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Keratin intermediate filaments mechanically position melanin pigments for genome photoprotection

Nature Cell Biology volume 28, pages 98–112 (2026) Cite this article

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Abstract

Melanin pigments block genotoxic agents by positioning on the sun-exposed side of the nucleus in human skin keratinocytes. How this positioning is regulated and its role in genome photoprotection remain unknown. Here, by developing a model of human keratinocytes internalizing extracellular melanin into pigment organelles, we show that keratin 5 and keratin 14 intermediate filaments and microtubules control the three-dimensional perinuclear position of pigments, shielding DNA from photodamage. Imaging and microrheology in a human-disease-related model identify structural keratin cages surrounding pigment organelles to stiffen their microenvironment and maintain their three-dimensional position. Optimum supranuclear spatialization of pigment organelles is required for DNA photoprotection and relies on intermediate filaments and microtubules bridged by plectin cytolinkers. Thus, the mechanically driven proximity of pigment organelles to the nucleus is a key photoprotective parameter. Uncovering how human skin counteracts solar radiation by positioning the melanin microparasol next to the genome anticipates that dynamic spatialization of organelles is a physiological response to ultraviolet stress.

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Fig. 1: MCs are the secreted luminal melanin content of intracellular melanosomes.
The alternative text for this image may have been generated using AI.
Fig. 2: Internalized MCs are perinuclearly positioned in lysosome-like organelles.
The alternative text for this image may have been generated using AI.
Fig. 3: MC+ organelles are surrounded by KRT5 IFs in cage-like structures.
The alternative text for this image may have been generated using AI.
Fig. 4: 3D positioning of MC+ organelles relies on KRT5 IFs and the MT network.
The alternative text for this image may have been generated using AI.
Fig. 5: The KRT5DDD mutation decreases the stiffness of the cytosolic microenvironment.
The alternative text for this image may have been generated using AI.
Fig. 6: DNA photoprotection relies on MC+ organelles and their perinuclear 3D positioning.
The alternative text for this image may have been generated using AI.

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Data availability

The KRT5 c.2 T > C mutation was published and characterized previously86 (accession code rs267607444; dbSNP). All unique and stable reagents generated in this study are available from the corresponding author upon receipt of a completed materials transfer agreement. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

Code availability

The original codes used for pigment detection and analysis, as well as DNA damage quantification, are available from GitHub (https://github.com/Anne-SophieMACE/PigmentDetectionAndQuantification) and archived at https://doi.org/10.5281/zenodo.17347181 (ref. 97). The original code used for the 3D detection and quantification of γH2AX nuclear foci is available from GitHub (https://github.com/laurasalavessa/3D-particles-nucleus.git) and archived at https://doi.org/10.5281/zenodo.17371442 (ref. 98).

