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Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis

Cells communicate with their environments via the plasma membrane and various membrane proteins. Clathrin-mediated endocytosis (CME) plays a central role in such communication and proceeds with a series of multiprotein assembly, deformation of the plasma membrane, and production of a membrane vesicle that delivers extracellular signaling molecules into the cytoplasm.*

In the article “Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis”, Aiko Yoshida, Nobuaki Sakai, Yoshitsugu Uekusa, Yuka Imaoka, Yoshitsuna Itagaki, Yuki Suzuki and Shige H. Yoshimura describe how they utilized their home-built correlative imaging system comprising high-speed atomic force microscopy (HS-AFM) and confocal fluorescence microscopy to simultaneously image morphological changes of the plasma membrane and protein localization during CME in a living cell.*

Overlaying AFM and fluorescence images revealed the dynamics of protein assembly and concomitant morphological changes of the plasma membrane with high spatial resolution. In particular, the authors elucidate the role of actin in the closing step of CME.*

The results revealed a tight correlation between the size of the pit and the amount of clathrin assembled. Actin dynamics play multiple roles in the assembly, maturation, and closing phases of the process, and affects membrane morphology, suggesting a close relationship between endocytosis and dynamic events at the cell cortex. Knock down of dynamin also affected the closing motion of the pit and showed functional correlation with actin.*

An AFM tip-scan–type HS-AFM unit combined with an inverted fluorescence/optical microscope equipped with a phase contrast system and a confocal unit was used for this study.*

The modulation method was set to phase modulation mode to detect AFM tip–sample interactions. A customized NanoWorld Ultra-Short AFM cantilever with an electron beam–deposited sharp AFM tip with a spring constant of 0.1 N m−1 (USC-F0.8-k0.1-T12) was used. *

All observations were performed at 28 °C. The AFM tip was aligned with confocal views as described in the Results section of the article. The images from the confocal microscope and AFM were simultaneously acquired at a scanning rate of 10 s/frame. The captured sequential images were overlaid by using AviUTL (http://spring-fragrance.mints.ne.jp/aviutl/) based on the AFM tip position.
The fluorescence intensity was quantified by Image J software (http://rsbweb.nih.gov/ij/). *

Fig 1 from Aiko Yoshida et al 2018 “Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis” :Aligning the confocal image and the AFM image. (A) Schematic illustration of the sample stage. A cross-shaped movable XY-stage (orange) is mounted on the base plate (light green) of the inverted optical microscope (IX83) via a stage guide (gray) equipped at each of the 4 ends of the cross. A 3-point support plate (purple) for mounting the AFM scanner unit is fixed on the base plate with a configuration that does not hinder the sliding of the XY-stage along the x-axis and y-axis. These setups allow the sample stage to move independently of the AFM unit and the optical axis. (B) Side view of the HS-AFM unit mounted on the stage illustrated in panel A. (C) Overlaying a confocal image and an AFM image. COS-7 cells expressing EGFP-CLCa were fixed with 5% paraformaldehyde and subjected to AFM (left) and CLSM (middle) imaging. The x-y position of the probe tip was determined as described in S1 Fig. Two images were overlaid (right) based on the x-y center position. Scale bar: 1 μm. Autofluorescence of the probe was much weaker than clathrin spot and could not be detected during the fast scanning. (D) AFM images of CCP on the cytoplasmic surface of the plasma membrane. COS-7 cells were “unroofed” by mild sonication as described in Materials and methods and then fixed with glutaraldehyde. Scale bar: 0.1 μm. AFM, atomic force microscopy; CCP, clathrin-coated pit; CLSM, confocal laser scanning microscopy; COS-7, CV-1 in origin with SV40 gene line 7; EGFP, enhanced green fluorescent protein; EGFP-CLCa, EGFP-fused clathrin light chain a; HS-AFM, high-speed AFM. https://doi.org/10.1371/journal.pbio.2004786.g001 customized NanoWorld Ultra-Short AFM cantilevers with an electron beam–deposited sharp AFM tip with a spring constant of 0.1 N m−1 (USC-F0.8-k0.1-T12) were used — Fig 1 from Aiko Yoshida et al 2018 “Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis” :
Aligning the confocal image and the AFM image.
(A) Schematic illustration of the sample stage. A cross-shaped movable XY-stage (orange) is mounted on the base plate (light green) of the inverted optical microscope (IX83) via a stage guide (gray) equipped at each of the 4 ends of the cross. A 3-point support plate (purple) for mounting the AFM scanner unit is fixed on the base plate with a configuration that does not hinder the sliding of the XY-stage along the x-axis and y-axis. These setups allow the sample stage to move independently of the AFM unit and the optical axis. (B) Side view of the HS-AFM unit mounted on the stage illustrated in panel A. (C) Overlaying a confocal image and an AFM image. COS-7 cells expressing EGFP-CLCa were fixed with 5% paraformaldehyde and subjected to AFM (left) and CLSM (middle) imaging. The x-y position of the probe tip was determined as described in S1 Fig. Two images were overlaid (right) based on the x-y center position. Scale bar: 1 μm. Autofluorescence of the probe was much weaker than clathrin spot and could not be detected during the fast scanning. (D) AFM images of CCP on the cytoplasmic surface of the plasma membrane. COS-7 cells were “unroofed” by mild sonication as described in Materials and methods and then fixed with glutaraldehyde. Scale bar: 0.1 μm. AFM, atomic force microscopy; CCP, clathrin-coated pit; CLSM, confocal laser scanning microscopy; COS-7, CV-1 in origin with SV40 gene line 7; EGFP, enhanced green fluorescent protein; EGFP-CLCa, EGFP-fused clathrin light chain a; HS-AFM, high-speed AFM.
https://doi.org/10.1371/journal.pbio.2004786.g001

