Adherens junctions organize size-selective proteolytic hotspots critical for Notch signalling

Minsuk Kwak; Kaden M. Southard; Woon Ryoung Kim; Annie Lin; Nam Hyeong Kim; Ramu Gopalappa; Hyun Jung Lee; Minji An; Seo Hyun Choi; Yunmin Jung; Kunwoo Noh; Justin Farlow; Anastasios Georgakopoulos; Nikolaos K. Robakis; Min K. Kang; Matthew L. Kutys; Daeha Seo; Hyongbum Henry Kim; Yong Ho Kim; Jinwoo Cheon; Zev J. Gartner; Young wook Jun

doi:10.1038/s41556-022-01031-6

Adherens junctions organize size-selective proteolytic hotspots critical for Notch signalling

Minsuk Kwak, Kaden M. Southard, Woon Ryoung Kim, Annie Lin, Nam Hyeong Kim, Ramu Gopalappa, Hyun Jung Lee, Minji An, Seo Hyun Choi, Yunmin Jung, Kunwoo Noh, Justin Farlow, Anastasios Georgakopoulos, Nikolaos K. Robakis, Min K. Kang, Matthew L. Kutys, Daeha Seo, Hyongbum Henry Kim, Yong Ho Kim, Jinwoo CheonZev J. Gartner, Young wook Jun

Research output: Contribution to journal › Article › peer-review

1 Citation (Scopus)

Abstract

Adherens junctions (AJs) create spatially, chemically and mechanically discrete microdomains at cellular interfaces. Here, using a mechanogenetic platform that generates artificial AJs with controlled protein localization, clustering and mechanical loading, we find that AJs also organize proteolytic hotspots for γ-secretase with a spatially regulated substrate selectivity that is critical in the processing of Notch and other transmembrane proteins. Membrane microdomains outside of AJs exclusively organize Notch ligand–receptor engagement (LRE microdomains) to initiate receptor activation. Conversely, membrane microdomains within AJs exclusively serve to coordinate regulated intramembrane proteolysis (RIP microdomains). They do so by concentrating γ-secretase and primed receptors while excluding full-length Notch. AJs induce these functionally distinct microdomains by means of lipid-dependent γ-secretase recruitment and size-dependent protein segregation. By excluding full-length Notch from RIP microdomains, AJs prevent inappropriate enzyme–substrate interactions and suppress spurious Notch activation. Ligand-induced ectodomain shedding eliminates size-dependent segregation, releasing Notch to translocate into AJs for processing by γ-secretase. This mechanism directs radial differentiation of ventricular zone-neural progenitor cells in vivo and more broadly regulates the proteolysis of other large cell-surface receptors such as amyloid precursor protein. These findings suggest an unprecedented role of AJs in creating size-selective spatial switches that choreograph γ-secretase processing of multiple transmembrane proteins regulating development, homeostasis and disease.

Original language	English
Pages (from-to)	1739-1753
Number of pages	15
Journal	Nature Cell Biology
Volume	24
Issue number	12
DOIs	https://doi.org/10.1038/s41556-022-01031-6
Publication status	Published - 2022 Dec

