle found in a specific cell. To further investigate the relationship between cell morphology and spindle morphology, we studied metaphase spindles in root cells of the cytokinesis defective1 (cyd1) mutant.⁵ Because the root cells in cyd1 were larger than corresponding cells in the wild type, presumably due to abnormal polyploidization prior to the collection of the root cells,^5,6 this investigation might reveal a relationship between increasing cell size and altered spindle morphology. A pattern of different spindle types in different cell types similar to that in the wild type was observed in cyd1 (Fig. 2). Figures 2A–C show narrow cells that contained spindles with pointed poles even though the spindles differed in size and focus. Figure 2D shows a barrel-shaped spindle in a wide cell, resembling Figure 1C in overall appearance. The large number of chromosomes at metaphase (more than the diploid number of 10) in Figure 2F indicates that the cells in Figure 2 were polyploid. These figures thus demonstrate that the enlargement in cell size did not alter the pattern of types 1 and 2 spindles in narrow cells, as well as type 3 spindles in wide cells. Moreover, the edges of the spindles in Figure 2B and E were similarly distanced to the cell walls in the equatorial plane, and yet they differ greatly in shape with the former being type 2 and the latter being type 3. This finding argues against that the cell width in the equatorial direction dictates the spindle shape. On the other hand, the cells in Figure 2B and E are obviously of different types. Taken together, these observations suggest that the spindle diversity in both wild type and cyd1 is associated with cell-type diversity.It is unclear whether the different spindle types have different functions in their respective cell types, in addition to the usual role for chromosome segregation. One possibility is that, at the ensuing telophase, the pointed spindles result in compact chromosomal congregation at the poles whereas the barrel-shaped spindles result in loose chromosomal congregation at the poles, which in turn may differentially affect the shape of the subsequently formed daughter nuclei and their organization. Different nuclear shape and organization are likely to be integrated into the processes that confer cell differentiation.     

Testicular Sclerosing Sertoli Cell Tumor: A Case Report and Review of the Literature

Sertoli cell tumors are very rare testicular tumors, representing 0.4% to 1.5% of all testicular malignancies. They are subclassified as classic, large-cell calcifying, and sclerosing Sertoli cell tumors (SSCT) based on distinct clinical features. Only 42 cases of SSCTs have been reported in the literature. We present a case of a 23-year-old man diagnosed with SSCT.Key words: Testicular neoplasm, Sertoli cell tumor, Sclerosing Sertoli cell tumorA 23-year-old man was referred to the Cleveland Clinic Department of Urology (Cleveland, OH) for an incidentally detected right testicular mass. The mass was identified during a work-up for transient left testicular discomfort. His only notable medical history was nephrolithiasis. There was no personal or family history of testicular cancer or cryptorchidism. On physical examination, he was a well-nourished, well-masculinized young man without gynecomastia. Testicular examination revealed normal volume and consistency bilaterally without other relevant findings. Testicular ultrasonography demonstrated an 8 mm × 6 mm × 6 mm hypoechoic, solid mass in the posterior right testicle with peripheral flow on color Doppler (Figure 1).Open in a separate windowFigure 1Testicular ultrasound demonstrating an 8 mm × 6 mm × 6 mm hypoechoic, solid mass in the posterior right testicle (blue arrows).The remainder of the ultrasound examination yielded normal results. Lactic dehydrogenase, B-human chorionic gonadotropin, and α-fetoprotein levels were all within the normal range. After a thorough review of the options, the patient was then taken to the operating room for inguinal exploration. Intraoperative ultrasound confirmed a superficial 8-mm hypoechoic testis lesion. A whiteyellow, well-demarcated nodule was widely excised and a frozen section was sent to pathology for examination. The frozen section examination revealed the lesion to be a neoplasm with differential diagnosis including sclerosing Sertoli cell tumor (SSCT), adenomatoid tumor, and a variant of Leydig cell tumor. Because the final diagnosis could not be determined from frozen section, the decision was made to perform a right radical orchiectomy. Pathologic examination revealed a grossly unifocal, well-circumscribed, white, firm mass of 0.8 cm. Microscopically the lesion was composed of solid and hollow tubules and occasional anastomosing cords distributed within the hypocellular, densely collagenous stroma. Although the lesion was somewhat well circumscribed, entrapped seminiferous tubules with Sertoli-only cells were