Abstract:Background: For the investigation of the molecular mechanisms involved in neurite outgrowth and differentiation, accurate and reproducible segmentation and quantification of neuronal processes are a prerequisite. To facilitate this task, we developed a semiautomatic neurite tracing technique. This article describes the design and validation of the technique.
Methods:The technique was compared to fully manual delineation. Four observers repeatedly traced selected neurites in 20 fluorescence microscopy images of… Show more
“…Six days after germination, seedlings with the same root length were transferred for additional 9 d to MS plates optionally supplemented with 300 mM mannitol. Primary roots were measured using the ImageJ plug-in NeuronJ (Meijering et al, 2004) to determine growth rates. For ABA treatment, 3-week-old hydroponically grown plants were sprayed with 100 mM ABA-KOH in 0.01% (v/v) Tween 20, or a mock solution, starting after 3 h of light, and rosettes were harvested at different time points for further analyses.…”
Section: Plant Materials and Growth Conditionsmentioning
Starch serves functions that range over a timescale of minutes to years, according to the cell type from which it is derived. In guard cells, starch is rapidly mobilized by the synergistic action of b-AMYLASE1 (BAM1) and a-AMYLASE3 (AMY3) to promote stomatal opening. In the leaves, starch typically accumulates gradually during the day and is degraded at night by BAM3 to support heterotrophic metabolism. During osmotic stress, starch is degraded in the light by stress-activated BAM1 to release sugar and sugar-derived osmolytes. Here, we report that AMY3 is also involved in stress-induced starch degradation. Recently isolated Arabidopsis thaliana amy3 bam1 double mutants are hypersensitive to osmotic stress, showing impaired root growth. amy3 bam1 plants close their stomata under osmotic stress at similar rates as the wild type but fail to mobilize starch in the leaves. 14 C labeling showed that amy3 bam1 plants have reduced carbon export to the root, affecting osmolyte accumulation and root growth during stress. Using genetic approaches, we further demonstrate that abscisic acid controls the activity of BAM1 and AMY3 in leaves under osmotic stress through the AREB/ABF-SnRK2 kinase-signaling pathway. We propose that differential regulation and isoform subfunctionalization define starch-adaptive plasticity, ensuring an optimal carbon supply for continued growth under an ever-changing environment.
“…Six days after germination, seedlings with the same root length were transferred for additional 9 d to MS plates optionally supplemented with 300 mM mannitol. Primary roots were measured using the ImageJ plug-in NeuronJ (Meijering et al, 2004) to determine growth rates. For ABA treatment, 3-week-old hydroponically grown plants were sprayed with 100 mM ABA-KOH in 0.01% (v/v) Tween 20, or a mock solution, starting after 3 h of light, and rosettes were harvested at different time points for further analyses.…”
Section: Plant Materials and Growth Conditionsmentioning
Starch serves functions that range over a timescale of minutes to years, according to the cell type from which it is derived. In guard cells, starch is rapidly mobilized by the synergistic action of b-AMYLASE1 (BAM1) and a-AMYLASE3 (AMY3) to promote stomatal opening. In the leaves, starch typically accumulates gradually during the day and is degraded at night by BAM3 to support heterotrophic metabolism. During osmotic stress, starch is degraded in the light by stress-activated BAM1 to release sugar and sugar-derived osmolytes. Here, we report that AMY3 is also involved in stress-induced starch degradation. Recently isolated Arabidopsis thaliana amy3 bam1 double mutants are hypersensitive to osmotic stress, showing impaired root growth. amy3 bam1 plants close their stomata under osmotic stress at similar rates as the wild type but fail to mobilize starch in the leaves. 14 C labeling showed that amy3 bam1 plants have reduced carbon export to the root, affecting osmolyte accumulation and root growth during stress. Using genetic approaches, we further demonstrate that abscisic acid controls the activity of BAM1 and AMY3 in leaves under osmotic stress through the AREB/ABF-SnRK2 kinase-signaling pathway. We propose that differential regulation and isoform subfunctionalization define starch-adaptive plasticity, ensuring an optimal carbon supply for continued growth under an ever-changing environment.