Bibliographical note

Funding Information:
The authors thank S. Blacklow (Harvard U.), C. Miller (King’s College London) and K. Shimamura (Kumamoto U.) for the kind gifts of Notch, APP and DN-cadherin plasmids, respectively. We also thank A. Balmain, M. Moasser and E. Collison (UCSF) for sharing cell lines. D. Fletcher (UC Berkeley), A. Joffe (UC Berkeley) and D. Al-Rawi (Stanford U.) provided insightful discussion. For reagents, technical support, and discussions, we thank the Kim, Cheon, Gartner and Jun laboratories, as well as the Nikon Imaging Center and Wynton at UCSF. M.K. was supported by a Life Science Research Foundation fellowship as the Shurl and Kay Curci Foundation fellow, and by Burroughs Wellcome Travel Fund. This work was supported by NRF, NRF-2021R1F1A1063378 (M.K.), NRF-2018R1A5A1025511 (D.S.) and NRF-2017R1A2B3004198 (H.H.K.), HI17C0676 from Korean Ministry of Health and Welfare (H.H.K.), 5R01NS047229 from National Institute on Aging (NIA) and the National Institute of Health (NIH) (A.G.), 5R01AG008200 from National Institute on Aging (NIA) and the National Institute of Health (NIH) (N.K.R.), IBS-R026-D1 from IBS (M.K., H.H.K. and J.C.), NRF-2019R1A2C1085712 (Y.H.K.), the UCSF Center for Cellular Construction (an NSF Science and Technology Center, no. DBI-1548297) (Z.J.G.), U01CA244109 from the National Cancer Institute (Z.J.G.), 1R01GM112081, 1R01GM126542-01 and R35GM134948 from the National Institute of General Medical Science (NIGMS) and the NIH (Y.Jun), 1R21AG072232-01 from the National Institute on Aging (NIA) and the NIH (M.L.K. and Y.Jun), R00CA226366 from the National Cancer Institute (M.L.K) and NIH, and the UCSF Program for Breakthrough Biomedical Research (PBBR) funded in part by the Sandler Foundation (M.L.K and Y.Jun). Z.J.G. is a Chan Zuckerberg BioHub Investigator.

Funding Information:
The authors thank S. Blacklow (Harvard U.), C. Miller (King’s College London) and K. Shimamura (Kumamoto U.) for the kind gifts of Notch, APP and DN-cadherin plasmids, respectively. We also thank A. Balmain, M. Moasser and E. Collison (UCSF) for sharing cell lines. D. Fletcher (UC Berkeley), A. Joffe (UC Berkeley) and D. Al-Rawi (Stanford U.) provided insightful discussion. For reagents, technical support, and discussions, we thank the Kim, Cheon, Gartner and Jun laboratories, as well as the Nikon Imaging Center and Wynton at UCSF. M.K. was supported by a Life Science Research Foundation fellowship as the Shurl and Kay Curci Foundation fellow, and by Burroughs Wellcome Travel Fund. This work was supported by NRF, NRF-2021R1F1A1063378 (M.K.), NRF-2018R1A5A1025511 (D.S.) and NRF-2017R1A2B3004198 (H.H.K.), HI17C0676 from Korean Ministry of Health and Welfare (H.H.K.), 5R01NS047229 from National Institute on Aging (NIA) and the National Institute of Health (NIH) (A.G.), 5R01AG008200 from National Institute on Aging (NIA) and the National Institute of Health (NIH) (N.K.R.), IBS-R026-D1 from IBS (M.K., H.H.K. and J.C.), NRF-2019R1A2C1085712 (Y.H.K.), the UCSF Center for Cellular Construction (an NSF Science and Technology Center, no. DBI-1548297) (Z.J.G.), U01CA244109 from the National Cancer Institute (Z.J.G.), 1R01GM112081, 1R01GM126542-01 and R35GM134948 from the National Institute of General Medical Science (NIGMS) and the NIH (Y.Jun), 1R21AG072232-01 from the National Institute on Aging (NIA) and the NIH (M.L.K. and Y.Jun), R00CA226366 from the National Cancer Institute (M.L.K) and NIH, and the UCSF Program for Breakthrough Biomedical Research (PBBR) funded in part by the Sandler Foundation (M.L.K and Y.Jun). Z.J.G. is a Chan Zuckerberg BioHub Investigator.

Publisher Copyright:
© 2022, The Author(s), under exclusive licence to Springer Nature Limited.

All Science Journal Classification (ASJC) codes

Cell Biology

Access to Document

10.1038/s41556-022-01031-6

Cite this

Kwak, M., Southard, K. M., Kim, W. R., Lin, A., Kim, N. H., Gopalappa, R., Lee, H. J., An, M., Choi, S. H., Jung, Y., Noh, K., Farlow, J., Georgakopoulos, A., Robakis, N. K., Kang, M. K., Kutys, M. L., Seo, D., Kim, H. H., Kim, Y. H., ... Jun, Y. W. (2022). Adherens junctions organize size-selective proteolytic hotspots critical for Notch signalling. Nature Cell Biology, 24(12), 1739-1753. https://doi.org/10.1038/s41556-022-01031-6