Supporting figure 1 from Aiko Yoshida et al 2018 “Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis”:1 Fig. Aligning confocal and AFM images. (A) Scanning electron microscopy (SEM) images of a cantilever equipped with an EBD tip with tilt angle of 12°. Scale bar, 5 μm. Note that the cantilever is held on the AFM head unit with a tilt angle of 102° (from the x-y plane) so that the relative tip–sample angle (θ) is 90°. This setup makes it possible to precisely determine the position of the AFM tip. Scale bar, 2 μm. (B) Determining the position of the AFM probe in a fluorescence image. While the AFM probe was attached on the glass surface without scanning, the autofluorescence signal of the probe was imaged by the confocal scanning unit. The observed fluorescence spot (arrowhead in the middle panel) is defined as an origin of the fluorescence image plane (x = 0, y = 0) and used to define the optical axis (left panel). The position of a fluorescence spot derived from EGFP-CLCa was determined on this axis. On the other hand, the scanning area of the AFM scanner covers the area of (−3, 2.25) (left top), (3, 2.25) (right top), (3, −2.25) (right bottom), and (−3, −2.25) (left bottom) (all right panel). By aligning the axis from both images, the x, y position of the AFM image and that of the confocal fluorescence image could be merged. AFM, atomic force microscopy; EBD, electron beam–deposited; EGFP, enhanced green fluorescent protein; EGFP-CLCa, EGFP-fused clathrin light chain a. https://doi.org/10.1371/journal.pbio.2004786.s001 customized NanoWorld Ultra-Short AFM cantilevers with an electron beam–deposited sharp AFM tip with a spring constant of 0.1 N m−1 (USC-F0.8-k0.1-T12) were used — Supporting figure 1 from Aiko Yoshida et al 2018 “Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis”:
1 Fig. Aligning confocal and AFM images.
(A) Scanning electron microscopy (SEM) images of a cantilever equipped with an EBD tip with tilt angle of 12°. Scale bar, 5 μm. Note that the cantilever is held on the AFM head unit with a tilt angle of 102° (from the x-y plane) so that the relative tip–sample angle (θ) is 90°. This setup makes it possible to precisely determine the position of the AFM tip. Scale bar, 2 μm. (B) Determining the position of the AFM probe in a fluorescence image. While the AFM probe was attached on the glass surface without scanning, the autofluorescence signal of the probe was imaged by the confocal scanning unit. The observed fluorescence spot (arrowhead in the middle panel) is defined as an origin of the fluorescence image plane (x = 0, y = 0) and used to define the optical axis (left panel). The position of a fluorescence spot derived from EGFP-CLCa was determined on this axis. On the other hand, the scanning area of the AFM scanner covers the area of (−3, 2.25) (left top), (3, 2.25) (right top), (3, −2.25) (right bottom), and (−3, −2.25) (left bottom) (all right panel). By aligning the axis from both images, the x, y position of the AFM image and that of the confocal fluorescence image could be merged. AFM, atomic force microscopy; EBD, electron beam–deposited; EGFP, enhanced green fluorescent protein; EGFP-CLCa, EGFP-fused clathrin light chain a.
https://doi.org/10.1371/journal.pbio.2004786.s001

