---
_id: '7572'
abstract:
- lang: eng
text: The polymerization–depolymerization dynamics of cytoskeletal proteins play
essential roles in the self-organization of cytoskeletal structures, in eukaryotic
as well as prokaryotic cells. While advances in fluorescence microscopy and in
vitro reconstitution experiments have helped to study the dynamic properties of
these complex systems, methods that allow to collect and analyze large quantitative
datasets of the underlying polymer dynamics are still missing. Here, we present
a novel image analysis workflow to study polymerization dynamics of active filaments
in a nonbiased, highly automated manner. Using treadmilling filaments of the bacterial
tubulin FtsZ as an example, we demonstrate that our method is able to specifically
detect, track and analyze growth and shrinkage of polymers, even in dense networks
of filaments. We believe that this automated method can facilitate the analysis
of a large variety of dynamic cytoskeletal systems, using standard time-lapse
movies obtained from experiments in vitro as well as in the living cell. Moreover,
we provide scripts implementing this method as supplementary material.
alternative_title:
- Methods in Cell Biology
article_processing_charge: No
author:
- first_name: Paulo R
full_name: Dos Santos Caldas, Paulo R
id: 38FCDB4C-F248-11E8-B48F-1D18A9856A87
last_name: Dos Santos Caldas
orcid: 0000-0001-6730-4461
- first_name: Philipp
full_name: Radler, Philipp
id: 40136C2A-F248-11E8-B48F-1D18A9856A87
last_name: Radler
orcid: '0000-0001-9198-2182 '
- first_name: Christoph M
full_name: Sommer, Christoph M
id: 4DF26D8C-F248-11E8-B48F-1D18A9856A87
last_name: Sommer
orcid: 0000-0003-1216-9105
- first_name: Martin
full_name: Loose, Martin
id: 462D4284-F248-11E8-B48F-1D18A9856A87
last_name: Loose
orcid: 0000-0001-7309-9724
citation:
ama: 'Dos Santos Caldas PR, Radler P, Sommer CM, Loose M. Computational analysis
of filament polymerization dynamics in cytoskeletal networks. In: Tran P, ed.
Methods in Cell Biology. Vol 158. Elsevier; 2020:145-161. doi:10.1016/bs.mcb.2020.01.006'
apa: Dos Santos Caldas, P. R., Radler, P., Sommer, C. M., & Loose, M. (2020).
Computational analysis of filament polymerization dynamics in cytoskeletal networks.
In P. Tran (Ed.), Methods in Cell Biology (Vol. 158, pp. 145–161). Elsevier.
https://doi.org/10.1016/bs.mcb.2020.01.006
chicago: Dos Santos Caldas, Paulo R, Philipp Radler, Christoph M Sommer, and Martin
Loose. “Computational Analysis of Filament Polymerization Dynamics in Cytoskeletal
Networks.” In Methods in Cell Biology, edited by Phong Tran, 158:145–61.
Elsevier, 2020. https://doi.org/10.1016/bs.mcb.2020.01.006.
ieee: P. R. Dos Santos Caldas, P. Radler, C. M. Sommer, and M. Loose, “Computational
analysis of filament polymerization dynamics in cytoskeletal networks,” in Methods
in Cell Biology, vol. 158, P. Tran, Ed. Elsevier, 2020, pp. 145–161.
ista: 'Dos Santos Caldas PR, Radler P, Sommer CM, Loose M. 2020.Computational analysis
of filament polymerization dynamics in cytoskeletal networks. In: Methods in Cell
Biology. Methods in Cell Biology, vol. 158, 145–161.'
mla: Dos Santos Caldas, Paulo R., et al. “Computational Analysis of Filament Polymerization
Dynamics in Cytoskeletal Networks.” Methods in Cell Biology, edited by
Phong Tran, vol. 158, Elsevier, 2020, pp. 145–61, doi:10.1016/bs.mcb.2020.01.006.
short: P.R. Dos Santos Caldas, P. Radler, C.M. Sommer, M. Loose, in:, P. Tran (Ed.),
Methods in Cell Biology, Elsevier, 2020, pp. 145–161.