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Acknowledgements

This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Curie, Centre National de la Recherche Scientifique (CNRS), Agence Nationale de la Recherche projects MYOACTIONS (ANR-17-CE11-0029-03 to A. Houdusse and C. Delevoye), CILIOPHAGING (ANR-22-CE14-0019-01 to E.M.) and MOBIDIC (ANR-23-CE14-0041-02 to C. Delevoye and E.M.), Fondation ARC pour la Recherche Sur le Cancer (ARCPJA22020060002267 to C. Delevoye), Fondation pour la Recherche Médicale (FRM Team label EQU201903007827 to G.R., SPF201909009097 to J.S.-C. and FDT202001010801 to S.B.-M.), National Institutes of Health (grant R01 EY015625 to M. S. Marks and G.R.), Biomolecular Analyses for Tailored Medicine in AcneiNversa (BATMAN) project (funded by ERA PerMed (JTC_2018) to M.B.) and Ligue Nationale Contre le Cancer and Worldwide Research Cancer (to S.E.-M.). This work was also supported by the French National Research Agency through the Investments for the Future programme (ANR-10-INSB-04; France-BioImaging) and we acknowledge the PICT-IBiSA, a member of the France-BioImaging national research infrastructure supported by the CelTisPhyBio Labex (ANR-10-LBX-0038), part of the IDEX PSL (ANR-10-IDEX-0001-02 PSL), and the Structure Fédérative de Recherche Necker Necker technical imaging platform. N.L. received funding from Université Paris-Saclay through a Contrat Doctoral Spécifique pour Normaliens from Ecole Normale Supérieure Paris-Saclay. M. Rouabah received funding from Université Paris Cité through Contrat Doctoral. S.B.-M. received funding from the European Union’s Horizon 2020 Research and Innovation programme under Marie Sklodowska–Curie grant 666003 (Institut Curie 3-I PhD Program, Marie Sklodowska–Curie Actions). This publication reflects only the authors’ views and the European Research Executive Agency is not responsible for any use that may be made of the information it contains. We thank all members of the laboratories, as well as A. Alcover and N. Sauvonnet (Institut Pasteur, INSERM U1221 and the Biomaterials and Microfluidics platform), F. Niedergang (Institut Cochin, INSERM U1016) and F. Perez (Institut Curie, CNRS UMR144) for insightful discussions during the project; C. Kikuti (Institut Curie, CNRS UMR144) for discussions during the analysis of MC-enriched fractions; H. Moreiras and D. C. Barral (NOVA Medical School, Faculdade de Ciências Médicas, Universidade NOVA de Lisboa) for sharing initial protocols for MC isolation; F. Rajabi (Gustave Roussy Cancer Center, INSERM U981) for sharing the anti-CPD antibody; E. Rubinstein (Sorbonne Université, INSERM, CNRS, CIMI-Paris) for sharing the anti-CD63 antibody; R. Basto (Institut Curie, CNRS UMR144) for sharing the anti-α-tubulin antibody; R. Leube and N. Schwarz (Institute of Molecular and Cellular Anatomy, RWTH Aachen University) for sharing the KRT5–mCh plasmid; and B. Boëda for technical advice during KRT5MITO–mCh plasmid generation.

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Author notes

  1. These authors contributed equally: Silvia Benito-Martínez, Laura Salavessa.

Authors and Affiliations

  1. Institut Curie, PSL University, Sorbonne Université, CNRS UMR144, Cell Biology and Cancer, Structure and Membrane Compartments, Paris, France

    Silvia Benito-Martínez, Laura Salavessa, Julia Sirés-Campos, Riddhi Atul Jani, Maryse Romao, Ilse Hurbain, Graça Raposo & Cédric Delevoye

  2. Université Paris Cité, INSERM UMR-S1151, CNRS UMR-S8253, Institut Necker Enfants Malades, Paris, France

    Laura Salavessa, Myckaëla Rouabah, Etienne Morel & Cédric Delevoye

  3. Institut Curie, PSL University, Sorbonne Université, CNRS UMR144, Cell Biology and Cancer, Cell and Tissue Imaging Facility (PICT-IBiSA), Paris, France

    Anne-Sophie Macé, Vincent Fraisier, Maryse Romao, Ilse Hurbain, Graça Raposo & Cédric Delevoye

  4. Institut Curie, PSL University, Sorbonne Université, CNRS UMR144, Cell Biology and Cancer, Molecular Mechanisms of Intracellular Transport, Paris, France

    Nathan Lardier & Jean-Baptiste Manneville

  5. Cell Polarity, Migration and Cancer Unit, Équipe Labellisée Ligue Contre le Cancer 2023, Institut Pasteur, UMR3691 CNRS, Université Paris, Paris, France

    Vanessa Roca & Sandrine Etienne-Manneville

  6. L’Oréal Research and Innovation, Aulnay-sous-Bois, France

    Charlène Gayrard, Marion Plessis, Françoise Bernerd & Christine Duval

  7. Université Paris-Est Créteil, INSERM, IMRB, Translational Neuropsychiatry, Créteil, France

    Cécile Nait-Meddour & Michele Boniotto

  8. Laboratoire Matière et Systèmes Complexes (MSC), Université Paris Cité, CNRS, UMR7057, Paris, France

    Jean-Baptiste Manneville

Authors
  1. Silvia Benito-Martínez
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  2. Laura Salavessa
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  3. Anne-Sophie Macé
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  4. Myckaëla Rouabah
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  5. Nathan Lardier
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  6. Vincent Fraisier
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  7. Julia Sirés-Campos
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  8. Riddhi Atul Jani
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  9. Maryse Romao
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  10. Vanessa Roca
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  11. Charlène Gayrard
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  12. Marion Plessis
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  13. Ilse Hurbain
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  14. Cécile Nait-Meddour
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  15. Sandrine Etienne-Manneville
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  16. Etienne Morel
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  17. Michele Boniotto
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  18. Jean-Baptiste Manneville
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  19. Françoise Bernerd
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  20. Christine Duval
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  21. Graça Raposo
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  22. Cédric Delevoye
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Contributions

