Radial Organization Research Articles

Hyperbranched Polyesters with Carboxylic or Sulfonic Acid Functional Groups for Crystallization Modification of Calcium Carbonate

AbstractHyperbranched aliphatic polyesters functionalized with carboxylic or sulfonic acid groups have been synthesized and employed as crystallization modifiers for calcium carbonate. The functionalized polyesters exhibit remarkable polymorph selectivity and morphology control. Stable spherical vaterite particles of diameter less than 10 µm with narrow size distribution were easy to produce on a large scale. With an increase in the concentration and monomer/core ratio of the polyester, the vaterite content increases accordingly with promoted morphology control to monodisperse spheres. The CaCO3 concentration and the initial pH can also influence the morphology and polymorphism of the produced calcium carbonate. Under certain conditions, well‐defined core‐shell structured vaterite spheres are obtained with obvious radial organization by nano‐aggregation of about 70 ± 10 nm crystallites, which may mediate and combine the nano‐aggregation and crystal growth formation mechanism of vaterite polymorph.magnified image

Evolutionarily conserved, cell type and species-specific higher order chromatin arrangements in interphase nuclei of primates

Several studies demonstrated a gene-density-correlated radial organization of chromosome territories (CTs) in spherically shaped nuclei of human lymphocytes or lymphoblastoid cells, while CT arrangements in flat-ellipsoidal nuclei of human fibroblasts are affected by both gene density and chromosome size. In the present study, we performed fluorescence in situ hybridization (FISH) experiments to three-dimensionally preserved nuclei (3D-FISH) from human and nonhuman primate cultured lymphoblastoid cells and fibroblasts. We investigated apes, Old, and New World monkeys showing either evolutionarily conserved karyotypes, multiple translocations, fusions, or serial fissions. Our goal was to test whether cell type specific differences of higher order chromatin arrangements are evolutionarily conserved in different primate lineages. Whole genome painting experiments and further detailed analyses of individual chromosomes indicate a gene-density-correlated higher order organization of chromatin in lymphoblastoid cell nuclei of all studied primate species, despite evolutionary chromosome reshuffling. In contrast, in primate fibroblast nuclei evolutionary translocations, fissions and fusions resulted in positional shifts of orthologous chromosome segments, thus arguing against a functional role of chromosome size-dependent spatial chromatin arrangements and for geometrical constraints in flat-ellipsoidal fibroblast nuclei. Notably, in both cell types, regions of rearranged chromosomes with distinct differences in gene density showed polarized arrangements with the more gene-dense segment oriented towards the nuclear interior. Our results indicate that nonrandom breakage and rejoining of preferentially gene-dense chromosomes or chromosome segments may have occurred during evolution.

Defects in lamin B1 expression or processing affect interphase chromosome position and gene expression

Radial organization of nuclei with peripheral gene-poor chromosomes and central gene-rich chromosomes is common and could depend on the nuclear boundary as a scaffold or position marker. To test this, we studied the role of the ubiquitous nuclear envelope (NE) component lamin B1 in NE stability, chromosome territory position, and gene expression. The stability of the lamin B1 lamina is dependent on lamin endoproteolysis (by Rce1) but not carboxymethylation (by Icmt), whereas lamin C lamina stability is not affected by the loss of full-length lamin B1 or its processing. Comparison of wild-type murine fibroblasts with fibroblasts lacking full-length lamin B1, or defective in CAAX processing, identified genes that depend on a stable processed lamin B1 lamina for normal expression. We also demonstrate that the position of mouse chromosome 18 but not 19 is dependent on such a stable nuclear lamina. The results implicate processed lamin B1 in the control of gene expression as well as chromosome position.

