The type IV collagen triple helix, composed of three ⍺-chains, is a core basement membrane (BM) component that assembles into a network within BMs. Endogenous tagging of all ⍺-chains with genetically encoded fluorophores has remained elusive, limiting our understanding of this crucial BM component. Through genome editing, we show that the C termini of the C. elegans type IV collagen ⍺-chains EMB-9 and LET-2 can be fused to a variety of fluorophores to create a strain toolkit with wild-type health. Using quantitative imaging, our results suggest a preference for LET-2-LET-2-EMB-9 trimer construction, but also tissue-specific flexibility in trimers assembled driven by differences in ⍺-chain expression levels. By tagging emb-9 and let-2 mutants that model human Gould syndrome, a complex multitissue disorder, we further discover defects in extracellular accumulation and turnover that might help explain disease pathology. Together, our findings identify a permissive tagging site in C. elegans that will allow diverse studies on type IV collagen regulation and function in animals.
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March 18 2025
A collagen IV fluorophore knock-in toolkit reveals trimer diversity in C. elegans basement membranes
Sandhya Srinivasan
,
Sandhya Srinivasan
*
(Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Validation, Visualization, Writing - original draft, Writing - review & editing)
1Department of Biology,
Duke University
, Durham, NC, USA
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William Ramos-Lewis
,
William Ramos-Lewis
*
(Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing)
1Department of Biology,
Duke University
, Durham, NC, USA
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Mychel R.P.T. Morais
,
Mychel R.P.T. Morais
(Formal analysis, Investigation, Visualization, Writing - review & editing)
2Division of Cell-Matrix Biology and Regenerative Medicine,
Wellcome Centre for Cell-Matrix Research, School of Biological Sciences, Faculty of Biology Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester
, Manchester, UK
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Qiuyi Chi
,
Qiuyi Chi
(Investigation, Methodology)
1Department of Biology,
Duke University
, Durham, NC, USA
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Adam W.J. Soh
,
Adam W.J. Soh
(Investigation)
1Department of Biology,
Duke University
, Durham, NC, USA
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Emily Williams
,
Emily Williams
(Investigation, Writing - review & editing)
2Division of Cell-Matrix Biology and Regenerative Medicine,
Wellcome Centre for Cell-Matrix Research, School of Biological Sciences, Faculty of Biology Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester
, Manchester, UK
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Rachel Lennon
,
Rachel Lennon
(Resources, Supervision, Writing - original draft, Writing - review & editing)
2Division of Cell-Matrix Biology and Regenerative Medicine,
Wellcome Centre for Cell-Matrix Research, School of Biological Sciences, Faculty of Biology Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester
, Manchester, UK
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David R. Sherwood
(Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Writing - original draft, Writing - review & editing)
1Department of Biology,
Duke University
, Durham, NC, USA
Correspondence to David R. Sherwood: [email protected]
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Sandhya Srinivasan
Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Validation, Visualization, Writing - original draft, Writing - review & editing
*
1Department of Biology,
Duke University
, Durham, NC, USA
William Ramos-Lewis
Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing
*
1Department of Biology,
Duke University
, Durham, NC, USA
Mychel R.P.T. Morais
Formal analysis, Investigation, Visualization, Writing - review & editing
2Division of Cell-Matrix Biology and Regenerative Medicine,
Wellcome Centre for Cell-Matrix Research, School of Biological Sciences, Faculty of Biology Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester
, Manchester, UK
Qiuyi Chi
Investigation, Methodology
1Department of Biology,
Duke University
, Durham, NC, USA
Adam W.J. Soh
Investigation
1Department of Biology,
Duke University
, Durham, NC, USA
Emily Williams
Investigation, Writing - review & editing
2Division of Cell-Matrix Biology and Regenerative Medicine,
Wellcome Centre for Cell-Matrix Research, School of Biological Sciences, Faculty of Biology Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester
, Manchester, UK
Rachel Lennon
Resources, Supervision, Writing - original draft, Writing - review & editing
2Division of Cell-Matrix Biology and Regenerative Medicine,
Wellcome Centre for Cell-Matrix Research, School of Biological Sciences, Faculty of Biology Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester
, Manchester, UK
Correspondence to David R. Sherwood: [email protected]
*
S. Srinivasan and W. Ramos-Lewis are joint first author.
Disclosures: The authors declare no competing interests exist.
Received:
December 14 2024
Revision Received:
February 20 2025
Accepted:
February 28 2025
Online ISSN: 1540-8140
Print ISSN: 0021-9525
Funding
Funder(s):
National Institutes of Health
- Award Id(s): P40 OD010440,R35GM118049,R21OD032430,F32AG077907
Funder(s):
Wellcome Trust
- Award Id(s): 227417/Z/23/Z,226804/Z/22/Z,301803/Z/23/Z
Funder(s):
Jane Coffin Childs Memorial Fund
Funder(s):
Kidney Research UK
Funder(s):
Stoneygate Trust
Funder(s):
National Institute for Health Research
© 2025 Srinivasan et al.
2025
Srinivasan et al.
This article is distributed under the terms as described at https://rupress.org/pages/terms102024/.
J Cell Biol (2025) 224 (6): e202412118.
Article history
Received:
December 14 2024
Revision Received:
February 20 2025
Accepted:
February 28 2025
Citation
Sandhya Srinivasan, William Ramos-Lewis, Mychel R.P.T. Morais, Qiuyi Chi, Adam W.J. Soh, Emily Williams, Rachel Lennon, David R. Sherwood; A collagen IV fluorophore knock-in toolkit reveals trimer diversity in C. elegans basement membranes. J Cell Biol 2 June 2025; 224 (6): e202412118. doi: https://doi.org/10.1083/jcb.202412118
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