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

Mark R. Winter, Miri Morgulis, Tsvia Gildor, Andrew R. Cohen, Smadar Ben-Tabou de-Leon

Preprint posted on February 05, 2021 https://www.biorxiv.org/content/10.1101/2020.08.14.244053v3.full

Article now published in PLOS Computational Biology at http://dx.doi.org/10.1371/journal.pcbi.1008780

In the latest preprint from the Ben-Tabou de-Leon lab, Mark R Winter et al. use gorgeous 3D live imaging to study how calcium makes its way to the embryonic skeleton in sea urchins.

Selected by Sophia Friesen

Background and context:

From bacterial magnetosomes to human bones, from the intricate cell walls of diatoms to the abrasive silica particles that protect grasses from herbivores, organisms across every kingdom of life have evolved ways of synthesizing solid mineral structures. This process, called biomineralization, can produce materials that are far more precisely patterned than is possible with modern human technology. It’s unknown how minerals are trafficked to the site where they crystallize. Here, Mark Winter et al. take advantage of the optical clarity and simple skeleton of sea urchin embryos to study how calcium, taken up from the inner cavity of the embryo, moves to the developing skeleton to be deposited. The researchers adopted two main strategies to answer this question: live imaging the movement of calcium vesicles in three dimensions, and investigating the cytoskeletal features of calcium-transporting cells.

Main findings:

  1. Skeleton-producing cells contain specialized calcium-rich vesicles

To see the movement of calcium in living sea urchin embryos, the authors began by introducing two fluorescent dyes into the surrounding seawater: green calcein, which binds calcium ions, and dextran-red, which can only enter cells through endocytosis. In ectodermal cells, which do not directly contribute to skeleton production, most of the observed vesicles were labeled with both dyes. In contrast, in skeletogenic mesoderm cells, a third of the vesicles were only labeled with calcein, indicating that they had been processed to remove dextran and retain calcium.

  1. Calcium vesicle dynamics are consistent with active diffusion

In sea urchins, minerals are deposited and crystallize in dedicated biomineralization compartments, which are tubes made up of skeletogenic cells. The two calcium carbonate spines that make up the embryonic skeleton form within these tubes. To see how calcium-containing vesicles were transported from the skeletogenic cells to the biomineralization compartments, the researchers live imaged embryos in which calcium was labeled with calcein and cell membranes were labeled with a lipophilic dye. By using lattice light-sheet microscopy, they were able to image in three dimensions and on fast timescales (about 6 seconds per frame), while limiting the amount of light-induced damage to embryos.

The researchers used automated segmentation and tracking to measure the volume and movement of calcium vesicles. The calcium vesicles moved more slowly than vesicles that are actively trafficked along microtubules, and they lacked the “stop-and-start” motion which is typical of such active transport, leading the researchers to suspect that the vesicles moved by diffusion. However, bigger calcium vesicles didn’t more any slower than small calcium vesicles, implying that their movement isn’t driven by passive diffusion. Having ruled out motor-driven transport and passive diffusion, the authors proposed that calcium vesicle movement is driven by active diffusion, in which diffusive movement is modulated by the continuous reorganization of the cytoskeleton. Intriguingly, calcium vesicles move more quickly within skeletogenic cells, which have lower levels of active myosin-II and f-actin, than in ectodermal cells, which have higher levels of these cytoskeletal components. This difference in cytoskeletal composition could explain the difference in active diffusion speed.

  1. Calcium vesicle movement isn’t directed towards the forming skeleton, but does slow near the skeleton

Perhaps surprisingly, calcium vesicle velocity towards the spicule averages zero, which means that vesicle movement isn’t directed towards the forming skeleton. However, vesicles that are within a few microns of the spicule move at considerably slower speeds than vesicles which are farther away. This could indicate that vesicles slow down as they bind to the cell membrane adjacent to the biomineralization compartment.

Why I liked this paper:

This paper is a fantastic showcase of the power and beauty of live imaging. The researchers captured not just the vesicle dynamics which are the focus of the manuscript, but also the movements of skeletogenic cells as they migrate to their proper positions and send out long cellular projections. By automating analysis of hundreds of vesicles across multiple movies, the researchers were able to detect subtle differences in vesicle speed and size. Plus, the movies are gorgeous – 2D versions of the 3D recordings are available in the supplemental data of this paper, and they’re well worth a watch!

Questions for the authors:

  1. Did you consider staining with dextran-red as well as calcein during your measurements of vesicle dynamics? Even in skeletogenic cells, most calcium-containing vesicles were also marked with dextran-red, indicating that they are not processed to enrich for calcium and are perhaps not involved with skeletogenesis at all. How do you account for the impact of these potentially extraneous vesicles on your analysis?
  2. When measuring vesicle directionality, you found that it was about the same as that of actively trafficked vesicles, which I found surprising. Is this high level of directionality typical of active diffusion?
  3. Why did you measure vesicle dynamics in one species of sea urchin, and use a second species to detect differences in the cytoskeleton?

Tags: biomineralization, bone, echinoderm, sea urchin, skeletal development, skeletogenesis, skeleton

Posted on: 15th February 2021 , updated on: 22nd February 2021

doi: https://doi.org/10.1242/prelights.27396

Read preprint (1 votes)




Author's response

Smadar Ben-Tabou de-Leon shared

Dear Sophia,

Thank you very much for your positive cover of our paper. I’m happy to say that the paper was just accepted to Plos Computational biology and will be available online later today here: https://doi.org/10.1371/journal.pcbi.1008780

You ask very good questions and I am happy to answer:

1. This is a great idea to use dextran to identify vesicles that their content is specifically processed in the skeletogenic cells. However, in this work, it was important for us to detect and visualize the cells in the embryo and therefore we used the membrane marker that is in the same wave-length range of dextran-red. Maybe in future works, we will do that!

2. I think it is hard to tell what is high or low directionality within a cell. The directionality index we measured was 0.4 which is closer to 0 (no directionality) than 1 (fully directed motion). I think that this partial directionality could be due to the physical constraints in the cell, such as organelles and the actomyosin network, that possibly limit the propagation in some directions (please see https://currentprotocols.onlinelibrary.wiley.com/doi/abs/10.1002/cpcb.88 for more explanations). So the movement fits a random walk, but it is partially confined in space.

3. I am happy you asked that – We should have explained that in the paper! It was simply species availability that dictated our choices. The 3D-imaging of calcium dynamics was done in the Janelia research campus in Virginia, USA. The species that was available there was the Atlantic sea urchin – Lytechinus variegatus. The part on the cytoskeleton was added thanks to the reviewers of Plos Comp Biol and was done in our lab which is located at the University of Haifa, Israel. The species we use here is Paracentrotus lividus. These two species have a similar developmental program and their larval skeletogenesis is regulated by similar gene regulatory networks, so they are comparable.

Thank you very much for your excellent questions and for liking our paper!
Best,
Smadar.

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