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Objectives: Alzheimer's disease (AD) is a neurodegenerative disorder resulting from the accumulation of toxic β-amyloid (Aβ) aggregates in the human brain. Epidemiological studies have shown that elevated cholesterol plasma levels are associated with the development of AD and we have previously shown that cholesterol restriction reduces the Aβ-induced paralysis in an Alzheimer model of the nematode Caenorhabditis elegans. In the present study we investigated the effects of the cholesterol homolog cholecalciferol, i.e. vitamin D, on Aβ-induced paralysis in C. elegans and its interference with the steroid-signaling pathway. Methods: Aβ-induced paralysis was assessed in the C. elegans strain CL2006, expressing human Aβ1-42 under control of a muscle-specific promoter. Knockdown of members of the steroid-signaling pathway was achieved by RNA interference (RNAi). Nuclear translocation of foxo transcription factor DAF-16 was visualized using the strain TJ356, carrying a daf-16::gfp transgene. Results: Cholecalciferol at a concentration of 1 µM reduced the Aβ-induced paralysis in CL2006 significantly, which was reverted by increasing the cholesterol concentration in the medium. Knockdown of nhr-8, daf-36, daf-9 or daf-12, all reduced Aβ-induced paralysis to the same extent as cholecalciferol with no additional or synergistic effects under co-application. Functional DAF-16 proved to be crucial for the effects of cholecalciferol and DAF-16 nuclear translocation was increased by cholecalciferol and also RNAi versus nhr-8, daf-36, daf-9 or daf-12 with no additive or synergistic effects. Conclusions: Our results suggest, that cholecalciferol inhibits Aβ-induced paralysis in C. elegans through inhibition of steroid-signaling and the concomitant nuclear translocation of DAF-16. [ABSTRACT FROM AUTHOR]

During cytokinesis, aurora B kinase (ABK) relocalizes from centromeres to the spindle midzone, where it is thought to provide a spatial cue for cytokinesis. While global ABK inhibition in Drosophila S2 cells results in macro- and multi-nucleated large cells, mislocalization of midzone ABK (mABK) by depletion of Subito (Drosophila MKLP2) does not cause notable cytokinesis defects. Subito depletion was, therefore, used to investigate the contribution of other molecules and redundant pathways to cytokinesis in the absence of mABK. Inhibiting potential polar relaxation pathways via removal of centrosomes (CNN RNAi) or a kinetochore-based phosphatase gradient (Sds22 RNAi) did not result in cytokinesis defects on their own or in combination with loss of mABK. Disruption of aurora A kinase (AAK) activity resulted in midzone assembly defects, but did not significantly affect contractile ring positioning or cytokinesis. Live-cell imaging of a Förster resonance energy transfer (FRET)-based aurora kinase phosphorylation sensor revealed that midzone substrates were less phosphorylated in AAK-inhibited cells, despite the fact that midzone levels of active phosphorylated ABK (pABK) were normal. Interestingly, in the absence of mABK, an increased number of binucleated cells were observed following AAK inhibition. The data suggest that equatorial stimulation rather than polar relaxation mechanisms is the major determinant of contractile ring positioning and high-fidelity cytokinesis in Drosophila S2 cells. Furthermore, we propose that equatorial stimulation is mediated primarily by the delivery of factors to the cortex by noncentrosomal microtubules (MTs), as well as a midzone-derived phosphorylation gradient that is amplified by the concerted activities of mABK and a soluble pool of AAK. [ABSTRACT FROM AUTHOR]

Dual-view inverted selective plane illumination microscopy (diSPIM) enables high-speed, long-term, fourdimensional (4D) imaging with isotropic spatial resolution. It is also compatible with conventional sample mounting on glass coverslips. However, broadening of the light sheet at distances far from the beam waist and sample-induced scattering degrades diSPIM contrast and optical sectioning. We describe two simple improvements that address both issues and entail no additional hardware modifications to the base diSPIM. First, we demonstrate improved diSPIM sectioning by keeping the light sheet and detection optics stationary, and scanning the sample through the stationary light sheet (rather than scanning the broadening light sheet and detection plane through the stationary sample, as in conventional diSPIM). This stage-scanning approach allows a thinner sheet to be used when imaging laterally extended samples, such as fixed microtubules or motile mitochondria in cell monolayers, and produces finer contrast than does conventional diSPIM. We also used stage-scanning diSPIM to obtain high-quality, 4D nuclear datasets derived from an uncompressed nematode embryo, and performed lineaging analysis to track 97% of cells until twitching. Second, we describe the improvement of contrast in thick, scattering specimens by synchronizing light-sheet synthesis with the rolling, electronic shutter of our scientific complementary metal-oxide-semiconductor (sCMOS) detector. This maneuver forms a virtual confocal slit in the detection path, partially removing out-of-focus light. We demonstrate the applicability of our combined stage- and slit-scanningmethods by imaging pollen grains and nuclear and neuronal structures in live nematode embryos. All [ABSTRACT FROM AUTHOR]

Lamin A/C is part of the nuclear lamina, a meshwork of intermediate filaments underlying the inner nuclear membrane. The lamin network is anchoring a complex set of structural and linker proteins and is either directly or through partner proteins also associated or interacting with a number of signaling protein and transcription factors. During mitosis the nuclear lamina is dissociated by well established phosphorylation- dependent mechanisms. A-type lamins are, however, also phosphorylated during interphase. A recent study identified 20 interphase phosphorylation sites on lamin A/C and explored their functions related to lamin dynamics; movements, localization and solubility. Here we discuss these findings in the light of lamin functions in health and disease. [ABSTRACT FROM PUBLISHER]

