A minimal constraint device for imaging nuclei in live<i>Drosophila</i>contractile larval muscles reveals novel nuclear mechanical dynamics

Lorber, Dana; Rotkopf, Ron; Volk, Talila

doi:10.1039/d0lc00214c

Cited by 11 publications

(18 citation statements)

References 51 publications

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“…For imaging live nuclei in their intrinsic environment, a minimal constraint device for Drosophila larvae was designed in our laboratory, to be placed on top of a confocal microscope stage, as previously described (28). Briefly, the center of the device is a thin plastic bar positioned in a larger frame, with a groove crossing the frame and bar from side to side.…”

Section: Drosophila Larvaementioning

confidence: 99%

“…Here, we combined a custom-made device placed on top of a confocal microscope stage (28), with genetic labeling of the chromatin and the nuclear lamina, to characterize in vivo nuclear scale 3D chromatin organization in fully differentiated muscles of live, intact Drosophila larva. Imaging of live 3D chromatin organization was reported for various cell lines (2,29,30), as well as in the isolated Drosophila larva salivary gland (31).…”

Section: Introductionmentioning

confidence: 99%

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Live imaging of chromatin distribution reveals novel principles of nuclear architecture and chromatin compartmentalization

et al. 2021

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The three-dimensional organization of chromatin contributes to transcriptional control, but information about native chromatin distribution is limited. Imaging chromatin in live Drosophila larvae, with preserved nuclear volume, revealed that active and repressed chromatin separates from the nuclear interior and forms a peripheral layer underneath the nuclear lamina. This is in contrast to the current view that chromatin distributes throughout the nucleus. Furthermore, peripheral chromatin organization was observed in distinct Drosophila tissues, as well as in live human effector T lymphocytes and neutrophils. Lamin A/C up-regulation resulted in chromatin collapse toward the nuclear center and correlated with a significant reduction in the levels of active chromatin. Physical modeling suggests that binding of lamina-associated domains combined with chromatin self-attractive interactions recapitulate the experimental chromatin distribution profiles. Together, our findings reveal a novel mode of mesoscale organization of peripheral chromatin sensitive to lamina composition, which is evolutionary conserved.

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Section: Drosophila Larvaementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Live imaging of chromatin distribution reveals novel principles of nuclear architecture and chromatin compartmentalization

et al. 2021

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“…To study 3D chromatin organization under physiological conditions in a live organism, we have developed a setup to visualize nuclei and chromatin in live, intact Drosophila larva. This setup is based on a minimally constrained device designed for imaging stationary or contractile muscles and their nuclei in an intact Drosophila larva, preserving tissue and nuclear native environment 29 . Figure 1A shows a 3D view of a single muscle nucleus, cut through the middle, of live, intact 3 rd instar Drosophila larva.…”

Section: Live Imaging Of 3d Chromatin Distribution In Intact Drosophimentioning

confidence: 99%

“…Drosophila larvae was designed in our lab, to be placed on top of a confocal microscope stage, as previously described 29…”

Section: The Minimal Constraint Device For Imaging Live Intact Drosopmentioning

confidence: 99%

“…Here we combined a custom-made device placed on top of a confocal microscope stage 29 , with genetic labeling of the chromatin and the nuclear lamina, to characterize in vivo nuclear scale 3D chromatin organization in fully differentiated muscle of live, intact Drosophila larva. Imaging of live 3D chromatin organization was reported for various cell lines 2,30,31 , as well as in the isolated Drosophila larva salivary gland 32 .…”

Section: Introductionmentioning

confidence: 99%

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Live imaging of chromatin distribution in muscle nuclei reveals novel principles of nuclear architecture and chromatin compartmentalization

Pavlov¹,

Lorber²,

Bajpai³

et al. 2020

Preprint

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Packaging of the chromatin within the nucleus serves as an important factor in the regulation of transcriptional output. However, information on chromatin architecture on nuclear scale in fully differentiated cells, under physiological conditions and in live organisms, is largely unavailable. Here, we imaged nuclei and chromatin in muscle fibers of live, intact Drosophila larvae. In contrast to the common view that chromatin is distributed throughout the nuclear volume, we show that the entire chromatin, including active and repressed regions, forms a peripheral layer underneath the nuclear lamina, leaving a chromatin-devoid compartment at the nucleus center. Importantly, visualization of nuclear compartmentalization required imaging of un-fixed nuclei embedded within their intrinsic tissue environment, with preserved nuclear volume. Upon fixation of similar muscle nuclei, we observed an average of three-fold reduction in nuclear volume caused by dehydration and evidenced by nuclear flattening. In these conditions, the peripheral chromatin layer was not detected anymore, demonstrating the importance of preserving native biophysical tissue environment. We further show that nuclear compartmentalization is sensitive to the levels of lamin C, since over-expression of lamin C-GFP in muscle nuclei resulted in detachment of the peripheral chromatin layer from the lamina and its collapse into the nuclear center. Computer simulations of chromatin distribution recapitulated the peripheral chromatin organization observed experimentally, when binding of lamina associated domains (LADs) was incorporated with chromatin selfattractive interactions. Reducing the number of LADs led to collapse of the chromatin, similarly to our observations following lamin C over-expression. Taken together, our findings reveal a novel mode of mesoscale organization of chromatin within the nucleus in a live organism, in which the chromatin forms a peripheral layer separated from the nuclear interior.This architecture may be essential for robust transcriptional regulation in fully differentiated cells.

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Mechanics and functional consequences of nuclear deformations

Kalukula

Stephens

Lammerding

et al. 2022

Nat Rev Mol Cell Biol

221

180

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A minimal constraint device for imaging nuclei in liveDrosophilacontractile larval muscles reveals novel nuclear mechanical dynamics

Abstract: Muscle contractions produce reiterated cytoplasmic mechanical variations, which potentially influence nuclear mechanotransduction, however information regarding the dynamics of muscle nuclei in the course of muscle contraction is still missing.

Cited by 11 publications

References 51 publications

Live imaging of chromatin distribution reveals novel principles of nuclear architecture and chromatin compartmentalization

Live imaging of chromatin distribution reveals novel principles of nuclear architecture and chromatin compartmentalization

Live imaging of chromatin distribution in muscle nuclei reveals novel principles of nuclear architecture and chromatin compartmentalization

Mechanics and functional consequences of nuclear deformations

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