Tag Archives: force curves

Male-female communication enhances release of extracellular vesicles leading to high fertility in Drosophila

The female reproductive tract (female-RT) must decipher the repertoire of molecular cues received from the male during copulation in order to activate and coordinate tract functionality necessary for high fertility. In Drosophila, this modulation is partially driven by spermathecal secretory cells (SSC). The SSC are a layer of cuboidal secretory glandular cells surrounding the spermatheca capsule where sperm is stored. It is unclear, however, how the SSC regulate the system’s activity. *

In the article “Male-female communication enhances release of extracellular vesicles leading to high fertility in Drosophila” Javier Arturo Sanchez-Lopez, Shai Twena, Ido Apel, Shani Chen Kornhaeuser, Michael Chasnitsky, Andras G. Miklosi, Perla J. Vega-Dominguez, Alex Shephard, Amir Hefetz and Yael Heifetz show that mating activates the secretory machinery of the SSC.*

The SSC release a heterogeneous population of extracellular vesicles (EVs) which is involved in initiating and managing the increase in egg-laying, and possibly sperm storage. Moreover, sperm and male accessory gland proteins are essential for such mating-mediated SSC activity. Thus, mating regulates secretory/endocytic pathways required for trafficking of vesicles to SSC-female-RT target sites, which modulate and coordinate reproductive tract activity to achieve high fertility. *

The authors used atomic force microscopy to scan the extracellular vesicles (EVs).

The samples were scanned in liquid using AFM cantilever beam 3 (CB3) of the NANOSENSORS uniqprobe qp-BioAC-CI AFM probes. The qp-BioAC-CI AFM tips have been rounded to a nominal AFM tip radius of 30nm and are dedicated for cell imaging applications and measurements on soft and life science samples. The uniqprobe qp-BioAC-CI AFM probes feature three different rectangular AFM cantilevers on one side of the support chip and the cantilevers unite fairly high resonance frequencies with low force constants.

The reflective gold coating deposited on the detector side of the AFM cantilevers covers only the free end above where the AFM tip is located. Main advantages of the uniqprobe coating are considerably less AFM cantilever bending and reduced thermal drift particularly for measurements in liquid environments.

The samples were also scanned in QI mode, in which a force curve is measured on every pixel.*

From these force curves, the authors calculated the adhesion force and Young’s modulus. *

The profiles of adhesion and Young’s modulus were created by measuring the mean value of the particle in three different areas of 5 × 5 µm. The diameter (nm) was obtained by creating a cross section of each EV and measuring the base of the profile. *

The involvement of EVs in SSC-female-RT routes of communication has added another layer of complexity to the process driving the switch towards a functional female-RT, and to high fertility. The precise mechanism by which male-derived signals, including EVs, affect SSC-derived trafficking has yet to be resolved. Deciphering male-female communication via EVs in Drosophila and other organisms will contribute greatly to our understanding of the different combinations of input modalities and output networks leading to high fertility. *

