Tag: Arrow-TL1

Cell surface fluctuations regulate early embryonic lineage sorting

In development, lineage segregation is coordinated in time and space. An important example is the mammalian inner cell mass, in which the primitive endoderm (PrE, founder of the yolk sac) physically segregates from the epiblast (EPI, founder of the fetus). While the molecular requirements have been well studied, the physical mechanisms determining spatial segregation between EPI and PrE remain elusive.*

In the article “Cell surface fluctuations regulate early embryonic lineage sorting” Ayaka Yanagida, Elena Corujo-Simon, Christopher K. Revell, Preeti Sahu, Giuliano G. Stirparo, Irene M. Aspalter, Alex K. Winkel, Ruby Peters, Henry De Belly, Davide A.D. Cassani, Sarra Achouri     Raphael Blumenfeld, Kristian Franze, Edouard Hannezo, Ewa K. Paluch, Jennifer Nichols and Kevin J. Chalut investigate the mechanical basis of EPI and PrE sorting. *

The authors find that rather than the differences in static cell surface mechanical parameters as in classical sorting models, it is the differences in surface fluctuations that robustly ensure physical lineage sorting.*

These differential surface fluctuations systematically correlate with differential cellular fluidity, which Ayaka Yanagida et al. propose together constitute a non-equilibrium sorting mechanism for EPI and PrE lineages. By combining experiments and modeling, A. Yanagida et al. identify cell surface dynamics as a key factor orchestrating the correct spatial segregation of the founder embryonic lineages.*

The surface tension of cells was measured using an Atomic Force Microscopy (AFM) based technique with a commercially available stand-alone platform for cell adhesion and cytomechanics studies mounted on an inverted confocal microscope.*

pEPI (epiblast , EPI, founder of the fetus) and pPrE (primitive endoderm, founder of the yolk sac ) tension measurements were performed using NanoWorld ARROW-TL1Au tipless silicon AFM cantilevers (nominal spring constant of 0.03 N/m).*
Sensitivity was calibrated by acquiring a force curve on a glass coverslip. Spring constant was calibrated by the thermal noise fluctuation method. Z-length parameter and setpoint force were set at 30 μm and 10 nN, respectively. Constant height mode was selected. The measurement was carried on by lowering the tipless AFM cantilever onto an empty area next to a target cell. Once the cantilever retracted (by roughly 30 μm), it was positioned above the target cell and run a compression for 200 seconds. During the constant height compression, the force acting on the AFM cantilever was recorded. After initial force relaxation, the resulting force value was used to extract surface tension.*

ES cells tension measurements were performed using the same commercial platform for cell adhesion and cytomechanics studies and a DSD2 Differential Spinning Disk both mounted on an inverted microscope.*

NanoWorld tipless silicon AFM cantilevers of the ARROW-TL1 type were chosen (nominal spring constant of 0.03 N/m). Sensitivity was calibrated by acquiring a force curve on glass. Spring constant was calibrated by the thermal noise fluctuation method. Z-length parameter and setpoint force were set at 80 μm and 4 nN, respectively. Constant height mode was selected. The measurement was carried on by lowering the tipless AFM cantilever onto an empty area next to a target cell. Once the AFM cantilever retracted (by roughly 80 μm), it was positioned above the target cell and a compression was run for 50 seconds. During the constant height compression, the force acting on the AFM cantilever was recorded. After initial force relaxation, the resulting force value was used to extract surface tension. A confocal stack was acquired using a ×40/1.1 NA water immersion objective.*

