STED microscopy: A simplified method for liver sinusoidal endothelial <i>fenestrae</i> analysis

Martino, Julie Di; Mascalchi, Patrice; Legros, Philippe; Lacomme, Sabrina; Gontier, Étienne; Bioulac‐Sage, Paulette; Balabaud, Charles; Moreau, Violaine; Saltel, Frédéric

doi:10.1111/boc.201800016

Cited by 8 publications

(5 citation statements)

References 24 publications

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“…CytB heavily increased pMLC and at the same time, had no effect on fenestration diameter, other than increasing the number of fenestrations. The results are in agreement with previous reports [ 15 , 46 ]. In addition to this, we showed that CytB can be used to exacerbate the effect of other drugs on fenestrations.…”

Section: Discussionsupporting

confidence: 94%

“…Cytochalasin B (CytB) is used to depolymerise filamentous actin, and increases the number of fenestrations [ 15 ]. CytB is therefore used as a gold standard to easily induce fenestrations in LSEC (up to 300% increase in their number) within 15–60 min [ 13 , 50 ], without affecting the fenestration size [ 15 , 46 ]. We applied it to decrease the length of actin fibres.…”

Section: Methodsmentioning

confidence: 99%

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Tuning of Liver Sieve: The Interplay between Actin and Myosin Regulatory Light Chain Regulates Fenestration Size and Number in Murine Liver Sinusoidal Endothelial Cells

Zapotoczny

Lekka

Ahluwalia

et al. 2022

IJMS

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Liver sinusoidal endothelial cells (LSECs) facilitate the efficient transport of macromolecules and solutes between the blood and hepatocytes. The efficiency of this transport is realized via transcellular nanopores, called fenestrations. The mean fenestration size is 140 ± 20 nm, with the range from 50 nm to 350 nm being mostly below the limits of diffraction of visible light. The cellular mechanisms controlling fenestrations are still poorly understood. In this study, we tested a hypothesis that both Rho kinase (ROCK) and myosin light chain (MLC) kinase (MLCK)-dependent phosphorylation of MLC regulates fenestrations. We verified the hypothesis using a combination of several molecular inhibitors and by applying two high-resolution microscopy modalities: structured illumination microscopy (SIM) and scanning electron microscopy (SEM). We demonstrated precise, dose-dependent, and reversible regulation of the mean fenestration diameter within a wide range from 120 nm to 220 nm and the fine-tuning of the porosity in a range from ~0% up to 12% using the ROCK pathway. Moreover, our findings indicate that MLCK is involved in the formation of new fenestrations—after inhibiting MLCK, closed fenestrations cannot be reopened with other agents. We, therefore, conclude that the Rho-ROCK pathway is responsible for the control of the fenestration diameter, while the inhibition of MLCK prevents the formation of new fenestrations.

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Section: Discussionsupporting

confidence: 94%

Section: Methodsmentioning

confidence: 99%

Tuning of Liver Sieve: The Interplay between Actin and Myosin Regulatory Light Chain Regulates Fenestration Size and Number in Murine Liver Sinusoidal Endothelial Cells

Zapotoczny

Lekka

Ahluwalia

et al. 2022

IJMS

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“…LSEC fenestrae are important structures for liver physiology, and their loss is involved in several liver pathologies . Recently, we demonstrated that STED microscopy was suitable for LSEC fenestrae observation . Using this methodology in combination with TEM and an in vitro LSEC dedifferentiation model that mimics capillarization observed in vivo , we tested actin cytoskeleton involvement in fenestrae formation.…”

Section: Discussionmentioning

confidence: 99%

“…(12) Recently, we demonstrated that STED microscopy was suitable for LSEC fenestrae observation. (8) Using this methodology in combination with TEM and an in vitro LSEC dedifferentiation model that mimics capillarization observed in vivo, we tested actin cytoskeleton involvement in fenestrae formation. We revealed that the cytoskeleton is closely associated with fenestrae and is organized in a dense ring surrounding them.…”

Section: Discussionmentioning

confidence: 99%

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Actin Depolymerization in Dedifferentiated Liver Sinusoidal Endothelial Cells Promotes Fenestrae Re‐Formation

et al. 2018

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Liver sinusoidal endothelial cells (LSECs) possess fenestrae, which are key for the exchange between blood and hepatocytes. Alterations in their number or diameter have important implications for hepatic function in liver diseases. They are lost early in the development of hepatic fibrosis through a process called capillarization. In this study, we aimed to demonstrate whether in vitro dedifferentiated LSECs that have lost fenestrae are able to re‐form these structures. Using stimulated emission depletion super‐resolution microscopy in combination with transmission electron microscopy, we analyzed fenestrae formation in a model mimicking the capillarization process in vitro. Actin is known to be involved in fenestrae regulation in differentiated LSECs. Using cytochalasin D, an actin‐depolymerizing agent, we demonstrated that dedifferentiated LSECs remain capable of forming fenestrae. Conclusion: We provide a new insight into the complex role of actin in fenestrae formation and in the control of their size and show that LSEC fenestrae re‐formation is possible, suggesting that this process could be used during fibrosis regression to try to restore exchanges and hepatocyte functions.

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The Liver Sinusoidal Endothelial Cell

Sørensen

Smedsrød

2020

The Liver

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STED microscopy: A simplified method for liver sinusoidal endothelial fenestrae analysis

Abstract: This new way of analysing LSEC fenestrae will allow for expedited investigation of their dynamics, molecular composition and functions to better understand their function in liver pathophysiology.

Cited by 8 publications

References 24 publications

Tuning of Liver Sieve: The Interplay between Actin and Myosin Regulatory Light Chain Regulates Fenestration Size and Number in Murine Liver Sinusoidal Endothelial Cells

Tuning of Liver Sieve: The Interplay between Actin and Myosin Regulatory Light Chain Regulates Fenestration Size and Number in Murine Liver Sinusoidal Endothelial Cells

Actin Depolymerization in Dedifferentiated Liver Sinusoidal Endothelial Cells Promotes Fenestrae Re‐Formation

The Liver Sinusoidal Endothelial Cell

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