Untangling Irregular Actin Cytoskeleton Architectures in Tomograms of the Cell with Struwwel Tracer
Abstract
:1. Introduction
- Among the cryo-ET-related actin tracing methods, some are only applicable for the tracing of well-ordered filaments [4,6,7]. Among these, one noteworthy tool is our Spaghetti Tracer approach [7]. Spaghetti Tracer introduced a paradigm shift in the tracing of semi-regular actin filaments because it is a dynamic-programming-based method at the voxel level that does not require an expensive missing wedge correction, template convolution, or deconvolution. Therefore, it yields a substantial improvement in time efficiency over template convolution [8,10] or deconvolution methods [4], enabling fast and accurate tracing of such filament arrangements. (The accuracy of Spaghetti Tracer was validated in a rigorous statistical analysis, achieving F1-scores of 0.86–0.95 on phantom tomograms under experimental conditions.) The success of Spaghetti Tracer motivated us to extend its capabilities to randomly oriented actin filaments in the present work.
- There are very few earlier algorithms that are agnostic of the relative orientations and distances of the actin filaments, so that they can trace central lines of irregular filaments individually without leveraging the information of adjacent filaments or requiring a mean direction. Volume Tracer [8] utilized an expensive genetic-algorithm-based search employing a population of cylindrical templates (combined with a bi-directional tracing) to detect randomly oriented filaments in Dictyostelium discoideum filopodia. The genetic algorithm was implemented as part of our group’s free, open-source Situs and Sculptor packages, but it required extensive computational time on the order of several days when applied to a complete tomogram, without guaranteeing convergence (leading to false negatives when a user is impatient). Co-author Rigort also developed a similar template-matching method independently [10]. This approach was implemented in Amira, a commercial software requiring a paid license and limiting any algorithmic modifications by third parties or end users.
- Recently, a number of deep-learning-based approaches have been proposed for the segmentation of diverse biological assemblies, including actin [18,19]. For example, Chen et al. [18] presented a deep-learning-based segmentation approach for a voxel-level classification of shapes of interest in the tomogram and integrated the approach into the EMAN2 [20] package. However, these segmentation tools are generic in nature and are not specifically designed for filamentous shape structures. Besides, they require users to annotate training data and fine-tune the deep learning model, which could be a laborious process. These segmentation approaches only provide a voxel-level density segmentation, without any tracing of central lines. Recent studies that used these segmentation methods subsequently required separate approaches, such as the above template matching, for the additional tracing [11,12,13].
2. Results and Discussion
2.1. Statistical Evaluation of Tracing Accuracy in Simulated Tomograms
2.2. Measuring Filament Center Lines in a Previously Untraced Experimental Tomogram
2.3. Computation Time and Manual Intervention
2.4. Software Implementation and Dissemination
3. Materials and Methods
3.1. Simulated Phantom Tomograms
3.2. Automatic Seed Selection
3.3. CFS Creation
3.3.1. CFS Generation
3.3.2. CFS Refinement
3.4. CFS Segmentation
3.5. CFS Fusion
3.5.1. Fusion Based on Physical Proximity
3.5.2. Fusion by Extension
4. Conclusions
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Noise Level | Precision | Recall | F1-Score |
---|---|---|---|
0.35 | 0.97 | 0.85 | 0.90 |
0.50 | 0.97 | 0.81 | 0.88 |
0.65 | 0.96 | 0.84 | 0.89 |
0.80 | 0.95 | 0.79 | 0.87 |
0.95 | 0.95 | 0.78 | 0.85 |
Parameter Name | strwtrc Argument | Description | Default Value | Program Stages |
---|---|---|---|---|
Required Parameter | ||||
Threshold | -thr | Threshold for partitioning the CFS by the normalized path density. | N/A (user-defined based on the pruning map) | CFS segmentation |
Optional Parameters | ||||
Length | -len | Length (infinity norm) of the CFS in voxel units. Internally, this also defines the spacing of the cubic grid for placing seed points (half this value; see the text) and the extension length of the CFS (same value). | 10 | Automatic seed selection, CFS generation, and CFS fusion |
Gap Spacing | -gap | Maximum gap spacing, in voxels, tolerated while fusing adjacent CFSs. | 10 | CFS fusion |
Fusion Angle | -ang | Maximum angle, in degrees, tolerated while fusing adjacent CFSs. | 30 | CFS fusion |
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Sazzed, S.; Scheible, P.; He, J.; Wriggers, W. Untangling Irregular Actin Cytoskeleton Architectures in Tomograms of the Cell with Struwwel Tracer. Int. J. Mol. Sci. 2023, 24, 17183. https://doi.org/10.3390/ijms242417183
Sazzed S, Scheible P, He J, Wriggers W. Untangling Irregular Actin Cytoskeleton Architectures in Tomograms of the Cell with Struwwel Tracer. International Journal of Molecular Sciences. 2023; 24(24):17183. https://doi.org/10.3390/ijms242417183
Chicago/Turabian StyleSazzed, Salim, Peter Scheible, Jing He, and Willy Wriggers. 2023. "Untangling Irregular Actin Cytoskeleton Architectures in Tomograms of the Cell with Struwwel Tracer" International Journal of Molecular Sciences 24, no. 24: 17183. https://doi.org/10.3390/ijms242417183