present within the tumor (Figure 2). Tumor cells had pale or eosinophilic cytoplasm with small and dark nuclei with inconspicuous nucleoli. The tumor was confined to the testis and margins were negative. A diagnosis of SSCT was reached, supported by positive immunostain results for steroidogenic factor 1, focal inhibin, and calretinin expression, and negative stain results for cytokeratin AE1/AE3 and epithelial membrane antigen in the tumor (Figure 3). The postoperative course was unremarkable. Computed tomography scan of the abdomen and pelvis and chest radiograph were negative for metastatic disease.Open in a separate windowFigure 2Low-power examination revealing a well-circumscribed tumor composed of solid and hollow tubules and occasional anastomosing cords distributed within the hypocellular, densely collagenous stroma. Hematoxylin and eosin stain, original magnification ×40. (B) High-power examination. Note entrapped seminiferous tubules lacking spermatogenesis. Hematoxylin and eosin stain, original magnification ×100.Open in a separate windowFigure 3Nuclear expression of steroidogenic factor 1 in the tumor as well as benign Sertoli cells in entrapped seminiferous tubules (original magnification ×200). (B) Focal calretinin expression in the tumor (inhibin had a similar staining pattern; original magnification ×100).     

Mutational Biosynthesis of Novel Rapamycins by a Strain of Streptomyces hygroscopicus NRRL 5491 Disrupted in rapL,Encoding a Putative Lysine Cyclodeaminase

The gene rapL lies within the region of the Streptomyces hygroscopicus chromosome which contains the biosynthetic gene cluster for the immunosuppressant rapamycin. Introduction of a frameshift mutation into rapL by ΦC31 phage-mediated gene replacement gave rise to a mutant which did not produce significant amounts of rapamycin. Growth of this rapL mutant on media containing added l-pipecolate restored wild-type levels of rapamycin production, consistent with a proposal that rapL encodes a specific l-lysine cyclodeaminase important for the production of the l-pipecolate precursor. In the presence of added proline derivatives, rapL mutants synthesized novel rapamycin analogs, indicating a relaxed substrate specificity for the enzyme catalyzing pipecolate incorporation into the macrocycle.Rapamycin is a 31-member macrocyclic polyketide produced by Streptomyces hygroscopicus NRRL 5491 which, like the structurally related compounds FK506 and immunomycin (Fig. ​(Fig.1),1), has potent immunosuppressive properties (24). Such compounds are potentially valuable in the treatment of autoimmune diseases and in preventing the rejection of transplanted tissues (16). The biosynthesis of rapamycin requires a modular polyketide synthase, which uses a shikimate-derived starter unit (11, 20) and which carries out a total of fourteen successive cycles of polyketide chain elongation that resemble the steps in fatty acid biosynthesis (2, 27). l-Pipecolic acid is then incorporated (21) into the chain, followed by closure of the macrocyclic ring, and both these steps are believed to be catalyzed by a pipecolate-incorporating enzyme (PIE) (18), the product of the rapP gene (8, 15). Further site-specific oxidations and O-methylation steps (15) are then required to produce rapamycin. Open in a separate windowFIG. 1Structures of rapamycin, FK506, and immunomycin.The origin of the pipecolic acid inserted into rapamycin has been previously established (21) to be free l-pipecolic acid derived from l-lysine (although the possible role of d-lysine as a precursor must also be borne in mind) (9). Previous work with other systems has suggested several alternative pathways for pipecolate formation from lysine (22), but the results of the incorporation of labelled lysine into the pipecolate moiety of immunomycin (Fig. ​(Fig.1)1) clearly indicate loss of the α-nitrogen atom (3). More recently, the sequencing of the rap gene cluster revealed the presence of the rapL gene (Fig. ​(Fig.2),2), whose deduced gene product bears striking sequence similarity to two isoenzymes of ornithine deaminase from Agrobacterium tumefaciens (25, 26). Ornithine deaminase catalyzes the deaminative cyclization of ornithine to proline, and we have proposed (15) that the rapL gene product catalyzes the analogous conversion of l-lysine to l-pipecolate (Fig. ​(Fig.3).3). Open in a separate windowFIG. 2A portion of the rapamycin biosynthetic gene cluster which contains ancillary (non-polyketide synthase) genes (15, 27). PKS, polyketide synthase.Open in a separate windowFIG. 3(A) The conversion of l-ornithine to l-proline by ornithine cyclodeaminase (17). (B) Proposed conversion of l-lysine to l-pipecolic acid by the rapL gene product.Here, we report the use of ΦC31 phage-mediated gene replacement (10) to introduce a frameshift mutation into rapL and the ability of the mutant to synthesize rapamycins in the absence or presence of added pipecolate or pipecolate analogs.     