@article{8579b49acce149398405c7b4b3bc7481,

title = "Adherens junctions organize size-selective proteolytic hotspots critical for Notch signalling",

abstract = "Adherens junctions (AJs) create spatially, chemically and mechanically discrete microdomains at cellular interfaces. Here, using a mechanogenetic platform that generates artificial AJs with controlled protein localization, clustering and mechanical loading, we find that AJs also organize proteolytic hotspots for γ-secretase with a spatially regulated substrate selectivity that is critical in the processing of Notch and other transmembrane proteins. Membrane microdomains outside of AJs exclusively organize Notch ligand–receptor engagement (LRE microdomains) to initiate receptor activation. Conversely, membrane microdomains within AJs exclusively serve to coordinate regulated intramembrane proteolysis (RIP microdomains). They do so by concentrating γ-secretase and primed receptors while excluding full-length Notch. AJs induce these functionally distinct microdomains by means of lipid-dependent γ-secretase recruitment and size-dependent protein segregation. By excluding full-length Notch from RIP microdomains, AJs prevent inappropriate enzyme–substrate interactions and suppress spurious Notch activation. Ligand-induced ectodomain shedding eliminates size-dependent segregation, releasing Notch to translocate into AJs for processing by γ-secretase. This mechanism directs radial differentiation of ventricular zone-neural progenitor cells in vivo and more broadly regulates the proteolysis of other large cell-surface receptors such as amyloid precursor protein. These findings suggest an unprecedented role of AJs in creating size-selective spatial switches that choreograph γ-secretase processing of multiple transmembrane proteins regulating development, homeostasis and disease.",

author = "Minsuk Kwak and Southard, {Kaden M.} and Kim, {Woon Ryoung} and Annie Lin and Kim, {Nam Hyeong} and Ramu Gopalappa and Lee, {Hyun Jung} and Minji An and Choi, {Seo Hyun} and Yunmin Jung and Kunwoo Noh and Justin Farlow and Anastasios Georgakopoulos and Robakis, {Nikolaos K.} and Kang, {Min K.} and Kutys, {Matthew L.} and Daeha Seo and Kim, {Hyongbum Henry} and Kim, {Yong Ho} and Jinwoo Cheon and Gartner, {Zev J.} and Jun, {Young wook}",

