---
_id: '14502'
abstract:
- lang: eng
text: A precise quantitative description of the ultrastructural characteristics
underlying biological mechanisms is often key to their understanding. This is
particularly true for dynamic extra- and intracellular filamentous assemblies,
playing a role in cell motility, cell integrity, cytokinesis, tissue formation
and maintenance. For example, genetic manipulation or modulation of actin regulatory
proteins frequently manifests in changes of the morphology, dynamics, and ultrastructural
architecture of actin filament-rich cell peripheral structures, such as lamellipodia
or filopodia. However, the observed ultrastructural effects often remain subtle
and require sufficiently large datasets for appropriate quantitative analysis.
The acquisition of such large datasets has been enabled by recent advances in
high-throughput cryo-electron tomography (cryo-ET) methods. This also necessitates
the development of complementary approaches to maximize the extraction of relevant
biological information. We have developed a computational toolbox for the semi-automatic
quantification of segmented and vectorized fila- mentous networks from pre-processed
cryo-electron tomograms, facilitating the analysis and cross-comparison of multiple
experimental conditions. GUI-based components simplify the processing of data
and allow users to obtain a large number of ultrastructural parameters describing
filamentous assemblies. We demonstrate the feasibility of this workflow by analyzing
cryo-ET data of untreated and chemically perturbed branched actin filament networks
and that of parallel actin filament arrays. In principle, the computational toolbox
presented here is applicable for data analysis comprising any type of filaments
in regular (i.e. parallel) or random arrangement. We show that it can ease the
identification of key differences between experimental groups and facilitate the
in-depth analysis of ultrastructural data in a time-efficient manner.
author:
- first_name: Georgi A
full_name: Dimchev, Georgi A
id: 38C393BE-F248-11E8-B48F-1D18A9856A87
last_name: Dimchev
orcid: 0000-0001-8370-6161
- first_name: Behnam
full_name: Amiri, Behnam
last_name: Amiri
- first_name: Florian
full_name: Fäßler, Florian
id: 404F5528-F248-11E8-B48F-1D18A9856A87
last_name: Fäßler
orcid: 0000-0001-7149-769X
- first_name: Martin
full_name: Falcke, Martin
last_name: Falcke
- first_name: Florian KM
full_name: Schur, Florian KM
id: 48AD8942-F248-11E8-B48F-1D18A9856A87
last_name: Schur
orcid: 0000-0003-4790-8078
citation:
ama: Dimchev GA, Amiri B, Fäßler F, Falcke M, Schur FK. Computational toolbox for
ultrastructural quantitative analysis of filament networks in cryo-ET data. 2023.
doi:10.15479/AT:ISTA:14502
apa: Dimchev, G. A., Amiri, B., Fäßler, F., Falcke, M., & Schur, F. K. (2023).
Computational toolbox for ultrastructural quantitative analysis of filament networks
in cryo-ET data. Institute of Science and Technology Austria. https://doi.org/10.15479/AT:ISTA:14502
chicago: Dimchev, Georgi A, Behnam Amiri, Florian Fäßler, Martin Falcke, and Florian
KM Schur. “Computational Toolbox for Ultrastructural Quantitative Analysis of
Filament Networks in Cryo-ET Data.” Institute of Science and Technology Austria,
2023. https://doi.org/10.15479/AT:ISTA:14502.
ieee: G. A. Dimchev, B. Amiri, F. Fäßler, M. Falcke, and F. K. Schur, “Computational
toolbox for ultrastructural quantitative analysis of filament networks in cryo-ET
data.” Institute of Science and Technology Austria, 2023.
ista: Dimchev GA, Amiri B, Fäßler F, Falcke M, Schur FK. 2023. Computational toolbox
for ultrastructural quantitative analysis of filament networks in cryo-ET data,
Institute of Science and Technology Austria, 10.15479/AT:ISTA:14502.
mla: Dimchev, Georgi A., et al. Computational Toolbox for Ultrastructural Quantitative
Analysis of Filament Networks in Cryo-ET Data. Institute of Science and Technology
Austria, 2023, doi:10.15479/AT:ISTA:14502.
short: G.A. Dimchev, B. Amiri, F. Fäßler, M. Falcke, F.K. Schur, (2023).
date_created: 2023-11-08T19:40:54Z
date_published: 2023-11-21T00:00:00Z
date_updated: 2023-11-21T08:36:02Z
day: '21'
ddc:
- '570'
department:
- _id: FlSc
doi: 10.15479/AT:ISTA:14502
file:
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content_type: application/zip
creator: fschur
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date_updated: 2023-11-08T20:23:07Z
file_id: '14503'
file_name: Computational_Toolbox_v1.2.zip
file_size: 347641117
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success: 1
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checksum: 14db2addbfca61a085ba301ed6f2900b
content_type: text/plain
creator: dernst
date_created: 2023-11-21T08:20:23Z
date_updated: 2023-11-21T08:20:23Z
file_id: '14586'
file_name: Readme.txt
file_size: 1522
relation: main_file
success: 1
file_date_updated: 2023-11-21T08:20:23Z
has_accepted_license: '1'
keyword:
- cryo-electron tomography
- actin cytoskeleton
- toolbox
license: https://choosealicense.com/licenses/agpl-3.0/
month: '11'
oa: 1
project:
- _id: 9B954C5C-BA93-11EA-9121-9846C619BF3A
grant_number: P33367
name: Structure and isoform diversity of the Arp2/3 complex
publisher: Institute of Science and Technology Austria
related_material:
record:
- id: '10290'
relation: used_for_analysis_in
status: public
status: public
title: Computational toolbox for ultrastructural quantitative analysis of filament
networks in cryo-ET data
tmp:
legal_code_url: https://www.gnu.org/licenses/agpl-3.0.html
name: GNU Affero General Public License v3.0
short: 'GNU AGPLv3 '
type: software
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
year: '2023'
...
