scholarly journals RoPod, a customizable toolkit for non-invasive root imaging, reveals cell type-specific dynamics of plant autophagy

2021 ◽  
Author(s):  
Marjorie Guichard ◽  
Sanjana Holla ◽  
Dasa Wernerova ◽  
Guido E.A. Grossmann ◽  
Alyona E.A. Minina
Keyword(s):  
Complex Dynamics ◽  
Time Lapse ◽  
Automated Image Analysis ◽  
Cell Type ◽  
Time Resolved ◽  
Living Plant ◽  
Fluorescent Reporters ◽  
Drug Treatments ◽  
Cell Type Specific ◽  
Time Lapse Imaging

Autophagy is the major catabolic process in eukaryotes and a key regulator of plant fitness. It enables rapid response to stress stimuli, essential for plastic adaptation of plants to changes in the environment. Fluorescent reporters and confocal microscopy are among the most frequently used methods for assessing plant autophagic activity. However, detection of dynamic changes in the pathway activity has been hampered by stresses imposed on living plant tissues during sample mounting and imaging. Here we implemented RoPod, a toolkit optimized for minimally-invasive time-lapse imaging of Arabidopsis roots, to reveal a time-resolved response of plant autophagy to drug treatments typically used for pathway modulation and discovered previously overlooked cell type-specific changes in the pathway response. These results not only give an insight into the complex dynamics of plant autophagy, but also provide necessary information for choosing sampling time for the end-point assays currently employed in plant autophagy research. RoPods are inexpensive and easy-to-use devices that are based on commercial or custom designed chambers, compatible with inverted microscopes. We describe a detailed protocol for the fabrication and use of RoPods and provide a complete pipeline including semi-automated image analysis for root hair growth assays, demonstrating the broader applicability of the RoPod toolkit.

scholarly journals 3DeFDR: statistical methods for identifying cell type-specific looping interactions in 5C and Hi-C data

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Lindsey R. Fernandez ◽  
Thomas G. Gilgenast ◽  
Jennifer E. Phillips-Cremins
Keyword(s):  
Cell Type ◽  
Statistical Tool ◽  
Chromosome Conformation ◽  
Developmental Models ◽  
Disease States ◽  
Genetic Perturbations ◽  
Drug Treatments ◽  
Cell Type Specific ◽  
Carbon Copy ◽  
Chromatin Biology

Abstract An important unanswered question in chromatin biology is the extent to which long-range looping interactions change across developmental models, genetic perturbations, drug treatments, and disease states. Computational tools for rigorous assessment of cell type-specific loops across multiple biological conditions are needed. We present 3DeFDR, a simple and effective statistical tool for classifying dynamic loops across biological conditions from Chromosome-Conformation-Capture-Carbon-Copy (5C) and Hi-C data. Our work provides a statistical framework and open-source coding libraries for sensitive detection of cell type-specific loops in high-resolution 5C and Hi-C data from multiple cellular conditions.

295 THE SHAPE OF PORCINE NEURAL PROGENITOR CELL CELLULAR GENEALOGIES REVEALED BY TIME-LAPSE IMAGING

2011 ◽  
Vol 23 (1) ◽  
pp. 245
Author(s):  
I. Faerge ◽  
A. Egeskov-Madsen ◽  
P. Holm
Keyword(s):  
Cell Cycle ◽  
Growth Factor ◽  
Cycle Length ◽  
Individual Cell ◽  
Time Lapse ◽  
Cell Type ◽  
Epidermal Growth ◽  
Cellular Development ◽  
Time Lapse Imaging ◽  
Cell Cycle Length