Arabinogalactan proteins (AGPs) related to pollen tube guidance into the embryo sac in Arabidopsis

Some AGP molecules or their sugar moieties are probably related to the guidance of the pollen tube into the embryo sac, in the final part of its pathway, when arriving at the ovules. The specific labelling of the synergid cells and its filiform apparatus, which are the cells responsible for pollen tube attraction, and also the specific labelling of the micropyle and micropylar nucellus, which constitutes the pollen tube entryway into the embryo sac, are quite indicative of this role. We also discuss the possibility that AGPs in the sperm cells are probably involved in the double fertilization process.Key words: Arabidopsis, arabinogalactan proteins, AGP 6, gametic cells, pollen tube guidanceThe selective labelling obtained by us with monoclonal antibodies directed to the glycosidic parts of AGPs, in Arabidopsis and in other plant species, namely Amaranthus hypochondriacus,¹ Actinidia deliciosa² and Catharanthus roseus, shows that some AGP molecules or their sugar moieties are probably related to the guidance of the pollen tube into the embryo sac, in the final part of its pathway, when arriving at the ovules. The evaluation of the selective labelling obtained with AGP-specific monoclonal antibodies (Mabs) JIM 8, JIM 13, MAC 207 and LM 2, during Arabidopsis pollen development, led us to postulate that some AGPs, in particular those with sugar epitopes identified by JIM 8 and JIM 13, can be classified as molecular markers for generative cell differentiation and development into male gametes.Likewise, we also postulated that the AGP epitopes recognized by Mabs JIM 8 and JIM 13 are also molecular markers for the development of the embryo sac in Arabidopsis thaliana. Moreover, these AGP epitopes are also present along the pollen tube pathway, predominantly in its last stage, the micropyle, which constitutes the region of the ovule in the immediate vicinity of the pollen tube target, the embryo sac.³We have recently shown the expression of AGP genes in Arabidopsis pollen grains and pollen tubes and also the presence of AGPs along Arabidopsis pollen tube cell surface and tip region, as opposed to what had been reported earlier. We have also shown that only a subset of AGP genes is expressed in pollen grain and pollen tubes, with prevalence for Agp6 and Agp11, suggesting a specific and defined role for some AGPs in Arabidopsis sexual reproduction (Pereira et al., 2006).⁴Therefore we continued by using an Arabidopsis line expressing GFP under the command of the Agp6 gene promoter sequence. These plants were studied under a low-power binocular fluorescence microscope. GFP labelling was only observed in haploid cells, pollen grains (Fig. 1) and pollen tubes (Fig. 2); all other tissues clearly showed no labelling. These observations confirmed the specific expression of Agp6 in pollen grains and pollen tubes. As shown in the Figures 1 and ​and2,2, the labelling with GFP is present in all pollen tube extension, so probably, AGP 6 is not one of the AGPs identified by JIM 8 and JIM 13, otherwise GFP light emission would localize more specifically in the sperm cells.⁵ So we think that MAC 207 which labels the entire pollen tube wall (Fig. 3) may indeed be recognizing AGP6, which seems to be expressed in the vegetative cell. In other words, the specific labelling obtained for the generative cell and for the two male gametes, is probably given by AGPs that are present in very low quantities, apparently not the case for AGP 6 or AGP 11.Open in a separate windowFigure 1Low-power binocular fluorescence microscope image of an Arabidopsis flower with the AGP 6 promoter:GFP construct. The labelling is evident in pollen grains that are being released and in others that are already in the stigma papillae.Open in a separate windowFigure 2Low-power binocular fluorescence microscope image of an Arabidopsis ovary with the AGP6 promoter:GFP construct. The ovary was partially opened to show the pollen tubes growing in the septum, and into the ovules. The pollen tubes are also labelled by GFP.Open in a separate windowFigure 3Imunofluorescence image of a pollen tube growing in vitro, and labeled by MAC 207 monoclonal antibody. The labelling is evident all over the pollen tube wall.After targeting an ovule, the pollen tube growth arrests inside a synergid cell and bursts, releasing the two sperm cells. It has recently been shown that sperm cells, for long considered to be passive cargo, are involved in directing the pollen tube to its target. In Arabidopsis, HAP2 is expressed only in the haploid sperm and is required for efficient pollen tube guidance to the ovules.