note = "Funding Information: The authors thank S. Blacklow (Harvard U.), C. Miller (King{\textquoteright}s College London) and K. Shimamura (Kumamoto U.) for the kind gifts of Notch, APP and DN-cadherin plasmids, respectively. We also thank A. Balmain, M. Moasser and E. Collison (UCSF) for sharing cell lines. D. Fletcher (UC Berkeley), A. Joffe (UC Berkeley) and D. Al-Rawi (Stanford U.) provided insightful discussion. For reagents, technical support, and discussions, we thank the Kim, Cheon, Gartner and Jun laboratories, as well as the Nikon Imaging Center and Wynton at UCSF. M.K. was supported by a Life Science Research Foundation fellowship as the Shurl and Kay Curci Foundation fellow, and by Burroughs Wellcome Travel Fund. This work was supported by NRF, NRF-2021R1F1A1063378 (M.K.), NRF-2018R1A5A1025511 (D.S.) and NRF-2017R1A2B3004198 (H.H.K.), HI17C0676 from Korean Ministry of Health and Welfare (H.H.K.), 5R01NS047229 from National Institute on Aging (NIA) and the National Institute of Health (NIH) (A.G.), 5R01AG008200 from National Institute on Aging (NIA) and the National Institute of Health (NIH) (N.K.R.), IBS-R026-D1 from IBS (M.K., H.H.K. and J.C.), NRF-2019R1A2C1085712 (Y.H.K.), the UCSF Center for Cellular Construction (an NSF Science and Technology Center, no. DBI-1548297) (Z.J.G.), U01CA244109 from the National Cancer Institute (Z.J.G.), 1R01GM112081, 1R01GM126542-01 and R35GM134948 from the National Institute of General Medical Science (NIGMS) and the NIH (Y.Jun), 1R21AG072232-01 from the National Institute on Aging (NIA) and the NIH (M.L.K. and Y.Jun), R00CA226366 from the National Cancer Institute (M.L.K) and NIH, and the UCSF Program for Breakthrough Biomedical Research (PBBR) funded in part by the Sandler Foundation (M.L.K and Y.Jun). Z.J.G. is a Chan Zuckerberg BioHub Investigator. Funding Information: The authors thank S. Blacklow (Harvard U.), C. Miller (King{\textquoteright}s College London) and K. Shimamura (Kumamoto U.) for the kind gifts of Notch, APP and DN-cadherin plasmids, respectively. We also thank A. Balmain, M. Moasser and E. Collison (UCSF) for sharing cell lines. D. Fletcher (UC Berkeley), A. Joffe (UC Berkeley) and D. Al-Rawi (Stanford U.) provided insightful discussion. For reagents, technical support, and discussions, we thank the Kim, Cheon, Gartner and Jun laboratories, as well as the Nikon Imaging Center and Wynton at UCSF. M.K. was supported by a Life Science Research Foundation fellowship as the Shurl and Kay Curci Foundation fellow, and by Burroughs Wellcome Travel Fund. This work was supported by NRF, NRF-2021R1F1A1063378 (M.K.), NRF-2018R1A5A1025511 (D.S.) and NRF-2017R1A2B3004198 (H.H.K.), HI17C0676 from Korean Ministry of Health and Welfare (H.H.K.), 5R01NS047229 from National Institute on Aging (NIA) and the National Institute of Health (NIH) (A.G.), 5R01AG008200 from National Institute on Aging (NIA) and the National Institute of Health (NIH) (N.K.R.), IBS-R026-D1 from IBS (M.K., H.H.K. and J.C.), NRF-2019R1A2C1085712 (Y.H.K.), the UCSF Center for Cellular Construction (an NSF Science and Technology Center, no. DBI-1548297) (Z.J.G.), U01CA244109 from the National Cancer Institute (Z.J.G.), 1R01GM112081, 1R01GM126542-01 and R35GM134948 from the National Institute of General Medical Science (NIGMS) and the NIH (Y.Jun), 1R21AG072232-01 from the National Institute on Aging (NIA) and the NIH (M.L.K. and Y.Jun), R00CA226366 from the National Cancer Institute (M.L.K) and NIH, and the UCSF Program for Breakthrough Biomedical Research (PBBR) funded in part by the Sandler Foundation (M.L.K and Y.Jun). Z.J.G. is a Chan Zuckerberg BioHub Investigator. Publisher Copyright: {\textcopyright} 2022, The Author(s), under exclusive licence to Springer Nature Limited.",

year = "2022",

month = dec,

doi = "10.1038/s41556-022-01031-6",

language = "English",

volume = "24",

pages = "1739--1753",

journal = "Nature Cell Biology",

issn = "1465-7392",

publisher = "Nature Publishing Group",

number = "12",

}

Kwak, M, Southard, KM, Kim, WR, Lin, A, Kim, NH, Gopalappa, R, Lee, HJ, An, M, Choi, SH, Jung, Y, Noh, K, Farlow, J, Georgakopoulos, A, Robakis, NK, Kang, MK, Kutys, ML, Seo, D, Kim, HH, Kim, YH, Cheon, J, Gartner, ZJ & Jun, YW 2022, 'Adherens junctions organize size-selective proteolytic hotspots critical for Notch signalling', Nature Cell Biology, vol. 24, no. 12, pp. 1739-1753. https://doi.org/10.1038/s41556-022-01031-6

TY - JOUR

T1 - Adherens junctions organize size-selective proteolytic hotspots critical for Notch signalling

AU - Kwak, Minsuk

AU - Southard, Kaden M.

AU - Kim, Woon Ryoung

AU - Lin, Annie

AU - Kim, Nam Hyeong

AU - Gopalappa, Ramu

AU - Lee, Hyun Jung

AU - An, Minji

AU - Choi, Seo Hyun

AU - Jung, Yunmin

AU - Noh, Kunwoo

AU - Farlow, Justin

AU - Georgakopoulos, Anastasios

AU - Robakis, Nikolaos K.

AU - Kang, Min K.