---
_id: '12334'
abstract:
- lang: eng
text: Regulation of the Arp2/3 complex is required for productive nucleation of
branched actin networks. An emerging aspect of regulation is the incorporation
of subunit isoforms into the Arp2/3 complex. Specifically, both ArpC5 subunit
isoforms, ArpC5 and ArpC5L, have been reported to fine-tune nucleation activity
and branch junction stability. We have combined reverse genetics and cellular
structural biology to describe how ArpC5 and ArpC5L differentially affect cell
migration. Both define the structural stability of ArpC1 in branch junctions and,
in turn, by determining protrusion characteristics, affect protein dynamics and
actin network ultrastructure. ArpC5 isoforms also affect the positioning of members
of the Ena/Vasodilator-stimulated phosphoprotein (VASP) family of actin filament
elongators, which mediate ArpC5 isoform–specific effects on the actin assembly
level. Our results suggest that ArpC5 and Ena/VASP proteins are part of a signaling
pathway enhancing cell migration.
acknowledged_ssus:
- _id: ScienComp
- _id: LifeSc
- _id: Bio
- _id: EM-Fac
acknowledgement: "We would like to thank K. von Peinen and B. Denker (Helmholtz Centre
for Infection Research, Braunschweig, Germany) for experimental and technical assistance,
respectively.\r\nThis research was supported by the Scientific Service Units (SSUs)
of ISTA through resources provided by Scientific Computing (SciComp), the Life Science
Facility (LSF), the Imaging and Optics facility (IOF), and the Electron Microscopy
Facility (EMF). We acknowledge support from ISTA and from the Austrian Science Fund
(FWF) (P33367) to F.K.M.S., from the Research Training Group GRK2223 and the Helmholtz
Society to K.R,. and from the Deutsche Forschungsgemeinschaft (DFG) to J.F. and
K.R."
article_number: add6495
article_processing_charge: No
article_type: original
author:
- first_name: Florian
full_name: Fäßler, Florian
id: 404F5528-F248-11E8-B48F-1D18A9856A87
last_name: Fäßler
orcid: 0000-0001-7149-769X
- first_name: Manjunath
full_name: Javoor, Manjunath
id: 305ab18b-dc7d-11ea-9b2f-b58195228ea2
last_name: Javoor
- first_name: Julia
full_name: Datler, Julia
id: 3B12E2E6-F248-11E8-B48F-1D18A9856A87
last_name: Datler
orcid: 0000-0002-3616-8580
- first_name: Hermann
full_name: Döring, Hermann
last_name: Döring
- first_name: Florian
full_name: Hofer, Florian
id: b9d234ba-9e33-11ed-95b6-cd561df280e6
last_name: Hofer
- first_name: Georgi A
full_name: Dimchev, Georgi A
id: 38C393BE-F248-11E8-B48F-1D18A9856A87
last_name: Dimchev
orcid: 0000-0001-8370-6161
- first_name: Victor-Valentin
full_name: Hodirnau, Victor-Valentin
id: 3661B498-F248-11E8-B48F-1D18A9856A87
last_name: Hodirnau
- first_name: Jan
full_name: Faix, Jan
last_name: Faix
- first_name: Klemens
full_name: Rottner, Klemens
last_name: Rottner
- first_name: Florian KM
full_name: Schur, Florian KM
id: 48AD8942-F248-11E8-B48F-1D18A9856A87
last_name: Schur
orcid: 0000-0003-4790-8078
citation:
ama: Fäßler F, Javoor M, Datler J, et al. ArpC5 isoforms regulate Arp2/3 complex–dependent
protrusion through differential Ena/VASP positioning. Science Advances.
2023;9(3). doi:10.1126/sciadv.add6495
apa: Fäßler, F., Javoor, M., Datler, J., Döring, H., Hofer, F., Dimchev, G. A.,
… Schur, F. K. (2023). ArpC5 isoforms regulate Arp2/3 complex–dependent protrusion
through differential Ena/VASP positioning. Science Advances. American Association
for the Advancement of Science. https://doi.org/10.1126/sciadv.add6495
chicago: Fäßler, Florian, Manjunath Javoor, Julia Datler, Hermann Döring, Florian
Hofer, Georgi A Dimchev, Victor-Valentin Hodirnau, Jan Faix, Klemens Rottner,
and Florian KM Schur. “ArpC5 Isoforms Regulate Arp2/3 Complex–Dependent Protrusion
through Differential Ena/VASP Positioning.” Science Advances. American
Association for the Advancement of Science, 2023. https://doi.org/10.1126/sciadv.add6495.
ieee: F. Fäßler et al., “ArpC5 isoforms regulate Arp2/3 complex–dependent
protrusion through differential Ena/VASP positioning,” Science Advances,
vol. 9, no. 3. American Association for the Advancement of Science, 2023.
ista: Fäßler F, Javoor M, Datler J, Döring H, Hofer F, Dimchev GA, Hodirnau V-V,
Faix J, Rottner K, Schur FK. 2023. ArpC5 isoforms regulate Arp2/3 complex–dependent
protrusion through differential Ena/VASP positioning. Science Advances. 9(3),
add6495.
mla: Fäßler, Florian, et al. “ArpC5 Isoforms Regulate Arp2/3 Complex–Dependent Protrusion
through Differential Ena/VASP Positioning.” Science Advances, vol. 9, no.
3, add6495, American Association for the Advancement of Science, 2023, doi:10.1126/sciadv.add6495.
short: F. Fäßler, M. Javoor, J. Datler, H. Döring, F. Hofer, G.A. Dimchev, V.-V.
