---
OA_place: publisher
OA_type: hybrid
_id: '22925'
abstract:
- lang: eng
  text: Hybridization Chain Reaction (HCR) enables highly sensitive and multiplexed
    detection of mRNA with subcellular spatial resolution. It employs fluorophore-tagged
    DNA hairpins that self-assemble on target-bound probe pairs, amplifying the signal
    without enzymatic reactions. Here, we describe a protocol for performing HCR fluorescence
    in situ hybridization (FISH) on Xenopus tissue sections. We also outline a semi-automated
    image analysis pipeline that enables per-cell quantification of probe expression
    and co-localization. This method provides a robust and quantitative approach for
    visualizing gene expression patterns in situ.
acknowledgement: "This work was supported by a Horizon Europe ERC Starting Grant\r\nNumber
  101041551 (L.B.S., D.V., S.P.), Special Research Program\r\n(SFB) of the Austrian
  Science Fund (FWF) F7814-B (L.B.S., S.P.),\r\nAustrian Science Fund (FWF) 10.55776/COE16
  (L.B.S.), FTI\r\nStrategy Lower Austria Dissertation Grant Number FT121-D-046\r\n(D.V.),
  and Austrian Academy of Sciences DOC Fellowship 27229\r\n(S.P.). We also thank the
  Imaging & Optics and Scientific Computing Facilities at ISTA for their support in
  developing the quantification pipeline."
alternative_title:
- Methods in Molecular Biology
article_processing_charge: No
author:
- first_name: David
  full_name: Vijatovic, David
  id: cf391e77-ec3c-11ea-a124-d69323410b58
  last_name: Vijatovic
  orcid: 0000-0002-5494-0941
- first_name: Stavros
  full_name: Papadopoulos, Stavros
  id: 40606b92-f128-11eb-9611-bf66a98cfa5c
  last_name: Papadopoulos
- first_name: Marco
  full_name: Dalla Vecchia, Marco
  id: 02a7a869-ff06-11ed-a87f-86649d6077e5
  last_name: Dalla Vecchia
- first_name: Lora Beatrice Jaeger
  full_name: Sweeney, Lora Beatrice Jaeger
  id: 56BE8254-C4F0-11E9-8E45-0B23E6697425
  last_name: Sweeney
  orcid: 0000-0001-9242-5601
citation:
  ama: 'Vijatovic D, Papadopoulos S, Dalla Vecchia M, Sweeney LB. Fluorescent in situ
    mRNA hybridization (FISH) using Hybridization Chain Reaction (HCR) in Xenopus
    cryosections. In: Beck CW, ed. <i>Xenopus</i>. Vol 3049. MIMB. Springer; 2026:245-259.
    doi:<a href="https://doi.org/10.1007/978-1-0716-5360-9_11">10.1007/978-1-0716-5360-9_11</a>'
  apa: Vijatovic, D., Papadopoulos, S., Dalla Vecchia, M., &#38; Sweeney, L. B. (2026).
    Fluorescent in situ mRNA hybridization (FISH) using Hybridization Chain Reaction
    (HCR) in Xenopus cryosections. In C. W. Beck (Ed.), <i>Xenopus</i> (Vol. 3049,
    pp. 245–259). Springer. <a href="https://doi.org/10.1007/978-1-0716-5360-9_11">https://doi.org/10.1007/978-1-0716-5360-9_11</a>
  chicago: Vijatovic, David, Stavros Papadopoulos, Marco Dalla Vecchia, and Lora B.