⁶ The same could be happening with the AGPs identified in the sperm cells by JIM 8 and JIM 13. We are now working on tagging these AGPs and using transgenic plants aiming to answer to such questions.Pollen tube guidance in the ovary has been shown to be in the control of signals produced by the embryo sac. When pollen tubes enter ovules bearing feronia or sirene mutations (the embryo sac is mutated), they do not stop growing and do not burst. In Zea mays a pollen tube attractant was recently identified in the egg apparatus and synergids.⁷ Chimeric ZmEA1 fused to green fluorescent protein (ZmEA1:GFP) was first visible within the filiform apparatus and later was localized to nucellar cell walls below the micropylar opening of the ovule. This is the same type of labelling that we have shown in Arabidopsis ovules, using Mabs JIM 8 and JIM 13. We are now involved in the identification of the specific AGPs associated with the labellings that we have been showing.     

Actin bundling via LIM domains

The LIM domain is defined as a protein-protein interaction module involved in the regulation of diverse cellular processes including gene expression and cytoskeleton organization. We have recently shown that the tobacco WLIM1, a two LIM domain-containing protein, is able to bind to, stabilize and bundle actin filaments, suggesting that it participates to the regulation of actin cytoskeleton structure and dynamics. In the December issue of the Journal of Biological Chemistry we report a domain analysis that specifically ascribes the actin-related activities of WLIM1 to its two LIM domains. Results suggest that LIM domains function synergistically in the full-length protein to achieve optimal activities. Here we briefly summarize relevant data regarding the actin-related properties/functions of two LIM domain-containing proteins in plants and animals. In addition, we provide further evidence of cooperative effects between LIM domains by transiently expressing a chimeric multicopy WLIM1 protein in BY2 cells.Key words: Actin-binding proteins, actin-bundling, cysteine-rich proteins, cytoskeleton, LIM domainThe LIM domain is a ≈55 amino acid peptide domain that was first identified in 1990 as a common cystein-rich sequence found in the three homeodomain proteins LIN-11, Isl1 and MEC-3. It has since been found in a wide variety of eukaryotic proteins of diverse functions. Animals possess several families of LIM proteins, with members containing 1–5 LIM domains occasionally linked to other catalytic or protein-binding domains such as homeodomain, kinase and SH3 domains. In contrast, plants only possess two distinct sets of LIM proteins. One is plant-specific and has not been functionally characterized yet. The other one comprises proteins that exhibit the same overall structure as the animal cystein rich proteins (CRPs), i.e., two very similar LIM domains separated by a ≈50 amino acid-long interLIM domain and a relatively short and variable C-terminal domain (Fig. 1A). The mouse CRP2 protein was the first CRP reported to interact directly with actin filaments (AF) and to stabilize the latter.¹ Identical observations were subsequently described for the chicken CRP1 and tobacco WLIM1 proteins.^2,3 In addition, these two proteins were shown to arrange AF into cables both in vitro and in vivo and thus join the list of actin bundlers.Open in a separate windowFigure 1Domain maps for wild-type WLIM1 (A) and GFP-fused chimeric 3xWLIM1 (B). A. WLIM1 basically comprises a short N-terminal domain (Nt), two LIM domains (LIM1 and LIM2), an interLIM spacer (IL) and a C-terminal domain (Ct). B. 3xWLIM1 consists of three tandem WLIM1 copies. This chimeric protein has been fused in C-terminus to GFP and transiently expressed in tobacco BY2 cells.To identify the peptide domains of WLIM1 responsible for its actin-related properties/activities, we generated domain-deleted and single domain variants and submitted them to a series of in vivo and in vitro assays.