scholarly journals Guidance of zoospores by potassium gradient sensing mediates aggregation

2018 ◽  
Author(s):  
Eric Galiana ◽  
Celine Cohen ◽  
Philippe Thomen ◽  
Catherine Etienne ◽  
Xavier Noblin
Keyword(s):  
Time Lapse ◽  
Support Surface ◽  
Cell Distribution ◽  
Aggregate Formation ◽  
Motion Velocity ◽  
Gradient Sensing ◽  
Geometric Arrangements ◽  
Gradient Generation ◽  
Potassium Gradient ◽  
Threshold Range

ABSTRACTBiflagellate zoospores of some phytopathogenic Phytophthora species spontaneously aggregate within few minutes in suspension. Depending on species auto-aggregate formation results from bioconvection or from a sequence bioconvection-positive chemotaxis. In this study, we show that P. parasitica zoospores may form aggregates upon application of a K+ gradient in particular geometric arrangements. Based on the use of macro- and microfluidic devices, in addition to time-lapse live imaging both in the vertical and horizontal planes, we defined (i) the spatiotemporal and concentration scale evolution within the gradient in correlation with (ii) cell distribution and (iii) metrics of zoospore motion (velocity, trajectory). The results indicated that K+-induced aggregates result from a single bi-phasic temporal sequence involving negative chemotaxis and then bioconvection in a K+ gradient concentration scale [0-17 mM]. Each K+-sensing cell undergoes a forward-to-backward movement within a threshold range of 1-4mM, thereby forming progressively a swarm. Once a critical population density is achieved zoospores form a plume which undergoes a downward migration leading to aggregate formation on the support surface. We discuss putative sources for K+ gradient generation in natural environment (zoospore population, microbiota, plant roots, soil particles), and implication for events preceding inoculum formation on host plant.

scholarly journals Guidance of zoospores by potassium gradient sensing mediates aggregation

2019 ◽  
Vol 16 (157) ◽  
pp. 20190367 ◽  
Author(s):  
Eric Galiana ◽  
Celine Cohen ◽  
Philippe Thomen ◽  
Catherine Etienne ◽  
Xavier Noblin
Keyword(s):  
Host Plants ◽  
Potassium Concentration ◽  
Time Lapse ◽  
Support Surface ◽  
Aggregate Formation ◽  
Motion Velocity ◽  
Gradient Sensing ◽  
Spatio Temporal ◽  
Potassium Gradient ◽  
Threshold Range

The biflagellate zoospores of some phytopathogenic Phytophthora species spontaneously aggregate within minutes in suspension. We show here that Phytophthora parasitica zoospores can form aggregates in response to a K + gradient with a particular geometric arrangement. Using time-lapse live imaging in macro- and microfluidic devices, we defined (i) spatio-temporal and concentration-scale changes in the gradient, correlated with (ii) the cell distribution and (iii) the metrics of zoospore motion (velocity, trajectory). In droplets, we found that K + -induced aggregates resulted from a single biphasic temporal sequence involving negative chemotaxis followed by bioconvection over a K + gradient concentration scale [0–17 mM]. Each K + -sensing cell moved into a region in which potassium concentration is below the threshold range of 1–4 mM, resulting in swarming. Once a critical population density had been achieved, the zoospores formed a plume that migrated downward, with fluid advection in its wake and aggregate formation on the support surface. In the microfluidic device, the density of zoospores escaping potassium was similar to that achieved in droplets. We discuss possible sources of K + gradients in the natural environment (zoospore population, microbiota, plant roots, soil particles), and implications for the events preceding inoculum formation on host plants.

scholarly journals Dynamic redistribution of raft domains as an organizing platform for signaling during cell chemotaxis

2004 ◽  
Vol 164 (5) ◽  
pp. 759-768 ◽  
Author(s):  
Concepción Gómez-Moutón ◽  
Rosa Ana Lacalle ◽  
Emilia Mira ◽  
Sonia Jiménez-Baranda ◽  
Domingo F. Barber ◽  
...  
Keyword(s):  
Lipid Rafts ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Leading Edge ◽  
Time Lapse ◽  
Cholesterol Depletion ◽  
Dependent Manner ◽  
Gradient Sensing ◽  
Raft Domains ◽  
Green Fluorescent