Fig. 5 from “Male-female communication enhances release of extracellular vesicles leading to high fertility in Drosophila” by Javier Arturo Sanchez-Lopez et al.: The spermatheca releases EVs with specific characteristics. a–e The spermathecae (SSC > CD63-GFP) were cultured ex vivo to characterize the particles that were released to the spent media: a size distribution of CD63-GFP-positive particles (n = 25 spermathecae from 15 flies) using single-particle tracking of the Nanoimager. b Characterization of CD63-GFP particles by ExoView (100 spermathecae from 50-60 flies) using anti-GFP or anti-CD63. c Representative dSTORM image of EVs found in the spent media of SSC expressing CD63-GFP (n = 25 spermathecae from 15 flies), stained with AlexaFluor555 conjugated anti-CD63 primary antibody. The image shows CD63-GFP-positive EVs and single CD63 AlexaFluor555 molecules the EV’s surface. Scale bars = 1 µm and insets = 20 nm. d–e The morphology of EVs in the spent media was observed by e negative staining in STEM; scale bar = 200 nm; and by d Cryo-TEM; Scale bar = 100 nm (see also Supplementary Fig. 5a-c). f–i The spent media of ex vivo cultured spermathecae were analyzed for the presence of EVs: f Representative AFM scans of SSC-EVs isolated by acoustic sorting (n = 100 spermathecae from 70 flies), showing from left to right: height (nm), adhesion (pN) and Young’s modulus images (MPa). Scale bars = 100 nm (see Supplementary Fig. 5d–g). g–i Single-particle tracking in the nanoimager. g CellMaskTM-positive particle size distribution and concentration profiles of EVs in the spent media of spermathecae incubated in media alone (No ecdy) or with ecdysone (Ecdy) (see also Supplementary Movie 5 for a time lapse of spermathecae end apparatus and endosomal activity post-ecdysone stimulation and methods, ex vivo spermatheca culture section); media with only CellMaskTM stain served as a control for the formation of dye aggregates. h Particle concentration and i mean particle diameter (nm) from g; Box plots are the measurements of particles from four frames of spent media from 25 spermathecae; boxes represent maximum, median and minimum values with outliers; one-way ANOVA, with nonparametric Wilcoxon multiple comparison post-hoc test; *p < 0.0001. NANOSENSORS uniqprobe qp-BioAC-CI AFM probes (CB3) were used for the Atomic Force Microscopy in liquid
Fig. 5 from “Male-female communication enhances release of extracellular vesicles leading to high fertility in Drosophila” by Javier Arturo Sanchez-Lopez et al.:
The spermatheca releases EVs with specific characteristics.
a–e The spermathecae (SSC > CD63-GFP) were cultured ex vivo to characterize the particles that were released to the spent media: a size distribution of CD63-GFP-positive particles (n = 25 spermathecae from 15 flies) using single-particle tracking of the Nanoimager. b Characterization of CD63-GFP particles by ExoView (100 spermathecae from 50-60 flies) using anti-GFP or anti-CD63. c Representative dSTORM image of EVs found in the spent media of SSC expressing CD63-GFP (n = 25 spermathecae from 15 flies), stained with AlexaFluor555 conjugated anti-CD63 primary antibody. The image shows CD63-GFP-positive EVs and single CD63 AlexaFluor555 molecules the EV’s surface. Scale bars = 1 µm and insets = 20 nm. d–e The morphology of EVs in the spent media was observed by e negative staining in STEM; scale bar = 200 nm; and by d Cryo-TEM; Scale bar = 100 nm (see also Supplementary Fig. 5a-c). f–i The spent media of ex vivo cultured spermathecae were analyzed for the presence of EVs: f Representative AFM scans of SSC-EVs isolated by acoustic sorting (n = 100 spermathecae from 70 flies), showing from left to right: height (nm), adhesion (pN) and Young’s modulus images (MPa). Scale bars = 100 nm (see Supplementary Fig. 5d–g). g–i Single-particle tracking in the nanoimager. g CellMaskTM-positive particle size distribution and concentration profiles of EVs in the spent media of spermathecae incubated in media alone (No ecdy) or with ecdysone (Ecdy) (see also Supplementary Movie 5 for a time lapse of spermathecae end apparatus and endosomal activity post-ecdysone stimulation and methods, ex vivo spermatheca culture section); media with only CellMaskTM stain served as a control for the formation of dye aggregates. h Particle concentration and i mean particle diameter (nm) from g; Box plots are the measurements of particles from four frames of spent media from 25 spermathecae; boxes represent maximum, median and minimum values with outliers; one-way ANOVA, with nonparametric Wilcoxon multiple comparison post-hoc test; *p < 0.0001.

