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Automated characterization of cell shape changes during amoeboid motility by skeletonization.

Xiong Y, Kabacoff C, Franca-Koh J, Devreotes PN, Robinson DN, Iglesias PA - BMC Syst Biol (2010)

Bottom Line: The method uses skeletonization, a technique from morphological image processing to reduce a shape into a series of connected lines.We illustrate the algorithms on movies of chemotaxing Dictyostelium cells and show that our method makes it possible to capture the spatial and temporal dynamics as well as the stochastic features of the pseudopodial behavior.Thus, the method provides a powerful tool for investigating amoeboid motility.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Electrical and Computer Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.

ABSTRACT

Background: The ability of a cell to change shape is crucial for the proper function of many cellular processes, including cell migration. One type of cell migration, referred to as amoeboid motility, involves alternating cycles of morphological expansion and retraction. Traditionally, this process has been characterized by a number of parameters providing global information about shape changes, which are insufficient to distinguish phenotypes based on local pseudopodial activities that typify amoeboid motility.

Results: We developed a method that automatically detects and characterizes pseudopodial behavior of cells. The method uses skeletonization, a technique from morphological image processing to reduce a shape into a series of connected lines. It involves a series of automatic algorithms including image segmentation, boundary smoothing, skeletonization and branch pruning, and takes into account the cell shape changes between successive frames to detect protrusion and retraction activities. In addition, the activities are clustered into different groups, each representing the protruding and retracting history of an individual pseudopod.

Conclusions: We illustrate the algorithms on movies of chemotaxing Dictyostelium cells and show that our method makes it possible to capture the spatial and temporal dynamics as well as the stochastic features of the pseudopodial behavior. Thus, the method provides a powerful tool for investigating amoeboid motility.

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Localizations of myosin and Dynacortin in chemotaxing cells. Fluorescent images of a chemotaxing Dictyostelium cell expressing mCherry-dynacortin and GFP-myosin-II. A chemoattractant gradient was created using a micropipette needle on the left as previously described [34]. Small arrows placed near cell membrane point to the pseudopodial activities. The numbers at the right upper corners represent the time (in seconds) from the beginning of the movie. The scale bar represents 5 μm. B, D. Fluorescent intensity of mCherry-dynacortin (B) or GFP-myosin-II (D) as a function of time around the cell perimeter for the cell in panel A. The data is normalized between minimum (0) and maximum (1). C. Pseudopod activity as a function of time for the cell in panel A using the same color scheme as in Figure 5D-F. E. Cross-correlations between the two fluorescently-tagged proteins and protrusion or retraction activities as a function of time and angle. Left panels: averaged over 37 cells; right panels: averaged over 100 cells. F. Changes of local protrusion or retraction length in one frame as a function of the local intensity of GFP-myosin-II (100 cells, 4254 frames) or mCherry-dynacortin (37 cells, 1533 frames). Error bars represent standard errors.
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Figure 8: Localizations of myosin and Dynacortin in chemotaxing cells. Fluorescent images of a chemotaxing Dictyostelium cell expressing mCherry-dynacortin and GFP-myosin-II. A chemoattractant gradient was created using a micropipette needle on the left as previously described [34]. Small arrows placed near cell membrane point to the pseudopodial activities. The numbers at the right upper corners represent the time (in seconds) from the beginning of the movie. The scale bar represents 5 μm. B, D. Fluorescent intensity of mCherry-dynacortin (B) or GFP-myosin-II (D) as a function of time around the cell perimeter for the cell in panel A. The data is normalized between minimum (0) and maximum (1). C. Pseudopod activity as a function of time for the cell in panel A using the same color scheme as in Figure 5D-F. E. Cross-correlations between the two fluorescently-tagged proteins and protrusion or retraction activities as a function of time and angle. Left panels: averaged over 37 cells; right panels: averaged over 100 cells. F. Changes of local protrusion or retraction length in one frame as a function of the local intensity of GFP-myosin-II (100 cells, 4254 frames) or mCherry-dynacortin (37 cells, 1533 frames). Error bars represent standard errors.

Mentions: To investigate the molecular drivers of pseudopod formation, the correlation between the time and location of pseudopod activities with fluorescently-labeled proteins is calculated. An AX3-based cell strain is created where myosin-II is tagged using GFP, and, simultaneously, dynacortin is tagged using mCherry. At the same time, both endogenous expressions are confirmed to be depleted completely. It is commonly accepted that myosin-II enriched at the posterior during chemotaxis, generating contractile force at the back and squeezing the cell body forward [7,35]. On the other hand, dynacortin is normally concentrated at the leading edge where it is thought to cooperate with actin to influence cortical viscoelasticity [34]. In our study, developed cells are placed in a chemoattractant gradient, and images of chemotaxing cells are obtained using a dual-emission microscope (Figure 8A). The respective fluorescent concentrations are detected around the cellular membrane (Figures 8B, D), and pseudopods are identified and classified as to whether they are expanding or contracting (Figure 8C). The mCherry-dynacortin is found consistently at the front of the cell, aligned with the chemoattractant gradient. In contrast, GFP-myosin-II is found to be mostly at the side.