⁴ Localization experiments established that both LIM domains are required to efficiently target the actin cytoskeleton in tobacco BY2 cells. High-speed (200,000 g) cosedimentation data confirmed that the actin-binding activity of WLIM1 relies on its LIM domains. Indeed, the deletion of either the first or the second LIM domain respectively resulted in a 5-fold and 10-fold decrease of the protein affinity for AF. Importantly, each single LIM domain was found able to interact with AF in an autonomous manner, although with a reduced affinity compared to the wild-type WLIM1. Low-speed (12,500 g) cosedimentation data and electron microscopy observations revealed that the actin bundling activity of WLIM1 is also triggered by its LIM domains. Surprisingly each single LIM domain was able to bundle AF in an autonomous manner, suggesting that WLIM1 has two discrete actin-bundling sites. However, the bundles induced by the variants containing only one LIM domain, i.e., LIM domain-deleted mutants and single LIM domains, differed from those induced by the full-length WLIM1. They appeared more wavy and loosely packed and formed only at relatively high protein:actin ratios. Together these data suggest that LIM domains are autonomous actin-binding and -bundling modules that function in synergy in wild-type WLIM1 to achieve optimal activities.To further assess the mechanism of cooperation between the LIM domains of plant CRP-related proteins, we generated a chimeric protein composed of three WLIM1 copies in tandem (3 × WLIM1, Fig. 1B), and transiently expressed it as a GFP-fusion in tobacco BY2 cells. We anticipated that such a six LIM domain-containing protein displays an even higher actin-bundling activity. (Fig. 2A) shows the typical actin cytoskeleton pattern in an expanding BY2 cell as visualized using the actin marker GFP-fABD2.⁵ As previously reported by Sheahan et al.,⁵ GFP-fABD2 decorated dense, transversely oriented, cortical networks as well as transvacuolar strands connecting the subcortical-perinuclear region to the cortex. Ectopic expression of WLIM1-GFP (BY2 cells normally do not express the WLIM1 gene) induced moderate but perceptible modifications of the actin cytoskeleton structure (Fig. 2B). Most AF are arranged in bundles thicker than those observed in GFP-fABD2 expressing cells and fine AF arrays are less frequently observed. As expected, this phenotype was significantly enhanced in cells transformed with the 3xWLIM1-GFP protein (Fig. 2C). Indeed, cells were almost devoided of fine AF arrays and exhibited very thick actin cables (Fig. 2C) that, at times (≈30 %), form atypical long looped structures (Fig. 2D). The appearance of such structures may result from the increase of cable stability and thickness induced by the 3xWLIM1-GFP protein, as these parameters are likely to determine, at least partially, the maximal length of actin bundles. Together the present observations support earlier data showing that LIM domains work in concert in LIM proteins to regulate actin bundling in plant cells. Strikingly, vertebrate and plant CRPs invariably contain two LIM domains. The lack, in these organisms, of CRP-related proteins combining more than two LIM domains may be explained by the fact that very thick cables, such as those induced by the artificial 3xWLIM1, may be too stable structures incompatible with the necessary high degree of actin cytoskeleton plasticity. As an exception, a muscle CRP-related protein with five LIM domains (Mlp84B) has been identified in Drosophila.⁶ However, rather than decorating actin filaments in an homogenous manner, this protein has been found to concentrate in a specialized region of the Z-discs where it stabilizes, in concert with D-titin, muscle sarcomeres.⁷Open in a separate windowFigure 2Typical actin cytoskeleton patterns in tobacco BY2 cells that have been transiently transformed, using a particle gun, with GFP-fABD2 (A), WLIM1-GFP (B), and 3xWLIM1-GFP (C and D). For each construct, more than 60 cells were analyzed by confocal microscopy. In the case of 3xWLIM1-GFP, two prevalent patterns have been observed (C and D). Bars = 20 µm.The relatively well conserved spacer length (≈50 amino acids) that separates the two LIM domains in vertebrate CRPs and related plant LIM proteins remains an intriguing feature the importance of which in actin cable organization remains to be established. Using electron microscopy we are currently evaluating the effects of the modification of the interLIM domain length on the structural properties of actin cables.