Optic nerve sectioning does not affect the development of the retina

The development of the retina of the albino rat was studied after sectioning of the optic nerves on the 2nd postnatal day. The 2nd day represents a stage at which the retina shows only the ganglion cell layer clearly delineated from an undifferentiated mass. Section of optic nerves at this stage did not affect the subsequent retinal development. Both control and experimental eyes developed at the same pace. Some minor degrees of 'retardation' e.g. the sizes of outer segments, appeared to deviate in the experimental retinae.     

Golgi-localized UDP-glucose transporter is required for cell wall integrity in rice

Cell wall-related nucleotide sugar transporters (NSTs) theoretically supply the cytosolic nucleotide sugars for glycosyltransferases (GTs) to carry out ploysaccharide synthesis and modification in the Golgi apparatus. However, the regulation of cell wall synthesis by NSTs remains undescribed. Recently, we have reported the functional characterization of Oryza sativa nucleotide sugar transport (Osnst1) mutant and its corresponding gene. OsNST1/BC14 is localized in the Golgi apparatus and transports UDP-glucose. This mutant provides us with a unique opportunity for evaluation of its broad impacts on cell wall structure and components. We previously examined cell wall composition of bc14 and wild type plants. Here, the spatial distribution of these cell wall alterations was analyzed by immunolabeling approach. Analysis of the sugar yield in different cell wall fractions indicated that this mutation improves the extractability of cell wall components. Field emission scanning electron microscopy further showed that the orientation of microfibrils in bc14 is irregular when compared to that in wild type. Therefore, this UDP-glucose transporter, making substrates available for polysaccharide biosynthesis, plays a critical role in maintaining cell wall integrity.Key words: UDP-glucose transporter, Golgi apparatus, cell wall polysaccharides, xylan, riceNucleotide sugars mainly generated in cytosol are the substrates for the synthesis of cell wall polysaccharides. Supply of nucleotide sugars is thus a key level for regulation of cell wall components and structure. Mutation in MUR1, an isoform of GDP-D-mannose-4,6-dehydratase, causes reduced amount of GDP-fucose and abnormal xyloglucan structure.^1,2 Disturbance of UDP-rhamnose synthesis via the mutation in RHM2/MUM4 decreases the rhamnogalacturonan I contents in Arabidopsis seeds. Cellulose synthase catalytic subunits (CESAs) generally use cytosolic UDP-glucoses to synthesize cellulose on the plasma membrane. UDP-glucose can be produced either via the catalysis of sucrose by sucrose synthase (SuSy) or through the phosphorylation of glucose-1-phosphate by UDP-glucose pyrophosphorylase (UGPase).³ Suppression of SuSy function in cotton inhibited fiber initiation and elongation.⁴ For the synthesis of noncellulosic polysaccharides occurring inside the Golgi lumen, the cytosolic nucleotide sugars should be translocated inwards by Golgi nucleotide sugar transporters (NSTs).⁵ However, this hypothesis remains to be confirmed, although transport activities have been identified in some plant NSTs.^6–10 Altering the precursor supply may also affect the overall carbon allocation in plants. It is reasonable that substrate regulation often causes pleiotropic effects on cell wall biosynthesis and plant growth. Without genetic resources or mutants on cell wall related NST, the exact evaluation of NSTs'' impacts on cell wall structure and composition is largely delayed. Until recently, we identified a Golgi-localized transporter OsNST1 mutant in rice. This transporter has been found to supply UDP-glucose for the formation of matrix polysaccharides, thereby modulating cellulose biosynthesis.¹¹ Here, we examine these alterations of cell wall polymers at the cellular level. The orientation of cellulose microfibrils and extractability of wall polysaccharides were also compared between the mutant and wild type. All those further our understandings of the functions of NSTs and the synergetic synthesis of different polymers.     

The KEX2 gene of Candida glabrata is required for cell surface integrity

Candida glabrata has emerged as one of the most common causes of candidosis. In order to identify factors that are necessary for viability and pathogenicity of this fungal pathogen, we analysed the role of the KEX2 gene, which codes for a regulatory endoproteinase that is known to process certain virulence factors in Candida albicans. The KEX2 gene from C. glabrata was cloned and found to have 51% and 62% identity and high structural similarities to the homologous counterparts in C. albicans and Saccharomyces cerevisiae. KEX2 was expressed at all time points investigated during growth in complex medium. In order to investigate the role of this putative regulatory proteinase, Kex2-deficient mutants were produced. In addition to known kex2 phenotypes, such as pH and calcium hypersensitivity, the mutants grew in cellular aggregates and were found to be hypersensitive to several antifungal drugs that target the cell membrane, including azoles, amorolfine and amphotericin B. Ultrastructural investigation after exposure to low doses of itraconazole showed azole-specific alterations such as enlarged vacuoles and proliferation of the cytoplasmatic membrane in the kex2 mutants, but not in the control strains. In contrast, antifungals such as 5-flucytosine and hydroxypyridones inhibited growth of the kex2 mutants and the control strains to the same extent. In an in vitro model of oral candidosis, kex2 mutants showed reduced tissue damage in the presence of itraconazole compared with the control infections. These data suggest that Kex2 is involved in the processing of proteins that are essential for cell surface integrity of C. glabrata.     

