Immobilization of the Glycosylphosphatidylinositol-anchored Gas1 protein into the chitin ring and septum is required for proper morphogenesis in yeast

Gas1p is a glucan-elongase that plays a crucial role in yeast morphogenesis. It is predominantly anchored to the plasma membrane through a glycosylphosphatidylinositol but a fraction was also found covalently bound to the cell wall. We have used fusions with the green or red fluorescent proteins (GFP or RFP) to determine its localization. Gas1p was present in microdomains of the plasma membrane, at the mother-bud neck and in the bud scars. By exploiting the instability of RFP-Gas1p we identified mobile and immobile pools of Gas1p. Moreover, in chs3\uf044 cells the chitin ring and the cross-linked Gas1p were missing but this unveiled an additional unexpected localization of Gas1p along the septum line in cells at cytokinesis. Localization of Gas1p was also perturbed in a chs2\uf044 mutant where a remedial septum is produced. Phenotypic analysis of cells expressing a fusion of Gas1p to a trans-membrane domain unmasked new roles of the cell wall-bound Gas1p in the maintenance of the bud neck size and in cell separation. We present evidence that the Crh1p and Crh2p are required for tethering Gas1p to the chitin ring and bud scar. These results reveal a new mechanism of protein immobilization at specific sites of the cell envelop

Metamaterial architecture from a self-shaping carnivorous plant

As meticulously observed and recorded by Darwin, the leaves of the carnivorous plant Drosera capensis L. slowly fold around insects trapped on their sticky surface in order to ensure their digestion. While the biochemical signaling driving leaf closure has been associated with plant growth hormones, how mechanical forces actuate the process is still unknown. Here, we combine experimental tests of leaf mechanics with quantitative measurements of the leaf microstructure and biochemistry to demonstrate that the closure mechanism is programmed into the cellular architecture of D. capensis leaves, which converts a homogeneous biochemical signal into an asymmetric response. Inspired by the leaf closure mechanism, we devise and test a mechanical metamaterial, which curls under homogeneous mechanical stimuli. This kind of metamaterial could find possible applications as a component in soft robotics and provides an example of bio-inspired design

Functional characterization of wild-type and a mutated form of SLC26A4 identified in a patient with pendred syndrome

BACKGROUND: Malfunction of the SLC26A4 protein leads to prelingual deafness often associated with mild thyroid dysfunction and goiter. It is assumed that SLC26A4 acts as a chloride/anion exchanger responsible for the iodide organification in the thyroid gland, and conditioning of the endolymphatic fluid in the inner ear. METHODS: Chloride uptake studies were made using HEK293-Phoenix cells expressing human wild type SLC26A4 (pendrin) and a mutant (SLC26A4(S28R)) we recently described in a patient with hypothyroidism, goiter and sensorineural hearing loss. RESULTS: Experiments are summarized showing the functional characterization of wild type SLC26A4 and a mutant (S28R), which we described recently. This mutant protein is transposed towards the cell membrane, however, its transport capability is markedly reduced if compared to wild-type SLC26A4. Furthermore, we show that the SLC26A4 induced chloride uptake in HEK293-Phoenix cells competes with iodide, and, in addition, that the chloride uptake can be blocked by NPPB and niflumic acid, whereas DIDS is ineffective. CONCLUSIONS: The functional characteristics of SLC26A4(S28R) we describe here, are consistent with the clinical phenotype observed in the patient from which the mutant was derived

Mass production of basal bodies in paraspermiogenesis of Tubificinae (Annelida, Oligochaeta)

The oligochaete annelids, belonging to the subfamily Tubificinae, produce two types of spermatozoa: eusperm (the fertilising ones) and parasperm, protecting and carrying the eusperm. The pathway for the production of the two types is common until the onset of meiosis, but then a regular meiosis produces eusperm, whereas parasperm is generated through a peculiar mechanism of nuclear fragmentation giving rise to an irregular, but very high, number of paraspermatozoa. Since every parasperm has its own flagellum, this entails the necessity of producing a very high number of basal bodies. In the present paper, we describe how basal bodies are generated through a mechanism similar to that producing the basal bodies in ciliated epithelia, but never observed up to now during the genesis of a uniflagellated cell. Basal bodies form in close proximity to a precursor structure called deuterosome, which originates de novo in the cytoplasm from fibrogranular material. The various stages of centriologenesis are positive to anti-centrin antibodies and, observed by electron microscopy, correspond closely to the ones described for ciliated epithelia. However, once formed, the basal bodies migrate to their final position and produce the parasperm flagellum

Gentamicin ototoxicity in the saccule of the lizard Podarcis sicula induces hair cell recovery and regeneration

There is little information available on the susceptibility of reptilian saccule hair cells to ototoxin-induced sensory damage. In this study, we report morphological evidence of hair cell recovery and regeneration after damage induced by gentamicin in the saccule of a lizard. We perform morphological analysis using scanning electron microscopy and confocal laser scanning microscopy with actin and calbindin as markers for hair cells and tubulin as a marker for supporting cells. The data were consistent: gentamicin induced damage in the hair cells, and the damage increased with increasing duration of treatment. Initially, the saccule appeared unhealthy. Subsequently, the sensory hair cells became compromised, with fused stereovilli, followed by widespread loss of hair cell bundles from the hair cells. Finally, numerous hair cells were lost. Morphologically, the saccule appeared normal 28 days after gentamicin treatment. Using a mitogenic marker, we tested whether or not there is hair cell regeneration following administration of gentamicin. We found evidence of bromodeoxyuridine incorporation first in supporting cell nuclei and subsequently in hair cell nuclei. This indicates that a process of sensory epithelium repair and hair cell regeneration occurred, in both extrastriolar and striolar regions, and that the recovery was due to both the proliferation of supporting cells and, as seems likely, self-repair of hair cell bundles

Are the peripheric cells of macula to produce its increase?