    Sweeney. “Fluorescent in Situ MRNA Hybridization (FISH) Using Hybridization Chain
    Reaction (HCR) in Xenopus Cryosections.” In <i>Xenopus</i>, edited by Caroline
    W. Beck, 3049:245–59. MIMB. Springer, 2026. <a href="https://doi.org/10.1007/978-1-0716-5360-9_11">https://doi.org/10.1007/978-1-0716-5360-9_11</a>.
  ieee: D. Vijatovic, S. Papadopoulos, M. Dalla Vecchia, and L. B. Sweeney, “Fluorescent
    in situ mRNA hybridization (FISH) using Hybridization Chain Reaction (HCR) in
    Xenopus cryosections,” in <i>Xenopus</i>, vol. 3049, C. W. Beck, Ed. Springer,
    2026, pp. 245–259.
  ista: 'Vijatovic D, Papadopoulos S, Dalla Vecchia M, Sweeney LB. 2026.Fluorescent
    in situ mRNA hybridization (FISH) using Hybridization Chain Reaction (HCR) in
    Xenopus cryosections. In: Xenopus. Methods in Molecular Biology, vol. 3049, 245–259.'
  mla: Vijatovic, David, et al. “Fluorescent in Situ MRNA Hybridization (FISH) Using
    Hybridization Chain Reaction (HCR) in Xenopus Cryosections.” <i>Xenopus</i>, edited
    by Caroline W. Beck, vol. 3049, Springer, 2026, pp. 245–59, doi:<a href="https://doi.org/10.1007/978-1-0716-5360-9_11">10.1007/978-1-0716-5360-9_11</a>.
  short: D. Vijatovic, S. Papadopoulos, M. Dalla Vecchia, L.B. Sweeney, in:, C.W.
    Beck (Ed.), Xenopus, Springer, 2026, pp. 245–259.
corr_author: '1'
das_tickbox: '0'
date_created: 2026-09-13T22:01:55Z
date_published: 2026-09-02T00:00:00Z
date_updated: 2026-09-17T07:31:16Z
day: '02'
ddc:
- '570'
department:
- _id: LoSw
- _id: GradSch
- _id: IAS
doi: 10.1007/978-1-0716-5360-9_11
editor:
- first_name: Caroline W.
  full_name: Beck, Caroline W.
  last_name: Beck
external_id:
  pmid:
  - '42681228'
file:
- access_level: open_access
  checksum: e7a075a9ee1b91d5824f8f2b9b1f3c9b
  content_type: application/pdf
  creator: dernst
  date_created: 2026-09-17T07:28:53Z
  date_updated: 2026-09-17T07:28:53Z
  file_id: '22938'
  file_name: 2026_MIMB_Vijatovic.pdf
  file_size: 1004024
  relation: main_file
  success: 1
file_date_updated: 2026-09-17T07:28:53Z
fulldoi: https://doi.org/10.1007/978-1-0716-5360-9_11
has_accepted_license: '1'
intvolume: '      3049'
keyword:
- mRNA FISH
- Hybridization chain reaction
- In situ hybridization
- Fluorescence microscopy
language:
- iso: eng
month: '09'
oa: 1
oa_version: Published Version
page: 245-259
pmid: 1
project:
- _id: ebb66355-77a9-11ec-83b8-b8ac210a4dae
  grant_number: '101041551'
  name: Development and Evolution of Tetrapod Motor Circuits
- _id: 8da85f50-16d5-11f0-9cad-eab8b0ff6c9e
  grant_number: F7814
  name: 'Stem Cell Modulation in Neural Development and Regeneration/ P14-Swim-to-limb
    transition: cell type to connection diversity'
- _id: cf428362-b037-11f1-b015-8277a8a2f63d
  grant_number: COE16
  name: Neuronal circuits in health and disease (Sweeney)
- _id: bd73af52-d553-11ed-ba76-912049f0ac7a
  grant_number: FTI21-D-046
  name: Development of V1 interneuron diversity during swim-to-walk transition of
    Xenopus metamorphosis
- _id: 907b765e-16d5-11f0-9cad-fef108a945b1
  grant_number: '27229'
  name: 'A Tale of Two Circuits: Rostrocaudal spinal cord patterning during the swim-to-limb
    transition of Xenopus metamorphosis'
publication: Xenopus
publication_identifier:
  eissn:
  - 1940-6029
publication_status: published
publisher: Springer
quality_controlled: '1'
researchdata_availability: no
scopus_import: '1'
series_title: MIMB
status: public
supplementarymaterial: no
title: Fluorescent in situ mRNA hybridization (FISH) using Hybridization Chain Reaction
  (HCR) in Xenopus cryosections
tmp:
  image: /images/cc_by_nc_nd.png
  legal_code_url: https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode
  name: Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
    (CC BY-NC-ND 4.0)
  short: CC BY-NC-ND (4.0)
type: book_chapter
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 3049
year: '2026'
...
