Anat Cell Biol.  2022 Jun;55(2):190-204. 10.5115/acb.21.168.

Antimicrobial peptide nisin induces spherical distribution of macropinocytosis-like cytokeratin 5 and cytokeratin 17 following immediate derangement of the cell membrane

• Kitagawa N

• KMID: 2531206
• DOI: http://doi.org/10.5115/acb.21.168

Abstract

The anti-aging effects of Lactococcus lactis are extensively investigated. Nisin is an antimicrobial peptide produced by L. lactis subsp. lactis. We previously reported that 24-hour nisin treatment disturbs the intermediate filament distribution in human keratinocytes. Additionally, we showed that the ring-like distribution of the intermediate filament proteins, cytokeratin (CK) 5 and CK17 is a marker of nisin action. However, two questions remained unanswered: 1) What do the CK5 and CK17 ring-like distributions indicate? 2) Is nisin ineffective under the experimental conditions wherein CK5 and CK17 do not exhibit a ring-like distribution? Super resolution microscopy revealed that nisin treatment altered CK5 and CK17 distribution, making them spherical rather than ring-like, along with actin incorporation. This spherical distribution was not induced by the suppression of endocytosis. The possibility of a macropinocytosis-like phenomenon was indicated, because the spherical distribution was >1 µm in diameter and the spherical distribution was suppressed by macropinocytosis inhibiting conditions, such as the inclusion of an actin polymerization inhibitor and cell migration. Even when the spherical distribution of CK5 and CK17 was not induced, nisin induced derangement of the cell membrane. Nisin treatment for 30 minutes deranged the regular arrangement of the lipid layer (flip-flop); the transmembrane structure of the CK5–desmosome or CK17–desmosome protein complex was disturbed. To the best of our knowledge, this is the first study to report that CK5 and CK17 in a spherical distribution could be involved in a macropinosome-like structure, under certain conditions of nisin action in keratinocytes.

Keyword

Aging; Nisin; Pinocytosis; Keratin-5; Keratin-17

Figure

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  Fig. 1 The 24-hour nisin treatment induces a spherical distribution composed of cytokeratin (CK) 5, CK17, and actin. (A) The 24-hour nisin-treated cells exhibit a spherical distribution similar to that of a fishbowl (asterisk or double asterisk at no. 4 or 8) and slightly sparse CK5 and CK17 intermediate filament distribution, except in the area of the spherical distribution. 3D images consist of stacked super resolution microscopy images (green) and fluorescence microscopy (blue) along the z-axis. (B) Cut surface of no. 4 and 8 in Fig. 1A. Difference between no. 4’ and no. 4” or no. 8’ and no. 8” are the height of the Z-axis direction in the X-Y plane, which is shown in the upper left window. Upper left window is the white line height section of the lower window. Upper left window can also be explained as the red line height section of the right window. Two arrows at the right lower corner of each window indicate the axis of each plane; (B) rotated approximately 45 degrees counterclockwise compared to (A). The meaning of each indicator is illustrated. Scale bars represent 10 μm. *Same spherical distribution as shown in no. 4 of Fig. 1A and no. 4’, 4’’ of Fig. 1B. **Same spherical distribution as shown in no. 8 of Fig. 1A and no. 8’, 8’’ of Fig. 1B.

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  Fig. 2 Under 4°C culture, nisin did not induce a spherical distribution that is composed of cytokeratin (CK) 5 and CK17. Slightly sparse CK5 and CK17 intermediate filament distribution were also not induced. 3D images consisting of stacked super resolution microscopy images (green) and fluorescence microscopy (blue) along the z-axis. Scale bar represent 10 μm.

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  Fig. 3 In ultralow calcium concentration medium, nisin did not cause a spherical distribution that is composed of cytokeratin (CK) 5 and CK17. Slightly sparse CK5 and CK17 intermediate filament distribution were also not induced. 3D images consist of stacked super resolution microscopy images (green) and fluorescence microscopy (blue) along the z-axis. Scale bar represent 10 μm.