Spatially restricted activation of signaling molecules governs critical aspects of cell migration; the mechanism by which this is achieved nonetheless remains unknown. Using time-lapse confocal microscopy, we analyzed dynamic redistribution of lipid rafts in chemoattractant-stimulated leukocytes expressing glycosyl phosphatidylinositol–anchored green fluorescent protein (GFP-GPI). Chemoattractants induced persistent GFP-GPI redistribution to the leading edge raft (L raft) and uropod rafts of Jurkat, HL60, and dimethyl sulfoxide–differentiated HL60 cells in a pertussis toxin–sensitive, actin-dependent manner. A transmembrane, nonraft GFP protein was distributed homogeneously in moving cells. A GFP-CCR5 chimera, which partitions in L rafts, accumulated at the leading edge, and CCR5 redistribution coincided with recruitment and activation of phosphatidylinositol-3 kinase γ in L rafts in polarized, moving cells. Membrane cholesterol depletion impeded raft redistribution and asymmetric recruitment of PI3K to the cell side facing the chemoattractant source. This is the first direct evidence that lipid rafts order spatial signaling in moving mammalian cells, by concentrating the gradient sensing machinery at the leading edge.

scholarly journals Nuclear Aggresomes Form by Fusion of PML-associated Aggregates

2005 ◽  
Vol 16 (10) ◽  
pp. 4905-4917 ◽  
Author(s):  
Lianwu Fu ◽  
Ya-sheng Gao ◽  
Albert Tousson ◽  
Anish Shah ◽  
Tung-Ling L. Chen ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Fluorescence Recovery After Photobleaching ◽  
Nuclear Architecture ◽  
Time Lapse ◽  
Promyelocytic Leukemia ◽  
Live Cells ◽  
Aggregate Formation ◽  
Pml Bodies ◽  
Nuclear Domains ◽  
Time Lapse Imaging

Nuclear aggregates formed by proteins containing expanded poly-glutamine (poly-Q) tracts have been linked to the pathogenesis of poly-Q neurodegenerative diseases. Here, we show that a protein (GFP170*) lacking poly-Q tracts forms nuclear aggregates that share characteristics of poly-Q aggregates. GFP170*aggregates recruit cellular chaperones and proteasomes, and alter the organization of nuclear domains containing the promyelocytic leukemia (PML) protein. These results suggest that the formation of nuclear aggregates and their effects on nuclear architecture are not specific to poly-Q proteins. Using GFP170*as a model substrate, we explored the mechanistic details of nuclear aggregate formation. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching analyses show that GFP170*molecules exchange rapidly between aggregates and a soluble pool of GFP170*, indicating that the aggregates are dynamic accumulations of GFP170*. The formation of cytoplasmic and nuclear GFP170*aggregates is microtubule-dependent. We show that within the nucleus, GFP170*initially deposits in small aggregates at or adjacent to PML bodies. Time-lapse imaging of live cells shows that small aggregates move toward each other and fuse to form larger aggregates. The coalescence of the aggregates is accompanied by spatial rearrangements of the PML bodies. Significantly, we find that the larger nuclear aggregates have complex internal substructures that reposition extensively during fusion of the aggregates. These studies suggest that nuclear aggregates may be viewed as dynamic multidomain inclusions that continuously remodel their components.

scholarly journals Preferred mitotic orientation in pattern formation by vascular mesenchymal cells

2012 ◽  
Vol 303 (12) ◽  
pp. H1411-H1417 ◽  
Author(s):  
Margaret N. Wong ◽  
Timothy P. Nguyen ◽  
Ting-Hsuan Chen ◽  
Jeffrey J. Hsu ◽  
Xingjuan Zeng ◽  
...  
Keyword(s):  
Cell Division ◽  
Pattern Formation ◽  
Time Lapse ◽  
Mesenchymal Cells ◽  
Self Organization ◽  
Aggregate Formation ◽  
Cell Alignment ◽  
Periodic Patterns ◽  
Daughter Cells ◽  
Local Cell