After the birth, in the inferior Vertebrates the inner ear increase continuously and progressively. How happens such increase? Our studies have performed on the inner ear of Rana esculenta by immunofluorescence techniques and by SEM observations. In order to determine the new sensory cells’ derivation, we have given a mitogenic marker, the BrdU, to label cells that were in the process of dividing. We have evidenced that the sensory cells, with morphologic characteristics of young cells, are present in all three of maculae mostly to the periphery of the sensory epithelium. The young cells are rare between the mature sensory cells of central part of the maculae. For these data we can assume that the increase of the sensory areas happens from the periphery to the middle of the maculae by forming the new sensory and supporting cells. These cells, probably, have been originated by the division of support cells, subsequently they differ in sensory cells

Evidence for hair cell regeneration in the papilla basilaris of the lizard Podarcis sicula.

Post embryonic hair cell production occurs continually throughout life in the vestibular and auditory sensory epithelium in fishes and amphibians and in the vestibular end organs of birds (Corwin, 1985, PNAS, USA 82: 3911-15; Roberson et al., 1992, Hear. Res., 57: 166-74; Stone et al., 1999, J. Neurocytol. 28: 863-76). In the mature hearing organ of birds and in the vestibular sensory epithelium of mammalian only after hair cell loss by treatment with ototoxic drugs or trauma, new hair cells reappeared in sensory epithelia (Cotanche, 1987, Hear. Res., 30: 181-96; Forge et al., 1993, Science 259: 1616-19; Zheng and Gao, 1997 J Neurosci., 17 (21): 8270-82). According to Bagger-Sjoback and Wersäll (1976), lizards treated with gentamicin for 21 days showed a severely damaged papilla basilaris. We investigated recovery phenomena in the sensory epithelium occurring in the gentamicin-treated lizard papilla basilaris by SEM. Using 5-bromodeoxyuridine we have studied the hair cells regeneration in the papilla basilaris both in normal conditions and at early and late time intervals following 3 doses of aminoglycoside gentamicin. Finally, in order to define if the regenerated cells become sensory cells, we also analysed the presence of calbindin, used as a marker of hair cells, by CLSM. The SEM and CLSM data are consistent: gentamicin induced damage of the hair cells in a dose-dependent manner and the two segments of papilla basilaris differed in their susceptibility to gentamicin. Initially the apical type A cells were relatively intact while the type B cell-population in the basal segment of the organ showed sensory hair fusions and cytoplasmic herniations. Successively a wide spread loss of hair of the sensory cells, 8 days after treatment, extrusion of entire hair cell was observed and the surface of the basal part of the organ instead was covered by supporting cells. The papilla basilaris, however, showed an apparently normal morphology after 28 days from treatment. Our immunocytochemistry studies showed that in the papilla basilaris of Podarcis sicula regeneration phenomena are present also in normal conditions and that regenerative processes, limited or slow in normal conditions, are very amplified by treatment with gentamicin. Therefore, in a different way to chick in which the regeneration occur only after treatment with ototoxic drugs or trauma, in some orders of reptiles, in the auditory organ, the regeneration is a spontaneous phenomenon also in normal conditions

Exposure of pig oocytes to PCBs during in vitro maturation: effects on developmental competence, cytoplasmic remodelling and communications with cumulus cells

Polychlorinated biphenyls (PCBs) are one of the most persistent and widespread groups of endocrine disrupting compounds in the ecosystem. These substances are present in sewage sludge that is spread in increasing amounts on arable land and pasture as fertilizer, and are ingested by farm animals with food and drinking water. This study investigated the effect of different PCB concentrations on pig oocyte in vitro maturation and developmental competence as well as examined the possible mechanisms involved. A concentration ranging from 0 to 1 ?g/mL of Aroclor 1254 (A1254), a pool of more than 60 PCB congeners, was added to the maturation medium, as its composition is considered environmentally relevant. A1254 had no effect on maturation of pig oocytes and on the number of oocytes that cleaved following parthenogenetic activation at any of the doses tested. By contrast, a significant decrease in the number of zygotes that developed to blastocyst stage became evident at a concentration of 10 ng/mL. The number of blastocysts obtained decreased significantly, and in a dose response manner with higher concentrations. Exposure to PCBs altered mitochondria relocation during maturation and this was associated with the lack of a cytoplasmic microtubule network. No effect on mitochondria activity was observed. A1254 exposure also perturbed gap-junction mediated communications between oocytes and cumulus cells. In conclusion, PCB exposure of pig oocytes during in vitro maturation significantly decreased oocyte developmental competence, altered both their cytoplasmic remodelling and the communication with the somatic compartment. These data indicated that accumulation of PCBs in the pig organism may have a detrimental effect on the reproductive efficiency in this species

The neuron specific Ras-exchange factor Ras-GRF1 assembles with polymerized tubulin: microscopy analysis and biochemical studies

5noneForlani G.; Fascio U.; Baldassa S.; Sturani E.; Zippel R.Forlani, Greta; Fascio, U.; Baldassa, S.; Sturani, E.; Zippel, R