Figure 4 from Ayaka Yanagida et al. “Cell surface fluctuations regulate early embryonic lineage sorting”:Differences in ezrin-mediated surface fluctuations regulate cell sorting (A) Representative images of constitutively active Ezrin-IRES-mCherry (CA-EZR) ES cells, showing a high degree of pERM variability in the low mCherry-expressing ES cells. Surface fluctuations of single CA-EZR cells without Dox and WT H2B-BFP, and CA-EZR ES cells with or without Dox in 2i+LIF. L, M, and H indicate low, medium, and high expression of mCherry as assessed by the 3-quantiles of expression in the mCherry-expressing cells. Surface fluctuations were normalized by the mean of the Dox− surface fluctuations in each of the experiments or the mean of the WT H2B-BFP surface fluctuations. p values were calculated using one-way ANOVA, with the p values above each group representing the outcome of pairwise comparison with Dox−, and the p value above all values in CA-EZR Dox+ condition representing the comparison of all groups. (B) The surface tension of dissociated Dox-treated CA-EZR ES cells measured using the AFM technique presented in Chugh et al., 2017 is plotted against the intensity of mCherry to show that there is no correlation between CA-EZR expression and surface tension. On the right is the surface tension of dissociated WT H2B-BFP ES cells and Dox-treated CA-EZR ES cells. p value was calculated by two-way ANOVA using cell type and experimental replicate as variables. (C) θ of the homotypic doublets that can be formed from CA-EZR ES cells with or without Dox. (D) Representative images of CA-EZR ES cells and WT H2B-BFP ES cells aggregated with or without Dox. The line drawn through the center of the aggregates represents the line over which we found an intensity profile in (E). (E) Representative comparison of BFP and mCherry line scan signals in the CA-EZR and H2B-BFP ES cells aggregates with or without Dox, using the line across the images in (D). (F) Schematic showing how the radial average (dipole moment) R is calculated, along with model examples of R for distributions shown. (G) R of aggregates of CA-EZR and H2B-BFP ES cells. pEPI (epiblast , EPI, founder of the fetus) and pPrE (primitive endoderm, founder of the yolk sac ) tension measurements were performed using NanoWorld ARROW-TL1Au tipless silicon AFM cantilevers. ES cells tension measurements were performed using NanoWorld tipless silicon AFM cantilevers of the ARROW-TL1 type were chosen (nominal spring constant of 0.03 N/m).
Figure 4 from Ayaka Yanagida et al. “Cell surface fluctuations regulate early embryonic lineage sorting”:
Differences in ezrin-mediated surface fluctuations regulate cell sorting
(A) Representative images of constitutively active Ezrin-IRES-mCherry (CA-EZR) ES cells, showing a high degree of pERM variability in the low mCherry-expressing ES cells. Surface fluctuations of single CA-EZR cells without Dox and WT H2B-BFP, and CA-EZR ES cells with or without Dox in 2i+LIF. L, M, and H indicate low, medium, and high expression of mCherry as assessed by the 3-quantiles of expression in the mCherry-expressing cells. Surface fluctuations were normalized by the mean of the Dox− surface fluctuations in each of the experiments or the mean of the WT H2B-BFP surface fluctuations. p values were calculated using one-way ANOVA, with the p values above each group representing the outcome of pairwise comparison with Dox−, and the p value above all values in CA-EZR Dox+ condition representing the comparison of all groups.
(B) The surface tension of dissociated Dox-treated CA-EZR ES cells measured using the AFM technique presented in Chugh et al., 2017
is plotted against the intensity of mCherry to show that there is no correlation between CA-EZR expression and surface tension. On the right is the surface tension of dissociated WT H2B-BFP ES cells and Dox-treated CA-EZR ES cells. p value was calculated by two-way ANOVA using cell type and experimental replicate as variables.
(C) θ of the homotypic doublets that can be formed from CA-EZR ES cells with or without Dox.
(D) Representative images of CA-EZR ES cells and WT H2B-BFP ES cells aggregated with or without Dox. The line drawn through the center of the aggregates represents the line over which we found an intensity profile in (E).
(E) Representative comparison of BFP and mCherry line scan signals in the CA-EZR and H2B-BFP ES cells aggregates with or without Dox, using the line across the images in (D).
(F) Schematic showing how the radial average (dipole moment) R is calculated, along with model examples of R for distributions shown.
(G) R of aggregates of CA-EZR and H2B-BFP ES cells.

*Ayaka Yanagida, Elena Corujo-Simon, Christopher K. Revell, Preeti Sahu, Giuliano G. Stirparo, Irene M. Aspalter, Alex K. Winkel, Ruby Peters, Henry De Belly, Davide A.D. Cassani, Sarra Achouri     Raphael Blumenfeld, Kristian Franze, Edouard Hannezo, Ewa K. Paluch, Jennifer Nichols and Kevin J. Chalut
Cell surface fluctuations regulate early embryonic lineage sorting
Cell, Volume 185, Issue 5, 3 March 2022, Pages 777-793.e20
DOI: https://doi.org/10.1016/j.cell.2022.01.022

The article “Cell surface fluctuations regulate early embryonic lineage sorting” by Ayaka Yanagida, Elena Corujo-Simon, Christopher K. Revell, Preeti Sahu, Giuliano G. Stirparo, Irene M. Aspalter, Alex K. Winkel, Ruby Peters, Henry De Belly, Davide A.D. Cassani, Sarra Achouri     Raphael Blumenfeld, Kristian Franze, Edouard Hannezo, Ewa K. Paluch, Jennifer Nichols and Kevin J. Chalut is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third-party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/.