S.B.-M., L.S. and C. Delevoye conceived of the study. S.E.-M., E.M., M.B., J.-B.M., G.R. and C. Delevoye contributed to funding the project. S.B.-M., L.S., M. Rouabah, N.L., C.G., M.P., C.N.-M., S.E.-M., E.M., M.B., J.-B.M., C. Duval, F.B., G.R. and C. Delevoye designed the research. S.B.-M., L.S., A.-S.M., M. Rouabah, N.L., V.F., J.S.-C., R.A.J., M. Romao, V.R., C.G., M.P., I.H. and C.N.-M. developed the protocols. S.B.-M., L.S., A.-S.M., M. Rouabah, N.L., V.F., M. Romao, V.R., C.G., M.P., I.H. and C.N.-M. performed the experiments. S.B.-M., L.S., M. Rouabah, N.L., M. Romao, I.H. and C. Delevoye collected the data. S.B.-M., L.S., A.-S.M., M. Rouabah, N.L., J.-B.M. and C. Delevoye analysed the data. All authors provided intellectual support and interpreted the results. S.B.-M. and C. Delevoye wrote the initial paper with contributions from L.S., A.-S.M., C.G., M.B., J.-B.M., C. Duval, F.B. and G.R. The revised versions of the paper were designed, prepared and edited by L.S. and C. Delevoye. All authors contributed intellectual capital to the study and edited versions of the paper.

Corresponding author

Correspondence to Cédric Delevoye.

Ethics declarations

Competing interests

M.P., C.G., F.B. and C. Duval are full-time employees of L’Oreal Research and Innovation, which also provided financial support through a research contract agreement with the Structure and Membrane Compartments’s team (Institut Curie, PSL Research University, CNRS, UMR144, Paris, France). The other authors declare no competing interests.

Peer review

Peer review information

Nature Cell Biology thanks Thomas Magin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Collection and automatic optical detection of MCs.

(a) Steps illustrating the procedure for isolating MCs from MNT-1 cell culture medium collected and deposited in a column containing a porous filter (1), then centrifuged (2) to retain pigmented MCs in a pocket (3, arrow) followed by content transfer to a tube and centrifugation to form a pigmented MCs pellet (4). (b) Average estimated melanin concentration of MCs-enriched fractions. Each data point represents an individual MC preparation, from n = 41 independent biological replicates. (c) EM micrographs of MCs isolated from primary highly pigmented HEM cell culture medium. Arrowheads point to lipid vesicles associated to MC. (d) Maximum intensity of the inverted signal captured by brightfield illumination of individual MCs as a function of their maximum intensity of the fluorescent signal for HMB45 (n = 255 MCs). (e-f) IFM of isolated MCs captured by brightfield illumination (e, top) and stained with HMB45 antibody (f, top; red). HMB45+ and lighter MCs (e, bottom, arrowheads) not identified by automatic detection of MCs based on the brightfield image were subsequently identified using automatic detection of HMB45 signal (f, bottom, arrowheads). Automatically detected MCs are circled in yellow. Data are the average of at least three independent experiments presented as the mean ± SEM. See Supplementary Table 1 for details.

Source data

Extended Data Fig. 2 Position of MC+ organelles relative to nucleus and molecular composition.

(a) IFM of HEKs 10 min (top) or 7-days (bottom) after MCs (arrowheads) deposition and DAPI staining (blue). Automatically detected MCs are circled in yellow and the distance (d in insets) of individual MC to nucleus edge (dashed line in insets) is depicted in magenta. (b) Diagram showing the automatic measurement (by code2) of the total distance (D; double black arrow) from each MC (yellow circles) to the center of mass of the nucleus (blue ball). Further subtraction of the nuclear radius (r) from D gives the estimated 2D distance (d; magenta double arrow) from the individual MC to the nuclear membrane (NM, dashed black line). (c) Conventional EM micrographs of ultrathin sections of HEKs 30 min after MC deposition. The arrowheads point to an extracellular MC affixed to the plasma membrane (arrow), forming a finger-like protrusion (inset, arrow). (d) Conventional EM micrograph of ultrathin section of HEKs 1-day after MC deposition depicting the area used for analysis in Fig. 2d. (e) SR-IFM of HEKs 1-day after MCs deposition and stained with HMB45 (red), anti-LAMP1 (green) and -CD63 (cyan) antibodies. Insets (left) are consecutive z-stacks of the same MC+ organelle.