Cellular self-organization is essential to physiological tissue and organ development. We previously observed that vascular mesenchymal cells, a multipotent subpopulation of aortic smooth muscle cells, self-organize into macroscopic, periodic patterns in culture. The patterns are produced by cells gathering into raised aggregates in the shape of nodules or ridges. To determine whether these patterns are accounted for by an oriented pattern of cell divisions or postmitotic relocation of cells, we acquired time-lapse, videomicrographic phase-contrast, and fluorescence images during self-organization. Cell division events were analyzed for orientation of daughter cells in mitoses during separation and their angle relative to local cell alignment, and frequency distribution of the mitotic angles was analyzed by both histographic and bin-free statistical methods. Results showed a statistically significant preferential orientation of daughter cells along the axis of local cell alignment as early as day 8, just before aggregate formation. This alignment of mitotic axes was also statistically significant at the time of aggregate development ( day 11) and after aggregate formation was complete ( day 15). Treatment with the nonmuscle myosin II inhibitor, blebbistatin, attenuated alignment of mitotic orientation, whereas Rho kinase inhibition eliminated local cell alignment, suggesting a role for stress fiber orientation in this self-organization. Inhibition of cell division using mitomycin C reduced the macroscopic pattern formation. Time-lapse monitoring of individual cells expressing green fluorescent protein showed postmitotic movement of cells into neighboring aggregates. These findings suggest that polarization of mitoses and postmitotic migration of cells both contribute to self-organization into periodic, macroscopic patterns in vascular stem cells.

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

High-voltage Electron Microscopy of astroglial cell whole mounts

1986 ◽  
Vol 44 ◽  
pp. 316-317
Author(s):  
J.N. Turner ◽  
W.G. Shain ◽  
V. Madelian ◽  
R.A. Grassucci ◽  
D.L. Forman
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Time Lapse ◽  
Astroglial Cells ◽  
High Voltage Electron Microscopy ◽  
Rat Glioma ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Nervous System Function ◽  
Beta Adrenergic Agonist

Homogeneous cultures of astroglial cells have proved useful for studying biochemical, pharmacological, and toxicological responses of astrocytes to effectors of central nervous system function. LRM 55 astroglial cells, which were derived from a rat glioma and maintained in continuous culture, exhibit a number of astrocyte properties (1-3). Stimulation of LRM 55s and astrocytes in primary cell cultures with the beta-adrenergic agonist isoproterenol results in rapid changes of morphology. Studies with time lapse video light microscopy (VLM) and high-voltage electron microscopy (HVEM) have been correlated to changes in intracellular levels of c-AMP. This report emphasizes the HVEM results.

Measurement of spontaneous neuronal membrane activity by time lapse confocal microscopy

1995 ◽  
Vol 53 ◽  
pp. 972-973
Author(s):  
R H. Selinfreund ◽  
A. H. Cornell-Bell
Keyword(s):  
Membrane Potential ◽  
Confocal Microscopy ◽  
Patch Clamp ◽  
Electrical Activity ◽  
Time Lapse ◽  
Neuronal Firing ◽  
Neuronal Membrane ◽  
Pituitary Cells ◽  
Patch Clamp Recording ◽  
Spontaneous Electrical Activity

Cellular electrophysiological properties are normally monitored by standard patch clamp techniques . The combination of membrane potential dyes with time-lapse laser confocal microscopy provides a more direct, least destructive rapid method for monitoring changes in neuronal electrical activity. Using membrane potential dyes we found that spontaneous action potential firing can be detected using time-lapse confocal microscopy. Initially, patch clamp recording techniques were used to verify spontaneous electrical activity in GH4\C1 pituitary cells. It was found that serum depleted cells had reduced spontaneous electrical activity. Brief exposure to the serum derived growth factor, IGF-1, reconstituted electrical activity. We have examined the possibility of developing a rapid fluorescent assay to measure neuronal activity using membrane potential dyes. This neuronal regeneration assay has been adapted to run on a confocal microscope. Quantitative fluorescence is then used to measure a compounds ability to regenerate neuronal firing.The membrane potential dye di-8-ANEPPS was selected for these experiments. Di-8- ANEPPS is internalized slowly, has a high signal to noise ratio (40:1), has a linear fluorescent response to change in voltage.