Schizosaccharomyces pombe Pmr1p is essential for cell wall integrity and is required for polarized cell growth and cytokinesis

The cps5-138 fission yeast mutant shows an abnormal lemon-like morphology at 28 degrees C in minimal medium and a lethal thermosensitive phenotype at 37 degrees C. Cell growth is completely inhibited at 28 degrees C in a Ca2+-free medium, in which the wild type is capable of growing normally. Under these conditions, actin patches become randomly distributed throughout the cell, and defects in septum formation and subsequent cytokinesis appear. The mutant cell is hypersensitive to the cell wall-digesting enzymatic complex Novozym234 even under permissive conditions. The gene SPBC31E1.02c, which complements all the mutant phenotypes described above, was cloned and codes for the Ca2+-ATPase homologue Pmr1p. The gene is not essential under optimal growth conditions but is required under conditions of low Ca2+ (<0.1 mM) or high temperature (>35 degrees C). The green fluorescent protein-tagged Cps5 proteins, which are expressed under physiological conditions (an integrated single copy with its own promoter in the cps5Delta strain), display a localization pattern typical of endoplasmic reticulum proteins. Biochemical analyses show that 1,3-beta-D-glucan synthase activity in the mutant is decreased to nearly half that of the wild type and that the mutant cell wall contains no detectable galactomannan when the cells are exposed to a Ca2+-free medium. The mutant acid phosphatase has an increased electrophoretic mobility, suggesting that incomplete protein glycosylation takes place in the mutant cells. These results indicate that S. pombe Pmr1p is essential for the maintenance of cell wall integrity and cytokinesis, possibly by allowing protein glycosylation and the polarized actin distribution to take place normally. Disruption and complementation analyses suggest that Pmr1p shares its function with a vacuolar Ca2+-ATPase homologue, Pmc1p (SPAPB2B4.04c), to prevent lethal activation of calcineurin for cell growth.     

On the complex dynamics of intracellular ganglion cell light responses in the cat retina

We recorded intracellular responses from cat retinal ganglion cells to sinusoidal flickering lights, and compared the response dynamics with a theoretical model based on coupled nonlinear oscillators. Flicker responses for several different spot sizes were separated in a smooth generator (G) potential and corresponding spike trains. We have previously shown that the G-potential reveals complex, stimulus-dependent, oscillatory behavior in response to sinusoidally flickering lights. Such behavior could be simulated by a modified van der Pol oscillator. In this paper, we extend the model to account for spike generation as well, by including extended Hodgkin-Huxley equations describing local membrane properties. We quantified spike responses by several parameters describing the mean and standard deviation of spike burst duration, timing (phase shift) of bursts, and the number of spikes in a burst. The dependence of these response parameters on stimulus frequency and spot size could be reproduced in great detail by coupling the van der Pol oscillator and Hodgkin-Huxley equations. The model mimics many experimentally observed response patterns, including non-phase-locked irregular oscillations. Our findings suggest that the information in the ganglion cell spike train reflects both intraretinal processing, simulated by the van der Pol oscillator, and local membrane properties described by Hodgkin-Huxley equations. The interplay between these complex processes can be simulated by changing the coupling coefficients between the two oscillators. Our simulations therefore show that irregularities in spike trains, which normally are considered to be noise, may be interpreted as complex oscillations that might carry information.To the memory of Prof. Otto-Joachim Grusser     

