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Article ; Online: Multiplexed Proximity Biotinylation Coupled to Mass Spectrometry for Defining Integrin Adhesion Complexes.

Chastney, Megan R / Lawless, Craig / Humphries, Martin J

2020 Volume 88, Issue 1, Page(s) e113

Abstract: BioID, a proximity biotinylation technique, offers a valuable approach to examine the interactions occurring within protein complexes that complements traditional protein biochemical methods. BioID has various advantages that are beneficial to the study ... ...

Abstract	BioID, a proximity biotinylation technique, offers a valuable approach to examine the interactions occurring within protein complexes that complements traditional protein biochemical methods. BioID has various advantages that are beneficial to the study of complexes, including an ability to detect insoluble and transient proteins. We have applied BioID to the study of integrin adhesion complexes (IACs), which are located at the junction between the plasma membrane and actin cytoskeleton. The use of multiple BioID baits enables a complex-wide, spatial annotation of IACs, which in turn facilitates the detection of novel proximal interactors and provides insights into IAC architecture. This article describes the labeling and affinity purification of IAC-proximal proteins and their analysis by label-free quantitative mass spectrometry. The article also outlines steps to identify high-confidence proximity interactors, and to interrogate the topology and functional relevance of proximity interaction networks through bioinformatic analyses. © 2020 The Authors. Basic Protocol 1: Proximity biotinylation of integrin adhesion complex components Basic Protocol 2: Mass spectrometry data processing by MaxQuant and detection of high-confidence proximal interactors Basic Protocol 3: Bioinformatic analysis and data visualization.
MeSH term(s)	Biotinylation/methods ; Cell Membrane/metabolism ; Chromatography, Affinity/methods ; Humans ; Integrins/metabolism ; Mass Spectrometry/methods ; Protein Interaction Mapping/methods
Chemical Substances	Integrins
Language	English
Publishing date	2020-08-27
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2179048-6
ISSN	1934-2616 ; 1934-2500
ISSN (online)	1934-2616
ISSN	1934-2500
DOI	10.1002/cpcb.113
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Article ; Online: Talin2 and KANK2 functionally interact to regulate microtubule dynamics, paclitaxel sensitivity and cell migration in the MDA-MB-435S melanoma cell line.

Lončarić, Marija / Stojanović, Nikolina / Rac-Justament, Anja / Coopmans, Kaatje / Majhen, Dragomira / Humphries, Jonathan D / Humphries, Martin J / Ambriović-Ristov, Andreja

Cellular & molecular biology letters

2023 Volume 28, Issue 1, Page(s) 56

Abstract: Background: Focal adhesions (FAs) are integrin-containing, multi-protein structures that link intracellular actin to the extracellular matrix and trigger multiple signaling pathways that control cell proliferation, differentiation, survival and motility. ...

