Where and when is microtubule diversity generated inParamecium? Immunological properties of microtubular networks in the interphase and dividing cells

Summary Ciliates are highly differentiated cells which display extensive deployment of microtubular systems. Because genetic diversity of tubulin is extremely reduced in these cells, microtubule diversity is mostly generated at the post-translational level either through direct modification of tubulin or through the binding of associated proteins to microtubules. We have undertaken a systematic exploration of microtubule diversity in ciliates by way of production of monoclonal antibodies. Previously we reported the biochemical characterization of these antibodies. In addition to antibodies directed against primary sequences, we obtained antibodies directed against post-translational modifications. In this paper, we report a detailed analysis of the distribution of the various epitopes on the microtubular networks ofParamecium, both in interphase cells and during division morphogenesis. Each of these antibodies decorates a subset of microtubules. Acetylation, recognized by antibodies TEU 318 and TEU 348, is detected on stable microtubules early after microtubule assembly. Epitopes recognized by two other antibodies (TAP 952 and AXO 58) are found on a subset of stable microtubules; in addition, the TAP 952 antibody is also found on labile microtubules; both epitopes are detected as soon as microtubule assembly occurs. In contrast, the epitope of the antibody, AXO 49, is associated with only a restricted subset of stable microtubules in the interphase cell, and is detected a lag-time after microtubule assembly during division morphogenesis. These data show that microtubule diversity is generated through a time-dependent sequence and according to a definite spatial pattern.     

Apoptosis in the heart: when and why?

Since mammalian cardiac myocytes essentially rely on aerobic energy metabolism, it has been assumed that cardiocytes die in a catastrophic breakdown of cellular homeostasis (i.e. necrosis), if oxygen supply remains below a critical limit. Recent observations, however, indicate that a process of gene-directed cellular suicide (i.e. apoptosis) is activated in terminally differentiated cardiocytes of the adult mammalian heart by ischemia and reperfusion, and by cardiac overload as well. Apoptosis or programmed cell death is an actively regulated process of cellular self destruction, which requires energy and de novo gene expression, and which is directed by an inborn genetic program. The final result of this program is the fragmentation of nuclear DNA into typical nucleosomal ladders, while the functional integrity of the cell membrane and of other cellular organelles is still maintained. The critical step in this regulated apoptotic DNA fragmentation is the proteolytic inactivation of poly-[ADPribose]-polymerase (PARP) by a group of cysteine proteases with some structural homologies to interleukin-1-converting enzyme (ICE-related proteases [IRPs] such as apopain, yama and others). PARP catalyzes the ADP-ribosylation of nuclear proteins at the sites of spontaneous DNA strand breaks and thereby facilitates the repair of this DNA damage. IRP-mediated destruction of PARP, the supervisor of the genome, can be induced by activation of membrane receptors (e.g. FAS or APOI) and other signals, and is inhibited by activation of anti-death genes (e.g. bcl-2). Overload-triggered myocyte apoptosis appears to contribute to the transition to cardiac failure, which can be prevented by therapeutic hemodynamic unloading. In myocardial ischemia, the activation of the apoptotic program in cardiocytes does not exclude their final destiny to catastrophic necrosis with release of cytosolic enzymes, but might be considered as an adaptive process in hypoperfused ventricular zones, sacrificing some jeopardized myocytes to regulated apoptosis, which may by less arrhythmogenic than necrosis with the primary disturbance of membrane function.     

Where and when does a ring start and end? Testing the ring-species hypothesis in a species complex of Australian parrots

Speciation, despite ongoing gene flow can be studied directly in nature in ring species that comprise two reproductively isolated populations connected by a chain or ring of intergrading populations. We applied three tiers of spatio-temporal analysis (phylogeny/historical biogeography, phylogeography and landscape/population genetics) to the data from mitochondrial and nuclear genomes of eastern Australian parrots of the Crimson Rosella Platycercus elegans complex to understand the history and present genetic structure of the ring they have long been considered to form. A ring speciation hypothesis does not explain the patterns we have observed in our data (e.g. multiple genetic discontinuities, discordance in genotypic and phenotypic assignments where terminal differentiates meet). However, we cannot reject that a continuous circular distribution has been involved in the group's history or indeed that one was formed through secondary contact at the 'ring's' east and west; however, we reject a simple ring-species hypothesis as traditionally applied, with secondary contact only at its east. We discuss alternative models involving historical allopatry of populations. We suggest that population expansion shown by population genetics parameters in one of these isolates was accompanied by geographical range expansion, secondary contact and hybridization on the eastern and western sides of the ring. Pleistocene landscape and sea-level and habitat changes then established the birds' current distributions and range disjunctions. Populations now show idiosyncratic patterns of selection and drift. We suggest that selection and drift now drive evolution in different populations within what has been considered the ring.     

