Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA266

Poster Communications

Segmentation and quantitative determination of cellular-, neurite- and plasma membrane-motility speed.

A. W. Henkel1, Z. Redzic1

1. Physiology, Kuwait University, Kuwait - Safat, Kuwait.


  • Figure 1. Vesicle motility in chicken telencephalon glia cells. A. Fluorescence image of acidic organelles in a glia cell, labeled with acridine orange; B. overlay image of two consecutive frames, frame 40 (red) and 41 (green) before addition of staurosporine; C. overlay image frame 300 (red) and 301 (green) after addition of staurosporine; D. vesicle trafficking velocity trace, recorded with ‘COPRAMove' algorithm; E. vesicle trafficking velocity trace, recorded with ‘DiffMove' algorithm G. Vesicle motility reduction by staurosporine (mean and standard deviation, asterisks indicate significant difference to control, p < 0.05).

  • Figure 2. Motility measurements on segmented structures. A. COPRAMove motility traces, obtained from isolated somata, neurites and combination of both. "Complete cells" refers to unsegmented hippocampal cultures, "Somata + Neurites" refers to the summed traces of segmented neurites plus segmented somata; B. Quantification of motility results.; C. plasma membrane ruffling velocity of pericytes, measured on isolated cell perimeters; D. Pericyte perimeter outlined by automatic plasma membrane segmentation with the ‘Isolate cells from background' (ICB) algorithm; E. contractility and area/perimeter traces show alternation contraction and extension of typical pericyte plasma membrane. The 4 insets show segmented cell perimeters, acquired at corresponding times (arrows) of the experiment; F. moving average of membrane velocity trace, calculated with the ‘Trace and Quantify' module. The velocity corresponds to the 1st derivative of contractility trace in E.

Mobility quantification of single cells, plasma membranes or cellular processes in dense cell cultures is a challenge, because tracking of individual cells is not readily possible. We used the recently developed software "SynoQuant" (designed by AWH; accessible on www.synosoft.de) to analyze the speed of randomly moving cultured cells, the dynamics of plasma membrane contraction and organelle trafficking. Primary cultures of brain pericytes, hippocampal neurons and chicken telencephalon cells were produced from Sprague Dawley rats[1], Wistar rats[2] and chicken embryos[3], respectively. All experiments were carried out in accordance with the guidelines of laboratory animal care in Kuwait University and the protocols were approved by Animal Resource Center. In some cases, cells were exposed to oxygen glucose deprivation (OGD) as described earlier[1] or maintained in the presence of drugs that affect cell mobility. Images were collected at various intervals over 15-48h in a temperature-controlled chamber on the phase-contrast microscope. Speed quantifications were obtained by segmenting cellular components and determining their individual velocities separately. Algorithms that use image subtraction (DiffMove'), or image correlation analysis (‘COPRAMove') were used to analyse the motility speed of: 1. Intracellular vesicles in chicken telencephalon glia cells (CTG), 2. Somata and neurites in hippocampal neurons and 3. plasma membrane dynamics in pericytes. Differences between the groups were tested with Student's t-test or with Wilcoxon-Mann-Whitney U-test. The speed of vesicles in CTGs was measured with both algorithms (Fig.1); the velocity of the organelles was slowed down by addition of staurosporine, a drug that reduced vesicle movement in the frog neuromuscular junctions[4]. Figs. 1B and C visualize vesicle traffic in an overlay image that was composed of two consecutive images, assigned to different color channels. Neurites and somata of hippocampal neurons were measured separately and their motility velocities were compared to the unprocessed series. The visual impression that the neurites moved slower than the somata, was confirmed by the ‘COPRAMove' quantitative analysis (Fig. 2A, B). The motility of pericytes was examined to quantify membrane ruffling, cell constriction and pseudopodia extrusion. Figs. 2C-F show that the motility of pericytes was consistently and significantly (p < 0.01) decreased after 1-hour of OGD. Pericytes showed alternating periods of extension and retraction of pseudopodia under control conditions. In conclusion, this study has revealed that our new software could be used successfully to reconstitute and analyze cellular and subcellular movements in cell cultures.

Where applicable, experiments conform with Society ethical requirements