Extended Data Fig. 3 MC+ organelles in keratinocytes are partially surrounded by KRT IFs in vitro and in vivo.

(a) Conventional EM micrographs of ultrathin sections of HEKs from light (left, middle) or dark (right) skin donors 1 day after MC deposition (n = 3 for both skin type). Arrowheads point to keratin+ intermediate filaments surrounding MC+ organelles (arrows). (b) Conventional EM micrographs of ultrathin sections of human pigmented skins from dark (top; n = 3 skin samples) or light (bottom; n = 4 skin samples) skin donors showing keratin+ intermediate filaments (arrowheads) surrounding MC+ organelles in epidermal keratinocytes. Middle panels are magnified area of the boxed regions. (c) IFM of HEKs stained with anti-KRT5 (green) and -α-tubulin (red) antibodies, fluorescence-conjugated phalloidin (cyan), and DAPI (blue). Monochrome and merged images are shown in addition to the outer contour delineation of the different cytoskeletal staining (dashed lines, right). (d-e) IFM of HEKs 1-day after MCs deposition (d) and stained as in c, highlighting perinuclear MC+ organelles (captured by brightfield and pseudocolored in magenta). Automatically detected MCs are circled in yellow (e).

Extended Data Fig. 4 KRT14 controls the 3D-position of MC+ organelles, while plectin contributes to their vertical positioning.