Myoblast migration is essential for muscle development and repair; however, the factors that contribute to the polarity of migrating myoblasts are relatively unknown. We find that randomly migrating C2C12 myoblasts orient their centrosomes in the direction of migration. Using wounded monolayers, we further show that centrosome orientation is stimulated by the serum factor lysophosphatidic acid (LPA) and involves the rearward movement of the nucleus while the centrosome is maintained at the cell centroid. The rate of nuclear movement correlated with that of actin retrograde flow and both cytochalasin D and blebbistatin prevented nuclear movement and centrosome orientation. Actin-dependent rearward nuclear movement in fibroblasts is mediated by assembly of nuclear membrane nesprin-2G and SUN2 LINC complexes into transmembrane actin-associated nuclear (TAN) lines anchored by A-type lamins and emerin. In C2C12 myoblasts, depletion of nesprin-2G, SUN2 or lamin A/C prevented nuclear movement and endogenous nesprin-2G and a chimeric GFP-mini-nesprin-2G formed TAN lines during nuclear movement. Depleting nesprin-2G strongly interfered with directed cell migration and reduced the efficiency of myoblast fusion into multinucleated myotubes. Our results show that nuclear movement contributes to centrosome orientation and polarity for efficient migration and fusion of myoblasts. Given that mutations in the genes encoding A-type lamins, nesprin-2 and SUN2 cause Emery-Dreifuss muscular dystrophy and related myopathies, our results have implications for understanding the mechanism of disease pathogenesis. [ABSTRACT FROM PUBLISHER]

Nuclear pore complexes (NPCs) span the 2 membranes of the nuclear envelope (NE) and facilitate nucleocytoplasmic exchange of macromolecules. NPCs have a roughly tripartite structural organization with the so-called nuclear basket emanating from the NPC scaffold into the nucleoplasm. The nuclear basket is composed of the 3 nucleoporins Nup153, Nup50, and Tpr, but their specific role for the structural organization of this NPC substructure is, however, not well established. In this study, we have used thin-section transmission electron microscopy to determine the structural consequences of altering the expression of Nup153 in human cells. We show that the assembly and integrity of the nuclear basket is not affected by Nup153 depletion, whereas its integrity is perturbed in cells expressing high concentrations of the zinc-finger domain of Nup153. Moreover, even mild over-expression of Nup153 is coinciding with massive changes in nuclear organization and it is the excess of the zinc-finger domain of Nup153 that is sufficient to induce these rearrangements. Our data indicate a central function of Nup153 in the organization of the nucleus, not only at the periphery, but throughout the entire nuclear interior. [ABSTRACT FROM AUTHOR]

DBS/MCF2Lhas been recently identified as a risk locus for osteoarthritis. It encodes a guanine nucleotide exchange factor (Dbs) that has been shown to regulate both normal and tumor cell motility. In the current study, we have determined that endogenous Dbs is predominantly expressed as 2 isoforms, a 130 kDa form (Dbs-130) that is localized to the Golgi complex, and an 80 kDa form (Dbs-80) that is localized to the endoplasmic reticulum (ER). We have previously described an inhibitor that binds to the RhoGEF domain of Dbs and blocks its transforming activity. Here we show that the inhibitor localizes to the Golgi, where it specifically interacts with Dbs-130. Inhibition of endogenous Dbs-130 activity is associated with reduced levels of activated Cdc42, enlarged Golgi, and resistance to Brefeldin A-mediated Golgi dispersal, suggesting a role for Dbs in vesicle transport. Cells treated with the inhibitor exhibit normal protein transport from the ER to the Golgi, but are defective in transport from the Golgi to the plasma membrane. Inhibition of Dbs-130 in MDA-MB-231 human breast tumor cells limits motility in both transwell and wound healing assays, but appears to have no effect on the organization of the microtubule cytoskeleton. The reduced motility is associated with a failure to reorient the Golgi toward the leading edge. This is consistent with the Golgi localization, and suggests that the Dbs-130 regulates aspects of the secretory pathway that are required to support cell polarization during directed migration. [ABSTRACT FROM PUBLISHER]

Actin is a major actor in the determination of cell shape. On the one hand, site-directed assembly/disassembly cycles of actin filaments drive protrusive force leading to lamellipodia and filopodia dynamics. Force produced by actin similarly contributes in membrane scission in endocytosis or Golgi remodeling. On the other hand, cellular processes like adhesion, immune synapse, cortex dynamics or cytokinesis are achieved by combining acto-myosin contractility and actin assembly in a complex and not fully understood manner. New chemical compounds are therefore needed to disentangle acto-myosin and actin dynamics. We have found that synthetic, cell permeant, short polyamines are promising new actin regulators in this context. They generate growth and stabilization of lamellipodia within minutes by slowing down the actin assembly/disassembly cycle and facilitating nucleation. We now report that these polyamines also slow down cytokinetic ring closure in fission yeast. This shows that these synthetic compounds are active also in yeasts, and these experiments specifically highlight that actin depolymerization is involved in the ring closure. Thus, synthetic polyamines appear to be potentially powerful agents in a quantitative approach to the role of actin in complex processes in cell biology, developmental biology and potentially cancer research. [ABSTRACT FROM PUBLISHER]