Abstract	Background: Focal adhesions (FAs) are integrin-containing, multi-protein structures that link intracellular actin to the extracellular matrix and trigger multiple signaling pathways that control cell proliferation, differentiation, survival and motility. Microtubules (MTs) are stabilized in the vicinity of FAs through interaction with the components of the cortical microtubule stabilizing complex (CMSC). KANK (KN motif and ankyrin repeat domains) family proteins within the CMSC, KANK1 or KANK2, bind talin within FAs and thus mediate actin-MT crosstalk. We previously identified in MDA-MB-435S cells, which preferentially use integrin αVβ5 for adhesion, KANK2 as a key molecule enabling the actin-MT crosstalk. KANK2 knockdown also resulted in increased sensitivity to MT poisons, paclitaxel (PTX) and vincristine and reduced migration. Here, we aimed to analyze whether KANK1 has a similar role and to distinguish which talin isoform binds KANK2. Methods: The cell model consisted of human melanoma cell line MDA-MB-435S and stably transfected clone with decreased expression of integrin αV (3αV). For transient knockdown of talin1, talin2, KANK1 or KANK2 we used gene-specific siRNAs transfection. Using previously standardized protocol we isolated integrin adhesion complexes. SDS-PAGE and Western blot was used for protein expression analysis. The immunofluorescence analysis and live cell imaging was done using confocal microscopy. Cell migration was analyzed with Transwell Cell Culture Inserts. Statistical analysis using GraphPad Software consisted of either one-way analysis of variance (ANOVA), unpaired Student's t-test or two-way ANOVA analysis. Results: We show that KANK1 is not a part of the CMSC associated with integrin αVβ5 FAs and its knockdown did not affect the velocity of MT growth or cell sensitivity to PTX. The talin2 knockdown mimicked KANK2 knockdown i.e. led to the perturbation of actin-MT crosstalk, which is indicated by the increased velocity of MT growth and increased sensitivity to PTX and also reduced migration. Conclusion: We conclude that KANK2 functionally interacts with talin2 and that the mechanism of increased sensitivity to PTX involves changes in microtubule dynamics. These data elucidate a cell-type-specific role of talin2 and KANK2 isoforms and we propose that talin2 and KANK2 are therefore potential therapeutic targets for improved cancer therapy.
MeSH term(s)	Humans ; Actins/metabolism ; Adaptor Proteins, Signal Transducing/genetics ; Adaptor Proteins, Signal Transducing/metabolism ; Cell Movement ; Cytoskeletal Proteins/genetics ; Integrins/metabolism ; Melanoma ; Microtubules/metabolism ; Paclitaxel/pharmacology ; Protein Isoforms/metabolism ; Talin/genetics ; Talin/chemistry ; Talin/metabolism ; Cell Line, Tumor/metabolism
Chemical Substances	Actins ; Adaptor Proteins, Signal Transducing ; Cytoskeletal Proteins ; Integrins ; Paclitaxel (P88XT4IS4D) ; Protein Isoforms ; Talin ; Kank2 protein, human ; TLN2 protein, human
Language	English
Publishing date	2023-07-17
Publishing country	England
Document type	Journal Article
ZDB-ID	2108724-6
ISSN	1689-1392 ; 1689-1392
ISSN (online)	1689-1392
ISSN	1689-1392
DOI	10.1186/s11658-023-00473-6
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Article ; Online: Connections between the cell cycle, cell adhesion and the cytoskeleton.

Jones, Matthew C / Zha, Junzhe / Humphries, Martin J

Philosophical transactions of the Royal Society of London. Series B, Biological sciences

2019 Volume 374, Issue 1779, Page(s) 20180227

Abstract: Cell division, the purpose of which is to enable cell replication, and in particular to distribute complete, accurate copies of genetic material to daughter cells, is essential for the propagation of life. At a morphological level, division not only ... ...

Abstract	Cell division, the purpose of which is to enable cell replication, and in particular to distribute complete, accurate copies of genetic material to daughter cells, is essential for the propagation of life. At a morphological level, division not only necessitates duplication of cellular structures, but it also relies on polar segregation of this material followed by physical scission of the parent cell. For these fundamental changes in cell shape and positioning to be achieved, mechanisms are required to link the cell cycle to the modulation of cytoarchitecture. Outside of mitosis, the three main cytoskeletal networks not only endow cells with a physical cytoplasmic skeleton, but they also provide a mechanism for spatio-temporal sensing via integrin-associated adhesion complexes and site-directed delivery of cargoes. During mitosis, some interphase functions are retained, but the architecture of the cytoskeleton changes dramatically, and there is a need to generate a mitotic spindle for chromosome segregation. An economical solution is to re-use existing cytoskeletal molecules: transcellular actin stress fibres remodel to create a rigid cortex and a cytokinetic furrow, while unipolar radial microtubules become the primary components of the bipolar spindle. This remodelling implies the existence of specific mechanisms that link the cell-cycle machinery to the control of adhesion and the cytoskeleton. In this article, we review the intimate three-way connection between microenvironmental sensing, adhesion signalling and cell proliferation, particularly in the contexts of normal growth control and aberrant tumour progression. As the morphological changes that occur during mitosis are ancient, the mechanisms linking the cell cycle to the cytoskeleton/adhesion signalling network are likely to be primordial in nature and we discuss recent advances that have elucidated elements of this link. A particular focus is the connection between CDK1 and cell adhesion. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.
MeSH term(s)	Cell Adhesion/physiology ; Cell Cycle ; Cell Proliferation/physiology ; Cytoskeleton/physiology ; Humans ; Neoplasms/metabolism ; Signal Transduction/physiology ; Tumor Microenvironment/physiology
Language	English
Publishing date	2019-07-01
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	208382-6
ISSN	1471-2970 ; 0080-4622 ; 0264-3839 ; 0962-8436
ISSN (online)	1471-2970
ISSN	0080-4622 ; 0264-3839 ; 0962-8436
DOI	10.1098/rstb.2018.0227
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Article: KANK family proteins in cancer

