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/.

MACC1-Induced Collective Migration Is Promoted by Proliferation Rather Than Single Cell Biomechanics

High metastasis-associated in colon cancer 1 (MACC1) expression is associated with metastasis, tumor cell migration, and increased proliferation in colorectal cancer. Tumors with high MACC1 expression show a worse prognosis and higher invasion into neighboring structures. However, the mediation of the pro-migratory effects is still a matter of investigation.*

In their study “MACC1-Induced Collective Migration Is Promoted by Proliferation Rather Than Single Cell Biomechanics”  Tim Hohmann, Urszula Hohmann, Mathias Dahlmann,  Dennis Kobelt, Ulrike Stein and Faramarz Dehghani aim to elucidate the impact of single cell biomechanics and proliferation on MACC1-dependent migration.*

The authors found that MACC1 expression associated with increased collective migration, caused by increased proliferation, and no changes in single cell biomechanics. Thus, targeting proliferation in high-MACC1-expressing tumors may offer additional effects on cell migration.*

The mechanical properties of single cells were assessed in the form of the Young’s modulus and cortex tension; both were measured using atomic force microscopy. Briefly, cells were seeded on a petri dish and measured 15 min after seeding to avoid slippage of individual cells. Measurements were conducted using a tipless NanoWorld Arrow-TL2 AFM cantilever array to apply a force of 1 nN that led to deformations of 1–2 µm. The Young’s modulus was calculated using the Hertz model.*

NanoWorld tipless Arrow-TL2 AFM probe array with two tipless AFM cantilevers
NanoWorld® Arrow™ TL2 AFM probes are tipless AFM cantilevers for special applications. They can for example be used for attaching spheres and other objects to the free end of the AFM cantilever, or for functionalizing and sensing applications.
The Arrow™ TL2 probes are optionally available with a sample facing side gold coating (Arrow™ TL2Au).
Figure 1 from “MACC1-Induced Collective Migration Is Promoted by Proliferation Rather Than Single Cell Biomechanics” by Tim Hohmann et al. Single cell properties of high- and low-MACC1-expressing colon carcinoma cells. (A,B) depict the results of the biomechanical measurements for the Young´s modulus and the cortex tension. (C,D) show the results of live cell imaging of single cells for the mean speed and the contact area with the substrate. Sample sizes: (A) nSW480/EV = 35; nSW480/MACC1 = 33; nSW620/shMACC1 = 40; nSW620/shCTL = 40. (B) nSW480/EV = 33; nSW480/MACC1 = 31; nSW620/shMACC1 = 25; nSW620/shCTL = 26. (C,D) nSW480/EV = 66; nSW480/MACC1 = 98; nSW620/shMACC1 = 102; nSW620/shCTL = 111. Asterisk depicts statistically significant results with p < 0.05. Box plots show the median (red line), 25 and 75 percentile (box), non-outlier range (whiskers), and outliers (red dots). The mechanical properties of single cells were assessed in the form of the Young’s modulus and cortex tension; both were measured using atomic force microscopy. Briefly, cells were seeded on a petri dish and measured 15 min after seeding to avoid slippage of individual cells. Measurements were conducted using a NanoWorld Arrow-TL2 AFM cantilever to apply a force of 1 nN that led to deformations of 1–2 µm. The Young’s modulus was calculated using the Hertz model.
Figure 1 from “MACC1-Induced Collective Migration Is Promoted by Proliferation Rather Than Single Cell Biomechanics” by Tim Hohmann et al.
Single cell properties of high- and low-MACC1-expressing colon carcinoma cells. (A,B) depict the results of the biomechanical measurements for the Young´s modulus and the cortex tension. (C,D) show the results of live cell imaging of single cells for the mean speed and the contact area with the substrate. Sample sizes: (A) nSW480/EV = 35; nSW480/MACC1 = 33; nSW620/shMACC1 = 40; nSW620/shCTL = 40. (B) nSW480/EV = 33; nSW480/MACC1 = 31; nSW620/shMACC1 = 25; nSW620/shCTL = 26. (C,D) nSW480/EV = 66; nSW480/MACC1 = 98; nSW620/shMACC1 = 102; nSW620/shCTL = 111. Asterisk depicts statistically significant results with p < 0.05. Box plots show the median (red line), 25 and 75 percentile (box), non-outlier range (whiskers), and outliers (red dots).

*Tim Hohmann, Urszula Hohmann, Mathias Dahlmann,  Dennis Kobelt, Ulrike Stein and Faramarz Dehghani
MACC1-Induced Collective Migration Is Promoted by Proliferation Rather Than Single Cell Biomechanics
Cancers 2022, 14(12), 2857
DOI: https://doi.org/10.3390/cancers14122857

Open Access

The article “MACC1-Induced Collective Migration Is Promoted by Proliferation Rather Than Single Cell Biomechanics” by Tim Hohmann, Urszula Hohmann, Mathias Dahlmann,  Dennis Kobelt, Ulrike Stein and Faramarz Dehghani 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/.

Optimized positioning through maximized tip visibility – Arrow AFM probes screencast passes 500 views mark

The screencast about NanoWorld Arrow Silicon AFM probes held byNanoWorld AG CEO Manfred Detterbeck has just passed the 500 views mark. Congratulations Manfred!

NanoWorld Arrow™ AFM probes are designed for easy AFM tip positioning and high resolution AFM imaging and are very popular with AFM users due to the highly symetric scans that are possible with these AFM probes because of their special tip shape. They fit to all well-known commercial SPMs (Scanning Probe Microscopes) and AFMs (Atomic Force Microscopes). The Arrow AFM probe consists of an AFM probe support chip with an AFM cantilever which has a tetrahedral AFM tip at its triangular free end.

The Arrow AFM probe is entirely made of monolithic, highly doped silicon.

The unique Arrow™ shape of the AFM cantilever with the AFM tip always placed at the very end of the AFM cantilever allows easy positioning of the AFM tip on the area of interest.
The Arrow AFM probes are available for non-contact mode, contact mode and force modulation mode imaging and are also available with a conductive platinum iridum coating. Furthermore the Arrow™ AFM probe series also includes a range of tipless AFM cantilevers and AFM cantilever arrays as well as dedicated ultra-high frequency Arrow AFM probes for high speed AFM.

To find out more about the different variations please have a look at:

https://www.nanoworld.com/arrow-afm-tips

You can also find various application examples for the Arrow AFM probes in the NanoWorld blog. For a selection of these articles just click on the “Arrow AFM probes” tag on the bottom of this blog entry.