Where are the Subtropics?

The tropics are clearly defined while ‘subtropics’ and ‘subtropical’ have been applied to a variety of zones, climates, and ecosystems. However, in the recent terrestrial biology literature, they have been used mainly for the zones between the tropics (±23.4^o C) and ±30.0^o. Adopting this standard would facilitate pan‐subtropical comparisons.     

SPARC and tumor growth: Where the seed meets the soil?

Matricellular proteins mediate interactions between cells and their extracellular environment. This functional protein family includes several structurally unrelated members, such as SPARC, thrombospondin 1, tenascin C, and osteopontin, as well as some homologs of these proteins, such as thrombospondin 2 and tensascin X. SPARC, a prototypic matricellular protein, and its homolog hevin, have deadhesive effects on cultured cells and have been characterized as antiproliferative factors in some cellular contexts. Both proteins are produced at high levels in many types of cancers, especially by cells associated with tumor stroma and vasculature. In this Prospect article we summarize evidence for SPARC and hevin in the regulation of tumor cell growth, differentiation, and metastasis, and we propose that matricellular proteins such as these perform critical functions in desmoplastic responses of tumors that culminate in their dissemination and eventual colonization of other sites.     

Where is the difference between the genomes of humans and annelids?

The first systematic investigation of an annelid genome has revealed that the genes of the marine worm Platynereis dumerilii are more closely related to those of vertebrates than to those of insects or nematodes. For hundreds of millions of years vertebrates have preserved exon-intron structures descended from their last common ancestor with the annelids.     

Breaching the basement membrane: who, when and how?

The basement membrane (BM), a specialized network of extracellular matrix macromolecules, surrounds epithelial, endothelial, muscle, fat and nerve cells. During development, immune surveillance and disease states ranging from cancer to fibrosis, host cells penetrate the BM by engaging tissue-invasive programs, the identity of which remain largely undefined. Although it is commonly assumed that all cells employ similar mechanisms to cross BM barriers, accumulating evidence indicates that cells might selectively mobilize protease-dependent or -independent invasion programs. New data indicate that protease-dependent transmigration is largely reliant on a group of membrane-anchored metalloenzymes, termed the membrane-type matrix metalloproteinases, which irreversibly remodel BM structure. By contrast, mechanisms that enable protease-independent transmigration remain undefined and potentially involve the reversible disassembly of the BM network. Further characterization of the molecular mechanisms underlying BM transmigration should provide important insights into pathophysiologic tissue remodeling events and also enable the development of novel therapeutics.     

Where will the wood come from? Plantation forests and the role of biotechnology

Wood is almost as important to humanity as food, and the natural forests from which most of it is harvested from are of enormous environmental value. However, these slow-growing forests are unable to meet current demand, resulting in the loss and degradation of forest. Plantation forests have the potential to supply the bulk of humanity's wood needs on a long-term basis, and so reduce to acceptable limits the harvest pressures on natural forests. However, if they are to be successful, plantation forests must have a far higher yield of timber than their natural counterparts, on much shorter rotation times. To achieve this in reasonable time, biotechnology must be applied to the tree-improvement process, for which large increases in public and private capital investment are needed. However, additional obstacles exist in the form of opposition to plantations, some forest ecocertification schemes, and concerns about aspects of forest biotechnology, especially genetic engineering. It is the intention of this article to explain, in detail, why plantation forests are needed to sustainably meet the world's demand for wood, why they are not being developed fast enough, and why the application of biotechnology to tree improvement is essential to speeding up this process.     

Where and how does phototropin transduce light signals in the cell?