(a) Western blot of HEK lysates treated with control (CTRL), keratin 14 (KRT14) or keratin 5 (KRT5) siRNAs and probed with anti-KRT14 (top), -KRT5 (middle) or -GAPDH (loading control, bottom) antibodies. (b) Average expression levels of KRT14 (left) or KRT5 (right) in siRNA-treated HEKs (as in a) normalized to GAPDH levels and control. Each data point represents an independent biological replicate (n = 3 for all conditions). (c) Average fluorescence intensity of KRT14 in siCTRL- or siKRT14-treated HEKs imaged in (d) and analyzed in (e-f) and normalized to control. Each data point represents an independent biological replicate (n = 3 for both conditions). (d) IFM of siCTRL- (left) or siKRT14- (right) treated HEKs stained with anti-KRT14 antibody (green) and DAPI (blue). (e) Distance of MC+ organelles to the nucleus edge in siCTRL- or siKRT14-treated HEKs expressed per cell. Each data point represents the median distance per cell (siCTRL DMSO: n = 33; siKRT14 DMSO: n = 31), obtained from 3 independent experiments. (f) Distance of MC+ organelles from the nucleus mid-plane. Each data point represents the median distance per cell (siCTRL DMSO: n = 80; siKRT14 DMSO: n = 33), obtained from 3 independent experiments. (g) Average expression levels by immunoblotting of KRT5 and KRT14 protein relative to VIM in HEKs. Each data point represents an independent biological replicate (n = 4 for both KRT5 and KRT14). (h) Western blot of HEK lysates treated with CTRL or vimentin (VIM) siRNAs and probed for VIM (top) or GAPDH (loading control, bottom). (i) Average expression level of VIM in siCTRL- or siVIM-treated HEKs normalized to GAPDH levels and control. Each data point represents an independent biological replicate (n = 3 for both conditions). (j) Mean VIM fluorescence intensity in siCTRL- or siVIM-treated HEKs used for analysis in (k-m) and normalized to control. Each data point represents an independent biological replicate (n = 3 for both conditions). (k) IFM of siCTRL- (left) or siVIM- (right) treated HEKs stained with anti-VIM antibody (green) and DAPI (blue). (l) Distance of MC+ organelles to the nucleus edge in siCTRL- or siVIM-treated HEKs expressed per experiment. Each data point represents the median distance per cell (siCTRL: n = 44; siVIM: n = 27), obtained from 3 independent experiments. (m) Number of MC+ organelles in siCTRL- and siVIM-treated HEKs analyzed in (l). Each data point represents one cell (siCTRL: n = 44; siVIM: n = 27), obtained from 3 independent experiments. (n) Mean nuclei radius of HEKs treated with CTRL, KRT5, KRT14, VIM or PLEC siRNAs and incubated or not with DMSO or nocodazole. Each data point represents an independent biological replicate (n = 3 for all conditions). (o) IFM of DMSO- or nocodazole-treated HEKs stained with anti-α-tubulin (red) and -TGN46 (green) antibodies, and DAPI (blue). (p) Western blot of HEK lysates treated with CTRL or PLEC siRNAs and probed for anti-PLEC (top) or -GAPDH (loading control, bottom) antibodies. (q) Average expression level of PLEC in siCTRL- or siPLEC-treated HEKs normalized to GAPDH levels and control. Each data point represents an independent biological replicate (n = 3 for both conditions). (r) Mean PLEC fluorescence intensity in siCTRL- or siPLEC-treated HEKs used for analysis in (t-u) and normalized to control. Each data point represents an independent biological replicate (n = 3 for both conditions). (s) IFM of siCTRL- (left) or siPLEC- (right) treated HEKs stained with anti-PLEC antibody (green) and DAPI (blue). (t) Distance of MC+ organelles to the nucleus edge in siCTRL- or siPLEC-treated HEKs expressed per cell. Each data point represents the median distance per cell (siCTRL: n = 39; siPLEC: n = 33), obtained from 3 independent experiments. (u) Distance of MC+ organelles from the nucleus mid-plane expressed per cell. Each data point represents the median distance per cell (siCTRL: n = 83; siPLEC: n = 33), obtained from 3 independent experiments. (d, k, o, s) Cell outlines are delineated by dashed lines. (d, k, s) Automatically detected MCs captured 1-day after deposition by brightfield microscopy were pseudo-colored in cyan and circled in yellow. Data are shown as mean ± SEM. One-way ANOVA with Tukey post-hoc, with P = 0.5936 (siCTRL vs. siKRT5), P = 0.0285 (siCTRL vs. siKRT14) in (b) left; P = 0.0002 (siCTRL vs. siKRT5), P = 0.245 (siCTRL vs. siKRT14) in (b) right. Two-tailed unpaired t test with P = 0.0057 in (c); P = 0.0006 in (i); P < 0.0001 in (j); P < 0.0001 in (q); and P < 0.0001 in (r). Two-tailed Mann-Whitney test with P = 0.0007 in (e); P = 0.0163 in (f); P = 0.0621 in (l); P = 0.7933 in (m); P = 0.537 in (t); and P < 0.0001 in (u). Krustal-Wallis test with Dunn’s correction with P ≥ 0.392 in all comparisons; ns, non-significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. See Supplementary Table 1 for details.

Source data

Extended Data Fig. 5 KRT5 expression is required for MC+ organelles perinuclear clustering and mechanical confinement.