Developmental Changes and Injury Induced Disruption of the Radial Organization of the Cortex in the Immature Rat Brain Revealed by In Vivo Diffusion Tensor MRI

During brain development, morphological changes modify the cortex from its immature radial organization to its mature laminar appearance. Applying in vivo diffusion tensor imaging (DTI), the microstructural organization of the cortex in the immature rat was analyzed and correlated to neurohistopathology. Significant differences in apparent diffusion coefficient (ADC) and fractional anisotropy (FA) were detected between the external (I-III) and deep (IV-VI) cortical layers in postnatal day 3 (P3) and P6 pups. With cortical maturation, ADC was reduced in both cortical regions, whereas a decrease in FA was only seen in the deep layers. A distinct radial organization of the external cortical layers with the eigenvectors perpendicular to the pial surface was observed at both ages. Histology revealed maturational differences in the cortical architecture with increased neurodendritic density and reduction in the radial glia scaffolding. Early DTI after hypoxia-ischemia at P3 shows reduced ADC and FA in the ipsilateral cortex that persisted at P6. Cortical DTI eigenvector maps reveal microstructural disruption of the radial organization corresponding to regions of neuronal death, radial glial disruption, and astrocytosis. Thus, the combined use of in vivo DTI and histopathology can assist in delineating normal developmental changes and postinjury modifications in the immature rodent brain.

A Comparison Study of the Vertical Bias of Pyramidal Cells in the Hippocampus and Neocortex

In this study, we employed morphometric image analysis of the hippocampus proper and temporal lobe neocortex in postmortem tissue to determine vertical bias quantified as ΔΘ, angular dispersion, as well asan index of alignment of cellular elements relative to the radial plane. The radial alignment of cellular elements was consistent with a minicolumnar organization of the cortex. Photomicrographs were taken of the left-hemisphere hippocampal CA3/1 subfields of 13 fetal subjects ranging in gestational age from 19 weeks to 36 weeks and 19 normal individuals aged 4 months to 98 years. For comparison, micrographs from the temporal lobe (Brodmann areas 21 and 22) were similarly processed for layers III and V, where the x-axes of the transformed coordinate system were taken to be the layer III/IV and IV/V borders, respectively. Computerized image analysis measurements of the angular dispersion for the temporal lobe region and hippocampus proper differed significantly within the same brains (p < 0.001). The neocortical layer III exhibited the highest values for ΔΘ, indicating a high degree of columnar organization. Values for ΔΘ in the hippocampal CA subfields were lower but demonstrated significance for the radial alignment of neurons in this area. Values for ΔΘin layer V were intermediate between those of layer III and the hippocampus, consistent with increasing degrees of radial columnar organization of infragranular layers of the neocortex in comparison with the hippocampus and of supragranular in comparison with infragranular neocortical layers. Pyramidal cell arrays within allocortical areas and the neocortex constitute different modular arrangements. This morphological variability may be the expression of evolutionary differences in cortical development.

Spatial Organization of Embryonic Stem Cell Responsiveness to Autocrine Gp130 Ligands Reveals an Autoregulatory Stem Cell Niche

Highly ordered aggregates of cells, or niches, regulate stem cell fate. Specific tissue location need not be an obligatory requirement for a stem cell niche, particularly during embryogenesis, where cells exist in a dynamic environment. We investigated autoregulatory fixed-location-independent processes controlling cell fate by analyzing the spatial organization of embryonic stem cells (ESCs) using quantitative single-cell immunocytochemistry and a computational approach involving Delaunay triangulation. ESC colonies demonstrated radial organization of phosphorylated signal transducer and activator of transcription 3, Nanog, and Oct4 (among others) in the presence and absence of exogenous leukemia inhibitory factor (LIF). Endogenous self-renewal signaling resulted from autocrine non-LIF gp130 ligands, which buffered cells against differentiation upon exogenous LIF deprivation. Together with a radial organization of differential responsiveness to gp130 ligands within colonies, autocrine signaling produced a radial organization of self-renewal, generating a fixed-location-independent autoregulatory niche. These findings reveal fundamental properties of niches and elucidate mechanisms colonies of cells use to transition between fates during morphogenesis.