Hodirnau, J. Faix, K. Rottner, F.K. Schur, Science Advances 9 (2023).
date_created: 2023-01-23T07:26:42Z
date_published: 2023-01-20T00:00:00Z
date_updated: 2023-11-21T08:05:35Z
day: '20'
ddc:
- '570'
department:
- _id: FlSc
- _id: EM-Fac
doi: 10.1126/sciadv.add6495
external_id:
isi:
- '000964550100015'
file:
- access_level: open_access
checksum: ce81a6d0b84170e5e8c62f6acfa15d9e
content_type: application/pdf
creator: dernst
date_created: 2023-01-23T07:45:54Z
date_updated: 2023-01-23T07:45:54Z
file_id: '12335'
file_name: 2023_ScienceAdvances_Faessler.pdf
file_size: 1756234
relation: main_file
success: 1
file_date_updated: 2023-01-23T07:45:54Z
has_accepted_license: '1'
intvolume: ' 9'
isi: 1
issue: '3'
keyword:
- Multidisciplinary
language:
- iso: eng
month: '01'
oa: 1
oa_version: Published Version
project:
- _id: 9B954C5C-BA93-11EA-9121-9846C619BF3A
grant_number: P33367
name: Structure and isoform diversity of the Arp2/3 complex
publication: Science Advances
publication_identifier:
issn:
- 2375-2548
publication_status: published
publisher: American Association for the Advancement of Science
quality_controlled: '1'
related_material:
record:
- id: '14562'
relation: research_data
status: public
scopus_import: '1'
status: public
title: ArpC5 isoforms regulate Arp2/3 complex–dependent protrusion through differential
Ena/VASP positioning
tmp:
image: /images/cc_by.png
legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
short: CC BY (4.0)
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 9
year: '2023'
...
---
_id: '12421'
abstract:
- lang: eng
text: The actin cytoskeleton plays a key role in cell migration and cellular morphodynamics
in most eukaryotes. The ability of the actin cytoskeleton to assemble and disassemble
in a spatiotemporally controlled manner allows it to form higher-order structures,
which can generate forces required for a cell to explore and navigate through
its environment. It is regulated not only via a complex synergistic and competitive
interplay between actin-binding proteins (ABP), but also by filament biochemistry
and filament geometry. The lack of structural insights into how geometry and ABPs
regulate the actin cytoskeleton limits our understanding of the molecular mechanisms
that define actin cytoskeleton remodeling and, in turn, impact emerging cell migration
characteristics. With the advent of cryo-electron microscopy (cryo-EM) and advanced
computational methods, it is now possible to define these molecular mechanisms
involving actin and its interactors at both atomic and ultra-structural levels
in vitro and in cellulo. In this review, we will provide an overview of the available
cryo-EM methods, applicable to further our understanding of the actin cytoskeleton,
specifically in the context of cell migration. We will discuss how these methods
have been employed to elucidate ABP- and geometry-defined regulatory mechanisms
in initiating, maintaining, and disassembling cellular actin networks in migratory
protrusions.
acknowledgement: 'We apologize for not being able to mention and cite additional excellent
work that would have fit the scope of this review, due to space restraints. We thank
Jesse Hansen for comments on the manuscript. We acknowledge support from the Austrian
Science Fund (FWF): P33367 and the Institute of Science and Technology Austria.'
article_processing_charge: No
article_type: original
author:
- first_name: Florian
full_name: Fäßler, Florian
id: 404F5528-F248-11E8-B48F-1D18A9856A87
last_name: Fäßler
orcid: 0000-0001-7149-769X
- first_name: Manjunath
full_name: Javoor, Manjunath
id: 305ab18b-dc7d-11ea-9b2f-b58195228ea2
last_name: Javoor
- first_name: Florian KM
full_name: Schur, Florian KM
id: 48AD8942-F248-11E8-B48F-1D18A9856A87
last_name: Schur
orcid: 0000-0003-4790-8078
citation:
ama: Fäßler F, Javoor M, Schur FK. Deciphering the molecular mechanisms of actin
cytoskeleton regulation in cell migration using cryo-EM. Biochemical Society
Transactions. 2023;51(1):87-99. doi:10.1042/bst20220221
apa: Fäßler, F., Javoor, M., & Schur, F. K. (2023). Deciphering the molecular
mechanisms of actin cytoskeleton regulation in cell migration using cryo-EM. Biochemical
Society Transactions. Portland Press. https://doi.org/10.1042/bst20220221
chicago: Fäßler, Florian, Manjunath Javoor, and Florian KM Schur. “Deciphering the
Molecular Mechanisms of Actin Cytoskeleton Regulation in Cell Migration Using
Cryo-EM.” Biochemical Society Transactions. Portland Press, 2023. https://doi.org/10.1042/bst20220221.
ieee: F. Fäßler, M. Javoor, and F. K. Schur, “Deciphering the molecular mechanisms
of actin cytoskeleton regulation in cell migration using cryo-EM,” Biochemical
Society Transactions, vol. 51, no. 1. Portland Press, pp. 87–99, 2023.
ista: Fäßler F, Javoor M, Schur FK. 2023. Deciphering the molecular mechanisms of
actin cytoskeleton regulation in cell migration using cryo-EM. Biochemical Society
Transactions. 51(1), 87–99.
mla: Fäßler, Florian, et al. “Deciphering the Molecular Mechanisms of Actin Cytoskeleton
Regulation in Cell Migration Using Cryo-EM.” Biochemical Society Transactions,
vol. 51, no. 1, Portland Press, 2023, pp. 87–99, doi:10.1042/bst20220221.
short: F. Fäßler, M. Javoor, F.K. Schur, Biochemical Society Transactions 51 (2023)
87–99.
