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Tetraspanin-enriched microdomains regulate digitation junctions

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A Correction to this article was published on 08 September 2018

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Abstract

Tetraspanins co-emerged with multi-cellular organisms during evolution are typically localized at the cell–cell interface, and form tetraspanin-enriched microdomains (TEMs) by associating with each other and other membrane molecules. Tetraspanins affect various biological functions, but how tetraspanins engage in multi-faceted functions at the cellular level is largely unknown. When cells interact, the membrane microextrusions at the cell–cell interfaces form dynamic, digit-like structures between cells, which we term digitation junctions (DJs). We found that (1) tetraspanins CD9, CD81, and CD82 and (2) TEM-associated molecules integrin α3β1, CD44, EWI2/PGRL, and PI-4P are present in DJs of epithelial, endothelial, and cancer cells. Tetraspanins and their associated molecules also regulate the formation and development of DJs. Moreover, (1) actin cytoskeleton, RhoA, and actomyosin activities and (2) growth factor receptor-Src-MAP kinase signaling, but not PI-3 kinase, regulate DJs. Finally, we showed that DJs consist of various forms in different cells. Thus, DJs are common, interactive structures between cells, and likely affect cell adhesion, migration, and communication. TEMs probably modulate various cell functions through DJs. Our findings highlight that DJ morphogenesis reflects the transition between cell–matrix adhesion and cell–cell adhesion and involves both cell–cell and cell–matrix adhesion molecules.

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Change history

  • 08 September 2018

    In the original publication, abstract text, one of the co-author’s name and the legend to Table 1 were incorrectly published.

Abbreviations

DJ:

Digitation junction

DMEM:

Dulbecco’s modified eagle medium

FCS:

Fetal calf serum

GAMMA:

Global microarray meta-analysis

GEO:

Gene expression omnibus

HGF:

Hepatocyte growth factor

HMEC:

Human microvascular endothelial cell

IF:

Intermediate filament

mAb:

Monoclonal antibody

MβCD:

Methyl beta cyclodextrin

Memtubs:

Membrane tubular structures

MLCK:

Myosin light chain kinase

pAb:

Polyclonal antibody

TEMs:

Tetraspanin-enriched microdomains

TIRF:

Total internal reflection fluorescence

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Acknowledgements

This work was supported by National Institutes of Health Research Grants CA096991, HL132553, and HL137819, American Heart Association Grant-in-Aid 13GRNT17040028, Oklahoma Center for Advanced Science and Technology Grant, and Chapman Foundation (to X. A. Z.). We thank Ms. Kathy Kyler for English editing.

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Authors and Affiliations

  1. Stephenson Cancer Center and Department of Physiology, University of Oklahoma Health Sciences Center, BRC Building West Room 1474, 975 N.E. 10th Street, Oklahoma City, OK, 73104, USA

    Chao Huang, Chenying Fu, Xuejun Wang, Feng Zhang & Xin A. Zhang

  2. Oklahoma Medical Research Foundation, Oklahoma City, OK, USA

    Jonathan D. Wren

  3. University of Tennessee Health Science Center, Memphis, TN, USA

    Yanhui H. Zhang

  4. St. Jude Children’s Research Hospital, Memphis, TN, USA

    Samuel A. Connel & Taosheng Chen

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  1. Chao Huang
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Contributions

Conceived and designed the experiments: XAZ. Performed the experiments: CH, CF, XW, JDW, FZ, YHZ, SAC, and XAZ. Analyzed the data: CH, JDW, and XAZ. Contributed reagents/materials/analysis tools: TC. Wrote the paper: XAZ.

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Correspondence to Xin A. Zhang.

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The authors declare no competing financial interests.

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Video

: Nanotubule was formed between two MDCK cells that were expressing GFP-CD151 proteins. Live cell imaging was captured with fluorescent light microscopy. (AVI 7444 kb)

Figure S1. How TEMs regulate DJ morphogenesis: contributions of growth factor/receptor tyrosine kinase and MAP kinase signaling

. LnCap cells cultured on glass coverslips were treated with following activator or inhibitor in serum-free media at 37oC for 60 min, unless otherwise indicated, before they were fixed, incubated with CD44 mAb, DAPI and phalloidin, and then examined and imaged with confocal microscopy. A. Role of HGF and c-Met signaling in DJs. The cells were treated with either HGF (100 ng/ml) after 6-hour serum starvation or MK-2461 (200 nM) without serum starvation. B. Role of MEK signaling in DJs. The cells were treated with Trametinib (200 nM) for 3 h and PD98059 (25 µM). Quantification of microextrusion density in DJs (mean ± S.E., n = 3 individual experiments). ***: P < 0.001. Scale bars: 10 μm. (TIFF 9584 kb)

Figure S2. Spatial arrangement of DJs.

LnCap cells cultured on glass coverslips were fixed and incubated with CD44 mAb and Alexa594-conjugated secondary Ab. Confocal images were acquired as z-stacks comprising sequential optical x–y sections of 46.18 µm x 46.18 µm taken at 1.0 μm z-intervals. Images with maximum cell spreading were selected to represent basal membrane at 0 µm z position. Scale bar: 10 μm. (TIFF 3414 kb)

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Huang, C., Fu, C., Wren, J.D. et al. Tetraspanin-enriched microdomains regulate digitation junctions. Cell. Mol. Life Sci. 75, 3423–3439 (2018). https://doi.org/10.1007/s00018-018-2803-2

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