Light plays pivotal roles as an important environmental signal in plant growth and development. In Arabidopsis, phototropin 1 (phot1) and 2 (phot2) are the photoreceptors that mediate phototropism, chloroplast relocation, stomatal opening and leaf flattening, in response to blue light. However, little is known about how phototropins transduce the signals after the light is perceived. Changes induced by blue light in terms of intracellular localization patterns of phot2 in Arabidopsis were examined. Phot2 distributed uniformly in the plasma membrane under dark conditions. Upon irradiation with blue light, some of the phot2 associated with the Golgi apparatus. It was also shown that the kinase domain, but not the photosensory domain, is required for a plasma membrane and Golgi localization. Furthermore a kinase fragment, lacking the photosensory domain, constitutively triggered physiological responses in planta. Thus, the plasma membrane and the Golgi apparatus appear to be the most likely sites for the initial step of phot2 signal transduction. The Golgi apparatus facilitates vesicle trafficking and delivery of membrane proteins to the required locations in the cell. Therefore, this study implicates the regulation of vesicle trafficking by the Golgi apparatus as a mechanism by which phot2 elicits its cellular responses.Key words: Golgi apparatus, kinase, light signal transduction, photoreceptor, phototropin, vesicle traffickingA range of physiological responses in plants is brought about by blue (390–500 nm) and ultraviolet-A (320–390 nm) light. Phototropin, one of major classes of blue light photoreceptors in plants, mediates responses such as phototropism, chloroplast relocation, light-induced stomatal opening and leaf flattening.^1–6 The dicotyledon Arabidopsis, possesses two phototropins, termed phot1 and phot2, which have both overlapping and distinct functions.^5,7 Phototropins consist of two functional domains, a N-terminal photosensory domain, containing two LOV (Light, Oxygen, Voltage) domains (LOV1 and LOV2) and a flavin-mononucleotide (FMN) chromophore and a regulatory serine/threonine kinase domain at the C-terminus.⁸To understand the mechanism of phototropin signal transduction, we expressed phot2 derivatives with translationally-fused green fluorescent protein (GFP) in a phot1phot2 double mutant in a wild type background in Arabidopsis.^9,10 Phototropin is a membrane- associated protein lacking a membrane spanning domain.⁸ Phot1 fused to GFP (P1G) is mainly localized to the plasma membrane, regardless of the light conditions.⁶ This property was retained when phot2 was fused to GFP (P2G).⁹ A part of P2G associates with punctate structures in the cytoplasm in response to blue light. The punctate P2G colocalized with KAM1ΔC:mRFP, a Golgi marker, we therefore conclude that phot2 associated with the Golgi apparatus in a blue light-dependent manner.⁹ This association was observed even in the presence of brefeldin A (BFA), an inhibitor of the vesicle trafficking.⁹To determine which domain of phot2 is responsible for the Golgi association, fragments of phot2 were fused to GFP and expressed in protoplasts.⁹ The N-terminal fragment fused to GFP (P2NG) was distributed uniformly in the cytoplasm. By contrast, the C-terminal fragment fused to GFP (P2CG) localized to both plasma membrane and punctate structures. The latter was shown to be the Golgi apparatus with the aid of the Golgi marker, KAM1ΔC:mRFP.⁹ These observations were corroborated from data using transgenic plants.¹⁰ Hence the C-terminal kinase domain, but not the N-terminal photo-sensory domain, is essential for the association of phot2 with the plasma membrane and the Golgi apparatus.The Golgi network is a key player in vesicle trafficking, to and from ER, vacuoles, trans-Golgi network, endosome and the plasma membrane.¹¹ Membrane spanning proteins are delivered and recycled through the Golgi apparatus. Among the membrane spanning proteins that are especially interesting, with respect to phototropin function, are auxin carriers such as PIN proteins. Phototropic curvature, which is under the control of phototropin, is believed to be caused by an uneven distribution of auxin.¹² The intracellular distribution of PIN proteins is maintained and regulated by vesicle trafficking.¹³ Indeed, factors such as ADP-ribosylation factor1 (ARF1) and guanine-nucleotide exchange factors (GEFs), which are involved in vesicle trafficking, are indispensable for the proper distribution of PIN proteins.^14–17 It is intriguing that a light stimulus alters the distribution pattern of PIN proteins.¹⁸ Hence, a fascinating possibility arises that phot2 alters the intracellular distribution of PIN proteins by regulating vesicle trafficking at the level of the Golgi apparatus.Phototropins are members of the subfamily VIII of AGC kinases.¹⁹ Interestingly, PINOID, another member of the subfamily, is localized at the cell periphery and regulates the apical-basal polar distribution of PIN proteins.^20–22 Accordingly, overexpression of PINOID disturbs the auxin distribution in transgenic plants.^23,24 The kinase fragment of phototropin exhibits constitutive kinase activity in vitro.²⁵ Interestingly, the auxin distribution is disturbed in plants expressing P2CG, as is the case with PINOID.¹⁰ Hence, both PINOID and phot2 might alter the PIN protein distribution in the cell through a common mechanism, in response to distinct stimuli.To date, no authentic substrate has been described for any of the AGC VIII kinases.¹⁹ Considering the localization pattern of phototropins, the substrates are most likely to reside in the plasma membrane and/or the Golgi apparatus. NPH3, RPT2 and PKS1 are downstream factors for phototropic responses,^26–28 all associating with the plasma membrane. Although they interact preferentially with the N-terminal rather than the C-terminal domain of phot1,^26,29 it is also possible that the C-terminal kinase domain interacts transiently with these factors leading to their phosphorylation. However, at present the molecular functions of NPH3, RPT2 and PKS1 remain unclear and await future investigation.Although both phot1 and phot2 are localized to the plasma membrane, punctate structures are yet to be described for P1G. Instead, a part of phot1-GFP is released from the plasma membrane to the cytosol in response to a light stimulus.⁶ We recently reexamined the intracellular localization of P1G. A specific network-like structure in the cytoplasm in addition to intense plasma membrane staining was observed (Fig. 1). A similar pattern was observed for P2G although it is less clear.⁹ Hence, both phot1 and phot2 might be associating with a structure in the cytoplasm that has yet to be described, and which might be another site of phototropin signaling in the cell.Open in a separate windowFigure 1A light-induced network-like distribution pattern of P1G in the cytoplasm. The P1G seedlings grown under dark conditions⁶ were incubated in MS solution (diluted 50%) without (upper panels) or with (lower panels) 100 µM BFA. The cells were inspected with a confocal laser scanning microscope. Images taken before (left) or after (right) blue light illumination at 48 µmol m⁻² sec⁻¹ are shown. Bar = 10 µm.P2CG elicits some phototropin responses without a light stimulus.¹⁰ That is, chloroplasts were in the avoidance position and stomata opened without a blue light stimulus in the P2CG overexpressing plants. It is a fascinating possibility that phototropin elicits those responses through the regulation of vesicle trafficking, although other possibilities exist. Stomata open as the result of phosphorylation of the plasma membrane H⁺-ATPase³⁰ and it is unlikely that the vesicle trafficking is directly involved in this regulatory process. It is possible to conjecture that vesicle trafficking affects chloroplast positioning but how this would work remains to be determined. Overall how a single photoreceptor such as phototoropin might regulate diverse physiological responses awaits future study.     