Drosophila Dystrophin is required for integrity of the musculature

Duchenne muscular dystrophy is caused by mutations in the dystrophin gene and is characterized by progressive muscle wasting. The highly conserved dystrophin gene encodes a number of protein isoforms. The Dystrophin protein is part of a large protein assembly, the Dystrophin glycoprotein complex, which stabilizes the muscle membrane during contraction and acts as a scaffold for signaling molecules. How the absence of Dystrophin results in the onset of muscular dystrophy remains unclear. Here, we have used transgenic RNA interference to examine the roles of the Drosophila Dystrophin isoforms in muscle. We previously reported that one of the Drosophila Dystrophin orthologs, the DLP2 isoform, is not required to maintain muscle integrity, but plays a role in neuromuscular homeostasis by regulating neurotransmitter release. In this report, we show that reduction of all Dystrophin isoform expression levels in the musculature does not apparently affect myogenesis or muscle attachment, but results in progressive muscle degeneration in larvae and adult flies. We find that a recently identified Dystrophin isoform, Dp117, is expressed in the musculature and is required for muscle integrity. Muscle fibers with reduced levels of Dp117 display disorganized actin-myosin filaments and the cellular hallmarks of necrosis. Our results indicate the existence of at least two possibly separate roles of dystrophin in muscle, maintaining synaptic homeostasis and preserving the structural stability of the muscle.     

DCC is specifically required for the survival of retinal ganglion and displaced amacrine cells in the developing mouse retina

Netrin-1 and DCC are well known for their roles in neurite growth, axonal guidance, and neuronal migration. Recently, a number of studies showed that DCC is involved in the induction of apoptosis, and this proapoptotic activity can be blocked in the presence of Netrin-1. However, here, we found that DCC is required for the survival of two types of neurons selectively in the developing mouse retina where DCC is abundantly expressed. Our results showed that the DCC^−/− retina displayed a reduced ganglion cell layer with relatively normal neuroblastic layer. Immunostaining assays revealed that in DCC^−/− mice, initial neurogenesis within retina was unchanged while the numbers of differentiated retinal ganglion cells and displaced amacrine cells in ganglion cell layer were greatly reduced due to increased apoptosis. By contrast, other neuronal types including horizontal cells, bipolar cells, amacrine cells, photoreceptors, and Müller cells appeared normal in DCC mutant retinas. Moreover, DCC^kanga mice that lack the intracellular P3 domain of DCC receptor displayed the same defects as DCC^−/− mice. Thus, our findings suggest that DCC is a key regulator for the survival of specific types of neurons during retinal development and that DCC-P3 domain is essential for this developing event.     

hTPX2 is required for normal spindle morphology and centrosome integrity during vertebrate cell division

Bipolar spindle formation is essential for the accurate segregation of genetic material during cell division. Although centrosomes influence the number of spindle poles during mitosis, motor and non-motor microtubule-associated proteins (MAPs) also play key roles in determining spindle morphology. TPX2 is a novel MAP also characterized in Xenopus cell-free extracts. To examine hTPX2 (human TPX2) function in human cells, we used siRNA to knock-down its expression and found that cells lacking hTPX2 arrest in mitosis with multipolar spindles. NuMA, gamma-tubulin, and centrin localize to each pole, and nocodazole treatment of cells lacking hTPX2 demonstrates that the localization of gamma-tubulin to multiple spindle poles requires intact microtubules. Furthermore, we show that the formation of monopolar microtubule arrays in human cell extracts does not require hTPX2, demonstrating that the mechanism by which hTPX2 promotes spindle bipolarity is independent of activities focusing microtubule minus ends at spindle poles. Finally, inhibition of the kinesin Eg5 in hTPX2-depleted cells leads to monopolar spindles, indicating that Eg5 function is necessary for multipolar spindle formation in the absence of hTPX2. Our observations reveal a structural role for hTPX2 in spindles and provide evidence for a balance between microtubule-based motor forces and structural spindle components.     

The fission yeast ran GTPase is required for microtubule integrity

The microtubule cytoskeleton plays a pivotal role in cytoplasmic organization, cell division, and the correct transmission of genetic information. In a screen designed to identify fission yeast genes required for chromosome segregation, we identified a strain that carries a point mutation in the SpRan GTPase. Ran is an evolutionarily conserved eukaryotic GTPase that directly participates in nucleocytoplasmic transport and whose loss affects many biological processes. Recently a transport-independent effect of Ran on spindle formation in vitro was demonstrated, but the in vivo relevance of these findings was unclear. Here, we report the characterization of a Schizosaccharomyces pombe Ran GTPase partial loss of function mutant in which nucleocytoplasmic protein transport is normal, but the microtubule cytoskeleton is defective, resulting in chromosome missegregation and abnormal cell shape. These abnormalities are exacerbated by microtubule destabilizing drugs, by loss of the spindle checkpoint protein Mph1p, and by mutations in the spindle pole body component Cut11p, indicating that SpRan influences microtubule integrity. As the SpRan mutant phenotype can be partially suppressed by the presence of extra Mal3p, we suggest that SpRan plays a role in microtubule stability.     

