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KANK2 at focal adhesions regulates their maintenance and dynamics, while at fibrillar adhesions it influences cell migration via microtubule-dependent mechanism

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  • Published: 03 March 2026
  • article number , (2026)
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Cell Communication and Signaling Aims and scope Submit manuscript
KANK2 at focal adhesions regulates their maintenance and dynamics, while at fibrillar adhesions it influences cell migration via microtubule-dependent mechanism
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  • Nikolina Stojanović1 na1,
  • Anja Rac1 na1,
  • Marija Lončarić1,
  • Ana Tadijan1,2,
  • Mladen Paradžik1,3,
  • Marta Acman1,
  • Jonathan D. Humphries4,
  • Martin J. Humphries5 &
  • …
  • Andreja Ambriović-Ristov1 

  • 204 Accesses

  • Explore all metrics

We are providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply.

Abstract

Background

Integrins form focal adhesions (FAs) at the cell edge and fibrillar adhesions (FBs) located centrally. Talin1 is essential to FAs, while talin2 is found in FAs and FBs. KANK (kidney ankyrin repeat-containing) family proteins regulate adhesion dynamics and are recruited to adhesions through interaction with talins. Previously, we showed in MDA-MB-435S melanoma cells that KANK2 is part of integrin αVβ5 FAs, that its interaction with talin2 regulates actin-microtubule (MT) crosstalk and that KANK2 knockdown mimics the effect of integrin αV or β5 knockdown by reducing cell migration. Here, in another melanoma cell line RPMI-7951 we observed that KANK2 is part of FAs and FBs and that KANK2 knockdown increases cell migration. Therefore, we analyse integrin adhesion complexes in RPMI-7951 cells, explore the localization and role of KANK2 in FAs and FBs.

Methods

Knockdown in human melanoma RPMI-7951 cells was achieved by transfection with gene-specific siRNAs. Integrin adhesion complexes were isolated and analysed by mass spectrometry. Immunofluorescence analysis, live cell imaging and the proximity ligation assays were done using confocal microscopy. Cell migration was assessed using Transwell Cell Culture Inserts. MTT assays were performed to determine cell sensitivity to paclitaxel. Data were statistically evaluated using one-way or two-way ANOVA or unpaired Student’s t-tests in GraphPad Software.

Results

We demonstrate that RPMI-7951 melanoma cells use integrin αVβ5 FAs and integrin α5β1 FBs for adhesion, and that KANK2 is part of both structures. KANK2 is predominantly in proximity to talin1 at the cell edge (FAs) and in proximity to talin2 in the cell center (FBs). KANK2 in FAs functionally interacts with talin1 to maintain FAs, and with talin2 to regulate their dynamics. KANK2 is a component of FBs, and its knockdown mimics integrin α5 knockdown by increasing MT-dependent cell migration.

Conclusions

Our study reveals the distinct roles of KANK2 in FAs and FBs. We show that KANK2 is a component of FBs, linking them to MTs and promoting their stabilisation. Loss of integrin α5 or KANK2 from FBs increases cell migration, a process that relies on the MT cytoskeleton.

Plain English summary

Cells adhere to their surroundings using specialized structures called adhesions, which are formed by different types of integrins and have different cell localisations: focal adhesions at the cell edge and fibrillar adhesions in the cell center. Talin1 is essential for focal adhesions, while talin2 is present in focal and fibrillar adhesions. KANK (kidney ankyrin repeat-containing) family proteins regulate adhesion dynamics by connecting talins to the microtubule cytoskeleton. Our study explored the different roles of KANK2 in focal or fibrillar adhesions, by focusing on its interactions with integrins and talins. We show that in RPMI-7951 cells, which form αVβ5 focal and α5β1 fibrillar adhesions, KANK2 is present in both of these adhesions. KANK2 supports αVβ5 focal adhesion maintenance via talin1 and regulates their dynamics via talin2. KANK2 is also a component of α5β1 fibrillar adhesions, and its depletion mimics integrin α5 knockdown and enhances cell migration, a process dependent on the microtubule cytoskeleton. In conclusion, our study demonstrates that KANK2 plays distinct roles in different adhesion structures, contributing to both focal and fibrillar adhesion function.

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  • Integrin-Mediated Cell Adhesion Mechanisms

Data availability

The datasets supporting the conclusions of this article are available in the ProteomeXchange Consortium via the PRIDE partner repository, dataset identifier PXD064756, [https://www.ebi.ac.uk/pride/archive/](https:/www.ebi.ac.uk/pride/archive) . Other raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

Abbreviations

ECM:

Extracellular matrix

IAC:

Integrin adhesion complex

FA:

Focal adhesion

FB:

Fibrillar adhesion

RA:

Reticular adhesion

MT:

Microtubule

CMSC:

Cortical microtubule stabilizing complex

KANK:

KN motif and ankyrin repeat domain containing protein

ABS2:

F-actin binding site 2

DMEM:

Dulbecco's Modified Eagle Medium

FBS:

Fetal Bovine Serum

ITGAV:

Integrin Subunit αV

ITGA5:

Integrin Subunit α5

ITGB5:

Integrin Subunit β5

HEPES:

Hydroxyethylpiperazine ethanesulfonic acid

DTBP:

Dimethyl 3,3’-dithiobispropionimidate, Wang and Richard’s reagent

MS :

Mass spectrometry

STRING:

Search Tool for the Retrieval of Interacting Genes/Proteins

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide

PTX:

Paclitaxel

DMSO:

Dimethyl Sulfoxide

PBS:

Phosphate-Buffered Saline

IRM:

Interference reflection microscopy

IF:

Immunofluorescence analysis

PLA:

Proximity ligation assay

WB:

Western blotting

SD :

Standard Deviation

ANOVA:

One-way analysis of variance

RhoA GEF ARHGEF2 / GEF-H1:

RhoA Guanine Nucleotide Exchange Factor

PPI network:

Protein–Protein Interaction Network

HSP proteins:

Heat Shock Proteins

GO:

Gene Ontology

DAVID:

Database for Annotation, Visualization and Integrated Discovery

REVIGO:

Reduce and Visualize Gene Ontology

SLIT2:

Slit Homolog 2 Protein

PXDN:

Peroxidasin Homolog

TNC:

Tenascin C

TGFBI:

Transforming Growth Factor-β-Induced Protein IG-H3

PDLIM7:

PDZ and LIM Domain Protein 7

PDLIM5:

PDZ and LIM Domain Protein 5

MYH9/MYH10:

Non-Muscle Myosin Heavy Chain 9 / 10

ZYX:

Zyxin

EZR:

Ezrin

MSN:

Moesin

TNS3:

Tensin 3

TPM3:

Tropomyosin 3, Isoform 1

PALLD:

Palladin

CALD1:

Caldesmon 1

ACTB:

Beta-Actin

IQGAP1:

Ras GTPase-Activating-Like Protein 1

ILK:

Integrin-Linked Kinase

CLIC1:

Chloride Intracellular Channel 1

MACF1:

Microtubule-Actin Cross-Linking Factor 1

ELKS:

Protein Rich in Glutamate (E), Leucine (L), Lysine (K), and Serine (S)

CLASP:

CLIP-Associated Protein

KIF21A:

Kinesin Family Member 21A

LL5β PHLDB2:

(Pleckstrin Homology Like Domain Family B Member 2), commonly referred to as LL5β

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Acknowledgements

We thank Marina Šutalo of the Laboratory for Cell Biology and Signalling for technical assistance.

Funding

This work was supported by the Croatian Science Foundation Project (Grant No IP-2019-04-1577 to AA-R), a Cancer Research UK Programme Grant to MJH (DRCRPG-100002) and an Academy of Medical Science Springboard award to JDH (SBF008\1094).

Author information

Author notes

  1. Nikolina Stojanović and Anja Rac contributed equally to this work.

Authors and Affiliations

  1. Laboratory for Cell Biology and Signalling, Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia

    Nikolina Stojanović, Anja Rac, Marija Lončarić, Ana Tadijan, Mladen Paradžik, Marta Acman & Andreja Ambriović-Ristov

  2. Laboratory for Cell Biology, Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia

    Ana Tadijan

  3. Laboratory of Experimental Therapy, Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia

    Mladen Paradžik

  4. Department of Life Science, Manchester Metropolitan University, Manchester, UK

    Jonathan D. Humphries

  5. Manchester Cell-Matrix Centre, Faculty of Biology, Medicine & Health, University of Manchester, Manchester, UK

    Martin J. Humphries

Authors
  1. Nikolina Stojanović
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Contributions

Material preparation, data collection and analysis were performed by [NS], [AR], [ML], [AT], [MP], [MA], and [AA-R]. [NS] and [AR] contributed equally to the experimental work. The first draft of the manuscript was written by [NS] and [AA-R], corrections were made by [AR], [ML], [JDH] and [MJH]. All authors read and approved the final manuscript. [NS] and [AA-R] equally contributed to the study conception and design.

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Correspondence to Nikolina Stojanović or Andreja Ambriović-Ristov.

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Supplementary Information

Supplementary Material 1. Supplementary Table S1. (download PDF )

Supplementary Material 2. Supplementary Fig S1-S8. (download PDF )

Supplementary Material 3. Supplementary Fig S9-S13. (download PDF )

Supplementary Material 4. Supplementary Table S2. (download XLS )

Supplementary Material 5. Movie S1.

Supplementary Material 6. Movie S2.

Supplementary Material 7. Movie S3.

Supplementary Material 8. Movie S4.

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Stojanović, N., Rac, A., Lončarić, M. et al. KANK2 at focal adhesions regulates their maintenance and dynamics, while at fibrillar adhesions it influences cell migration via microtubule-dependent mechanism. Cell Commun Signal (2026). https://doi.org/10.1186/s12964-026-02771-w

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  • Received: 28 November 2025

  • Accepted: 25 February 2026

  • Published: 03 March 2026

  • DOI: https://doi.org/10.1186/s12964-026-02771-w

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Keywords

  • Focal Adhesion
  • Fibrillar Adhesion
  • Talin1
  • Talin2
  • KANK2
  • Migration
Supplementary Material 5. Movie S1.Supplementary Material 6. Movie S2.Supplementary Material 7. Movie S3.Supplementary Material 8. Movie S4.

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