Tadijan, Ana / Samaržija, Ivana / Humphries, Jonathan D / Humphries, Martin J / Ambriović-Ristov, Andreja

international journal of biochemistry & cell biology. 2021 Feb., v. 131

2021

Abstract: The Kank (kidney or KN motif and ankyrin repeat domain-containing) family of proteins has been described as essential for crosstalk between actin and microtubules. Kank1, 2, 3 and 4 arose by gene duplication and diversification and share conserved ... ...

Abstract	The Kank (kidney or KN motif and ankyrin repeat domain-containing) family of proteins has been described as essential for crosstalk between actin and microtubules. Kank1, 2, 3 and 4 arose by gene duplication and diversification and share conserved structural domains. KANK proteins are localised mainly to the plasma membrane in focal adhesions, indirectly affecting RhoA and Rac1 thus regulating actin cytoskeleton. In addition, Kank proteins are part of the cortical microtubule stabilisation complex regulating microtubules. Most of the data have been collected for Kank1 protein whose expression promotes apoptosis and cell-cycle arrest while Kank3 was identified as hypoxia-inducible proapoptotic target of p53. A discrepancy in Kanks role in regulation of cell migration and sensitivity to antitumour drugs has been observed in different cell models. Since expression of Kank1 and 3 correlate positively with tumour progression and patient outcome, at least in some tumour types, they are candidates for tumour suppressors.
Keywords	actin ; apoptosis ; cell cycle checkpoints ; cell movement ; gene duplication ; kidneys ; microfilaments ; microtubules ; neoplasm progression ; neoplasms ; patients ; plasma membrane
Language	English
Dates of publication	2021-02
Publishing place	Elsevier Ltd
Document type	Article
Note	NAL-AP-2-clean
ZDB-ID	1228429-4
ISSN	1878-5875 ; 1357-2725
ISSN (online)	1878-5875
ISSN	1357-2725
DOI	10.1016/j.biocel.2020.105903
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: The βI domain promotes active β1 integrin clustering into mature adhesion sites.

Mana, Giulia / Valdembri, Donatella / Askari, Janet A / Li, Zhenhai / Caswell, Patrick / Zhu, Cheng / Humphries, Martin J / Ballestrem, Christoph / Serini, Guido

Life science alliance

2022 Volume 6, Issue 2

Abstract: Modulation of integrin function is required in many physiological and pathological settings, such as angiogenesis and cancer. Integrin allosteric changes, clustering, and trafficking cooperate to regulate cell adhesion and motility on extracellular ... ...

Abstract	Modulation of integrin function is required in many physiological and pathological settings, such as angiogenesis and cancer. Integrin allosteric changes, clustering, and trafficking cooperate to regulate cell adhesion and motility on extracellular matrix proteins via mechanisms that are partly defined. By exploiting four monoclonal antibodies recognizing distinct conformational epitopes, we show that in endothelial cells (ECs), the extracellular βI domain, but not the hybrid or I-EGF2 domain of active β1 integrins, promotes their FAK-regulated clustering into tensin 1-containing fibrillar adhesions and impairs their endocytosis. In this regard, the βI domain-dependent clustering of active β1 integrins is necessary to favor fibronectin-elicited directional EC motility, which cannot be effectively promoted by β1 integrin conformational activation alone.
MeSH term(s)	Integrin beta1/metabolism ; Endothelial Cells/metabolism ; Cell Adhesion/physiology ; Integrins ; Cluster Analysis
Chemical Substances	Integrin beta1 ; Integrins
Language	English
Publishing date	2022-11-21
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ISSN	2575-1077
ISSN (online)	2575-1077
DOI	10.26508/lsa.202201388
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Article: Cell adhesion assays.

Humphries, Martin J

Methods in molecular biology (Clifton, N.J.)