*Javier Arturo Sanchez-Lopez, Shai Twena, Ido Apel, Shani Chen Kornhaeuser, Michael Chasnitsky, Andras G. Miklosi, Perla J. Vega-Dominguez, Alex Shephard, Amir Hefetz and Yael Heifetz
Male-female communication enhances release of extracellular vesicles leading to high fertility in Drosophila
Nature Communications Biology volume 5, Article number: 815 (2022)
DOI: https://doi.org/10.1038/s42003-022-03770-6

Please follow this external link to read the full article: https://rdcu.be/cYtUq

Open Access: The article “Male-female communication enhances release of extracellular vesicles leading to high fertility in Drosophila” by Javier Arturo Sanchez-Lopez, Shai Twena, Ido Apel, Shani Chen Kornhaeuser, Michael Chasnitsky, Andras G. Miklosi, Perla J. Vega-Dominguez, Alex Shephard, Amir Hefetz and Yael Heifetz 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 licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit https://creativecommons.org/licenses/by/4.0/.

Elastic shell theory for plant cell wall stiffness reveals contributions of cell wall elasticity and turgor pressure in AFM measurement

The stiffness of a plant cell in response to an applied force is determined not only by the elasticity of the cell wall but also by turgor pressure and cell geometry, which affect the tension of the cell wall. Although stiffness has been investigated using atomic force microscopy (AFM) and Young’s modulus of the cell wall has occasionally been estimated using the contact-stress theory (Hertz theory), the existence of tension has made the study of stiffness more complex. *

An alternative model is a contact model based on elastic shell theory, in which the cell wall is assumed to be a thin, curved surface pushed by turgor pressure. This theory enables one to infer turgor pressure from the apparent stiffness in some cases. In the unified formula from the elastic shell theory, AFM indentation is described as the contributions of cell wall elasticity and turgor pressure, while the estimation of elasticity and pressure remains ambiguous. *

In the article “Elastic shell theory for plant cell wall stiffness reveals contributions of cell wall elasticity and turgor pressure in AFM measurement” Satoru Tsugawa, Yuki Yamasaki, Shota Horiguchi, Tianhao Zhang, Takara Muto, Yosuke Nakaso, Kenshiro Ito, Ryu Takebayashi, Kazunori Okano, Eri Akita, Ryohei Yasukuni, Taku Demura, Tetsuro Mimura, Ken’ichi Kawaguchi and Yoichiroh Hosokawa describe how they used finite element method simulations to verify the formula of the elastic shell theory for onion (Allium cepa) cells and further optimized the formula to analyze the apparent stiffness observed from the AFM measurement based on the elastic shell theory.*

The authors applied the formula and simulations to successfully quantify the turgor pressure and elasticity of a cell in the plane direction using the cell curvature and apparent stiffness measured by atomic force microscopy. They conclude that tension resulting from turgor pressure regulates cell stiffness, which can be modified by a slight adjustment of turgor pressure in the order of 0.1 MPa. This theoretical analysis reveals a path for understanding forces inherent in plant cells. *

NANOSENSORS™ sphere AFM probes SD-Sphere-NCH-S from the NANOSENSORS™ Special Developments List were used for the force-indentation curve measurements described in the article. NANOSENSORS™ tipless AFM cantilevers of the TL-NCH type were used to evaluate the tip radius dependence. *