(a) Western blot of lysates of WT and KRT5DDD HaCaT cells probed with anti-KRT14 (top), -KRT5 (middle) or -GAPDH (loading control, bottom) antibodies. (b) FM of nocodazole-treated KRT5DDD HaCaT cells expressing or not (left) KRT5WT-mCh or KRT5MITO-mCh (middle and right, respectively) 1-day after MCs deposition and stained with DAPI (blue). MCs captured 1-day after deposition by brightfield microscopy were pseudo-colored in cyan. Automatically detected MCs are circled in yellow and cell outlines are delineated by dashed lines. (c) Distance of MC+ organelles to the nucleus edge as in (b). Each data point represents the median distance per cell (Mock: n = 99; KRT5WT: n = 95; KRT5MITO: n = 67), obtained from 3 independent experiments. Mean ± SEM. (d) IFM of KRT5DDD HaCaT cells either mock-transfected (top) or transfected with KRT5WT-mCh (middle) or KRT5MITO-mCh (bottom) plasmids, and stained with anti-KRT14 (green) and -TOMM20 (blue) antibodies. Note the co-distribution of endogenous KRT14 with KRT5WT-mCh, and its partial re-distribution to TOMM20+ mitochondria upon KRT5MITO-mCh expression. (e) Live imaging frame of WT- (left) or KRT5DDD- (right) HaCaT cells incubated with SiR-Tubulin probe (magenta). MC+ organelles (not shown) were captured by brightfield, and their movements tracked throughout >8 min of acquisition. The trajectories (colored lines) of detected MC+ organelles are shown. Arrowheads point the trajectory of a MC+ organelle over 7 min in HaCaT-KRT5DDD cells and its alignment along SiR-Tubulin+ MTs. Plot (right) shows instantaneous speed in between time frames of tracked MC+ organelles in WT (cyan) or KRT5DDD (red) HaCaT cells and presented as a frequency plot. (f) Diagram illustrating the microrheology experiment. (Top) A 2 µm-diameter bead internalized in the cell is trapped with an optical tweezer. At time t = 0 s, the microscope stage is moved in a Xs = 0.5 µm step displacement. After the initial rapid displacement of the bead from the trap center, the bead position xb(t) relaxes towards the center of the optical trap, which acts as a spring. (Bottom left) Single particle tracking of the bead allows determination of the viscoelastic relaxation curves (see Fig. 5b). (Bottom right) Diagram of the Standard Linear Liquid (SLL) viscoelastic model. (g) IFM of WT (left) and KRT5DDD (right) HaCaT cells having internalized beads (blue) and stained with anti-LAMP1 (green) and -CD63 (red) antibodies. (h) IFM of WT HaCaT cell having internalized beads (blue) and stained with anti-KRT5 (green) and -LAMP1 (red) antibodies. Insets show several z-optical sections of the boxed area. (i) Relaxation curves in Fig. 5b quantified using the Standard Linear Liquid (SLL) viscoelastic model and analysis of the viscosity (in Pa.s) of the cytosolic microenvironment of the bead in WT (blue) or KRT5DDD (red) HaCaT cells. Each data point represents an individual bead (WT: n = 34; KRT5DDD: n = 27), obtained from 4 independent experiments and one bead per cell. Mean ± SD are shown. (b, d, e, g, h) Cell outlines are delineated by dashed lines. Krustal-Wallis one-way ANOVA with Dunn’s post-hoc with P = 0.0011 (Mock vs. KRT5WT); P = 0.4341 (Mock vs. KRT5MITO) in (c). Two-tailed Mann-Whitney test with P = 0.1938 in (i); ns, non-significant; *, P < 0.05. See Supplementary Table 1 for details.

Source data

Extended Data Fig. 6 Perinuclear MC+ organelles have photoprotective activity.