date_created: 2023-01-27T10:08:19Z
date_published: 2023-02-01T00:00:00Z
date_updated: 2023-08-01T12:55:32Z
day: '01'
ddc:
- '570'
department:
- _id: FlSc
doi: 10.1042/bst20220221
external_id:
isi:
- '000926043100001'
file:
- access_level: open_access
checksum: 4e7069845e3dad22bb44fb71ec624c60
content_type: application/pdf
creator: dernst
date_created: 2023-03-16T07:58:16Z
date_updated: 2023-03-16T07:58:16Z
file_id: '12728'
file_name: 2023_BioChemicalSocietyTransactions_Faessler.pdf
file_size: 10045006
relation: main_file
success: 1
file_date_updated: 2023-03-16T07:58:16Z
has_accepted_license: '1'
intvolume: ' 51'
isi: 1
issue: '1'
keyword:
- Biochemistry
language:
- iso: eng
month: '02'
oa: 1
oa_version: Published Version
page: 87-99
project:
- _id: 9B954C5C-BA93-11EA-9121-9846C619BF3A
grant_number: P33367
name: Structure and isoform diversity of the Arp2/3 complex
publication: Biochemical Society Transactions
publication_identifier:
eissn:
- 1470-8752
issn:
- 0300-5127
publication_status: published
publisher: Portland Press
quality_controlled: '1'
scopus_import: '1'
status: public
title: Deciphering the molecular mechanisms of actin cytoskeleton regulation in cell
migration using cryo-EM
tmp:
image: /images/cc_by.png
legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
short: CC BY (4.0)
type: journal_article
user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8
volume: 51
year: '2023'
...
---
_id: '11351'
abstract:
- lang: eng
text: 'One hallmark of plant cells is their cell wall. They protect cells against
the environment and high turgor and mediate morphogenesis through the dynamics
of their mechanical and chemical properties. The walls are a complex polysaccharidic
structure. Although their biochemical composition is well known, how the different
components organize in the volume of the cell wall and interact with each other
is not well understood and yet is key to the wall’s mechanical properties. To
investigate the ultrastructure of the plant cell wall, we imaged the walls of
onion (Allium cepa) bulbs in a near-native state via cryo-focused ion beam milling
(cryo-FIB milling) and cryo-electron tomography (cryo-ET). This allowed the high-resolution
visualization of cellulose fibers in situ. We reveal the coexistence of dense
fiber fields bathed in a reticulated matrix we termed “meshing,” which is more
abundant at the inner surface of the cell wall. The fibers adopted a regular bimodal
angular distribution at all depths in the cell wall and bundled according to their
orientation, creating layers within the cell wall. Concomitantly, employing homogalacturonan
(HG)-specific enzymatic digestion, we observed changes in the meshing, suggesting
that it is—at least in part—composed of HG pectins. We propose the following model
for the construction of the abaxial epidermal primary cell wall: the cell deposits
successive layers of cellulose fibers at −45° and +45° relative to the cell’s
long axis and secretes the surrounding HG-rich meshing proximal to the plasma
membrane, which then migrates to more distal regions of the cell wall.'
acknowledgement: This work was supported by the Howard Hughes Medical Institute (HHMI)
and grant R35 GM122588 to G.J. and the Austrian Science Fund (FWF) P33367 to F.K.M.S.
We thank Noé Cochetel for his guidance and great help in data analysis, discovery,
and representation with the R software. We thank Hans-Ulrich Endress for graciously
providing us with the purified citrus pectin and Jozef Mravec for generating and
providing the COS488 probe. Cryo-EM work was done in the Beckman Institute Resource
Center for Transmission Electron Microscopy at Caltech. This article is subject
to HHMI’s Open Access to Publications policy. HHMI lab heads have previously granted
a nonexclusive CC BY 4.0 license to the public and a sublicensable license to HHMI
in their research articles. Pursuant to those licenses, the author accepted manuscript
of this article can be made freely available under a CC BY 4.0 license immediately
upon publication.
article_processing_charge: No
article_type: original
author:
- first_name: William J.
full_name: Nicolas, William J.
last_name: Nicolas
- first_name: Florian
full_name: Fäßler, Florian
id: 404F5528-F248-11E8-B48F-1D18A9856A87
last_name: Fäßler
orcid: 0000-0001-7149-769X
- first_name: Przemysław
full_name: Dutka, Przemysław
last_name: Dutka
- first_name: Florian KM
full_name: Schur, Florian KM
id: 48AD8942-F248-11E8-B48F-1D18A9856A87
last_name: Schur
orcid: 0000-0003-4790-8078
- first_name: Grant
full_name: Jensen, Grant
last_name: Jensen
- first_name: Elliot
full_name: Meyerowitz, Elliot
last_name: Meyerowitz
citation:
ama: Nicolas WJ, Fäßler F, Dutka P, Schur FK, Jensen G, Meyerowitz E. Cryo-electron
tomography of the onion cell wall shows bimodally oriented cellulose fibers and
reticulated homogalacturonan networks. Current Biology. 2022;32(11):P2375-2389.
doi:10.1016/j.cub.2022.04.024
apa: Nicolas, W. J., Fäßler, F., Dutka, P., Schur, F. K., Jensen, G., & Meyerowitz,
E. (2022). Cryo-electron tomography of the onion cell wall shows bimodally oriented
cellulose fibers and reticulated homogalacturonan networks. Current Biology.
Elsevier. https://doi.org/10.1016/j.cub.2022.04.024
chicago: Nicolas, William J., Florian Fäßler, Przemysław Dutka, Florian KM Schur,
Grant Jensen, and Elliot Meyerowitz. “Cryo-Electron Tomography of the Onion Cell
Wall Shows Bimodally Oriented Cellulose Fibers and Reticulated Homogalacturonan
Networks.” Current Biology. Elsevier, 2022. https://doi.org/10.1016/j.cub.2022.04.024.
ieee: W. J. Nicolas, F. Fäßler, P. Dutka, F. K. Schur, G. Jensen, and E. Meyerowitz,
“Cryo-electron tomography of the onion cell wall shows bimodally oriented cellulose
fibers and reticulated homogalacturonan networks,” Current Biology, vol.