Hook2 Localizes to the Centrosome, Binds Directly to Centriolin/CEP110 and Contributes to Centrosomal Function

Centrosomes serve as microtubule-organizing centers. However, centrosome function depends on microtubule organization and protein transport because the formation, positioning and maintenance of centrosomes require microtubule-dependent retrograde transport. Linker proteins that associate with the motor protein dynein, organelles and microtubules facilitate loading of cargos for retrograde transport and thus contribute to the composition and placement of the centrosome and other juxtanuclear protein complexes. Members of the hook family of proteins may function as adaptors to link various organelle cargos to dynein for transport and have also been implicated directly in centrosome positioning. Here, we show that mammalian hook2, a previously uncharacterized member of the hook family, localizes to the centrosome through all phases of the cell cycle, the C-terminal domain of hook2 directly binds to centriolin/CEP110, the expression of the C-terminal domain of centriolin/CEP110 alters the distribution of endogenous hook2 and mislocalized wild-type or mutant hook2 proteins perturb endogenous centrosomal and pericentrosomal proteins in cultured mammalian cells. In addition, interference with hook2 function results in the loss of the radial organization of microtubules and a defect in regrowth of microtubules following their nocodazole-induced depolymerization. Thus, we propose that hook2 contributes to the establishment and maintenance of centrosomal structure and function.

Collagen Organization during Cardiac Fibroblast‐mediated Collagen Gel Contraction

Collagen within the extracellular matrix (ECM) in the heart is responsible for connecting myocytes to one another and mechanically couples neighboring cells. In the developing heart, the collagen matrix accumulates rapidly during early post-natal life and develops an organization which is maintained in the adult heart. The mechanisms by which cardiac fibroblasts organize collagen within the cardiac ECM are unclear; although probably involve a combination of mechanical tension and enzymatic remodeling. To investigate this process, neonatal cardiac fibroblasts were dispersed within a collagen gel and cultured in either a mechanically loaded or unloaded environment. Using confocal reflection microscopy combined with circular statistical analysis, both cell distribution, cell morphology and collagen fiber distribution have been analyzed. While initially cells display a rounded morphology and are surrounded by randomly organized collagen, over time as the fibroblasts extend cell processes the collagen network develops a radial organization around the cell with some areas on the cell surface displaying numerous collagen attachments and others lacking any visible collagen interaction. In addition, areas devoid of collagen are also visible, suggesting enzymatic degradation of collagen. Fibroblasts appear to differentially orient within the gels, having a more circumferential orientation in the outer regions of the gels and a more random orientation in the center of the gel. These studies provide direct insight into the organizational changes which occur during collagen remodeling.

A statistical basis for visual field anisotropies

There exist numerous psychophysical paradigms for which performance varies with location of stimulus presentation within the visual field. The following considers potential bases for visual anisotropies, considering the possibility of a statistical basis for such effects in cases where basic sensory asymmetries are present. In particular, the relationship between scene statistics and both upper–lower and lateral visual field asymmetries is considered. Finally, an argument is put forth concerning the apparent radial organization of the visual field, with the suggestion that geometric perspective may give rise to the statistical bias responsible for this effect.