AU - Kutys, Matthew L.

AU - Seo, Daeha

AU - Kim, Hyongbum Henry

AU - Kim, Yong Ho

AU - Cheon, Jinwoo

AU - Gartner, Zev J.

AU - Jun, Young wook

N1 - Funding Information: The authors thank S. Blacklow (Harvard U.), C. Miller (King’s College London) and K. Shimamura (Kumamoto U.) for the kind gifts of Notch, APP and DN-cadherin plasmids, respectively. We also thank A. Balmain, M. Moasser and E. Collison (UCSF) for sharing cell lines. D. Fletcher (UC Berkeley), A. Joffe (UC Berkeley) and D. Al-Rawi (Stanford U.) provided insightful discussion. For reagents, technical support, and discussions, we thank the Kim, Cheon, Gartner and Jun laboratories, as well as the Nikon Imaging Center and Wynton at UCSF. M.K. was supported by a Life Science Research Foundation fellowship as the Shurl and Kay Curci Foundation fellow, and by Burroughs Wellcome Travel Fund. This work was supported by NRF, NRF-2021R1F1A1063378 (M.K.), NRF-2018R1A5A1025511 (D.S.) and NRF-2017R1A2B3004198 (H.H.K.), HI17C0676 from Korean Ministry of Health and Welfare (H.H.K.), 5R01NS047229 from National Institute on Aging (NIA) and the National Institute of Health (NIH) (A.G.), 5R01AG008200 from National Institute on Aging (NIA) and the National Institute of Health (NIH) (N.K.R.), IBS-R026-D1 from IBS (M.K., H.H.K. and J.C.), NRF-2019R1A2C1085712 (Y.H.K.), the UCSF Center for Cellular Construction (an NSF Science and Technology Center, no. DBI-1548297) (Z.J.G.), U01CA244109 from the National Cancer Institute (Z.J.G.), 1R01GM112081, 1R01GM126542-01 and R35GM134948 from the National Institute of General Medical Science (NIGMS) and the NIH (Y.Jun), 1R21AG072232-01 from the National Institute on Aging (NIA) and the NIH (M.L.K. and Y.Jun), R00CA226366 from the National Cancer Institute (M.L.K) and NIH, and the UCSF Program for Breakthrough Biomedical Research (PBBR) funded in part by the Sandler Foundation (M.L.K and Y.Jun). Z.J.G. is a Chan Zuckerberg BioHub Investigator. Funding Information: The authors thank S. Blacklow (Harvard U.), C. Miller (King’s College London) and K. Shimamura (Kumamoto U.) for the kind gifts of Notch, APP and DN-cadherin plasmids, respectively. We also thank A. Balmain, M. Moasser and E. Collison (UCSF) for sharing cell lines. D. Fletcher (UC Berkeley), A. Joffe (UC Berkeley) and D. Al-Rawi (Stanford U.) provided insightful discussion. For reagents, technical support, and discussions, we thank the Kim, Cheon, Gartner and Jun laboratories, as well as the Nikon Imaging Center and Wynton at UCSF. M.K. was supported by a Life Science Research Foundation fellowship as the Shurl and Kay Curci Foundation fellow, and by Burroughs Wellcome Travel Fund. This work was supported by NRF, NRF-2021R1F1A1063378 (M.K.), NRF-2018R1A5A1025511 (D.S.) and NRF-2017R1A2B3004198 (H.H.K.), HI17C0676 from Korean Ministry of Health and Welfare (H.H.K.), 5R01NS047229 from National Institute on Aging (NIA) and the National Institute of Health (NIH) (A.G.), 5R01AG008200 from National Institute on Aging (NIA) and the National Institute of Health (NIH) (N.K.R.), IBS-R026-D1 from IBS (M.K., H.H.K. and J.C.), NRF-2019R1A2C1085712 (Y.H.K.), the UCSF Center for Cellular Construction (an NSF Science and Technology Center, no. DBI-1548297) (Z.J.G.), U01CA244109 from the National Cancer Institute (Z.J.G.), 1R01GM112081, 1R01GM126542-01 and R35GM134948 from the National Institute of General Medical Science (NIGMS) and the NIH (Y.Jun), 1R21AG072232-01 from the National Institute on Aging (NIA) and the NIH (M.L.K. and Y.Jun), R00CA226366 from the National Cancer Institute (M.L.K) and NIH, and the UCSF Program for Breakthrough Biomedical Research (PBBR) funded in part by the Sandler Foundation (M.L.K and Y.Jun). Z.J.G. is a Chan Zuckerberg BioHub Investigator. Publisher Copyright: © 2022, The Author(s), under exclusive licence to Springer Nature Limited.