32, no. 11. Elsevier, pp. P2375-2389, 2022.
ista: Nicolas WJ, Fäßler F, Dutka P, Schur FK, Jensen G, Meyerowitz E. 2022. Cryo-electron
tomography of the onion cell wall shows bimodally oriented cellulose fibers and
reticulated homogalacturonan networks. Current Biology. 32(11), P2375-2389.
mla: Nicolas, William J., et al. “Cryo-Electron Tomography of the Onion Cell Wall
Shows Bimodally Oriented Cellulose Fibers and Reticulated Homogalacturonan Networks.”
Current Biology, vol. 32, no. 11, Elsevier, 2022, pp. P2375-2389, doi:10.1016/j.cub.2022.04.024.
short: W.J. Nicolas, F. Fäßler, P. Dutka, F.K. Schur, G. Jensen, E. Meyerowitz,
Current Biology 32 (2022) P2375-2389.
date_created: 2022-05-04T06:22:06Z
date_published: 2022-06-06T00:00:00Z
date_updated: 2023-08-03T07:05:36Z
day: '06'
ddc:
- '570'
department:
- _id: FlSc
doi: 10.1016/j.cub.2022.04.024
external_id:
isi:
- '000822399200019'
pmid:
- '35508170'
file:
- access_level: open_access
checksum: af3f24d97c016d844df237abef987639
content_type: application/pdf
creator: dernst
date_created: 2022-08-05T06:29:18Z
date_updated: 2022-08-05T06:29:18Z
file_id: '11730'
file_name: 2022_CurrentBiology_Nicolas.pdf
file_size: 12827717
relation: main_file
success: 1
file_date_updated: 2022-08-05T06:29:18Z
has_accepted_license: '1'
intvolume: ' 32'
isi: 1
issue: '11'
keyword:
- General Agricultural and Biological Sciences
- General Biochemistry
- Genetics and Molecular Biology
language:
- iso: eng
month: '06'
oa: 1
oa_version: Published Version
page: P2375-2389
pmid: 1
project:
- _id: 9B954C5C-BA93-11EA-9121-9846C619BF3A
grant_number: P33367
name: Structure and isoform diversity of the Arp2/3 complex
publication: Current Biology
publication_identifier:
issn:
- 0960-9822
publication_status: published
publisher: Elsevier
quality_controlled: '1'
scopus_import: '1'
status: public
title: Cryo-electron tomography of the onion cell wall shows bimodally oriented cellulose
fibers and reticulated homogalacturonan networks
tmp:
image: /images/cc_by.png
legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
short: CC BY (4.0)
type: journal_article
user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8
volume: 32
year: '2022'
...
---
_id: '10290'
abstract:
- lang: eng
text: A precise quantitative description of the ultrastructural characteristics
underlying biological mechanisms is often key to their understanding. This is
particularly true for dynamic extra- and intracellular filamentous assemblies,
playing a role in cell motility, cell integrity, cytokinesis, tissue formation
and maintenance. For example, genetic manipulation or modulation of actin regulatory
proteins frequently manifests in changes of the morphology, dynamics, and ultrastructural
architecture of actin filament-rich cell peripheral structures, such as lamellipodia
or filopodia. However, the observed ultrastructural effects often remain subtle
and require sufficiently large datasets for appropriate quantitative analysis.
The acquisition of such large datasets has been enabled by recent advances in
high-throughput cryo-electron tomography (cryo-ET) methods. This also necessitates
the development of complementary approaches to maximize the extraction of relevant
biological information. We have developed a computational toolbox for the semi-automatic
quantification of segmented and vectorized filamentous networks from pre-processed
cryo-electron tomograms, facilitating the analysis and cross-comparison of multiple
experimental conditions. GUI-based components simplify the processing of data
and allow users to obtain a large number of ultrastructural parameters describing
filamentous assemblies. We demonstrate the feasibility of this workflow by analyzing
cryo-ET data of untreated and chemically perturbed branched actin filament networks
and that of parallel actin filament arrays. In principle, the computational toolbox
presented here is applicable for data analysis comprising any type of filaments
in regular (i.e. parallel) or random arrangement. We show that it can ease the
identification of key differences between experimental groups and facilitate the
in-depth analysis of ultrastructural data in a time-efficient manner.
acknowledged_ssus:
- _id: ScienComp
- _id: LifeSc
- _id: Bio
- _id: EM-Fac
acknowledgement: 'This research was supported by the Scientific Service Units (SSUs)
of IST Austria through resources provided by Scientific Computing (SciComp), the
Life Science Facility (LSF), the BioImaging Facility (BIF), and the Electron Microscopy
Facility (EMF). We also thank Victor-Valentin Hodirnau for help with cryo-ET data
acquisition. The authors acknowledge support from IST Austria and from the Austrian
Science Fund (FWF): M02495 to G.D. and Austrian Science Fund (FWF): P33367 to F.K.M.S.'
article_number: '107808'
article_processing_charge: Yes (via OA deal)
article_type: original
author:
- first_name: Georgi A
full_name: Dimchev, Georgi A
id: 38C393BE-F248-11E8-B48F-1D18A9856A87
last_name: Dimchev
orcid: 0000-0001-8370-6161
- first_name: Behnam
full_name: Amiri, Behnam
last_name: Amiri
- first_name: Florian
full_name: Fäßler, Florian
id: 404F5528-F248-11E8-B48F-1D18A9856A87
last_name: Fäßler
orcid: 0000-0001-7149-769X
- first_name: Martin
full_name: Falcke, Martin
last_name: Falcke
- first_name: Florian KM
full_name: Schur, Florian KM
id: 48AD8942-F248-11E8-B48F-1D18A9856A87
last_name: Schur
orcid: 0000-0003-4790-8078
citation:
ama: Dimchev GA, Amiri B, Fäßler F, Falcke M, Schur FK. Computational toolbox for
ultrastructural quantitative analysis of filament networks in cryo-ET data. Journal
of Structural Biology. 2021;213(4). doi:10.1016/j.jsb.2021.107808
apa: Dimchev, G. A., Amiri, B., Fäßler, F., Falcke, M., & Schur, F. K. (2021).