Intravascular adhesion and recruitment of neutrophils in response to CXCL1 depends on their TRPC6 channels

Representing a central element of the innate immune system, neutrophils are recruited from the blood stream to a site of inflammation. The recruitment process follows a well-defined sequence of events including adhesion to the blood vessel walls, migration, and chemotaxis to reach the inflammatory focus. A common feature of the underlying signalling pathways is the utilization of Ca2+ ions as intracellular second messengers. However, the required Ca2+ influx channels are not yet fully characterized.*

In the article “Intravascular adhesion and recruitment of neutrophils in response to CXCL1 depends on their TRPC6 channels” Otto Lindemann, Jan Rossaint, Karolina Najder, Sandra Schimmelpfennig, Verena Hofschröer, Mike Wälte, Benedikt Fels, Hans Oberleithner, Alexander Zarbock and Albrecht Schwab report a novel role for TRPC6, a member of the transient receptor potential (TRPC) channel family, in the CXCL1-dependent recruitment of murine neutrophil granulocytes.*

The authors describe how they tested whether TRPC6 channels are central elements of the signalling cascade underlying CXCR2-mediated neutrophil recruitment. They combined intravital microscopy, single-cell force spectroscopy with atomic force microscopy, Ca2+ imaging, and microfluidic flow chamber assays to investigate the role of TRPC6 channels in murine neutrophils for their recruitment in renal ischemia-reperfusion and cremaster models as well as in in vitro assays.*

The study reveals that TRPC6 channels in neutrophils are crucial signalling modules in their recruitment from the blood stream in response to CXCL1.*

The single-cell force spectroscopy experiments were performed by using atomic force microscopy (AFM) with NanoWorld Arrow-TL1 tipless cantilevers which were incubated prior to experiments for 30 min in Cell-Tak to make the AFM cantilever sticky for neutrophils.*


Figure 4a from “Intravascular adhesion and recruitment of neutrophils in response to CXCL1 depends on their TRPC6 channels” by Otto Lindemann et al:
Force-distance curves were obtained by probing bEnd5 cells with the neutrophil-carrying cantilever using 1 nN maximal loading force
NanoWorld Arrow-TL1 Tipless AFM cantilever, single cantilever beam on a silicon support chip
NanoWorld Arrow-TL1
Tipless cantilever,
single cantilever beam on a silicon support chip

*Otto Lindemann, Jan Rossaint, Karolina Najder, Sandra Schimmelpfennig, Verena Hofschröer, Mike Wälte, Benedikt Fels, Hans Oberleithner, Alexander Zarbock and Albrecht Schwab
Intravascular adhesion and recruitment of neutrophils in response to CXCL1 depends on their TRPC6 channels
Journal of Molecular Medicine volume 98, pages349–360(2020)
DOI: https://doi.org/10.1007/s00109-020-01872-4

Please follow this external link to read the full article: https://link.springer.com/article/10.1007/s00109-020-01872-4

Open Access The article “ Intravascular adhesion and recruitment of neutrophils in response to CXCL1 depends on their TRPC6 channels “ by Otto Lindemann, Jan Rossaint, Karolina Najder, Sandra Schimmelpfennig, Verena Hofschröer, Mike Wälte, Benedikt Fels, Hans Oberleithner, Alexander Zarbock and Albrecht Schwab is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Rapid changes in tissue mechanics regulate cell behaviour in the developing embryonic brain

In their short report “Rapid changes in tissue mechanics regulate cell behaviour in the developing embryonic brain” published in January 2019, Amelia J Thompson, Eva K Pillai, Ivan B Dimov, Sarah K Foster, Christine E Holt, and Kristian Franze describe how they used time-lapse in vivo atomic force microscopy (tiv-AFM), a method that combines sensitive upright epi-fluorescence imaging of opaque samples, with iterated AFM indentation measurements of in vivo tissue at cellular resolution and at a time scale of tens of minutes, in order to enable time-resolved measurements of developmental tissue mechanics.*

The technique developed by Thompson, Pillai et al. is a useful tool that can help elucidate how variations in stiffness control the brain wiring process. It could also be used to look into how other developmental or regenerative processes, such as the way neurons reconnect after injuries to thebrain or spinal cord, may be regulated by mechanical tissue properties.*

NanoWorld Arrow-TL1 tipless cantilevers were used for the AFM-based stiffness measurements. (Monodisperse spherical polystyrene beads were glued to the cantilever ends as probes.)

NanoWorld Arrow-TL1 tipless cantilever for atomic force microscopy
NanoWorld Arrow-TL1 tipless AFM cantilever

*Amelia J Thompson, Eva K Pillai, Ivan B Dimov, Sarah K Foster, Christine E Holt, Kristian Franze
Rapid changes in tissue mechanics regulate cell behaviour in the developing embryonic brain
eLife 2019; 8:e39356
DOI: https://doi.org/10.7554/eLife.39356

Please follow this external link to the full article: https://cdn.elifesciences.org/articles/39356/elife-39356-v1.pdf

Open Access: The article « Rapid changes in tissue mechanics regulate cell behaviour in the developing embryonic brain » by Amelia J Thompson et al. is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.