Limits...
Quantification of local morphodynamics and local GTPase activity by edge evolution tracking.

Tsukada Y, Aoki K, Nakamura T, Sakumura Y, Matsuda M, Ishii S - PLoS Comput. Biol. (2008)

Bottom Line: By applying EET to fluorescence resonance energy transfer images of the Rho-family GTPases Rac1, Cdc42, and RhoA, we examined the cross-correlation between the local area difference and GTPase activity.The calculated correlations changed with time-shifts as expected, but surprisingly, the peak of the correlation coefficients appeared with a 6-8 min time shift of morphological changes and preceded the Rac1 or Cdc42 activities.Our method enables the quantification of the dynamics of local morphological change and local protein activity and statistical investigation of the relationship between them by considering time shifts in the relationship.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Systems Biology, Graduate School of Information Science, Nara Institute of Science and Technology, Nara, Japan.

ABSTRACT
Advances in time-lapse fluorescence microscopy have enabled us to directly observe dynamic cellular phenomena. Although the techniques themselves have promoted the understanding of dynamic cellular functions, the vast number of images acquired has generated a need for automated processing tools to extract statistical information. A problem underlying the analysis of time-lapse cell images is the lack of rigorous methods to extract morphodynamic properties. Here, we propose an algorithm called edge evolution tracking (EET) to quantify the relationship between local morphological changes and local fluorescence intensities around a cell edge using time-lapse microscopy images. This algorithm enables us to trace the local edge extension and contraction by defining subdivided edges and their corresponding positions in successive frames. Thus, this algorithm enables the investigation of cross-correlations between local morphological changes and local intensity of fluorescent signals by considering the time shifts. By applying EET to fluorescence resonance energy transfer images of the Rho-family GTPases Rac1, Cdc42, and RhoA, we examined the cross-correlation between the local area difference and GTPase activity. The calculated correlations changed with time-shifts as expected, but surprisingly, the peak of the correlation coefficients appeared with a 6-8 min time shift of morphological changes and preceded the Rac1 or Cdc42 activities. Our method enables the quantification of the dynamics of local morphological change and local protein activity and statistical investigation of the relationship between them by considering time shifts in the relationship. Thus, this algorithm extends the value of time-lapse imaging data to better understand dynamics of cellular function.

Show MeSH

Related in: MedlinePlus

GTPase cascades involved in morphological regulation and cytoskeleton organization.Various upstream signals trigger the activation of Cdc42, Rac, and Rho GTPases and induce morphological and cytoskeletal changes such as formation of filopodia, lamellipodia, and stress fibers, respectively. The ratio of the inactive GDP-bound state to active GTP-bound state is regulated by guanine nucleotide exchange factors (GEFs) and the GTPase-activating proteins (GAPs). Many studies have shown crosstalk between these GTPases; however, direct links between these GTPases are still to be clarified.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2573959&req=5

pcbi-1000223-g002: GTPase cascades involved in morphological regulation and cytoskeleton organization.Various upstream signals trigger the activation of Cdc42, Rac, and Rho GTPases and induce morphological and cytoskeletal changes such as formation of filopodia, lamellipodia, and stress fibers, respectively. The ratio of the inactive GDP-bound state to active GTP-bound state is regulated by guanine nucleotide exchange factors (GEFs) and the GTPase-activating proteins (GAPs). Many studies have shown crosstalk between these GTPases; however, direct links between these GTPases are still to be clarified.

Mentions: We focused on the Rho-family small GTPases, or Rho GTPases, as signaling molecules associated with cell morphodynamics. Rho GTPases, which act as binary switches by cycling between inactive and active states (Figure 2), play key roles in linking biochemical signaling with biophysical cellular behaviors [19],[20] mainly through reorganization of the actin and microtubule cytoskeleton [21]. It is well known that RhoA, Rac1, and Cdc42 have unique abilities to induce specific filamentous actin structures, i.e., stress fibers, lamellipodia, and filopodia, respectively [19]. Considerable evidence, mainly obtained using constitutively-active or dominant-negative mutants, supports a promotional role of Rac1 and Cdc42 and an inhibitory role of RhoA in cell protrusion [19],[21]. Although some researchers have challenged this widely-accepted notion in a variety of cell contexts [22]–[24], our current study has been motivated by this predominant view.