---
OA_place: publisher
_id: '14510'
abstract:
- lang: eng
  text: "Clathrin-mediated endocytosis (CME) is vital for the regulation of plant
    growth and\r\ndevelopment by controlling plasma membrane protein composition and
    cargo uptake. CME\r\nrelies on the precise recruitment control of protein regulators
    for vesicle maturation and\r\nrelease. During the early stages of endocytosis,
    an area of flat membrane is remodelled by\r\nproteins to create a spherical vesicle
    against intracellular forces. After the Clathrin-coated\r\nvesicle (CCV) is fully
    formed, scission machinery releases it from the plasma membrane,\r\nand cargo
    proceeds for recycling or degradation through early endosomes / Trans Golgi\r\nnetwork.
    Protein machineries that mediate membrane bending and vesicle release in plants\r\nare
    unknown. However, studies show, that plant endocytosis is actin independent, thus\r\nindicating
    that plants utilize a unique mechanism to mediate membrane bending against highturgor
    pressure compared to other model systems. First, by using biochemical and advanced\r\nlive
    microscopy approaches we investigate the TPLATE complex, a plant-specific\r\nendocytosis
    protein complex. We found that TPLATE is peripherally associated with\r\nclathrin-coated
    vesicles and localises at the rim of endocytosis events. Next, our study of\r\nplant
    Dynamin-related protein 1C (DRP1C), which was hypothesised previously to play
    a\r\nrole in vesicle release, shows the recruitment of the protein already at
    the early stages of\r\nendocytosis. Moreover, DRP1C assembles into organised ring-like
    structures and is able to\r\ninduce membrane deformation and tubulation, suggesting
    its role also in membrane bending\r\nduring early CME. Based on the data from
    mammalian and yeast systems, plant DynaminRelated Proteins 2 and SH3P2 protein
    are strong candidates to be part of the plant vesicle\r\nscission machinery; however,
    their precise role in plant CME has not been yet elucidated.\r\nHere, we characterised
    DRP2s and SH3P2 roles in CME by combining high-resolution\r\nimaging of endocytic
    events in vivo and protein characterisation. Although DRP2s and\r\nSH3P2 arrive
    together during late CME and physically interact, genetic analysis using\r\n∆sh3p1,2,3
    mutant and complementation with non-DRP2-interacting SH3P2 variants suggest\r\nthat
    SH3P2 does not directly recruit DRP2s to the site of endocytosis. Summarising
    our\r\nresearch, these observations provide new important insights into the mechanism
    of plant\r\nCME and show that, despite plants posses many homologues of mammalian
    and yeast CME\r\ncomponents, they do not necessarily act in the same manner. "
acknowledged_ssus:
- _id: EM-Fac
- _id: Bio
- _id: LifeSc
alternative_title:
- ISTA Thesis
article_processing_charge: No
author:
- first_name: Nataliia
  full_name: Gnyliukh, Nataliia
  id: 390C1120-F248-11E8-B48F-1D18A9856A87
  last_name: Gnyliukh
  orcid: 0000-0002-2198-0509
citation:
  ama: Gnyliukh N. Mechanism of clathrin-coated vesicle  formation during endocytosis
    in plants. 2023. doi:<a href="https://doi.org/10.15479/at:ista:14510">10.15479/at:ista:14510</a>
  apa: Gnyliukh, N. (2023). <i>Mechanism of clathrin-coated vesicle  formation during
    endocytosis in plants</i>. Institute of Science and Technology Austria. <a href="https://doi.org/10.15479/at:ista:14510">https://doi.org/10.15479/at:ista:14510</a>
  chicago: Gnyliukh, Nataliia. “Mechanism of Clathrin-Coated Vesicle  Formation during
    Endocytosis in Plants.” Institute of Science and Technology Austria, 2023. <a
    href="https://doi.org/10.15479/at:ista:14510">https://doi.org/10.15479/at:ista:14510</a>.