“…Neurite centerlines may alternatively be obtained directly from the grayscale images, by applying Hessian (58)(59)(60)(61) or Jacobian (62) based analysis of critical points, or by nonmaximum suppression (37,63). The result of the skeletonization step often contains errors (such as spurious gaps or branches) and (especially in 2D) ambiguities (spurious loops or crossings).…”
Section: Tree Segmentationmentioning
confidence: 99%
“…In high-magnification 3D images of neurites running largely in the axial direction, region-growing methods may be used to segment the neurites in one optical section, whose centroid positions can serve as seed points to initiate segmentation in the next section (66,67), reminiscent of mean-shift tracking (68,69) and activecontour based propagation approaches (70)(71)(72). More robustness can be expected from algorithms that constrain the search to given start and end points, by defining a cost or ''energy'' function that assigns a penalty to connecting any two neighboring points (computed from local image features at these points), and minimizing the cumulative cost from start to end point (27,58,(73)(74)(75)(76). A related approach is to fit an active-contour model (based on generalized cylinders) to the image data between given crucial points (77,78).…”
The study of the structure and function of neuronal cells and networks is of crucial importance in the endeavor to understand how the brain works. A key component in this process is the extraction of neuronal morphology from microscopic imaging data. In the past four decades, many computational methods and tools have been developed for digital reconstruction of neurons from images, with limited success. As witnessed by the growing body of literature on the subject, as well as the organization of challenging competitions in the field, the quest for a robust and fully automated system of more general applicability still continues. The aim of this work, is to contribute by surveying recent developments in the field for anyone interested in taking up the challenge. Relevant aspects discussed in the article include proposed image segmentation methods, quantitative measures of neuronal morphology, currently available software tools for various related purposes, and morphology databases. ' 2010 International Society for Advancement of Cytometry
“…Samples were photographed in a Zeiss Axioplan2 microscope with a 63ϫ Zeiss objective lens (NA 1.4) coupled with a Zeiss AxioCam HRc or in a Zeiss Leo 912AB electron microscope (Zeiss, Oberkochen, Germany). Fiber length was calculated with the Neuron J plugin (Meijering et al, 2004) using the Image J program (Rasband, W.S., NIH, Bethesda, MA; http://rsb.info.nih.gov/ij/).…”
The function of ␣-synuclein, a soluble protein abundant in the brain and concentrated at presynaptic terminals, is still undefined. Yet, ␣-synuclein overexpression and the expression of its A30P mutant are associated with familial Parkinson's disease. Working in cell-free conditions, in two cell lines as well as in primary neurons we demonstrate that ␣-synuclein and its A30P mutant have different effects on actin polymerization. Wild-type ␣-synuclein binds actin, slows down its polymerization and accelerates its depolymerization, probably by monomer sequestration; A30P mutant ␣-synuclein increases the rate of actin polymerization and disrupts the cytoskeleton during reassembly of actin filaments. Consequently, in cells expressing mutant ␣-synuclein, cytoskeleton-dependent processes, such as cell migration, are inhibited, while exo-and endocytic traffic is altered. In hippocampal neurons from mice carrying a deletion of the ␣-synuclein gene, electroporation of wild-type ␣-synuclein increases actin instability during remodeling, with growth of lamellipodia-like structures and apparent cell enlargement, whereas A30P ␣-synuclein induces discrete actin-rich foci during cytoskeleton reassembly. In conclusion, ␣-synuclein appears to play a major role in actin cytoskeletal dynamics and various aspects of microfilament function. Actin cytoskeletal disruption induced by the A30P mutant might alter various cellular processes and thereby play a role in the pathogenesis of neurodegeneration.
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