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  Fig. 4 Actin polymerization inhibitor cytochalasin (cyto)-D suppresses nisin-induced spherical distribution composed of cytokeratin (CK) 5 and CK17. Interspacing between CK17 intermediate filaments (asterisk) are presented as a 2D structure and it is different from the 3D spherical distribution in Fig. 1. This interspacing could only be observed in the X-Y plane. In the X-Z plane or Y-Z plane, the interspacing observed in the X-Y plane did not exist. (A–F) 3D images consist of stacked super resolution microscopy images (green) and fluorescence microscopy (blue) along the z-axis. Control conditions shown in panels (A) and (D) are with the same data as that in Fig 1A no. 3 and 7. (G) Cut surface of (F). Upper left window is the white line height section of the lower window. Upper left window can also be explained as the red line height section of the right window. Two arrows at right lower corner of each window indicate the axis of each plane. Panel (G) have been rotated approximately 45 degrees counterclockwise compared to (F). The meaning of each indicator is illustrated. Scale bars represent 10 μm. *Interspaces between CK17 intermediate filaments.

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  Fig. 5 Disruption of regular E-cadherin distribution in cell boundary following 24-hour nisin treatment. Thickness and density of E-cadherin distribution changed from homogenous to nonequivalent following nisin treatment. Some E-cadherin line-like distributions appeared at right angles to the other line-like E-cadherin distributions in nisin-treated cells. Confocal microscopy images. Difference in thickness, density, and direction of E-cadherin line-like distribution illustrated by indicator. The meaning of each indicator is illustrated. Scale bar represents 20 μm.

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  Fig. 6 The 24-hour nisin treatment increases cytoplasmic distribution of the lysosome marker, Lamp II. Except in the 24-hour nisin treatment, the distribution of Lamp II in the cytoplasm was lesser than that in the nucleus or periphery of the nucleus. (A) 3D images consist of stacked super resolution microscopy images (green) and fluorescence microscopy (blue) along the z-axis. (B) Bar graph comparing Lamp II distribution in the cytoplasm and nucleus. Scale bar represent 10 μm.

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  Fig. 7 The 30-minute and 24-hour nisin treatment both decrease proximity of cytokeratin (CK) 5 and desmoglein (DSG) 3 or CK17 and DSG3 or actin and E-cadherin (E-cad). (A) Maximum intensity projection images consist of stacked confocal microscopy images along the z-axis. When cytoskeleton and cell junction proteins exist proximately, red circular spots are observed. Arrows mean proximity point of indicated cell junction protein and indicated cytoskeleton protein. a)Non-specific fluorescence from cell debris. Scale bar=20 μm. (B) Q uantitative analyses of the proximity ligation assay images. The line within the box plot represents the median value. When proximity index of the image is 100, it means that the number of red circular spots is the same as the average spots in the control images. Values expressed as the mean±SD (n>15). *P<0.05 versus control.

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  Fig. 8 Increase in flip-flops following 30-minute nisin treatment. (A) Green fluorescence of annexin V induced by nisin indicates flip-flop. Fluorescence and bright field (BF) microscopy images in unfixed cells. Scale bar=20 μm. (B) Q uantitative analysis of flip-flops. When index of the image is 100, it means that the ratio of the annexin V positive cell is the same as the average ratio in the control images. (C) Viability after 30-minute nisin treatment or 24-hour after treatment with the apoptosis inducer, staurosporine. The line within the box plot represents the median value. Values are expressed as the mean±SD (n>15). *P<0.05 versus control.

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  Fig. 9 In migrating cells, nisin does not induce spherical distribution of cytokeratin (CK) 5 and CK17. This reaction is different from that in cells surrounded by other cells in Fig. 1. Nisin treatment did not cause a slightly sparse CK5 and CK17 intermediate filament distribution. 3D images consist of stacked super resolution microscopy images (green) and fluorescence microscopy (blue) along the z-axis. Scale bar represent 10 μm.

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  Fig. 10 Nisin blocked globose shape type 3D cell cluster formation. At 0 to 24 hours or 3 to 24 hours nisin treated cell clusters showed a sea urchin-shape, compared to that in the 6 to 24 hours treated cell cluster and control. HaCaT cells in minimal cell attachment plates were treated with nisin or the vehicle for indicated periods from seeding. Images of phase-contrast microscopy. Scale bar represent 500 μm.

Reference

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