Regulation of microtubule dynamics in 3T3 fibroblasts by Rho family GTPases

To get insight into the action of Rho GTPases on the microtubule system we investigated the effects of Cdc42, Rac1, and RhoA on the dynamics of microtubules in Swiss 3T3 fibroblasts. In control cells microtubule ends were dynamic: plus ends frequently switched between growth, shortening and pauses; the growth phase predominated over shortening. Free minus ends of microtubules depolymerized rapidly and never grew. Free microtubules were short-lived, and the microtubule network was organized into a radial array. In serum-starved cells microtubule ends became more stable: although plus ends still transited between growth and shortening, polymerization and depolymerization excursions became shorter and balanced each other. Microtubule minus ends were also stabilized. Consequently lifespan of free microtubules increased and microtubule array changed its radial pattern into a random one. Activation of Cdc42 and Rac1 in serum-starved cells promoted dynamic behavior of microtubule plus and minus ends, while inhibition of these GTPases in serum-grown cells suppressed microtubule dynamics and mimicked all effects of serum starvation. Activation of RhoA in serum-grown cells had effects similar to Cdc42 /Rac1 inactivation: it suppressed the dynamics of plus and minus ends, reduced the length of growth and shrinking episodes, and disrupted the radial organization of microtubules. However, in contrast to Cdc42 and Rac1 inactivation, active RhoA had no effect on the balance between microtubule growth and shortening. We conclude that Cdc42 and Rac1 have similar stimulating effects on microtubule dynamics while RhoA acts in an opposite way.

Bioconjugated Superstructures of CdTe Nanowires and Nanoparticles: Multistep Cascade Förster Resonance Energy Transfer and Energy Channeling

Nanoparticle/nanowire assemblies with a degree of radial organization were prepared around luminescent semiconducting CdTe nanowires using bioconjugation with streptavidin and D-biotin linkers. Red-emitting nanowires (6.62 +/- 1.55 nm diameter, 512 +/- 119 nm length) and green-emitting nanoparticles (3.2 +/- 0.7 nm diameter) were surface-modified with biotin, while orange-emitting nanoparticles (4.1 +/- 1.2 nm diameter) were decorated with streptavidin. CdTe nanocrystals produced two fuzzy layers around the nanowires in which the diameter of CdTe nanoparticles decreased with the distance from the nanowire axis. Förster resonance energy transfer (FRET) from the outside layer of nanoparticles to the central nanowire was observed for nanowires conjugated with 4.1 nm CdTe. Addition of 3.2 nm CdTe resulted in a red-orange-green optical progression with band gaps of CdTe decreasing toward the axis of the superstructure. In this case, 4-fold luminescence enhancement of the nanowire luminescence was observed and was attributed to multistep FRET. This observation indicated the accumulation of photogenerated excitons in the cascade terminal. A simple model of multiconjugated superstructure with cascade energy transfer is developed and used to describe and understand the experimental data. The experimental data and theoretical model suggest the possibility of utilization of the prepared superstructures with radial symmetry in several classes of optoelectronic devices including nanomaterials for energy collection. They can also be a convenient model object for the investigation of methods of energy funneling in nanoscale assemblies.

Distinctive nuclear organisation of centromeres and regions involved in pluripotency in human embryonic stem cells

Nuclear organisation is thought to be important in regulating gene expression. Here we investigate whether human embryonic stem cells (hES) have a particular nuclear organisation, which could be important for maintaining their pluripotent state. We found that whereas the nuclei of hES cells have a general gene-density-related radial organisation of chromosomes, as is seen in differentiated cells, there are also distinctive localisations for chromosome regions and gene loci with a role in pluripotency. Chromosome 12p, a region of the human genome that contains clustered pluripotency genes including NANOG, has a more central nuclear localisation in ES cells than in differentiated cells. On chromosome 6p we find no overall change in nuclear chromosome position, but instead we detect a relocalisation of the OCT4 locus, to a position outside its chromosome territory. There is also a smaller proportion of centromeres located close to the nuclear periphery in hES cells compared to differentiated cells. We conclude that hES cell nuclei have a distinct nuclear architecture, especially at loci involved in maintaining pluripotency. Understanding this level of hES cell biology provides a framework within which other large-scale chromatin changes that may accompany differentiation can be considered.