Automated characterization of cell shape changes during amoeboid motility by skeletonization.

Xiong Y, Kabacoff C, Franca-Koh J, Devreotes PN, Robinson DN, Iglesias PA - BMC Syst Biol (2010)

Localizations of myosin and Dynacortin in chemotaxing cells. Fluorescent images of a chemotaxing Dictyostelium cell expressing mCherry-dynacortin and GFP-myosin-II. A chemoattractant gradient was created using a micropipette needle on the left as previously described [34]. Small arrows placed near cell membrane point to the pseudopodial activities. The numbers at the right upper corners represent the time (in seconds) from the beginning of the movie. The scale bar represents 5 μm. B, D. Fluorescent intensity of mCherry-dynacortin (B) or GFP-myosin-II (D) as a function of time around the cell perimeter for the cell in panel A. The data is normalized between minimum (0) and maximum (1). C. Pseudopod activity as a function of time for the cell in panel A using the same color scheme as in Figure 5D-F. E. Cross-correlations between the two fluorescently-tagged proteins and protrusion or retraction activities as a function of time and angle. Left panels: averaged over 37 cells; right panels: averaged over 100 cells. F. Changes of local protrusion or retraction length in one frame as a function of the local intensity of GFP-myosin-II (100 cells, 4254 frames) or mCherry-dynacortin (37 cells, 1533 frames). Error bars represent standard errors.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 8: Localizations of myosin and Dynacortin in chemotaxing cells. Fluorescent images of a chemotaxing Dictyostelium cell expressing mCherry-dynacortin and GFP-myosin-II. A chemoattractant gradient was created using a micropipette needle on the left as previously described [34]. Small arrows placed near cell membrane point to the pseudopodial activities. The numbers at the right upper corners represent the time (in seconds) from the beginning of the movie. The scale bar represents 5 μm. B, D. Fluorescent intensity of mCherry-dynacortin (B) or GFP-myosin-II (D) as a function of time around the cell perimeter for the cell in panel A. The data is normalized between minimum (0) and maximum (1). C. Pseudopod activity as a function of time for the cell in panel A using the same color scheme as in Figure 5D-F. E. Cross-correlations between the two fluorescently-tagged proteins and protrusion or retraction activities as a function of time and angle. Left panels: averaged over 37 cells; right panels: averaged over 100 cells. F. Changes of local protrusion or retraction length in one frame as a function of the local intensity of GFP-myosin-II (100 cells, 4254 frames) or mCherry-dynacortin (37 cells, 1533 frames). Error bars represent standard errors.
Mentions: To investigate the molecular drivers of pseudopod formation, the correlation between the time and location of pseudopod activities with fluorescently-labeled proteins is calculated. An AX3-based cell strain is created where myosin-II is tagged using GFP, and, simultaneously, dynacortin is tagged using mCherry. At the same time, both endogenous expressions are confirmed to be depleted completely. It is commonly accepted that myosin-II enriched at the posterior during chemotaxis, generating contractile force at the back and squeezing the cell body forward [7,35]. On the other hand, dynacortin is normally concentrated at the leading edge where it is thought to cooperate with actin to influence cortical viscoelasticity [34]. In our study, developed cells are placed in a chemoattractant gradient, and images of chemotaxing cells are obtained using a dual-emission microscope (Figure 8A). The respective fluorescent concentrations are detected around the cellular membrane (Figures 8B, D), and pseudopods are identified and classified as to whether they are expanding or contracting (Figure 8C). The mCherry-dynacortin is found consistently at the front of the cell, aligned with the chemoattractant gradient. In contrast, GFP-myosin-II is found to be mostly at the side.

Bottom Line: The method uses skeletonization, a technique from morphological image processing to reduce a shape into a series of connected lines.We illustrate the algorithms on movies of chemotaxing Dictyostelium cells and show that our method makes it possible to capture the spatial and temporal dynamics as well as the stochastic features of the pseudopodial behavior.Thus, the method provides a powerful tool for investigating amoeboid motility.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Electrical and Computer Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.

ABSTRACT

Background: The ability of a cell to change shape is crucial for the proper function of many cellular processes, including cell migration. One type of cell migration, referred to as amoeboid motility, involves alternating cycles of morphological expansion and retraction. Traditionally, this process has been characterized by a number of parameters providing global information about shape changes, which are insufficient to distinguish phenotypes based on local pseudopodial activities that typify amoeboid motility.

Results: We developed a method that automatically detects and characterizes pseudopodial behavior of cells. The method uses skeletonization, a technique from morphological image processing to reduce a shape into a series of connected lines. It involves a series of automatic algorithms including image segmentation, boundary smoothing, skeletonization and branch pruning, and takes into account the cell shape changes between successive frames to detect protrusion and retraction activities. In addition, the activities are clustered into different groups, each representing the protruding and retracting history of an individual pseudopod.

Conclusions: We illustrate the algorithms on movies of chemotaxing Dictyostelium cells and show that our method makes it possible to capture the spatial and temporal dynamics as well as the stochastic features of the pseudopodial behavior. Thus, the method provides a powerful tool for investigating amoeboid motility.

Show MeSH
Related in: MedlinePlus