Cellular and Molecular Life Sciences

, Volume 75, Issue 18, pp 3423–3439 | Cite as

Tetraspanin-enriched microdomains regulate digitation junctions

  • Chao Huang
  • Chenying Fu
  • Jonathan D. Wren
  • Xuejun Wang
  • Feng Zhang
  • Yanhui H. Zhang
  • Samuel A. Connel
  • Taosheng Chen
  • Xin A. Zhang
Original Article


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.


Cell–cell adhesion Cell–cell communication Microextrusion Nanotubule Tetraspanin Integrin 



Digitation junction


Dulbecco’s modified eagle medium


Fetal calf serum


Global microarray meta-analysis


Gene expression omnibus


Hepatocyte growth factor


Human microvascular endothelial cell


Intermediate filament


Monoclonal antibody


Methyl beta cyclodextrin


Membrane tubular structures


Myosin light chain kinase


Polyclonal antibody


Tetraspanin-enriched microdomains


Total internal reflection fluorescence



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.

Author 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.

Compliance with ethical standards

Conflict of interest

The authors declare no competing financial interests.

Supplementary material

18_2018_2803_MOESM1_ESM.avi (7.3 mb)
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)
18_2018_2803_MOESM2_ESM.tif (9.4 mb)
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)
18_2018_2803_MOESM3_ESM.tif (3.3 mb)
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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Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Chao Huang
    • 1
  • Chenying Fu
    • 1
  • Jonathan D. Wren
    • 2
  • Xuejun Wang
    • 1
  • Feng Zhang
    • 1
  • Yanhui H. Zhang
    • 3
  • Samuel A. Connel
    • 4
  • Taosheng Chen
    • 4
  • Xin A. Zhang
    • 1
  1. 1.Stephenson Cancer Center and Department of PhysiologyUniversity of Oklahoma Health Sciences CenterOklahoma CityUSA
  2. 2.Oklahoma Medical Research FoundationOklahoma CityUSA
  3. 3.University of Tennessee Health Science CenterMemphisUSA
  4. 4.St. Jude Children’s Research HospitalMemphisUSA

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