(a) Matrixes of correlation between the mean fluorescence nuclear intensities of CPD and HMB45 per HEK exposed to UVB doses of 0.5 J/cm2 (top) or 1 J/cm2 (bottom) analyzed in Fig. 6a-b and showing the non-parametric two-tailed Spearman correlation coefficient (r). Data from n = 129 cells, obtained from 4 independent experiments (top) or from n = 47 cells, obtained from 3 independent experiments. (b) IFM 1-day after or without (left) MC uptake of siCTRL-DMSO or siKRT5-NOCO treated HEKs exposed or not to UVB (1 J/cm2) before staining with anti-KRT5 (magenta) and -γH2AX (yellow) antibodies, and DAPI (blue). MCs were captured by brightfield microscopy. Merge image (top) and γH2AX channel alone (bottom; inverted LUT) are shown. (c-d) Number of γH2AX nuclear foci (c) and their mean fluorescence intensity (d) per nucleus in cells treated as in (b). In (c), each data point represents a cell’s nucleus (siCTRL DMSO -UV -MCs: n = 55; siCTRL DMSO + UV -MCs: n = 60; siCTRL DMSO + UV +MCs: n = 70; siCTRL KRT5 + UV +MCs: n = 36), obtained from 3 independent experiments. Data are presented as median with 95% confidence interval. In (d), each data point represents a cell’s nucleus (siCTRL DMSO -UV -MCs: n = 58; siCTRL DMSO + UV -MCs: n = 64; siCTRL DMSO + UV +MCs: n = 74; siCTRL KRT5 + UV +MCs: n = 42), obtained from 3 independent experiments. Data are presented as mean ± SEM. (e) IFM 1-day after MCs uptake of siCTRL-NOCO (left) or siKRT5-DMSO (right) treated HEKs exposed to 1 J/cm2 UVB dose before staining with anti-CPD antibody (green) and DAPI (blue). MCs captured by brightfield microscopy were pseudo-colored in magenta. Cells are delineated by dashed lines. (f) Mean number of MCs per cell treated in Fig. 6d. Each data point represents a cell (siCTRL DMSO: n = 80; siCTRL NOCO: n = 51; siKRT5 DMSO: n = 37; siKRT5 NOCO: n = 84), obtained from 4 (for siCTRL DMSO and siKRT5 NOCO) or 3 (for siCTRL NOCO and siKRT5 DMSO) independent experiments. Data are presented as mean ± SEM. (g) Matrix of correlation between the mean nuclear CPD fluorescence intensity and the mean number of MCs per HEK analyzed in Fig. 6c-e and showing the non-parametric two-tailed Spearman correlation coefficient (r). Data of n = 252 cells, obtained from 7 independent experiments. Non-parametric Spearman correlationwith P = 0.00019 and CI of rs = -0.4738 to -0.1542 (0.5 J/cm2); P = 0.02671 and CI of rs = -0.5645 to -0.03097 (1 J/cm2) in (a); and P = 0.361 and CI of rs = -0.1836 to 0.06995 in (g). Krustal-Wallis one-way ANOVA statistical analysis with Dunn’s post-hoc with P = 0.0152 (siCTRL DMSO -UV -MCs vs. siCTRL DMSO + UV -MCs); P = 0.1746 (siCTRL DMSO -UV -MCs vs. siCTRL DMSO + UV +MCs); P = 0.0125 (siCTRL DMSO -UV -MCs vs. siKRT5 NOCO + UV +MCs) in (c); P = 0.008 (siCTRL DMSO -UV -MCs vs. siCTRL DMSO + UV -MCs); P > 0.9999 (siCTRL DMSO -UV -MCs vs. siCTRL DMSO + UV +MCs); P < 0.0001 (siCTRL DMSO -UV -MCs vs. siKRT5 NOCO + UV +MCs) in (d). One-way ANOVA with Tukey correction with P = 0.2414 (siCTRL DMSO vs. siCTRL NOCO), P = 0.4392 (siCTRL DMSO vs. siKRT5 DMSO); P = 0.2993 (siCTRL DMSO vs. siKRT5 NOCO); P = 0.9981 (siCTRL NOCO vs. siKRT5 DMSO); P = 0.9851 (siCTRL NOCO vs. siKRT5 NOCO); and P = 0.9993 (siKRT5 DMSO vs. siKRT5 NOCO) in (f); ns, non-significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. See Supplementary Table 1 for details.

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Supplementary information

Reporting Summary (download PDF )

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Supplementary Table 1 (download XLSX )

Statistical summary of quantitative data.

Supplementary Video 1 (download MOV )

3D organization of MC+ organelles in contact with KRT5+ intermediate filaments. Animation of the 3D surface rendering of the image shown in Fig. 3c. MC+ organelles (labelled with HMB45 antibody; red) are distributed in close proximity to KRT5+ intermediate filaments (green). Semi-transparency of the green channel was applied to better appreciate proximity. Scale bar, 5 µm.

Supplementary Video 2 (download MOV )

MC+ organelles are confined in KRT5-expressing keratinocyte cells. Spinning-disc confocal microscopy on HaCaT-WT cells that internalized MCs (not shown) for 1 day and incubated with SiR-tubulin probe (magenta). Trajectories of MC+ organelles (coloured lines) throughout >8 min of live imaging acquisition are shown. Acquisition parameters: 1 image (merge)/7 seconds. Video is shown at 15 frames/seconds. Scale bar, 15 µm. See also Extended Data Fig. 5e.

Supplementary Video 3 (download MOV )

MC+ organelles are mobile in KRT5-null keratinocyte cells. Spinning-disc confocal microscopy on HaCaT-KRT5DDD cells that internalized MCs (not shown) for 1 day and incubated with SiR-tubulin probe (magenta). Trajectories of MC+ organelles (coloured lines) throughout >8 min of live imaging acquisition are shown. Acquisition parameters: 1 image (merge)/3 seconds. Video is shown at 15 frames/second. Scale bar, 15 µm. See also Extended Data Fig. 5e.

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Benito-Martínez, S., Salavessa, L., Macé, AS. et al. Keratin intermediate filaments mechanically position melanin pigments for genome photoprotection. Nat Cell Biol 28, 98–112 (2026). https://doi.org/10.1038/s41556-025-01817-4

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