Porcine neural progenitor cells (pNPC) derived from embryonic stem cells are capable of self-renewal and differentiation into neural and glia lineages, rendering them promising candidates for cell-based therapy of neurodegenerative diseases in a large animal biomedical model. A prerequisite for the successful future therapeutic use of pNPC is a comprehensive characterisation and understanding of the neurogenic process. This is important for learning how to direct cell fates into required proportions of the cell type wanted for the specific brain disease to be treated, and it is crucial for avoiding uncontrolled cell proliferation leading to fatal tumour formations. Time-lapse analysis is a powerful tool to obtain live cell characterisation by analysing individual cell fate. Information on cellular development, division, and differentiation can be composed into a pedigree-like structure denoted as cellular genealogy giving an overview of the proliferation profile of a cell culture and the duration of each cell cycle (Al-Kofani et al. 2006). The aim of the study was to construct cellular genealogies of pNPC and differentiated neural lineages, respectively, by time-lapse imaging to evaluate the effect of external variables observed by changes in the topology of the cellular genealogy. Porcine NPC were derived from epiblast cells isolated from day-9 porcine blastocysts and cultured in DMEM/12, Pen/strep, B27 and N2 with basic fibroblast growth factor and epidermal growth factor, and differentiation was obtained by withdrawal of basic fibroblast growth factor and epidermal growth factor. The state of cellular development of undifferentiated and differentiated pNPC was verified immunohistochemically by the presence of SOX2, NESTIN, TUJI, and GFAB (Rasmussen et al. 2010). The time-lapse images were captured by a Nikon Biostation with a 10× resolution under phase contrast in a humidified chamber at 38°C with 5% CO2, 5% O2, and 90% N2. For each sequence, images were captured at intervals of 10 min in 16 frames. Sequences 1, 2, and 3 constituted passage 15 pNPC, passage 4 pNPC, and presumably differentiated cells, respectively. For each sequence, cell cycle length was calculated after manual tracking of selected cells. The cell cycle length of pNPC is shown in Table 1. Based on these data, cellular genealogies characteristic of each individual cell type have been constructed. Table 1.Cell cycle length of porcine neutral progenitor cells (pNPC) before and after differentiation

Cell-type-specific rescue of myosin function during Dictyostelium development defines two distinct cell movements required for culmination

Development ◽  
1998 ◽  
Vol 125 (19) ◽  
pp. 3895-3903 ◽  
Author(s):  
T.L. Chen ◽  
W.A. Wolf ◽  
R.L. Chisholm
Keyword(s):  
Fluorescent Protein ◽  
Video Recording ◽  
Time Lapse ◽  
Active Movement ◽  
Upward Movement ◽  
Wild Type ◽  
Cell Type ◽  
Developmental Defects ◽  
Distinct Cell ◽  
Cell Type Specific

Mutant Dictyostelium cells lacking any of the component polypeptides of myosin II exhibit developmental defects. To define myosin's role in establishing Dictyostelium's developmental pattern, we have rescued myosin function in a myosin regulatory light chain null mutant (mlcR-) using cell-type-specific promoters. While mlcR- cells fail to progress beyond the mound stage, expression of RLC from the prestalk promoter, ecmA, produces culminants with normal stalks but with defects in spore cell localization. When GFP-marked prestalk and prespore cells expressing ecmA-RLC are mixed with wild-type cells, the mislocalization of prestalk cells, but not prespore cells, is rescued. Time-lapse video recording of ecmA-RLC cells showed that the posterior prespore zone failed to undergo a contraction important for the upward movement of prespore cells. Prespore cells marked with green fluorescent protein (GFP) failed to move toward the tip with the spiral motion typical of wild type. In contrast, expression of RLC in prespore cells using the psA promoter produced balloon-like structures reminiscent of sorocarps but lacking stalks. GFP-labeled prespore cells showed a spiral movement toward the top of the structures. Expression of RLC from the psA promoter restores the normal localization of psA-GFP cells, but not ecmA-GFP cells. These results define two distinct, myosin-dependent movements that are required for establishing a Dictyostelium fruiting body: stalk extension and active movement of the prespore zone that ensures proper placement of the spores atop the stalk. The approach used in these studies provides a direct means of testing the role of cell motility in distinct cell types during a morphogenetic program.

scholarly journals Size segregation of irregular granular materials captured by time-resolved 3D imaging

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Parmesh Gajjar ◽  
Chris G. Johnson ◽  
James Carr ◽  
Kevin Chrispeels ◽  
J. M. N. T. Gray ◽  
...  
Keyword(s):  
Granular Materials ◽  
Complex Dynamics ◽  
Experimental Studies ◽  
Vertical Axis ◽  
Time Lapse ◽  
Brazil Nut ◽  
Size Segregation ◽  
Time Resolved ◽  
Brazil Nuts ◽  
Brazil Nut Effect

