Culture of and experiments with sea urchin embryo primary mesenchyme cells

Moreno, Bradley; DiCorato, Allessandra; Park, Alexander; Mobilia, Kellen C.; Knapp, Regina; Bleher, Reiner; Wilke, Charlene; Alvares, Keith; Joester, Derk

doi:10.1016/bs.mcb.2019.01.002

Cited by 9 publications

(8 citation statements)

References 78 publications

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“…Strong signal of f-actin is clearly detected around the spicule of normal embryos, indicating that an actomyosin network is assembled near the spicule membranes (Fig. 7A, C), in agreement with previous studies in sea urchin skeletogenic cells cultures [67]. f-actin is also observed near the cell membranes in normal and VEGFR inhibited embryos (Fig.…”

Section: Vesicle Motion Is Not Directed Toward the Spicule But The Vesupporting

confidence: 92%

Calcium-vesicles perform active diffusion in the sea urchin embryo during larval biomineralization

Winter

Morgulis

Gildor

et al. 2020

Preprint

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Biomineralization is the process by which organisms use minerals to harden their tissues and provide them with physical support. Biomineralizing cells concentrate the mineral in vesicles that they secret into a dedicated compartment where crystallization occurs. The dynamics of mineral-vesicle motion and the molecular mechanisms that regulate it, are not well understood. Sea urchin larval skeletogenesis provides an excellent platform for the analyses of vesicle kinetics. Here we used calcein labeling and lattice light-sheet microscopy to investigate the three-dimensional (3D) vesicle dynamics in control sea urchin embryos and in Vascular Endothelial Growth Factor Receptor (VEGFR) inhibited embryos, where skeletogenesis is blocked. We developed computational tools for displaying 3D-volumetric movies and for automatically quantifying vesicle dynamics in the different embryonic tissues. Our findings imply that calcium vesicles perform an active diffusion motion in all the cells of the embryo. This mode of diffusion is defined by the mechanical properties of the cells and the dynamic rearrangements of the cytoskeletal network. The diffusion coefficient is larger in the mesenchymal skeletogenic cells compared to the epithelial ectodermal cells, possibly due to the distinct mechanical properties of the two tissues. Vesicle motion is not directed toward the biomineralization compartment, but the vesicles slow down when they approach it, and probably bind for mineral deposition. Under VEGFR inhibition, vesicle volume increases and vesicle speed is reduced but the vesicles continue in their diffusive motion. Overall, our studies provide an unprecedented view of calcium vesicle 3D-dynamics and illuminate possible molecular mechanisms that control vesicle dynamics and deposition.

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Section: Vesicle Motion Is Not Directed Toward the Spicule But The Vesupporting

confidence: 92%

Calcium-vesicles perform active diffusion in the sea urchin embryo during larval biomineralization

Winter

Morgulis

Gildor

et al. 2020

Preprint

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“…3). P. lividus skeletogenic cells were isolated and cultured as described in [45]. The addition of ROCK inhibitor to the skeletogenic cell culture at 16hpf completely abolished spicule formation (Fig.…”

Section: Rock Activity In the Skeletogenic Cells Controls Spicule Ini...mentioning

confidence: 99%

“…Calcium bearing vesicles perform an active diffusion motion in the skeletogenic cells with a diffusion length that inversely correlates with the strength and activity of the actomyosin network (35). Actin filaments are formed around the spicule (35, 36) and F-actin signal is enriched at the tips of the growing skeletal rods in skeletogenic cell culture (37). Genetic and pharmacological perturbations of the GTPase, CDC42, prevent the formation of filopodia in the skeletogenic cells and inhibit spicule formation and elongation (38).…”

Section: Introductionmentioning

confidence: 99%

ROCK and the actomyosin network control biomineral growth and morphology during sea urchin skeletogenesis