Fig-3-S-Tsugawa-et-al-2022-Elastic-shell-theory-for-plant-cell-wall-stiffness-reveals-contributions-of-cell-wall-elasticity-and-turgor-pressure-in-AFM-measurement NANOSENSORS Sphere-AFM probes SD-Sphere-NCH-S from the NANOSENSORS™ Special Developments List were used for the force-indentation curve measurements with atomic force microscopy
Figure 3 from “Elastic shell theory for plant cell wall stiffness reveals contributions of cell wall elasticity and turgor pressure in AFM measurement” by Satoru Tsugawa et al.:
AFM measurement of an onion epidermal cell with laser perforation. (A) Photographs of the cell measured before (left) and after (right) perforation. Yellow arrow indicates the perforation point. Cell lengths along long- and short- axes are denoted by La and Lb, respectively. Bars, 50 µm. (B) Topographic images before (left) and after (right) perforation. Measurement area corresponds to the dashed box area in (A). Lower images are three-dimensional images of upper images. (C) Enlarged image of the perforation point. (D) Cross-sectional graph of the cell wall surface before (red line) and after (blue line) perforation, corresponding to the height of dashed lines in upper-left and -right images in (B), respectively. Bulge height of the cell surface is denoted by w. Dashed lines are curves for curvature calculated from Lb and w. (E) Quantities determined from AFM measurement. Mean curvature of the cell wall surface κM is calculated from La, Lb, and w. (F) Force–indentation curves of the cell wall before (red dots) and after (blue dots) perforation. Dashed lines are fitting curves by the Hertz model and solid lines are fitting lines by the shell model. (G) Apparent stiffness kas as a function of force F applied to the cell wall before (red dots) and after (blue dots) perforation. kas is estimated by linear least squares fitting of the force-indentation curve in the vicinity of the F, as shown in (F). Bars on dots represent root mean squared error. Solid lines are exponential plateau curves: kas = 35 × {1 − exp(− F/7)} (red line); kas = 10 × {1 − exp(− F/1.28)} (blue line).
Abstract
The stiffness of a plant cell in response to an applied force is determined not only by the elasticity of the cell wall but also by turgor pressure and cell geometry, which affect the tension of the cell wall. Although stiffness has been investigated using atomic force microscopy (AFM) and Young’s modulus of the cell wall has occasionally been estimated using the contact-stress theory (Hertz theory), the existence of tension has made the study of stiffness more complex. Elastic shell theory has been proposed as an alternative method; however, the estimation of elasticity remains ambiguous. Here, we used finite element method simulations to verify the formula of the elastic shell theory for onion (Allium cepa) cells. We applied the formula and simulations to successfully quantify the turgor pressure and elasticity of a cell in the plane direction using the cell curvature and apparent stiffness measured by AFM. We conclude that tension resulting from turgor pressure regulates cell stiffness, which can be modified by a slight adjustment of turgor pressure in the order of 0.1 MPa. This theoretical analysis reveals a path for understanding forces inherent in plant cells.

*Satoru Tsugawa, Yuki Yamasaki, Shota Horiguchi, Tianhao Zhang, Takara Muto, Yosuke Nakaso, Kenshiro Ito, Ryu Takebayashi, Kazunori Okano, Eri Akita, Ryohei Yasukuni, Taku Demura, Tetsuro Mimura, Ken’ichi Kawaguchi and Yoichiroh Hosokawa
Elastic shell theory for plant cell wall stiffness reveals contributions of cell wall elasticity and turgor pressure in AFM measurement
Nature Scientific Reports volume 12, Article number: 13044 (2022)
DOI: https://doi.org/10.1038/s41598-022-16880-2

Please follow this external link to read the full article: https://rdcu.be/cSWFR

Open Access: The article “Elastic shell theory for plant cell wall stiffness reveals contributions of cell wall elasticity and turgor pressure in AFM measurement” by Satoru Tsugawa, Yuki Yamasaki, Shota Horiguchi, Tianhao Zhang, Takara Muto, Yosuke Nakaso, Kenshiro Ito, Ryu Takebayashi, Kazunori Okano, Eri Akita, Ryohei Yasukuni, Taku Demura, Tetsuro Mimura, Ken’ichi Kawaguchi and Yoichiroh Hosokawa 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 licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

Supercritical carbon dioxide decellularization of plant material to generate 3D biocompatible scaffolds

Biocompatible scaffolds that can be repopulated with human cells have many uses such serving as replacement organs and tissues. Therefore there is an increasing interest in plant-based biomaterials for tissue engineering.*