date_created: 2020-03-08T23:00:47Z
date_published: 2020-02-27T00:00:00Z
date_updated: 2023-10-04T09:50:24Z
day: '27'
department:
- _id: MaLo
doi: 10.1016/bs.mcb.2020.01.006
ec_funded: 1
editor:
- first_name: 'Phong '
full_name: 'Tran, Phong '
last_name: Tran
external_id:
isi:
- '000611826500008'
intvolume: ' 158'
isi: 1
language:
- iso: eng
main_file_link:
- open_access: '1'
url: https://doi.org/10.1101/839571
month: '02'
oa: 1
oa_version: Preprint
page: 145-161
project:
- _id: 2595697A-B435-11E9-9278-68D0E5697425
call_identifier: H2020
grant_number: '679239'
name: Self-Organization of the Bacterial Cell
- _id: 260D98C8-B435-11E9-9278-68D0E5697425
name: Reconstitution of Bacterial Cell Division Using Purified Components
publication: Methods in Cell Biology
publication_identifier:
issn:
- 0091679X
publication_status: published
publisher: Elsevier
quality_controlled: '1'
related_material:
record:
- id: '8358'
relation: part_of_dissertation
status: public
scopus_import: '1'
status: public
title: Computational analysis of filament polymerization dynamics in cytoskeletal
networks
type: book_chapter
user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8
volume: 158
year: '2020'
...
---
_id: '153'
abstract:
- lang: eng
text: Cells migrating in multicellular organisms steadily traverse complex three-dimensional
(3D) environments. To decipher the underlying cell biology, current experimental
setups either use simplified 2D, tissue-mimetic 3D (e.g., collagen matrices) or
in vivo environments. While only in vivo experiments are truly physiological,
they do not allow for precise manipulation of environmental parameters. 2D in
vitro experiments do allow mechanical and chemical manipulations, but increasing
evidence demonstrates substantial differences of migratory mechanisms in 2D and
3D. Here, we describe simple, robust, and versatile “pillar forests” to investigate
cell migration in complex but fully controllable 3D environments. Pillar forests
are polydimethylsiloxane-based setups, in which two closely adjacent surfaces
are interconnected by arrays of micrometer-sized pillars. Changing the pillar
shape, size, height and the inter-pillar distance precisely manipulates microenvironmental
parameters (e.g., pore sizes, micro-geometry, micro-topology), while being easily
combined with chemotactic cues, surface coatings, diverse cell types and advanced
imaging techniques. Thus, pillar forests combine the advantages of 2D cell migration
assays with the precise definition of 3D environmental parameters.
article_processing_charge: No
author:
- first_name: Jörg
full_name: Renkawitz, Jörg
id: 3F0587C8-F248-11E8-B48F-1D18A9856A87
last_name: Renkawitz
orcid: 0000-0003-2856-3369
- first_name: Anne
full_name: Reversat, Anne
id: 35B76592-F248-11E8-B48F-1D18A9856A87
last_name: Reversat
orcid: 0000-0003-0666-8928
- first_name: Alexander F
full_name: Leithner, Alexander F
id: 3B1B77E4-F248-11E8-B48F-1D18A9856A87
last_name: Leithner
orcid: 0000-0002-1073-744X
- first_name: Jack
full_name: Merrin, Jack
id: 4515C308-F248-11E8-B48F-1D18A9856A87
last_name: Merrin
orcid: 0000-0001-5145-4609
- first_name: Michael K
full_name: Sixt, Michael K
id: 41E9FBEA-F248-11E8-B48F-1D18A9856A87
last_name: Sixt
orcid: 0000-0002-6620-9179
citation:
ama: 'Renkawitz J, Reversat A, Leithner AF, Merrin J, Sixt MK. Micro-engineered
“pillar forests” to study cell migration in complex but controlled 3D environments.
In: Methods in Cell Biology. Vol 147. Academic Press; 2018:79-91. doi:10.1016/bs.mcb.2018.07.004'
apa: Renkawitz, J., Reversat, A., Leithner, A. F., Merrin, J., & Sixt, M. K.