Computational toolbox for ultrastructural quantitative analysis of filament networks
in cryo-ET data. Journal of Structural Biology. Elsevier . https://doi.org/10.1016/j.jsb.2021.107808
chicago: Dimchev, Georgi A, Behnam Amiri, Florian Fäßler, Martin Falcke, and Florian
KM Schur. “Computational Toolbox for Ultrastructural Quantitative Analysis of
Filament Networks in Cryo-ET Data.” Journal of Structural Biology. Elsevier
, 2021. https://doi.org/10.1016/j.jsb.2021.107808.
ieee: G. A. Dimchev, B. Amiri, F. Fäßler, M. Falcke, and F. K. Schur, “Computational
toolbox for ultrastructural quantitative analysis of filament networks in cryo-ET
data,” Journal of Structural Biology, vol. 213, no. 4. Elsevier , 2021.
ista: Dimchev GA, Amiri B, Fäßler F, Falcke M, Schur FK. 2021. Computational toolbox
for ultrastructural quantitative analysis of filament networks in cryo-ET data.
Journal of Structural Biology. 213(4), 107808.
mla: Dimchev, Georgi A., et al. “Computational Toolbox for Ultrastructural Quantitative
Analysis of Filament Networks in Cryo-ET Data.” Journal of Structural Biology,
vol. 213, no. 4, 107808, Elsevier , 2021, doi:10.1016/j.jsb.2021.107808.
short: G.A. Dimchev, B. Amiri, F. Fäßler, M. Falcke, F.K. Schur, Journal of Structural
Biology 213 (2021).
date_created: 2021-11-15T12:21:42Z
date_published: 2021-11-03T00:00:00Z
date_updated: 2023-11-21T08:36:02Z
day: '03'
ddc:
- '572'
department:
- _id: FlSc
doi: 10.1016/j.jsb.2021.107808
external_id:
isi:
- '000720259500002'
file:
- access_level: open_access
checksum: 6b209e4d44775d4e02b50f78982c15fa
content_type: application/pdf
creator: cchlebak
date_created: 2021-11-15T13:11:27Z
date_updated: 2021-11-15T13:11:27Z
file_id: '10291'
file_name: 2021_JournalStructBiol_Dimchev.pdf
file_size: 16818304
relation: main_file
success: 1
file_date_updated: 2021-11-15T13:11:27Z
has_accepted_license: '1'
intvolume: ' 213'
isi: 1
issue: '4'
keyword:
- Structural Biology
language:
- iso: eng
month: '11'
oa: 1
oa_version: Published Version
project:
- _id: 9B954C5C-BA93-11EA-9121-9846C619BF3A
grant_number: P33367
name: Structure and isoform diversity of the Arp2/3 complex
- _id: 2674F658-B435-11E9-9278-68D0E5697425
call_identifier: FWF
grant_number: M02495
name: Protein structure and function in filopodia across scales
publication: Journal of Structural Biology
publication_identifier:
issn:
- 1047-8477
publication_status: published
publisher: 'Elsevier '
quality_controlled: '1'
related_material:
record:
- id: '14502'
relation: software
status: public
scopus_import: '1'
status: public
title: Computational toolbox for ultrastructural quantitative analysis of filament
networks in cryo-ET data
tmp:
image: /images/cc_by.png
legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
short: CC BY (4.0)
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 213
year: '2021'
...
---
_id: '8586'
abstract:
- lang: eng
text: Cryo-electron microscopy (cryo-EM) of cellular specimens provides insights
into biological processes and structures within a native context. However, a major
challenge still lies in the efficient and reproducible preparation of adherent
cells for subsequent cryo-EM analysis. This is due to the sensitivity of many
cellular specimens to the varying seeding and culturing conditions required for
EM experiments, the often limited amount of cellular material and also the fragility
of EM grids and their substrate. Here, we present low-cost and reusable 3D printed
grid holders, designed to improve specimen preparation when culturing challenging
cellular samples directly on grids. The described grid holders increase cell culture
reproducibility and throughput, and reduce the resources required for cell culturing.
We show that grid holders can be integrated into various cryo-EM workflows, including
micro-patterning approaches to control cell seeding on grids, and for generating
samples for cryo-focused ion beam milling and cryo-electron tomography experiments.