*Aiko Yoshida, Nobuaki Sakai, Yoshitsugu Uekusa, Yuka Imaoka, Yoshitsuna Itagaki, Yuki Suzuki and Shige H. Yoshimura
Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis
PLoS Biol 16(5) (2018): e2004786
DOI: https://doi.org/10.1371/journal.pbio.2004786

The article “Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis” by Aiko Yoshida, Nobuaki Sakai, Yoshitsugu Uekusa, Yuka Imaoka, Yoshitsuna Itagaki, Yuki Suzuki and Shige H. Yoshimura 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/.

Structural and optical variation of pseudoisocyanine aggregates nucleated on DNA substrates

The ability to maximize the range of exciton transport while minimizing energy loss has significant implications for the design of future nanoscale light harvesting, optoelectronic, and sensing applications. *

One method of achieving this would be to densely pack dyes into strongly coupled aggregates such that excitations can be coherently delocalized through the partial or full length of the aggregate. *

Coherently coupled aggregates enable exciton migration over discreet spatial distances with near unitary quantum efficiency. As a result, controlled dye aggregation has long been studied by chemists as a method of tuning the photonic and physical properties of the dyes and pigments in light harvesting devices. An example of this coherent coupling phenomenon can be observed in the cyanine dye family and specifically the prototypical example, pseudoisocyanine (PIC) dye. *

In the article «Structural and optical variation of pseudoisocyanine aggregates nucleated on DNA substrates” Matthew Chiriboga, Christopher M Green, Divita Mathur, David A Hastman, Joseph S Melinger, Remi Veneziano, Igor L Medintz and Sebastián A Díaz show that DNA-nucleated PIC aggregates have properties which correlate to different molecular structures and are directed by changing the DNA scaffold. *

To achieve this, they formed PIC aggregates through heterogeneous nucleation by mixing dissolved PIC dye with various DNA nanostructures ranging from a rigid DX-tile to more flexible DNA duplex (dsDNA) or single strand DNA oligonucleotide (ssDNA). *

Although the aggregates Matthew Chiriboga et al formed required elevated excess of PIC dye relative to previously reported J-bits, they exhibited sharper and brighter fluorescence peaks as well as longer Ncoh. *

Therefore, the authors refer to aggregates formed by this approach as super aggregate (SA) in their article, though they note SA formed with different DNA substrates result in unique properties. *

Complementary circular dichroism (CD) and atomic force microscopy (AFM) characterizations were used to analyze the SA and both indicated distinctions in the way each substrate and subsequent dye aggregate incorporates the individual PIC molecules. *

To achieve high resolution imaging of nucleic acid nanostructures, the DNA is often deposited onto a mica substrate, where mica electrostatically binds the DNA. Once deposited onto the mica, the imaging can be done in a hydrated environment as there is no additional required dehydration or staining of the DNA, a particularly convenient advantage of AFM. *

The AFM imaging was performed under AC fast imaging mode (liquid) with NanoWorld Ultra-Short Cantilevers (USC) for Fast/High-Speed AFM of the USC-F0.3-k0.3 AFM probe type.*