Hijaze

Gildor

Seidel

et al. 2022

Preprint

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Biomineralization, the ability of organisms to use minerals to harden their tissues, has attracted scientists from various disciplines to decipher the molecular mechanisms that control it. Many of these studies focus on the gene regulatory networks (GRNs) that control biomineralization and are apparently, phylum specific. Yet, downstream to the GRNs lays the cellular machinery that drives morphogenetic processes that are commonly used in biomineralization, such as, vesicular motion and secretion. The actomyosin network is a key regulator of these processes and participates in biomineralization across Eukaryotes, from unicellular organisms to vertebrates, yet, little is known about its regulation of biomineral growth. Here we reveal that the actomyosin remodeling protein, Rho-associated coiled-coil kinase (ROCK), controls the formation, growth and morphology of the calcite spicules in the sea urchin larva. We show that ROCK expression is elevated in the sea urchin skeletogenic cells and its inhibition impairs the organization of F-actin around the spicules and leads to skeletal loss. We discovered that ROCK inhibition after spicule formation, slows down mineral deposition, induces ectopic spicule branching and disrupts skeletogenic gene expression. We propose that ROCK and the actomyosin network are an essential part of the common biomineralization tool-kit in Eukaryotes.

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“…During late gastrulation susAC mRNA was primarily expressed in the PMC cluster that is associated with the initiation of spiculogenesis (Figure 1B). The pluteus larval stage is characterized by spicule extension (36) and also in this stage susAC mRNA expression was highest in PMC clusters of the post-oral tips and in the middle section of the body rods (Figure 1B). Lower expression levels of susAC were detected in the gut and ectodermal cells.…”

Section: Identification and Localization Of Susac Expression In The D...mentioning

confidence: 93%

“…Single-cell transcriptomics were applied to analyze spatiotemporal expression of susAC mRNA in mesenchyme blastula (24 hpf), late gastrula (40 hpf), and pluteus (72 hpf) larvae (Figure 1B). Specification of the calcifying primary mesenchyme cells (PMCs) takes place during mesenchyme blastula (36). At this stage, susAC mRNA expression was already evident in a few PMCs (Figure 1B).…”

Section: Identification and Localization Of Susac Expression In The D...mentioning

confidence: 99%

Soluble adenylyl cyclase coordinates intracellular pH homeostasis and biomineralization in calcifying cells of a marine animal

Chang

Thies

Tresguerres

et al. 2023

American Journal of Physiology-Cell Physiology

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Biomineralizing cells concentrate dissolved inorganic carbon (DIC) and remove protons from the site of mineral precipitation. However, the molecular regulatory mechanisms that orchestrate pH homeostasis and biomineralization of calcifying cells are poorly understood. Here we report that the acid-base sensing enzyme soluble adenylyl cyclase (sAC) coordinates intracellular pH (pHi) regulation in the calcifying primary mesenchyme cells (PMCs) of sea urchin larvae. Single cell transcriptomics, in situ hybridization, and immunocytochemistry elucidated the spatio-temporal expression of sAC during skeletogenesis. Live pHi imaging of PMCs revealed that down-regulation of sAC activity with two structurally unrelated small molecules inhibited pHi regulation of PMCs, an effect that was rescued by addition of cell-permeable cAMP. Pharmacological sAC inhibition also significantly reduced normal spicule growth as well as spicule regeneration, establishing a link between PMC pHi regulation and biomineralization. Finally, increased expression of sAC mRNA was detected during skeleton re-mineralization and during exposure to CO2-induced acidification. These findings suggest that transcriptional regulation of sAC is required to promote re-mineralization and compensate for acidic stress. The present work highlights a central role of sAC in coordinating acid-base regulation and biomineralization in calcifying cells of a marine animal.

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Culture of and experiments with sea urchin embryo primary mesenchyme cells

Cited by 9 publications

References 78 publications

Calcium-vesicles perform active diffusion in the sea urchin embryo during larval biomineralization

Calcium-vesicles perform active diffusion in the sea urchin embryo during larval biomineralization

ROCK and the actomyosin network control biomineral growth and morphology during sea urchin skeletogenesis

Soluble adenylyl cyclase coordinates intracellular pH homeostasis and biomineralization in calcifying cells of a marine animal

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