Their adaptable design allows for the generation of specialized grid holders customized
to a large variety of applications.
acknowledged_ssus:
- _id: ScienComp
- _id: LifeSc
- _id: Bio
- _id: EM-Fac
acknowledgement: This work was supported by the Austrian Science Fund (FWF, P33367)
to FKMS. BZ acknowledges support by the Niederösterreich Fond. This research was
also supported by the Scientific Service Units (SSU) of IST Austria through resources
provided by Scientific Computing (SciComp), the Life Science Facility (LSF), the
BioImaging Facility (BIF) and the Electron Microscopy Facility (EMF). We thank Georgi
Dimchev (IST Austria) and Sonja Jacob (Vienna Biocenter Core Facilities) for testing
our grid holders in different experimental setups and Daniel Gütl and the Kondrashov
group (IST Austria) for granting us repeated access to their 3D printers. We also
thank Jonna Alanko and the Sixt lab (IST Austria) for providing us HeLa cells, primary
BL6 mouse tail fibroblasts, NIH 3T3 fibroblasts and human telomerase immortalised
foreskin fibroblasts for our experiments. We are thankful to Ori Avinoam and William
Wan for helpful comments on the manuscript and also thank Dorotea Fracchiolla (Art&Science)
for illustrating the graphical abstract.
article_number: '107633'
article_processing_charge: Yes (via OA deal)
article_type: original
author:
- first_name: Florian
full_name: Fäßler, Florian
id: 404F5528-F248-11E8-B48F-1D18A9856A87
last_name: Fäßler
orcid: 0000-0001-7149-769X
- first_name: Bettina
full_name: Zens, Bettina
id: 45FD126C-F248-11E8-B48F-1D18A9856A87
last_name: Zens
- first_name: Robert
full_name: Hauschild, Robert
id: 4E01D6B4-F248-11E8-B48F-1D18A9856A87
last_name: Hauschild
orcid: 0000-0001-9843-3522
- first_name: Florian KM
full_name: Schur, Florian KM
id: 48AD8942-F248-11E8-B48F-1D18A9856A87
last_name: Schur
orcid: 0000-0003-4790-8078
citation:
ama: Fäßler F, Zens B, Hauschild R, Schur FK. 3D printed cell culture grid holders
for improved cellular specimen preparation in cryo-electron microscopy. Journal
of Structural Biology. 2020;212(3). doi:10.1016/j.jsb.2020.107633
apa: Fäßler, F., Zens, B., Hauschild, R., & Schur, F. K. (2020). 3D printed
cell culture grid holders for improved cellular specimen preparation in cryo-electron
microscopy. Journal of Structural Biology. Elsevier. https://doi.org/10.1016/j.jsb.2020.107633
chicago: Fäßler, Florian, Bettina Zens, Robert Hauschild, and Florian KM Schur.
“3D Printed Cell Culture Grid Holders for Improved Cellular Specimen Preparation
in Cryo-Electron Microscopy.” Journal of Structural Biology. Elsevier,
2020. https://doi.org/10.1016/j.jsb.2020.107633.
ieee: F. Fäßler, B. Zens, R. Hauschild, and F. K. Schur, “3D printed cell culture
grid holders for improved cellular specimen preparation in cryo-electron microscopy,”
Journal of Structural Biology, vol. 212, no. 3. Elsevier, 2020.
ista: Fäßler F, Zens B, Hauschild R, Schur FK. 2020. 3D printed cell culture grid
holders for improved cellular specimen preparation in cryo-electron microscopy.
Journal of Structural Biology. 212(3), 107633.
mla: Fäßler, Florian, et al. “3D Printed Cell Culture Grid Holders for Improved
Cellular Specimen Preparation in Cryo-Electron Microscopy.” Journal of Structural
Biology, vol. 212, no. 3, 107633, Elsevier, 2020, doi:10.1016/j.jsb.2020.107633.
short: F. Fäßler, B. Zens, R. Hauschild, F.K. Schur, Journal of Structural Biology
212 (2020).
date_created: 2020-09-29T13:24:06Z
date_published: 2020-12-01T00:00:00Z
date_updated: 2024-03-25T23:30:04Z
day: '01'
ddc:
- '570'
department:
- _id: FlSc
doi: 10.1016/j.jsb.2020.107633
external_id:
isi:
- '000600997800008'
file:
- access_level: open_access
checksum: c48cbf594e84fc2f91966ffaafc0918c
content_type: application/pdf
creator: dernst
date_created: 2020-12-10T14:01:10Z
date_updated: 2020-12-10T14:01:10Z
file_id: '8937'
file_name: 2020_JourStrucBiology_Faessler.pdf
file_size: 7076870
relation: main_file
success: 1
file_date_updated: 2020-12-10T14:01:10Z
has_accepted_license: '1'
intvolume: ' 212'
isi: 1
issue: '3'
keyword:
- electron microscopy
- cryo-EM
- EM sample preparation
- 3D printing
- cell culture
language:
- iso: eng
month: '12'
oa: 1
oa_version: Published Version
project:
- _id: 9B954C5C-BA93-11EA-9121-9846C619BF3A
grant_number: P33367
name: Structure and isoform diversity of the Arp2/3 complex
- _id: 059B463C-7A3F-11EA-A408-12923DDC885E
name: NÖ-Fonds Preis für die Jungforscherin des Jahres am IST Austria
publication: Journal of Structural Biology
publication_identifier:
issn:
- 1047-8477
publication_status: published
publisher: Elsevier
quality_controlled: '1'
related_material:
record:
- id: '14592'
relation: used_in_publication
status: public
- id: '12491'
relation: dissertation_contains
status: public
scopus_import: '1'
status: public
title: 3D printed cell culture grid holders for improved cellular specimen preparation
in cryo-electron microscopy
tmp:
image: /images/cc_by.png
legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
short: CC BY (4.0)
type: journal_article
user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8
volume: 212
year: '2020'
...