On a segment of freshly cleaved mica mounted to a magnetic puck, 15 μl of PIC-DANN solution was deposited immediately before measurement. A 25 μl droplet of imaging buffer was deposited on the AFM tip, then the AFM tip mount was lowered into the sample buffer to create a liquid ‘chamber’ for imaging. *

When introducing various DNA scaffolds for SA formation and subsequent AFM imaging, Matthew Chiriboga et al. observed significant changes in the aggregates structure. *

The AFM imaging highlighted the stark differences in aggregate formation resulting from the DNA substrates. *

To the author’s knowledge this is the first visualization of DNA-based PIC aggregates. Results from the field have been pointing towards fiber-like or nanotube-like networks of polymerized PIC as a structural model for aggregates suspended in solution. *

On the other hand, other AFM studies demonstrate that PIC aggregates formed on mica substrates adopt a leafy island morphology. *

Interestingly, Matthew Chiriboga et al. observe evidence of both PIC fibers as well as leafy islands that exhibit distinct growth patterns, again depending on the DNA substrate. *

Although this work contributes to the growing body of evidence that solution-based PIC aggregates form fibrous networks structures, the AFM measurements presented in the article highlight the multiplicity of PIC aggregation modes when introduced to DNA scaffolds. *

The results presented in the research article suggest modification of the DNA substrate results in significant changes to how the DNA and companion dye molecules are integrated into larger form PIC aggregates. *

Bearing in mind that the broader motivation for studying DNA based PIC aggregates is to integrate strongly coupled dyes onto modular DNA structural units, PIC SAs should be given due consideration as a versatile option. *

In fact, similar work is being done with other cyanine dyes where DNA template modification is used to switch between quenching and energy transfer. *

Ultimately this could be a path for the PIC SA and one which possibly leads towards applications in optical microcavities for quantum electrodynamical devices and optical switching, molecular plasmonics, biosensors, and light-harvesting arrays. *

Figure 6 from Matthew Chiriboga et al. “Structural and optical variation of pseudoisocyanine aggregates nucleated on DNA substrates”:Atomic Force Microscopy visualisation of pseudoisocyanine aggregates – super aggregates (SA) nucleated on DNA substrates. AFM visualizations of pseudoisocyanine (PIC) aggregates formed in the (A) AT, (B) dsDNA, and (C) ssDNA nanostructures. Each of the samples was formed immediately before measurement by mixing 160 μM PIC dye with 500 nM DNA normalized to the dye-labeled strand concentration (i.e. 320-fold excess). When the SA was formed using an AT DX-tile template (figures 6(A) the authors observed the formation of large and long rod-like aggregates with a relatively isotropic growth axis. This supports the hypothesis proposed by Yoa et al which suggested aggregation along preferential axis due to a preferred interaction between the PIC and the mica NanoWorld USC-F0.3-k0.3 AFM probes were used for the under AC fast imaging mode in liquid. — Figure 6 from Matthew Chiriboga et al. “Structural and optical variation of pseudoisocyanine aggregates nucleated on DNA substrates”:
AFM visualization of SA formations. AFM visualizations of PIC aggregates formed in the (A) AT, (B) dsDNA, and (C) ssDNA nanostructures. Each of the samples was formed immediately before measurement by mixing 160 μM PIC dye with 500 nM DNA normalized to the dye-labeled strand concentration (i.e. 320-fold excess).

*Matthew Chiriboga, Christopher M Green, Divita Mathur, David A Hastman, Joseph S Melinger, Remi Veneziano, Igor L Medintz and Sebastián A Díaz
Structural and optical variation of pseudoisocyanine aggregates nucleated on DNA substrates
Methods and Applications in Fluorescence (2023) 11 014003
DOI: https://doi.org/10.1088/2050-6120/acb2b4

The article “Active self-assembly of piezoelectric biomolecular films via synergistic nanoconfinement and in-situ poling” by Matthew Chiriboga, Christopher M Green, Divita Mathur, David A Hastman, Joseph S Melinger, Remi Veneziano, Igor L Medintz and Sebastián A Díaz 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/.

