Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells

Lachuer, Hugo; Mathur, Pallavi; Bleakley, Kevin; Schauer, Kristine

doi:10.3791/60801

Cited by 2 publications

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“…1 C ). Such patterns can be the result of an uniformly random process (Complete Spatial Randomness, CSR) or reflects either clustering (i.e., aggregation) or dispersion (i.e., ordering with an inhibition surface around each point of exocytotic event) ( 48 , 49 ). We obtained a large dataset of 183 cells showing 32.880 exocytosis events to test the CSR hypothesis and to characterize the spatiotemporal properties of lysosomal exocytosis.…”

Section: Resultsmentioning

confidence: 99%

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Spatial organization of lysosomal exocytosis relies on membrane tension gradients

Lachuer

Lévêque‐Fort

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

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Lysosomal exocytosis is involved in many key cellular processes but its spatiotemporal regulation is poorly known. Using total internal reflection fluorescence microscopy (TIRFM) and spatial statistics, we observed that lysosomal exocytosis is not random at the adhesive part of the plasma membrane of RPE1 cells but clustered at different scales. Although the rate of exocytosis is regulated by the actin cytoskeleton, neither interfering with actin or microtubule dynamics by drug treatments alters its spatial organization. Exocytosis events partially co-appear at focal adhesions (FAs) and their clustering is reduced upon removal of FAs. Changes in membrane tension following a hypo-osmotic shock or treatment with methyl-β-cyclodextrin were found to increase clustering. To investigate the link between FAs and membrane tension, cells were cultured on adhesive ring-shaped micropatterns, which allow to control the spatial organization of FAs. By using a combination of TIRFM and fluorescence lifetime imaging microscopy (FLIM), we revealed the existence of a radial gradient in membrane tension. By changing the diameter of micropatterned substrates, we further showed that this gradient as well as the extent of exocytosis clustering can be controlled. Together, our data indicate that the spatial clustering of lysosomal exocytosis relies on membrane tension patterning controlled by the spatial organization of FAs.

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Section: Resultsmentioning

confidence: 99%

“…Cell seeding on micropatterns was described in Lachuer et al. ( 49 ). Briefly, coverslips were maintained in magnetic chamlides for live imaging or kept in a P6 wells for fixation.…”

Section: Methodsmentioning

confidence: 99%

Spatial organization of lysosomal exocytosis relies on membrane tension gradients

Lachuer

Lévêque‐Fort

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

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“…aggregation) or dispersion (i.e. ordering with an inhibition surface around each point of exocytotic event) (Diggle, 1983; Lachuer et al, 2020). We obtained a large dataset of 183 cells showing 32 880 exocytosis events to test the CSR hypothesis and to characterize the spatio-temporal properties of lysosomal exocytosis.…”

Section: Resultsmentioning

confidence: 99%

“…Cell seeding on micropatterns was described in Lachuer et al . (Lachuer et al, 2020). Briefly, coverslips were maintained in magnetic chamlides for live imaging or kept in a P6 wells for fixation.…”

Section: Methodsmentioning

confidence: 99%

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Membrane tension spatially organizes lysosomal exocytosis

Lachuer

Lévêque‐Fort

et al. 2022

Preprint

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Lysosomal exocytosis is involved in many key cellular processes but its spatio-temporal regulation is poorly known. Using total internal reflection fluorescence microscopy (TIRFM) and spatial statistics, we observed that lysosomal exocytosis is not random at the adhesive part of the plasma membrane of RPE1 cells but clustered at different scales. Although the rate of exocytosis is regulated by the actin cytoskeleton, neither interfering with actin or microtubule dynamics by drug treatments alters its spatial organization. Exocytosis events partially co-appear at focal adhesions (FAs) and their clustering is reduced upon removal of FAs. Changes in membrane tension following a hypo-osmotic shock or treatment with methyl-β-cyclodextrin was found to increase clustering. To investigate the link between FAs and membrane tension, cells were cultured on adhesive ring-shaped micropatterns, which allows to control the spatial organization of FAs. By using a combination of TIRFM and fluorescence lifetime imaging microscopy (FLIM) microscopy, we revealed the existence of a radial gradient in membrane tension. By changing the diameter of micropatterned substrates, we further showed that this gradient as well as the extent of exocytosis clustering can be controlled. Together, our data indicate that the spatial clustering of lysosomal exocytosis relies on membrane tension patterning controlled by the spatial organization of FAs.

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Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells

Cited by 2 publications

References 0 publications

Spatial organization of lysosomal exocytosis relies on membrane tension gradients

Spatial organization of lysosomal exocytosis relies on membrane tension gradients

Membrane tension spatially organizes lysosomal exocytosis

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