Adaptive tracking algorithm for trajectory analysis of cells and layer-by-layer assessment of motility dynamics

Qureshi, Mohammad Haroon; Özlü, Nurhan; Bayraktar, Halil

doi:10.1016/j.compbiomed.2022.106193

Cited by 1 publication

(3 citation statements)

References 92 publications

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“…Their centroids and attributes were acquired from segmented images for the tracking analysis. An adaptive tracking method, described previously [ 73 ], was applied to determine the cell linkages in consecutive frames and their fluorescence signal in consecutive frames. After the fluorescence signal for all three channels were determined, we employed them to compute the normalized or netFRET signal values.…”

Section: Methodsmentioning

confidence: 99%

“…The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/bios13090890/s1, Figure S1: The section of nucleotide sequence of C-Cytc-V encoding region; Figure S2: The computation of netFRET and normFRET intensity of single cells after the image acquisition and processing of raw data [73]; Figure S3: The fluorescence emission of C17V and C + V co-expressed samples; Figure S4: The evaluation of FRET efficiency by using C + V and C17V samples that are used as an internal standard in all experiments.…”

Section: Supplementary Materialsmentioning

confidence: 99%

“…We studied its interaction with heme and CCHL where the fluorescence signal was acquired by using the three-channel fluorescence imaging method. The adaptive tracking method demonstrated previously to track single cells was applied to acquire the fluorescence signal changes [ 73 ]. The signal intensities for different conformational states were determined by computing the corrected FRET (netFRET) and normalized FRET (normFRET) magnitudes.…”

Section: Introductionmentioning

confidence: 99%

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Genetically Encoded Fluorescent Probe for Detection of Heme-Induced Conformational Changes in Cytochrome c

Genceroglu,

Cavdar,

Manioglu

et al. 2023

Biosensors

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Cytochrome c (Cytc) is a key redox protein for energy metabolism and apoptosis in cells. The activation of Cytc is composed of several steps, including its transfer to the mitochondrial membrane, binding to cytochrome c heme lyase (CCHL) and covalent attachment to heme. The spectroscopic methods are often applied to study the structural changes of Cytc. However, they require the isolation of Cytc from cells and have limited availability under physiological conditions. Despite recent studies to elucidate the tightly regulated folding mechanism of Cytc, the role of these events and their association with different conformational states remain elusive. Here, we provide a genetically encoded fluorescence method that allows monitoring of the conformational changes of Cytc upon binding to heme and CCHL. Cerulean and Venus fluorescent proteins attached at the N and C terminals of Cytc can be used to determine its unfolded, intermediate, and native states by measuring FRET amplitude. We found that the noncovalent interaction of heme in the absence of CCHL induced a shift in the FRET signal, indicating the formation of a partially folded state. The higher concentration of heme and coexpression of CCHL gave rise to the recovery of Cytc native structure. We also found that Cytc was weakly associated with CCHL in the absence of heme. As a result, a FRET-based fluorescence approach was demonstrated to elucidate the mechanism of heme-induced Cytc conformational changes with spatiotemporal resolution and can be applied to study its interaction with small molecules and other protein partners in living cells.

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Section: Methodsmentioning

confidence: 99%

Section: Supplementary Materialsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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