AbstractWhen opening a box of mixed nuts, a common experience is to find the largest nuts at the top. This well-known effect is the result of size-segregation where differently sized ‘particles’ sort themselves into distinct layers when shaken, vibrated or sheared. Colloquially this is known as the ‘Brazil-nut effect’. While there have been many studies into the phenomena, difficulties observing granular materials mean that we still know relatively little about the process by which irregular larger particles (the Brazil nuts) reach the top. Here, for the first time, we capture the complex dynamics of Brazil nut motion within a sheared nut mixture through time-lapse X-ray Computed Tomography (CT). We have found that the Brazil nuts do not start to rise until they have first rotated sufficiently towards the vertical axis and then ultimately return to a flat orientation when they reach the surface. We also consider why certain Brazil nuts do not rise through the pack. This study highlights the important role of particle shape and orientation in segregation. Further, this ability to track the motion in 3D will pave the way for new experimental studies of segregating mixtures and will open the door to even more realistic simulations and powerful predictive models. Understanding the effect of size and shape on segregation has implications far beyond food products including various anti-mixing behaviors critical to many industries such as pharmaceuticals and mining.

scholarly journals Airpart: Interpretable statistical models for analyzing allelic imbalance in single-cell datasets

2021 ◽  
Author(s):  
Wancen Mu ◽  
Hirak Sarkar ◽  
Avi Srivastava ◽  
Kwangbom Choi ◽  
Rob Patro ◽  
...  
Keyword(s):  
Single Cell ◽  
Allelic Imbalance ◽  
Genetic Regulation ◽  
Real Data ◽  
Cell Types ◽  
Cell Type ◽  
Time Resolved ◽  
Bulk Data ◽  
Cell Type Specific ◽  
Cell Data

Motivation: Allelic expression analysis aids in detection of cis-regulatory mechanisms of genetic variation which produce allelic imbalance (AI) in heterozygotes. Measuring AI in bulk data lacking time or spatial resolution has the limitation that cell-type-specific (CTS), spatial-, or time-dependent AI signals may be dampened or not detected. Results: We introduce a statistical method airpart for identifying differential CTS AI from single-cell RNA-sequencing (scRNA-seq) data, or other spatially- or time-resolved datasets. airpart outputs discrete partitions of data, pointing to groups of genes and cells under common mechanisms of cis-genetic regulation. In order to account for low counts in single-cell data, our method uses a Generalized Fused Lasso with Binomial likelihood for partitioning groups of cells by AI signal, and a hierarchical Bayesian model for AI statistical inference. In simulation, airpart accurately detected partitions of cell types by their AI and had lower RMSE of allelic ratio estimates than existing methods. In real data, airpart identified differential AI patterns across cell states and could be used to define trends of AI signal over spatial or time axes. Availability: The airpart package is available as a R/Bioconductor package at https://bioconductor.org/packages/airpart.

IFIH1-deficiency results in a cell-type specific alteration of autophagic activity in response to S. typhimurium

2017 ◽  
Vol 55 (05) ◽  
pp. e28-e56
Author(s):  
S Macheiner ◽  
R Gerner ◽  
A Pfister ◽  
A Moschen ◽  
H Tilg
Keyword(s):  
Cell Type ◽  
A Cell ◽  
Cell Type Specific ◽  
Autophagic Activity ◽  
Specific Alteration

Cleavage of Human Embryos: Options and Diversity

Acta Naturae ◽  
2016 ◽  
Vol 8 (3) ◽  
pp. 88-96
Author(s):  
Yu. K. Doronin ◽  
I. V. Senechkin ◽  
L. V. Hilkevich ◽  
M. A. Kurcer
Keyword(s):  
Time Lapse ◽  
Reproductive Technologies ◽  
Human Embryos ◽  
Imaging Data ◽  
Noninvasive Methods ◽  
Topographic Features ◽  
Time Parameters ◽  
Embryo Cleavage ◽  
Time Lapse Imaging ◽  
Developmental Dynamics

In order to estimate the diversity of embryo cleavage relatives to embryo progress (blastocyst formation), time-lapse imaging data of preimplantation human embryo development were used. This retrospective study is focused on the topographic features and time parameters of the cleavages, with particular emphasis on the lengths of cleavage cycles and the genealogy of blastomeres in 2- to 8-cell human embryos. We have found that all 4-cell human embryos have four developmental variants that are based on the sequence of appearance and orientation of cleavage planes during embryo cleavage from 2 to 4 blastomeres. Each variant of cleavage shows a strong correlation with further developmental dynamics of the embryos (different cleavage cycle characteristics as well as lengths of blastomere cycles). An analysis of the sequence of human blastomere divisions allowed us to postulate that the effects of zygotic determinants are eliminated as a result of cleavage, and that, thereafter, blastomeres acquire the ability of own syntheses, regulation, polarization, formation of functional contacts, and, finally, of specific differentiation. This data on the early development of human embryos obtained using noninvasive methods complements and extend our understanding of the embryogenesis of eutherian mammals and may be applied in the practice of reproductive technologies.

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