Tubulin glycylation and glutamylation deficiencies in unconventional insect axonemes

Though the 9+2 axonemal organization has generally been conserved throughout metazoan evolution, insect spermatozoa possess a substantial variety in axoneme ultrastructure, displaying different axonemal patterns. Therefore, insects provide a wide range of models that may be useful for the study of the mechanisms of axoneme assembly. We have used antibodies specific for glutamylated, monoglycylated, and polyglycylated tubulin to investigate the tubulin isoform content expressed in the unorthodox sperm axonemes of four insect species belonging to both of the superorders Palaeoptera and Neoptera. Each one of these axonemal models exhibits distinctive structural features, either showing the typical radial organization endowed with a ninefold symmetry or consisting of an helical arrangement with up to 200 microtubular doublets, but in all cases these axonemes share the absence of a microtubule central pair. Our results showed that all these atypical patterns are characterized by a dramatic decrease in both tubulin glycylation and glutamylation levels or even lack of both polymodifications. These data provide the first examples of a simultaneous extreme reduction or even absence of both polymodifications in axonemal tubulin. Given the unrelated positions of the analyzed species in the insect phylogenetic tree, this common feature is probably not due to evolutionary relationships. Therefore, our findings support the hypothesis of the existence of a correlation between the low level of polymodifications and the lack of a microtubule central pair in these peculiar insect flagellar axonemes, similarly as was previously proposed for cilia of Tetrahymena glycylation site mutants.

Does radial nuclear organisation influence DNA damage?

It has been suggested that chromatin at the nuclear periphery could act to shield DNA sequences in the nuclear interior from damage. To test this hypothesis, we have examined the nuclear distribution of sites of DNA repair induced by oxidation or UV-C. We do not detect more damage (repair) at the nuclear periphery than in the nuclear interior. In fact, contrary to the body guard hypothesis, there is an excess of damage detected in the nuclear interior. This is further supported by sequence comparison between genes on human chromosomes 18 or 19, and their counterparts in the chimpanzee. The synonymous substitution rate for genes on chromosome 19, which is located towards the centre of the human nucleus, was higher than that for genes on chromosome 18, which is located at the nuclear periphery. We conclude that chromatin at the periphery of the human nucleus is not able to protect more internally located sequences from damage and mutation. We suggest that features of the chromatin structure, or base composition, of sequences in the nuclear centre make them more susceptible to damage.

Cortical Development: New Concepts

Cortical Development: New Concepts

Protein 4.1R regulates interphase microtubule organization at the centrosome

In human red blood cells, protein 4.1 (4.1R) stabilizes the spectrin-actin network and anchors it to the plasma membrane. To contribute to the characterization of functional roles of 4.1R in nonerythroid cells, we analysed the effect of ectopic expression of 4.1R isoforms on interphase microtubules in fibroblastic cells. We found that specific 4.1R isoforms disturbed the microtubule architecture but not the actin cytoskeleton. Biochemical sedimentation and/or confocal microscopy analyses showed that the pericentriolar components gamma-tubulin and pericentrin remained at centrosomes, whereas the distributions of proteins p150Glued and the dynein intermediate chain were altered. Remarkably, 4.1R was displaced from the centrosome. In microtubule depolymerizing-repolymerizing assays, 4.1R-transfected cells showed an ability to depolymerize and nucleate microtubules that was similar to that of untransfected cells; however, microtubules became disorganized soon after regrowth. In microtubule-depolymerized transfected cells and during the initial steps of microtubule regrowth, centrosomal 4.1R localized with gamma-tubulin but did not when microtubules became disorganized. To learn more about centrosomal 4.1R function, isolated centrosomes were examined by confocal microscopy, western blot and in vitro microtubule aster-assembly assays. The experiments showed that 4.1R was present in isolated centrosome preparations, that it remained in the center of in-vitro-assembled microtubule asters and that more asters were assembled by the addition of protein 4.1R fused to glutathione-S-transferase. Together, these results indicate that 4.1R plays a key role at the centrosome, contributing to the maintenance of a radial microtubule organization.