As the above mentioned scaffolds should mimic the in vivo tissue environment closely they need to provide a fitting structural and biomechanical support to the cells while at the same time promoting cell behaviour and tissue development. *

Currently the standard method to prepare plant tissue to serve as a biocompatible scaffold is to decellularize it with serial chemical treatment.*

In their article “Supercritical carbon dioxide decellularization of plant material to generate 3D biocompatible scaffolds” Ashlee F. Harris, Jerome Lacombe, Sumedha Liyanage, Margaret Y. Han, Emily Wallace, Sophia Karsunky, Noureddine Abidi and Frederic Zenhausern explore another method to produce biocompatible scaffolds.*

They use supercritical carbon dioxide (scCO2) together with 2% peracetic acid to decellularize plant material.*

Their subsequent investigations show that the process of decellularization, scaffold structure preservation and recellularization with human cells is less time consuming than with the standard chemical method.

In a further step the authors of the article describe how they use various scientific methods to evaluate the scaffolds they decellularized by the described scCO2 method.*

Ashlee F. Harris et al. use Atomic Force Microscopy (AFM) in order to find out if the scCO2 treatment had an impact on the mechanical properties of the scaffolds produced with this method.*

With AFM topography measurements they are able to establish that structures such as plant vasculature were preserved.*

The following determination of the Young’s Modulus calculated from multiple force curves of a homogeneous surface section of the produced scaffold shows it to be slightly lower than the one from a chemically decellularized scaffold.*

NANOSENSORS™ uniqprobe qp-BioAC AFM probes ( CB3 nominal values: 80 μm length, 30 μm mean width, 400 nm thickness, force constant 0.06 N/m, resonance frequency 30 kHz) were used for the scaffold measurements with Atomic Force Microscopy.

Figure 3 from “Supercritical carbon dioxide decellularization of plant material to generate 3D biocompatible scaffolds AFM imaging and spectrometry measurement” by Ashlee F. Harris et al.: 
They used AFM surface topography measurements to confirm that the structures such as plant vasculature were preserved after the scSO2 process and used  AFM force curves to calculate the  Young’s Modulus (YM) of the scCO2 decellularized scaffold. NANOSENSORS uniqprobe qp-BioAC AFM probes were used for the described AFM measurments. 
(a) Representative false colored three-dimensional surface mapping images and (b) Young’s modulus of scCO2 and chemically decellularized scaffolds (data as mean ± SEM; n = 5).
Figure 3 from “Supercritical carbon dioxide decellularization of plant material to generate 3D biocompatible scaffolds AFM imaging and spectrometry measurement” by Ashlee F. Harris et al.: (a) Representative false colored three-dimensional surface mapping images and (b) Young’s modulus of scCO2 and chemically decellularized scaffolds (data as mean ± SEM; n = 5).

While the scCo2 method promises to be a faster way to decellularize plant material and produce sterile and biocompatible scaffolds further research will be necessary to determine whether the differences the authors detected between the scaffolds produced with the scCO2 approach and the scaffolds produced with the chemical approach have a major influence on how repopulated cells behave in the achieved scaffolds.*

*Ashlee F. Harris, Jerome Lacombe, Sumedha Liyanage, Margaret Y. Han, Emily Wallace, Sophia Karsunky, Noureddine Abidi and Frederic Zenhausern
Supercritical carbon dioxide decellularization of plant material to generate 3D biocompatible scaffolds
Nature Scientific Reports 11, 3643 (2021)
DOI: https://doi.org/10.1038/s41598-021-83250-9

Please follow this external link to read the full article: https://rdcu.be/cAqW3

Open Access The article “Supercritical carbon dioxide decellularization of plant material to generate 3D biocompatible scaffolds” by Ashlee F. Harris, Jerome Lacombe, Sumedha Liyanage, Margaret Y. Han, Emily Wallace, Sophia Karsunky, Noureddine Abidi and Frederic Zenhausern 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 licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.