The Tracking of Individual Molecules in Cells and Tissues

Holtzer, Laurent; Schmidt, Thomas

doi:10.1002/9783527628360.ch2

Cited by 6 publications

(8 citation statements)

References 72 publications

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“…To avoid this issue, we adapted to 3D a threshold-free method for endosome segmentation, which is based on Gaussian fitting. In brief, signal-positive particles in both channels are first detected in 2D in each z plane by a 2D Gaussian fitting algorithm 60 , which does not rely on an intensity threshold, but rather on the fact that particles are characterized by fluorescent signals with a spatial Gaussian distribution with an offset which correspond to the local background. Then, the particles detected in each plane (2D), but corresponding to the same object in 3D, are connected based on the point spread function (PSF) of the microscope.…”

Section: Letter Researchmentioning

confidence: 99%

“…In brief, the 3D stack containing the iDelta 20 -Atto647N signal (3 μ m deep, Δ z = 0.5 μ m) was z projected (maximumintensity projection). Particles were detected using a 2D Gaussian fitting algorithm, then tracked using a modified Vogel algorithm, as previously described 60 . Tracks were rendered using the ImageJ plugin mTrackJ 63 .…”

Section: Letter Researchmentioning

confidence: 99%

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Polarized endosome dynamics by spindle asymmetry during asymmetric cell division

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Seum

Daeden

et al. 2015

Nature

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122

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During asymmetric division, fate determinants at the cell cortex segregate unequally into the two daughter cells. It has recently been shown that Sara (Smad anchor for receptor activation) signalling endosomes in the cytoplasm also segregate asymmetrically during asymmetric division. Biased dispatch of Sara endosomes mediates asymmetric Notch/Delta signalling during the asymmetric division of sensory organ precursors in Drosophila. In flies, this has been generalized to stem cells in the gut and the central nervous system, and, in zebrafish, to neural precursors of the spinal cord. However, the mechanism of asymmetric endosome segregation is not understood. Here we show that the plus-end kinesin motor Klp98A targets Sara endosomes to the central spindle, where they move bidirectionally on an antiparallel array of microtubules. The microtubule depolymerizing kinesin Klp10A and its antagonist Patronin generate central spindle asymmetry. This asymmetric spindle, in turn, polarizes endosome motility, ultimately causing asymmetric endosome dispatch into one daughter cell. We demonstrate this mechanism by inverting the polarity of the central spindle by polar targeting of Patronin using nanobodies (single-domain antibodies). This spindle inversion targets the endosomes to the wrong cell. Our data uncover the molecular and physical mechanism by which organelles localized away from the cellular cortex can be dispatched asymmetrically during asymmetric division.

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Section: Letter Researchmentioning

confidence: 99%

Section: Letter Researchmentioning

confidence: 99%

Polarized endosome dynamics by spindle asymmetry during asymmetric cell division

Derivery

Seum

Daeden

et al. 2015

Nature

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122

127

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“…Detection of endosomes was performed as previously described 26 , and trajectories of endosomes were reconstructed by connecting the measured positions using a modified Vogel-algorithm 26 . For a complete description of the image analysis and statistical analysis procedures, see the see Supplementary Methods section.…”

Section: Methodsmentioning

confidence: 99%

Sara phosphorylation state controls the dispatch of endosomes from the central spindle during asymmetric division

et al. 2017

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During asymmetric division, fate assignation in daughter cells is mediated by the partition of determinants from the mother. In the fly sensory organ precursor cell, Notch signalling partitions into the pIIa daughter. Notch and its ligand Delta are endocytosed into Sara endosomes in the mother cell and they are first targeted to the central spindle, where they get distributed asymmetrically to finally be dispatched to pIIa. While the processes of endosomal targeting and asymmetry are starting to be understood, the machineries implicated in the final dispatch to pIIa are unknown. We show that Sara binds the PP1c phosphatase and its regulator Sds22. Sara phosphorylation on three specific sites functions as a switch for the dispatch: if not phosphorylated, endosomes are targeted to the spindle and upon phosphorylation of Sara, endosomes detach from the spindle during pIIa targeting.

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“…The position of the fluorescence centroid of all particles is detected in both channels using a 2D Gaussian fitting algorithm (Holtzer & Schmidt, 2010). 2D Gaussian fitting is the method of choice to accurately segment diffractionlimited particles since it does not rely on an intensity threshold, but rather on the fact that the signal has a Gaussian shape and a minimum intensity above local background.…”

Section: Figure 2 Direct Observation Of Wash Localization In Endosomementioning

confidence: 99%

“…2. Detected particles are tracked over time using a modified Vogel algorithm (Holtzer & Schmidt, 2010). 3.…”

Section: Figure 2 Direct Observation Of Wash Localization In Endosomementioning

confidence: 99%

Quantitative analysis of endosome tubulation and microdomain organization mediated by the WASH complex

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Gautreau

2015

Sorting and Recycling Endosomes

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The Tracking of Individual Molecules in Cells and Tissues

Cited by 6 publications

References 72 publications

Polarized endosome dynamics by spindle asymmetry during asymmetric cell division

Polarized endosome dynamics by spindle asymmetry during asymmetric cell division

Sara phosphorylation state controls the dispatch of endosomes from the central spindle during asymmetric division

Quantitative analysis of endosome tubulation and microdomain organization mediated by the WASH complex

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