(2018). Micro-engineered “pillar forests” to study cell migration in complex but
controlled 3D environments. In Methods in Cell Biology (Vol. 147, pp. 79–91).
Academic Press. https://doi.org/10.1016/bs.mcb.2018.07.004
chicago: Renkawitz, Jörg, Anne Reversat, Alexander F Leithner, Jack Merrin, and
Michael K Sixt. “Micro-Engineered ‘Pillar Forests’ to Study Cell Migration in
Complex but Controlled 3D Environments.” In Methods in Cell Biology, 147:79–91.
Academic Press, 2018. https://doi.org/10.1016/bs.mcb.2018.07.004.
ieee: J. Renkawitz, A. Reversat, A. F. Leithner, J. Merrin, and M. K. Sixt, “Micro-engineered
‘pillar forests’ to study cell migration in complex but controlled 3D environments,”
in Methods in Cell Biology, vol. 147, Academic Press, 2018, pp. 79–91.
ista: 'Renkawitz J, Reversat A, Leithner AF, Merrin J, Sixt MK. 2018.Micro-engineered
“pillar forests” to study cell migration in complex but controlled 3D environments.
In: Methods in Cell Biology. vol. 147, 79–91.'
mla: Renkawitz, Jörg, et al. “Micro-Engineered ‘Pillar Forests’ to Study Cell Migration
in Complex but Controlled 3D Environments.” Methods in Cell Biology, vol.
147, Academic Press, 2018, pp. 79–91, doi:10.1016/bs.mcb.2018.07.004.
short: J. Renkawitz, A. Reversat, A.F. Leithner, J. Merrin, M.K. Sixt, in:, Methods
in Cell Biology, Academic Press, 2018, pp. 79–91.
date_created: 2018-12-11T11:44:54Z
date_published: 2018-07-27T00:00:00Z
date_updated: 2023-09-13T08:56:35Z
day: '27'
department:
- _id: MiSi
- _id: NanoFab
doi: 10.1016/bs.mcb.2018.07.004
external_id:
isi:
- '000452412300006'
pmid:
- '30165964'
intvolume: ' 147'
isi: 1
language:
- iso: eng
month: '07'
oa_version: None
page: 79 - 91
pmid: 1
publication: Methods in Cell Biology
publication_identifier:
issn:
- 0091679X
publication_status: published
publisher: Academic Press
publist_id: '7768'
quality_controlled: '1'
scopus_import: '1'
status: public
title: Micro-engineered “pillar forests” to study cell migration in complex but controlled
3D environments
type: book_chapter
user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1
volume: 147
year: '2018'
...
---
_id: '1213'
abstract:
- lang: eng
text: Bacterial cytokinesis is commonly initiated by the Z-ring, a dynamic cytoskeletal
structure that assembles at the site of division. Its primary component is FtsZ,
a tubulin-like GTPase, that like its eukaryotic relative forms protein filaments
in the presence of GTP. Since the discovery of the Z-ring 25 years ago, various
models for the role of FtsZ have been suggested. However, important information
about the architecture and dynamics of FtsZ filaments during cytokinesis is still
missing. One reason for this lack of knowledge has been the small size of bacteria,
which has made it difficult to resolve the orientation and dynamics of individual
FtsZ filaments in the Z-ring. While superresolution microscopy experiments have
helped to gain more information about the organization of the Z-ring in the dividing
cell, they were not yet able to elucidate a mechanism of how FtsZ filaments reorganize
during assembly and disassembly of the Z-ring. In this chapter, we explain how
to use an in vitro reconstitution approach to investigate the self-organization
of FtsZ filaments recruited to a biomimetic lipid bilayer by its membrane anchor
FtsA. We show how to perform single-molecule experiments to study the behavior
of individual FtsZ monomers during the constant reorganization of the FtsZ-FtsA
filament network. We describe how to analyze the dynamics of single molecules
and explain why this information can help to shed light onto possible mechanism
of Z-ring constriction. We believe that similar experimental approaches will be
useful to study the mechanism of membrane-based polymerization of other cytoskeletal
systems, not only from prokaryotic but also eukaryotic origin.