2009 Volume 522, Page(s) 203–210

Abstract: This chapter will outline in detail the two standard assays used in the author's laboratory for quantitating the adhesion of cells to an immobilized substrate. The attachment assay, which employs a colorimetric detection of bound cells, is based on Kueng ...

Abstract	This chapter will outline in detail the two standard assays used in the author's laboratory for quantitating the adhesion of cells to an immobilized substrate. The attachment assay, which employs a colorimetric detection of bound cells, is based on Kueng et al. (Anal Biochem 182:16-19, 1989), and the spreading assay, which employs phase contrast microscopy to measure the flattening of adherent cells, is based on the method of Yamada and Kennedy (J Cell Biol 99:29-36, 1984).It is important to realize that cell adhesion is a complex process that involves many different molecular interactions, including receptor-ligand binding, changes in the fluxes through intracellular signaling pathways, and modulation of cytoskeletal assembly. Consequently, adhesion assays not only measure the contacts between a cell and extracellular adhesion proteins, but also provide information about other cellular events. For this reason, care needs to be taken before choosing to perform adhesion assays. The most common uses of adhesion assays are (a) to test the ability of a specific type of cell or cell line to adhere to a specific adhesive substrate, and (b) to test the sensitivity of a specific cell-substrate interaction to inhibitors, but it is also apparent that adhesion assays can be used to probe the contribution of other cellular processes.
MeSH term(s)	Cell Adhesion ; Cells, Cultured
Language	English
Publishing date	2009
Publishing country	United States
Document type	Journal Article
ISSN	1064-3745
ISSN	1064-3745
DOI	10.1007/978-1-59745-413-1_14
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Article ; Online: Author Correction: Systemically administered wound-homing peptide accelerates wound healing by modulating syndecan-4 function.

Maldonado, Horacio / Savage, Bryan D / Barker, Harlan R / May, Ulrike / Vähätupa, Maria / Badiani, Rahul K / Wolanska, Katarzyna I / Turner, Craig M J / Pemmari, Toini / Ketomäki, Tuomo / Prince, Stuart / Humphries, Martin J / Ruoslahti, Erkki / Morgan, Mark R / Järvinen, Tero A H

Nature communications

2024 Volume 15, Issue 1, Page(s) 234

Language	English
Publishing date	2024-01-03
Publishing country	England
Document type	Published Erratum
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-023-44574-4
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Article ; Online: KANK family proteins in cancer.

Tadijan, Ana / Samaržija, Ivana / Humphries, Jonathan D / Humphries, Martin J / Ambriović-Ristov, Andreja

The international journal of biochemistry & cell biology

2020 Volume 131, Page(s) 105903

Abstract	The Kank (kidney or KN motif and ankyrin repeat domain-containing) family of proteins has been described as essential for crosstalk between actin and microtubules. Kank1, 2, 3 and 4 arose by gene duplication and diversification and share conserved structural domains. KANK proteins are localised mainly to the plasma membrane in focal adhesions, indirectly affecting RhoA and Rac1 thus regulating actin cytoskeleton. In addition, Kank proteins are part of the cortical microtubule stabilisation complex regulating microtubules. Most of the data have been collected for Kank1 protein whose expression promotes apoptosis and cell-cycle arrest while Kank3 was identified as hypoxia-inducible proapoptotic target of p53. A discrepancy in Kanks role in regulation of cell migration and sensitivity to antitumour drugs has been observed in different cell models. Since expression of Kank1 and 3 correlate positively with tumour progression and patient outcome, at least in some tumour types, they are candidates for tumour suppressors.
MeSH term(s)	Actin Cytoskeleton/drug effects ; Actin Cytoskeleton/metabolism ; Actin Cytoskeleton/ultrastructure ; Adaptor Proteins, Signal Transducing/chemistry ; Adaptor Proteins, Signal Transducing/genetics ; Adaptor Proteins, Signal Transducing/metabolism ; Antineoplastic Agents/therapeutic use ; Apoptosis/drug effects ; Carrier Proteins/chemistry ; Carrier Proteins/genetics ; Carrier Proteins/metabolism ; Cell Line, Tumor ; Cell Movement/drug effects ; Cell Proliferation/drug effects ; Cytoskeletal Proteins/chemistry ; Cytoskeletal Proteins/genetics ; Cytoskeletal Proteins/metabolism ; Focal Adhesions/drug effects ; Focal Adhesions/metabolism ; Focal Adhesions/pathology ; Gene Expression Regulation, Neoplastic ; Humans ; Microtubules/drug effects ; Microtubules/metabolism ; Microtubules/ultrastructure ; Neoplasms/drug therapy ; Neoplasms/genetics ; Neoplasms/metabolism ; Neoplasms/pathology ; Paclitaxel/therapeutic use ; Protein Domains ; Signal Transduction ; Treatment Outcome ; Vincristine/therapeutic use
Chemical Substances	Adaptor Proteins, Signal Transducing ; Antineoplastic Agents ; Carrier Proteins ; Cytoskeletal Proteins ; KANK1 protein, human ; KANK3 protein, human ; Vincristine (5J49Q6B70F) ; Paclitaxel (P88XT4IS4D)
Language	English
Publishing date	2020-12-10
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	1228429-4
ISSN	1878-5875 ; 1357-2725
ISSN (online)	1878-5875
ISSN	1357-2725
DOI	10.1016/j.biocel.2020.105903
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Article: Global proteomic analysis of insulin receptor interactors in glomerular podocytes.