Where is the vitamin D receptor?

The vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and plays a central role in the biological actions of vitamin D. VDR regulates the expression of numerous genes involved in calcium/phosphate homeostasis, cellular proliferation and differentiation, and immune response, largely in a ligand-dependent manner. To understand the global function of the vitamin D system in physiopathological processes, great effort has been devoted to the detection of VDR in various tissues and cells, many of which have been identified as vitamin D targets. This review focuses on the tissue- and cell type-specific distribution of VDR throughout the body.     

Where do fish go when stranded on land? Terrestrial orientation of the mangrove rivulus Kryptolebias marmoratus

The goal of the present study was to determine which sensory cues the mangrove rivulus Kryptolebias marmoratus, a quasi-amphibious, hermaphroditic fish, uses to orient in an unfamiliar terrestrial environment. In a laboratory setting, K. marmoratus were placed on a terrestrial test arena and were provided the opportunity to move toward reflective surfaces, water, dark colours v. light colours, and orange colouration. Compared with hermaphrodites, males moved more often toward an orange section of the test arena, suggesting that the response may be associated with camouflage or male–male competition, since only males display orange colouration. Younger individuals also moved more often toward the orange quadrant than older individuals, suggesting age-dependent orientation performance or behaviour. Sloped terrain also had a significant effect on orientation, with more movement downhill, suggesting the importance of the otolith-vestibular system in terrestrial orientation of K. marmoratus. By understanding the orientation of extant amphibious fishes, we may be able to infer how sensory biology and behaviour might have evolved to facilitate invasion of land by amphibious vertebrates millions of years ago.