acknowledged_ssus:
- _id: Bio
acknowledgement: Natalia Baranova is supported by an EMBO Long-Term Fellowship (EMBO
ALTF 1163-2015) and Martin Loose by an ERC Starting Grant (ERCStG-2015-SelfOrganiCell).
alternative_title:
- Methods in Cell Biology
article_processing_charge: No
author:
- first_name: Natalia
full_name: Baranova, Natalia
id: 38661662-F248-11E8-B48F-1D18A9856A87
last_name: Baranova
orcid: 0000-0002-3086-9124
- first_name: Martin
full_name: Loose, Martin
id: 462D4284-F248-11E8-B48F-1D18A9856A87
last_name: Loose
orcid: 0000-0001-7309-9724
citation:
ama: 'Baranova NS, Loose M. Single-molecule measurements to study polymerization
dynamics of FtsZ-FtsA copolymers. In: Echard A, ed. Cytokinesis. Vol 137.
Academic Press; 2017:355-370. doi:10.1016/bs.mcb.2016.03.036'
apa: Baranova, N. S., & Loose, M. (2017). Single-molecule measurements to study
polymerization dynamics of FtsZ-FtsA copolymers. In A. Echard (Ed.), Cytokinesis
(Vol. 137, pp. 355–370). Academic Press. https://doi.org/10.1016/bs.mcb.2016.03.036
chicago: Baranova, Natalia S., and Martin Loose. “Single-Molecule Measurements to
Study Polymerization Dynamics of FtsZ-FtsA Copolymers.” In Cytokinesis,
edited by Arnaud Echard, 137:355–70. Academic Press, 2017. https://doi.org/10.1016/bs.mcb.2016.03.036.
ieee: N. S. Baranova and M. Loose, “Single-molecule measurements to study polymerization
dynamics of FtsZ-FtsA copolymers,” in Cytokinesis, vol. 137, A. Echard,
Ed. Academic Press, 2017, pp. 355–370.
ista: 'Baranova NS, Loose M. 2017.Single-molecule measurements to study polymerization
dynamics of FtsZ-FtsA copolymers. In: Cytokinesis. Methods in Cell Biology, vol.
137, 355–370.'
mla: Baranova, Natalia S., and Martin Loose. “Single-Molecule Measurements to Study
Polymerization Dynamics of FtsZ-FtsA Copolymers.” Cytokinesis, edited by
Arnaud Echard, vol. 137, Academic Press, 2017, pp. 355–70, doi:10.1016/bs.mcb.2016.03.036.
short: N.S. Baranova, M. Loose, in:, A. Echard (Ed.), Cytokinesis, Academic Press,
2017, pp. 355–370.
date_created: 2018-12-11T11:50:45Z
date_published: 2017-12-01T00:00:00Z
date_updated: 2023-09-20T11:16:30Z
day: '01'
department:
- _id: MaLo
doi: 10.1016/bs.mcb.2016.03.036
ec_funded: 1
editor:
- first_name: 'Arnaud '
full_name: 'Echard, Arnaud '
last_name: Echard
external_id:
isi:
- '000403542900022'
intvolume: ' 137'
isi: 1
language:
- iso: eng
month: '12'
oa_version: None
page: 355 - 370
project:
- _id: 2596EAB6-B435-11E9-9278-68D0E5697425
grant_number: ALTF 2015-1163
name: Synthesis of bacterial cell wall
- _id: 25681D80-B435-11E9-9278-68D0E5697425
call_identifier: FP7
grant_number: '291734'
name: International IST Postdoc Fellowship Programme
publication: Cytokinesis
publication_identifier:
issn:
- 0091679X
publication_status: published
publisher: Academic Press
publist_id: '6134'
quality_controlled: '1'
scopus_import: '1'
status: public
title: Single-molecule measurements to study polymerization dynamics of FtsZ-FtsA
copolymers
type: book_chapter
user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1
volume: 137
year: '2017'
...