  ieee: N. Gnyliukh, “Mechanism of clathrin-coated vesicle  formation during endocytosis
    in plants,” Institute of Science and Technology Austria, 2023.
  ista: Gnyliukh N. 2023. Mechanism of clathrin-coated vesicle  formation during endocytosis
    in plants. Institute of Science and Technology Austria.
  mla: Gnyliukh, Nataliia. <i>Mechanism of Clathrin-Coated Vesicle  Formation during
    Endocytosis in Plants</i>. Institute of Science and Technology Austria, 2023,
    doi:<a href="https://doi.org/10.15479/at:ista:14510">10.15479/at:ista:14510</a>.
  short: N. Gnyliukh, Mechanism of Clathrin-Coated Vesicle  Formation during Endocytosis
    in Plants, Institute of Science and Technology Austria, 2023.
corr_author: '1'
date_created: 2023-11-10T09:10:06Z
date_published: 2023-11-10T00:00:00Z
date_updated: 2026-10-10T22:30:52Z
day: '10'
ddc:
- '570'
degree_awarded: PhD
department:
- _id: GradSch
- _id: JiFr
- _id: MaLo
doi: 10.15479/at:ista:14510
ec_funded: 1
file:
- access_level: closed
  checksum: 3d5e680bfc61f98e308c434f45cc9bd6
  content_type: application/vnd.openxmlformats-officedocument.wordprocessingml.document
  creator: ngnyliuk
  date_created: 2023-11-20T09:18:51Z
  date_updated: 2024-11-23T23:30:38Z
  embargo_to: open_access
  file_id: '14567'
  file_name: Thesis_Gnyliukh_final_08_11_23.docx
  file_size: 20824903
  relation: source_file
- access_level: open_access
  checksum: bfc96d47fc4e7e857dd71656097214a4
  content_type: application/pdf
  creator: ngnyliuk
  date_created: 2023-11-20T09:23:11Z
  date_updated: 2024-11-23T23:30:38Z
  embargo: 2024-11-23
  file_id: '14568'
  file_name: Thesis_Gnyliukh_final_20_11_23.pdf
  file_size: 24871844
  relation: main_file
file_date_updated: 2024-11-23T23:30:38Z
fulldoi: https://doi.org/10.15479/at:ista:14510
has_accepted_license: '1'
keyword:
- Clathrin-Mediated Endocytosis
- vesicle scission
- Dynamin-Related Protein 2
- SH3P2
- TPLATE complex
- Total internal reflection fluorescence microscopy
- Arabidopsis thaliana
language:
- iso: eng
license: https://creativecommons.org/licenses/by/4.0/
month: '11'
oa: 1
oa_version: Published Version
page: '180'
project:
- _id: 2564DBCA-B435-11E9-9278-68D0E5697425
  call_identifier: H2020
  grant_number: '665385'
  name: International IST Doctoral Program
publication_identifier:
  isbn:
  - 978-3-99078-037-4
  issn:
  - 2663-337X
publication_status: published
publisher: Institute of Science and Technology Austria
related_material:
  record:
  - id: '14591'
    relation: part_of_dissertation
    status: public
  - id: '9887'
    relation: part_of_dissertation
    status: public
  - id: '8139'
    relation: part_of_dissertation
    status: public
status: public
supervisor:
- first_name: Jiří
  full_name: Friml, Jiří
  id: 4159519E-F248-11E8-B48F-1D18A9856A87
  last_name: Friml
  orcid: 0000-0002-8302-7596
- first_name: Martin
  full_name: Loose, Martin
  id: 462D4284-F248-11E8-B48F-1D18A9856A87
  last_name: Loose
  orcid: 0000-0001-7309-9724
title: Mechanism of clathrin-coated vesicle  formation during endocytosis in plants
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: dissertation
user_id: ba8df636-2132-11f1-aed0-ed93e2281fdd
year: '2023'
...