Hosawi, Salman B / Humphries, Jonathan D / Coward, Richard J / Knight, David / Humphries, Martin J / Lennon, Rachel

Wellcome open research

2020 Volume 5, Page(s) 202

Abstract: Background: ...

Abstract	Background:
Language	English
Publishing date	2020-08-26
Publishing country	England
Document type	Journal Article
ISSN	2398-502X
ISSN	2398-502X
DOI	10.12688/wellcomeopenres.16072.1
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Article ; Online: Connexin 37 sequestering of activated-ERK in the cytoplasm promotes p27-mediated endothelial cell cycle arrest.

Acharya, Bipul R / Fang, Jennifer S / Jeffery, Erin D / Chavkin, Nicholas W / Genet, Gael / Vasavada, Hema / Nelson, Elizabeth A / Sheynkman, Gloria M / Humphries, Martin J / Hirschi, Karen K

Life science alliance

2023 Volume 6, Issue 8

Abstract: Connexin37-mediated regulation of cell cycle modulators and, consequently, growth arrest lack mechanistic understanding. We previously showed that arterial shear stress up-regulates Cx37 in endothelial cells and activates a Notch/Cx37/p27 signaling axis ... ...

Abstract	Connexin37-mediated regulation of cell cycle modulators and, consequently, growth arrest lack mechanistic understanding. We previously showed that arterial shear stress up-regulates Cx37 in endothelial cells and activates a Notch/Cx37/p27 signaling axis to promote G1 cell cycle arrest, and this is required to enable arterial gene expression. However, how induced expression of a gap junction protein, Cx37, up-regulates cyclin-dependent kinase inhibitor p27 to enable endothelial growth suppression and arterial specification is unclear. Herein, we fill this knowledge gap by expressing wild-type and regulatory domain mutants of Cx37 in cultured endothelial cells expressing the Fucci cell cycle reporter. We determined that both the channel-forming and cytoplasmic tail domains of Cx37 are required for p27 up-regulation and late G1 arrest. Mechanistically, the cytoplasmic tail domain of Cx37 interacts with, and sequesters, activated ERK in the cytoplasm. This then stabilizes pERK nuclear target Foxo3a, which up-regulates p27 transcription. Consistent with previous studies, we found this Cx37/pERK/Foxo3a/p27 signaling axis functions downstream of arterial shear stress to promote endothelial late G1 state and enable up-regulation of arterial genes.
MeSH term(s)	Endothelial Cells/metabolism ; Cell Cycle Checkpoints/genetics ; Connexins/genetics ; Connexins/metabolism ; G1 Phase Cell Cycle Checkpoints ; Cell Nucleus/metabolism ; Gap Junction alpha-4 Protein
Chemical Substances	Connexins
Language	English
Publishing date	2023-05-17
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
ISSN	2575-1077
ISSN (online)	2575-1077
DOI	10.26508/lsa.202201685
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