Intrinsically disordered regions in TRPV2 mediate protein-protein interactions

Transient receptor potential (TRP) ion channels are gated by diverse intra- and extracellular stimuli leading to cation inflow (Na+, Ca2+) regulating many cellular processes and initiating organismic somatosensation. *

Structures of most TRP channels have been solved. However, structural and sequence analysis showed that ~30% of the TRP channel sequences, mainly the N- and C-termini, are intrinsically disordered regions (IDRs). Unfortunately, very little is known about IDR ‘structure’, dynamics and function, though it has been shown that they are essential for native channel function. *

In the article “Intrinsically disordered regions in TRPV2 mediate protein-protein interactions”, Raghavendar R. Sanganna Gari, Grigory Tagiltsev, Ruth A. Pumroy, Yining Jiang, Martin Blackledge, Vera Y. Moiseenkova-Bell and Simon Scheuring imaged TRPV2 channels in membranes using high-speed atomic force microscopy (HS-AFM). *

The dynamic single molecule imaging capability of HS-AFM allowed the authors to visualize IDRs and revealed that N-terminal IDRs were involved in intermolecular interactions. Their work provides evidence about the ‘structure’ of the TRPV2 IDRs, and that the IDRs may mediate protein-protein interactions. *

In total, 1.5 µl of the TRPV2 reconstituted vesicles were deposited on a 1.5-mm2 freshly cleaved mica surface, which was glued with epoxy to the quartz sample stage. After 20–30 min incubation, the sample was gently rinsed with imaging buffer (20 mM Hepes, pH 8.0, 150 mM NaCl) and mounted in the HS-AFM fluid cell. All images in this study were taken using a HS-AFM operated in amplitude modulation mode using optimized scan and feedback parameters and lab-built amplitude detectors and free amplitude stabilizers. *

Short (8 µm) cantilevers (NanoWorld Ultra-Short Cantilevers for High-Speed AFM USC-F1.2-k0.15) with nominal spring constant of 0.15 N/m, resonance frequency of 0.6 MHz, and a quality factor of ∼1.5 in liquid were used. AFM probes were sharpened using oxygen plasma etching to obtain better resolution. *

Fig. 1 from “Intrinsically disordered regions in TRPV2 mediate protein-protein interactions” by Raghavendar R. Sanganna Gari et al. :TRPV2 reconstitution for HS-AFM analysis. b Overview HS-AFM images (Supplementary Movie 1) of TRPV2 (windmill-shaped molecules) in soy polar lipid membranes on mica (dark background areas). False color scale: 0–9 nm. The white oversaturated areas have a height of ~26 nm and represent likely non-ruptured small vesicles. NanoWorld-USC-F1.2-k0.15 AFM probes were used for the HS-AFM — Fig. 1 from “Intrinsically disordered regions in TRPV2 mediate protein-protein interactions” by Raghavendar R. Sanganna Gari et al. :
TRPV2 reconstitution for HS-AFM analysis.
b Overview HS-AFM images (Supplementary Movie 1) of TRPV2 (windmill-shaped molecules) in soy polar lipid membranes on mica (dark background areas). False color scale: 0–9 nm. The white oversaturated areas have a height of ~26 nm and represent likely non-ruptured small vesicles.

*Raghavendar R. Sanganna Gari, Grigory Tagiltsev, Ruth A. Pumroy, Yining Jiang, Martin Blackledge, Vera Y. Moiseenkova-Bell and Simon Scheuring
Intrinsically disordered regions in TRPV2 mediate protein-protein interactions
Communications Biology volume 6, Article number: 966 (2023)
DOI: https://doi.org/10.1038/s42003-023-05343-7

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

The article “Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response” by Raghavendar R. Sanganna Gari, Grigory Tagiltsev, Ruth A. Pumroy, Yining Jiang, Martin Blackledge, Vera Y. Moiseenkova-Bell and Simon Scheuring 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/.