PY - 2022/12

Y1 - 2022/12

N2 - Adherens junctions (AJs) create spatially, chemically and mechanically discrete microdomains at cellular interfaces. Here, using a mechanogenetic platform that generates artificial AJs with controlled protein localization, clustering and mechanical loading, we find that AJs also organize proteolytic hotspots for γ-secretase with a spatially regulated substrate selectivity that is critical in the processing of Notch and other transmembrane proteins. Membrane microdomains outside of AJs exclusively organize Notch ligand–receptor engagement (LRE microdomains) to initiate receptor activation. Conversely, membrane microdomains within AJs exclusively serve to coordinate regulated intramembrane proteolysis (RIP microdomains). They do so by concentrating γ-secretase and primed receptors while excluding full-length Notch. AJs induce these functionally distinct microdomains by means of lipid-dependent γ-secretase recruitment and size-dependent protein segregation. By excluding full-length Notch from RIP microdomains, AJs prevent inappropriate enzyme–substrate interactions and suppress spurious Notch activation. Ligand-induced ectodomain shedding eliminates size-dependent segregation, releasing Notch to translocate into AJs for processing by γ-secretase. This mechanism directs radial differentiation of ventricular zone-neural progenitor cells in vivo and more broadly regulates the proteolysis of other large cell-surface receptors such as amyloid precursor protein. These findings suggest an unprecedented role of AJs in creating size-selective spatial switches that choreograph γ-secretase processing of multiple transmembrane proteins regulating development, homeostasis and disease.

AB - Adherens junctions (AJs) create spatially, chemically and mechanically discrete microdomains at cellular interfaces. Here, using a mechanogenetic platform that generates artificial AJs with controlled protein localization, clustering and mechanical loading, we find that AJs also organize proteolytic hotspots for γ-secretase with a spatially regulated substrate selectivity that is critical in the processing of Notch and other transmembrane proteins. Membrane microdomains outside of AJs exclusively organize Notch ligand–receptor engagement (LRE microdomains) to initiate receptor activation. Conversely, membrane microdomains within AJs exclusively serve to coordinate regulated intramembrane proteolysis (RIP microdomains). They do so by concentrating γ-secretase and primed receptors while excluding full-length Notch. AJs induce these functionally distinct microdomains by means of lipid-dependent γ-secretase recruitment and size-dependent protein segregation. By excluding full-length Notch from RIP microdomains, AJs prevent inappropriate enzyme–substrate interactions and suppress spurious Notch activation. Ligand-induced ectodomain shedding eliminates size-dependent segregation, releasing Notch to translocate into AJs for processing by γ-secretase. This mechanism directs radial differentiation of ventricular zone-neural progenitor cells in vivo and more broadly regulates the proteolysis of other large cell-surface receptors such as amyloid precursor protein. These findings suggest an unprecedented role of AJs in creating size-selective spatial switches that choreograph γ-secretase processing of multiple transmembrane proteins regulating development, homeostasis and disease.

UR - http://www.scopus.com/inward/record.url?scp=85143270468&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85143270468&partnerID=8YFLogxK

U2 - 10.1038/s41556-022-01031-6

DO - 10.1038/s41556-022-01031-6

M3 - Article

C2 - 36456828

AN - SCOPUS:85143270468

SN - 1465-7392

VL - 24

SP - 1739

EP - 1753

JO - Nature Cell Biology

JF - Nature Cell Biology

IS - 12

ER -

Adherens junctions organize size-selective proteolytic hotspots critical for Notch signalling

Abstract

Bibliographical note

All Science Journal Classification (ASJC) codes

Access to Document

Other files and links

Fingerprint

Cite this