Quantification of local morphodynamics and local GTPase activity by edge evolution tracking.

Tsukada Y, Aoki K, Nakamura T, Sakumura Y, Matsuda M, Ishii S - PLoS Comput. Biol. (2008)

GTPase cascades involved in morphological regulation and cytoskeleton organization.Various upstream signals trigger the activation of Cdc42, Rac, and Rho GTPases and induce morphological and cytoskeletal changes such as formation of filopodia, lamellipodia, and stress fibers, respectively. The ratio of the inactive GDP-bound state to active GTP-bound state is regulated by guanine nucleotide exchange factors (GEFs) and the GTPase-activating proteins (GAPs). Many studies have shown crosstalk between these GTPases; however, direct links between these GTPases are still to be clarified.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2573959&req=5

pcbi-1000223-g002: GTPase cascades involved in morphological regulation and cytoskeleton organization.Various upstream signals trigger the activation of Cdc42, Rac, and Rho GTPases and induce morphological and cytoskeletal changes such as formation of filopodia, lamellipodia, and stress fibers, respectively. The ratio of the inactive GDP-bound state to active GTP-bound state is regulated by guanine nucleotide exchange factors (GEFs) and the GTPase-activating proteins (GAPs). Many studies have shown crosstalk between these GTPases; however, direct links between these GTPases are still to be clarified.
Mentions: We focused on the Rho-family small GTPases, or Rho GTPases, as signaling molecules associated with cell morphodynamics. Rho GTPases, which act as binary switches by cycling between inactive and active states (Figure 2), play key roles in linking biochemical signaling with biophysical cellular behaviors [19],[20] mainly through reorganization of the actin and microtubule cytoskeleton [21]. It is well known that RhoA, Rac1, and Cdc42 have unique abilities to induce specific filamentous actin structures, i.e., stress fibers, lamellipodia, and filopodia, respectively [19]. Considerable evidence, mainly obtained using constitutively-active or dominant-negative mutants, supports a promotional role of Rac1 and Cdc42 and an inhibitory role of RhoA in cell protrusion [19],[21]. Although some researchers have challenged this widely-accepted notion in a variety of cell contexts [22]–[24], our current study has been motivated by this predominant view.

Bottom Line: By applying EET to fluorescence resonance energy transfer images of the Rho-family GTPases Rac1, Cdc42, and RhoA, we examined the cross-correlation between the local area difference and GTPase activity.The calculated correlations changed with time-shifts as expected, but surprisingly, the peak of the correlation coefficients appeared with a 6-8 min time shift of morphological changes and preceded the Rac1 or Cdc42 activities.Our method enables the quantification of the dynamics of local morphological change and local protein activity and statistical investigation of the relationship between them by considering time shifts in the relationship.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Systems Biology, Graduate School of Information Science, Nara Institute of Science and Technology, Nara, Japan.

ABSTRACT
Advances in time-lapse fluorescence microscopy have enabled us to directly observe dynamic cellular phenomena. Although the techniques themselves have promoted the understanding of dynamic cellular functions, the vast number of images acquired has generated a need for automated processing tools to extract statistical information. A problem underlying the analysis of time-lapse cell images is the lack of rigorous methods to extract morphodynamic properties. Here, we propose an algorithm called edge evolution tracking (EET) to quantify the relationship between local morphological changes and local fluorescence intensities around a cell edge using time-lapse microscopy images. This algorithm enables us to trace the local edge extension and contraction by defining subdivided edges and their corresponding positions in successive frames. Thus, this algorithm enables the investigation of cross-correlations between local morphological changes and local intensity of fluorescent signals by considering the time shifts. By applying EET to fluorescence resonance energy transfer images of the Rho-family GTPases Rac1, Cdc42, and RhoA, we examined the cross-correlation between the local area difference and GTPase activity. The calculated correlations changed with time-shifts as expected, but surprisingly, the peak of the correlation coefficients appeared with a 6-8 min time shift of morphological changes and preceded the Rac1 or Cdc42 activities. Our method enables the quantification of the dynamics of local morphological change and local protein activity and statistical investigation of the relationship between them by considering time shifts in the relationship. Thus, this algorithm extends the value of time-lapse imaging data to better understand dynamics of cellular function.

Show MeSH
Related in: MedlinePlus