---
_id: '8971'
abstract:
- lang: eng
text: The actin-related protein (Arp)2/3 complex nucleates branched actin filament
networks pivotal for cell migration, endocytosis and pathogen infection. Its activation
is tightly regulated and involves complex structural rearrangements and actin
filament binding, which are yet to be understood. Here, we report a 9.0 Å resolution
structure of the actin filament Arp2/3 complex branch junction in cells using
cryo-electron tomography and subtomogram averaging. This allows us to generate
an accurate model of the active Arp2/3 complex in the branch junction and its
interaction with actin filaments. Notably, our model reveals a previously undescribed
set of interactions of the Arp2/3 complex with the mother filament, significantly
different to the previous branch junction model. Our structure also indicates
a central role for the ArpC3 subunit in stabilizing the active conformation.
acknowledged_ssus:
- _id: ScienComp
- _id: LifeSc
- _id: Bio
- _id: EM-Fac
acknowledgement: "This research was supported by the Scientific Service Units (SSUs)
of IST Austria through resources provided by Scientific Computing (SciComp), the
Life Science Facility (LSF), the BioImaging Facility (BIF), and the Electron Microscopy
Facility (EMF). We also thank Dimitry Tegunov (MPI for Biophysical Chemistry) for
helpful discussions\r\nabout the M software, and Michael Sixt (IST Austria) and
Klemens Rottner (Technical University Braunschweig, HZI Braunschweig) for critical
reading of the manuscript. We also thank Gregory Voth (University of Chicago) for
providing us the MD-derived branch junction model for comparison. The authors acknowledge
support from IST Austria and from the Austrian Science Fund (FWF): M02495 to G.D.
and Austrian Science Fund (FWF): P33367 to F.K.M.S. "
article_number: '6437'
article_processing_charge: No
article_type: original
author:
- first_name: Florian
full_name: Fäßler, Florian
id: 404F5528-F248-11E8-B48F-1D18A9856A87
last_name: Fäßler
orcid: 0000-0001-7149-769X
- first_name: Georgi A
full_name: Dimchev, Georgi A
id: 38C393BE-F248-11E8-B48F-1D18A9856A87
last_name: Dimchev
orcid: 0000-0001-8370-6161
- first_name: Victor-Valentin
full_name: Hodirnau, Victor-Valentin
id: 3661B498-F248-11E8-B48F-1D18A9856A87
last_name: Hodirnau
- first_name: William
full_name: Wan, William
last_name: Wan
- first_name: Florian KM
full_name: Schur, Florian KM
id: 48AD8942-F248-11E8-B48F-1D18A9856A87
last_name: Schur
orcid: 0000-0003-4790-8078
citation:
ama: Fäßler F, Dimchev GA, Hodirnau V-V, Wan W, Schur FK. Cryo-electron tomography
structure of Arp2/3 complex in cells reveals new insights into the branch junction.
Nature Communications. 2020;11. doi:10.1038/s41467-020-20286-x
apa: Fäßler, F., Dimchev, G. A., Hodirnau, V.-V., Wan, W., & Schur, F. K. (2020).
Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights
into the branch junction. Nature Communications. Springer Nature. https://doi.org/10.1038/s41467-020-20286-x
chicago: Fäßler, Florian, Georgi A Dimchev, Victor-Valentin Hodirnau, William Wan,
and Florian KM Schur. “Cryo-Electron Tomography Structure of Arp2/3 Complex in
Cells Reveals New Insights into the Branch Junction.” Nature Communications.
Springer Nature, 2020. https://doi.org/10.1038/s41467-020-20286-x.
ieee: F. Fäßler, G. A. Dimchev, V.-V. Hodirnau, W. Wan, and F. K. Schur, “Cryo-electron
tomography structure of Arp2/3 complex in cells reveals new insights into the
branch junction,” Nature Communications, vol. 11. Springer Nature, 2020.
ista: Fäßler F, Dimchev GA, Hodirnau V-V, Wan W, Schur FK. 2020. Cryo-electron tomography
structure of Arp2/3 complex in cells reveals new insights into the branch junction.
Nature Communications. 11, 6437.
mla: Fäßler, Florian, et al. “Cryo-Electron Tomography Structure of Arp2/3 Complex
in Cells Reveals New Insights into the Branch Junction.” Nature Communications,
vol. 11, 6437, Springer Nature, 2020, doi:10.1038/s41467-020-20286-x.
short: F. Fäßler, G.A. Dimchev, V.-V. Hodirnau, W. Wan, F.K. Schur, Nature Communications
11 (2020).
date_created: 2020-12-23T08:25:45Z
date_published: 2020-12-22T00:00:00Z
date_updated: 2023-08-24T11:01:50Z
day: '22'
ddc:
- '570'
department:
- _id: FlSc
- _id: EM-Fac
doi: 10.1038/s41467-020-20286-x
external_id:
isi:
- '000603078000003'
file:
- access_level: open_access
checksum: 55d43ea0061cc4027ba45e966e1db8cc
content_type: application/pdf
creator: dernst
date_created: 2020-12-28T08:16:10Z
date_updated: 2020-12-28T08:16:10Z
file_id: '8975'
file_name: 2020_NatureComm_Faessler.pdf
file_size: 3958727
relation: main_file
success: 1
file_date_updated: 2020-12-28T08:16:10Z
has_accepted_license: '1'
intvolume: ' 11'
isi: 1
keyword:
- General Biochemistry
- Genetics and Molecular Biology
- General Physics and Astronomy
- General Chemistry
language:
- iso: eng
month: '12'
oa: 1
oa_version: Published Version
project:
- _id: 9B954C5C-BA93-11EA-9121-9846C619BF3A
grant_number: P33367
name: Structure and isoform diversity of the Arp2/3 complex
- _id: 2674F658-B435-11E9-9278-68D0E5697425
call_identifier: FWF
grant_number: M02495
name: Protein structure and function in filopodia across scales
publication: Nature Communications
publication_identifier:
issn:
- 2041-1723
publication_status: published
publisher: Springer Nature
quality_controlled: '1'
related_material:
link:
- description: News on IST Homepage
relation: press_release
url: https://ist.ac.at/en/news/cutting-edge-technology-reveals-structures-within-cells/
scopus_import: '1'
status: public
title: Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights
into the branch junction
tmp:
image: /images/cc_by.png
legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
short: CC BY (4.0)
type: journal_article
user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8
volume: 11
year: '2020'
...