Five-Dimensional Microscopy using Widefield-Deconvolution: Practical Considerations and Biological Applications

1996 ◽  
Vol 54 ◽  
pp. 268-269
Author(s):  
W.F. Marshall ◽  
K. Oegema ◽  
J. Nunnari ◽  
A.F. Straight ◽  
D.A. Agard ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
High Speed ◽  
Laser Scanning ◽  
Ccd Camera ◽  
Image Plane ◽  
Time Lapse ◽  
Laser Scanning Confocal Microscopy ◽  
Three Dimensions ◽  
Excitation Light ◽  
Cooled Ccd

The ability to image cells in three dimensions has brought about a revolution in biological microscopy, enabling many questions to be asked which would be inaccessible without this capability. There are currently two major methods of three dimensional microscopy: laser-scanning confocal microscopy and widefield-deconvolution microscopy. The method of widefield-deconvolution uses a cooled CCD to acquire images from a standard widefield microscope, and then computationally removes out of focus blur. Using such a scheme, it is easy to acquire time-lapse 3D images of living cells without killing them, and to do so for multiple wavelengths (using computer-controlled filter wheels). Thus, it is now not only feasible, but routine, to perform five dimensional microscopy (three spatial dimensions, plus time, plus wavelength).Widefield-deconvolution has several advantages over confocal microscopy. The two main advantages are high speed of acquisition (because there is no scanning, a single optical section is acquired at a time by using a cooled CCD camera) and the use of low excitation light levels Excitation intensity can be much lower than in a confocal microscope for three reasons: 1) longer exposures can be taken since the entire 512x512 image plane is acquired in parallel, so that dwell time is not an issue, 2) the higher quantum efficiently of a CCD detect over those typically used in confocal microscopy (although this is expected to change due to advances in confocal detector technology), and 3) because no pinhole is used to reject light, a much larger fraction of the emitted light is collected. Thus we can typically acquire images with thousands of photons per pixel using a mercury lamp, instead of a laser, for illumination. The use of low excitation light is critical for living samples, and also reduces bleaching. The high speed of widefield microscopy is also essential for time-lapse 3D microscopy, since one must acquire images quickly enough to resolve interesting events.

A Simple and Rapid Method to Determine GPIIb/IIIa-Receptor Inhibitor Activity in Whole Blood

1999 ◽  
Vol 19 (03) ◽  
pp. 134-138
Author(s):  
Gitta Kühnel ◽  
A. C. Matzdorff
Keyword(s):  
Flow Cytometry ◽  
Platelet Count ◽  
Blood Sample ◽  
Platelet Activation ◽  
Whole Blood ◽  
Receptor Occupancy ◽  
Aggregate Formation ◽  
Inhibitor Activity ◽  
Receptor Inhibitor

SummaryWe studied the effect of GPIIb/IIIa-inhibitors on platelet activation with flow cytometry in vitro. Citrated whole blood was incubated with increasing concentrations of three different GPIIb/IIIa-inhibitors (c7E3, DMP728, XJ757), then thrombin or ADP were added and after 1 min the sample was fixed. Samples without c7E3 but with 0.1 U/ml thrombin had a decrease in platelet count. Samples with increasing concentrations of c7E3 had a lesser or no decrease in platelet count. The two other inhibitors (DMP 725, XJ757) gave similar results. GPIIb/IIIa-inhibitors prevent aggregate formation and more single platelets remain in the blood sample. The agonist-induced decrease in platelet count correlates closely with the concentration of the GPIIb/IIIa inhibitor and receptor occupancy. This correlation may be used as a simple measure for inhibitor activity in whole blood.

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