Getting an embryo into shape

Formation of a multicellular organism is a complex process involving differentiation and morphogenesis. During early vertebrate development, the radial symmetric organization of the egg is transferred into a bilateral symmetric organism with three distinct body axes: anteroposterior (AP), dorsoventral, and left-right. Due to cellular movements and proliferation, the body elongates along the AP axis. How are these processes coupled? Two recent publications now indicate that cell migration as well as orientated cell divisions contribute to axis elongation. The processes are coupled through the planar cell polarity pathway.1 At the same time, the AP axis is patterned independently of convergent extension. This process, however, is required for cell migration and represents a cue for polarized cell motility during gastrulation. Thus, it is AP polarity that instructs individual cells how to orientate with respect to the embryonic axis and provides positional information for the process of convergent extension.(2)

In Vivo Homodimerisation of HTLV-1 Gag and MA Gives Clues to the Retroviral Capsid and TM Envelope Protein Arrangement

During retroviral particle formation, the capsid precursors (Gag) associate with the cell membrane via their matrix (MA) domain to form viral assembling particles. After budding, Gag and its proteolytically matured MA, form a shell in the released immature and mature particles, respectively. Although the arrangement of Gag domains in vitro and their radial organisation in retroviral particles have been extensively studied, little is known concerning Gag inter-subunit interactions in authentic retroviruses. We report that human T-cell leukemia virus type 1 Gag homodimerises in the cell via a disulphide bonding at cysteine 61 in the MA domain. Most Gags are homodimeric after budding and MAs are also dimeric in mature authentic virions. Molecular modelling of the MA domain indicates that non-covalent interactions at the MA dimer interface may also be important for Gag (and MA) dimerisation. In addition, all amino acids previously reported to be involved in MA-transmembrane (TM) interactions are located on the MA face opposite to the dimer interface. The model reveals that homodimerisation is compatible with a hexameric network of Gag and MA dimers that look like the hexameric networks observed for other retroviruses. These data, together with previous studies, lead us to propose a supra-molecular arrangement model in which the transmembrane glycoproteins of the virion envelope are anchored in a hexameric cage hole formed by the MA.

Distant cortical locations of the upper and lower quadrants of the visual field represented by neurons with elongated and radially oriented receptive fields.

In our previous study of the cytoarchitectonic field 7 of cat cortex we had described neurons with extremely elongated receptive fields (RFs). The long axes of these RFs were oriented radially, towards the centre of the retina. These neurons represented only the lower contralateral part of visual field. They were surrounded from all sides by neurons with clearly different RF properties. We proposed that neurons with a similar radial organization and with RFs in the upper visual field also exist in the cortex but are localized in the area that was distant from the representation of the corresponding lower visual field. We expected to find these neurons in front of the representation of the upper visual field in areas V1, V2 and V3 (fields 17, 18 and 19), behind the central representation in area 21a. This cortical region was studied in five behaving cats. In all animals, neurons with radial RFs in the upper visual field were found in the expected location. As in the lower visual field, their RFs always spared the central visual field. Other RF properties of these neurons were also very similar to those found previously in the lower visual field. It became obvious that neurons with radial RFs are included into the fourth extrastriate crescent with complete contralateral representation. However, in the fourth crescent, RF properties in the central visual field differed significantly from those on the periphery. As a result, neurons with similar radial RFs in the upper and lower visual fields were located in the distant cortical regions, and were separated by the representation of the central visual field presented by the non-radial neurons of the cytoarchitectonic area 21a.

Radial organisation of neurons and dendrites in human cortical areas 25, 32, and 32′

Nissl cytoarchitectural and MAP-2 immunocytochemical evidence is presented for the radial organisation of neurons and neural processes in the human medial prefrontal cortex (mPFC). In Brodmann areas 25, 32, and 32′, neuronal cell bodies are organised into short vertical stacks of 15–19 somata with pyramidal cells apical dendrites being arranged into distinct vertically oriented units spaced 52–59 μm apart. Such architecture may underlie specific functional aspects of information processing in the human mPFC.