Obscurin is required for ankyrinB-dependent dystrophin localization and sarcolemma integrity

Obscurin is a large myofibrillar protein that contains several interacting modules, one of which mediates binding to muscle-specific ankyrins. Interaction between obscurin and the muscle-specific ankyrin sAnk1.5 regulates the organization of the sarcoplasmic reticulum in striated muscles. Additional muscle-specific ankyrin isoforms, ankB and ankG, are localized at the subsarcolemma level, at which they contribute to the organization of dystrophin and β-dystroglycan at costameres. In this paper, we report that in mice deficient for obscurin, ankB was displaced from its localization at the M band, whereas localization of ankG at the Z disk was not affected. In obscurin KO mice, localization at costameres of dystrophin, but not of β-dystroglycan, was altered, and the subsarcolemma microtubule cytoskeleton was disrupted. In addition, these mutant mice displayed marked sarcolemmal fragility and reduced muscle exercise tolerance. Altogether, the results support a model in which obscurin, by targeting ankB at the M band, contributes to the organization of subsarcolemma microtubules, localization of dystrophin at costameres, and maintenance of sarcolemmal integrity.     

A model of anuran retina relating interneurons to ganglion cell responses

A model is presented which accounts for many characteristic response properties used to classify anuran ganglion cell types while being consistent with data concerning interneurons. In the model color is ignored and input stimuli are assumed to be only black and white at high contrast. We show that accurate ganglion cell responses are obtained even with simplified receptors and horizontal cells: Receptors are modeled as responding with a step change, while horizontal cells respond only to global changes in intensity brought about by full field illumination changes. A hyperpolarizing and depolarizing bipolar cell are generated y subtracting local receptor and horizontal potentials. Two transient amacrine cells (On and Off) are generated using a high-pass filter like mechanism with a thresholded output which responds to positive going changes in the corresponding bipolar cell potentials. The model shows how a selective combination of bipolar and amacrine channels can account for many of the response properties used to classify the anuran ganglion cell types (class-0 through 4) and makes several experimental predictions.     

Fgf19 is required for zebrafish lens and retina development

Fgf signaling plays crucial roles in morphogenesis. Fgf19 is required for zebrafish forebrain development. Here, we examined the roles of Fgf19 in the formation of the lens and retina in zebrafish. Knockdown of Fgf19 caused a size reduction of the lens and the retina, failure of closure of the choroids fissure, and a progressive expansion of the retinal tissue to the midline of the forebrain. Fgf19 expressed in the nasal retina and lens was involved in cell survival but not cell proliferation during embryonic lens and retina development. Fgf19 was essential for the differentiation of lens fiber cells in the lens but not for the neuronal differentiation and lamination in the retina. Loss of nasal fate in the retina caused by the knockdown of Fgf19, expansion of nasal fate in the retina caused by the overexpression of Fgf19 and eye transplantation indicated that Fgf19 in the retina was crucial for the nasal-temporal patterning of the retina that is critical for the guidance of retinal ganglion cell axons. Knockdown of Fgf19 also caused incorrect axon pathfinding. The present findings indicate that Fgf19 positively regulates the patterning and growth of the retina, and the differentiation and growth of the lens in zebrafish.     

Exogenous gangliosides may affect methylation mechanisms in neuronal cell cultures

Primary neurons in culture from chick embryo cerebral hemispheres were treated with a mixture of gangliosides added to the growth medium (final concentration: 10^–5M and 10^–8M) from the 3rd to the 6th day in vitro. Under these conditions methylation processes measured with [³H] and [³⁵S] methionine and [³H]ethanolamine as precursors showed an increased methylation of [³H]ethanolamine containing phospholipids, a correspondent increased conversion of these compounds to [³H]choline containing phospholipids, and a general increased methylation of trichloroacetic acid precipitable macromolecules containing labeled methionine. A small increase in protein synthesis was observed after incubation of neurons with [³H]- and [³⁵S]methionine. This was confirmed after electrophoretic separation of a protein extract with increased³H-and³⁵S-labeling in protein bands with moecular weights between 50 and 60 KDaltons. A protein band of about 55 KDaltons appeared to be preferentially labelled when [³H] methionine was the precursor. The treatment with gangliosides increased the incorporation of [methyl-³H] label after incubation of neurons with [³H] methionine, into total DNA and decreased that of total RNA. The treatment of neurons in culture with exogenous gangliosides hence affects differently methylation processes, a finding which may confirm the involvement of gangliosides on the intracellular mediation of neuronal information mechanisms.