Simultaneous quantification of cell motility and protein‐membrane‐association using active contours

Abstract

We present a new method for the quantification of dynamic changes in fluorescence intensities at the cell membrane of moving cells. It is based on an active contour method for cell-edge detection, which allows tracking of changes in cell shape and position. Fluorescence intensities at specific cortical subregions can be followed in space and time and correlated with cell motility. The translocation of two GFP tagged proteins (CRAC and GRP1) from the cytosol to the membrane in response to stimulation with the chemoattractant cAMP during chemotaxis of Dictyostelium cells and studies of the spatio-temporal dynamics of this process exemplify the method: We show that the translocation can be correlated with motility parameters and that quantitative differences in the rate of association and dissociation from the membrane can be observed for the two PH domain containing proteins. The analysis of periodic CRAC translocation to the leading edge of a cell responding to natural cAMP waves in a mound demonstrates the power of this approach. It is not only capable of tracking the outline of cells within aggregates in front of a noisy background, but furthermore allows the construction of spatio-temporal polar plots, capturing the dynamics of the protein distribution at the cell membrane within the cells' moving co-ordinate system. Compilation of data by means of normalised polar plots is suggested as a future tool, which promises the so-far impossible practicability of extensive statistical studies and automated comparison of complex spatio-temporal protein distribution patterns.

Cell shape recognition and segmentation in fluorescence microscopy images.

“…In particular, there are software packages that show many userfriedly features like a graphical user interface (GUI) and compatibility with common image processing and graphics programs. A prominent example is the ImageJ plug-in QuimP, one of the most widely used tools for the processing of live-cell fluorescence images [4,6]. Table 1 shows a comparison between features of the QuimP software and our segmentation protocol.…”

“…QuimP is implemented as a set of plugins for ImageJ, one of the most widely used image processing tools [5]. QuimP uses an active contour to automatically detect the outline of a cell in order to quantify fluorescence signals along the cell membrane [6] and has recently been extended to correlate the cortical fluorescence signals with membrane movement, a typical problem in fluorescence microscopy of motile cells [7]. A similar problem has been addressed before based on morphodynamic profiling [8].…”

Comparative analysis of neural crest cell death, migration, and function during vertebrate embryogenesis

“…Disruption of Eph/ephrin signaling affects neuroblast cell migration (Conover et al, 2000). In another model system, the cellular slime mold Dictyostelium, individual cells form multicell-wide chains en route to assembling a multicellular organism by using a complex relay system of chemotactic signals (Dormann et al, 2002;Meili and Firtel, 2003). Thus, in the future, insights from each of these model systems will provide clues to help dissect the mechanisms of neural crest cell patterning and migration.…”

High Resolution Tracking of Cell Membrane Dynamics in Moving Cells: an Electrifying Approach

“…The mechanical spring model is analogous to using active contours for segmentation, but applied to tracking boundary regions. A similar approach has been used in the QuimP software package, previously developed by us [12], and discussed in more detail in the following section. This paper details the Electrostatic Contour Migration Method (ECMM), a new method for boundary tracking, which supersedes the current tracking in QuimP.…”

“…The QuimP software package [12] forms a framework for automating the measurement of fluorescent intensities of labeled cortical proteins while simultaneously tracking membrane deformations. QuimP (Quantitative imaging of membrane proteins) uses a simple active contour, implemented in ImageJ, to segment cells and define outlines as a chain of nodes ( Figure 2).…”