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Dynamics and segregation of cell–matrix adhesions in cultured fibroblasts

Nature Cell Biology volume 2, pages 191–196 (2000)Cite this article

Abstract

Here we use time-lapse microscopy to analyse cell–matrix adhesions in cells expressing one of two different cytoskeletal proteins, paxillin or tensin, tagged with green fluorescent protein (GFP). Use of GFP–paxillin to analyse focal contacts and GFP–tensin to study fibrillar adhesions reveals that both types of major adhesion are highly dynamic. Small focal contacts often translocate, by extending centripetally and contracting peripherally, at a mean rate of 19 micrometres per hour. Fibrillar adhesions arise from the medial ends of stationary focal contacts, contain α5β1 integrin and tensin but not other focal-contact components, and associate with fibronectin fibrils. Fibrillar adhesions translocate centripetally at a mean rate of 18 micrometres per hour in an actomyosin-dependent manner. We propose a dynamic model for the regulation of cell–matrix adhesions and for transitions between focal contacts and fibrillar adhesions, with the ability of the matrix to deform functioning as a mechanical switch.

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Figure 1: Relative distributions of α5 and αv integrins in HFFs at different times after plating on fibronectin-coated coverslips.
Figure 2: Distributions of different components of focal contacts and fibrillar adhesions.
Figure 3: Temporal changes in the pattern of GFP–paxillin labelling in transfected HFFs, as determined by FRIT.
Figure 4: Temporal (FRIT) analysis of GFP–tensin-transfected HFFs and FRIC analysis of phosphotyrosine residues and tensin.
Figure 5: High-magnification demonstration of the dynamics of tensin-containing structures and their phosphotyrosine content.
Figure 6: Effects of cytoskeletal drugs on the mobility of tensin-containing structures.
Figure 7: Model for the involvement of actomyosin-driven forces in the formation and segregation of fibrillar adhesions and focal contacts.

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Acknowledgements

This study was supported by the Israel Science Foundation, the Yad Abraham Center for Cancer Diagnosis and Therapy, and the Minerva Foundation. B.G. holds the Erwin Neter Chair in Cell and Tumor Biology. Z.K. holds the Israel Pollak Chair of Biophysics. We thank D. Rivelin and G. Tzur for advice regarding time-lapse recording and digital microscopy.

Correspondence and requests for materials should be addressed to B.G.

Supplementary Information is available on Nature Cell Biology’s World-Wide Web site ( www.nature.com/ncb).

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Authors and Affiliations

  1. Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, 76100, Israel

    Eli Zamir, Menachem Katz, Yehudit Posen, Noam Erez, Alexander Bershadsky, Zvi Kam & Benjamin Geiger

  2. Craniofacial Development Biology and Regeneration Branch, NIDCR, NIH, Bethesda, 20892-4370, Maryland, USA

    Kenneth M. Yamada

  3. The Hematology Institute, Tel-Aviv Medical Center, Tel-Aviv, 64239, Israel

    Ben-Zion Katz

  4. Department of Developmental and Cell Biology, University of California at Irvine, Irvine, 92697-7450, California, USA

    Shin Lin & Diane C. Lin

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Supplementary information

Movie 1

Time-lapse recording of an HFF cell expressing GFP-paxillin. Images were recorded at 1-min intervals starting 24 h after transfection. Time is indicated by the time bar. GFP-paxillin is localized in focal contacts, and their dynamic behaviour, namely assembly and growth, translocation and fading, can be appreciated. These changes are highlighted by FRIT analysis in Fig. 3 of the paper. Bar indicates 10 μm. (MOV 4214 kb)

Movie 2

Time-lapse recording of an HFF cell (and twofold-magnified inserts of the same and two other cells) expressing GFP-tensin. Images were recorded at 2-min intervals starting 24 h after transfection. Time is indicated by the time bar. GFP-tensin is localized in both focal contacts and fibrillar adhesions, and thus reveals the dynamic relationships between the two. Tensin-containing fibrils emerge from the medial ends of focal contacts and then translocate toward the cell centre. This dynamic behaviour is highlighted in Fig. 5 and by FRIT analysis in Fig. 4 of the paper. Scale bar represents 10 μm. (MOV 3346 kb)

Movie 3

3 Time-lapse recording of an HFF cell expressing GFP-tensin, before, during and after incubation with 2 μM latrunculin-A. Images were recorded at 2-min intervals starting 24 h after transfection. Time is indicated by the time bar, and the timing of drug addition and removal is marked by arrows. Scale bar represents 10 μm. (MOV 4095 kb)

Movie 4

Time-lapse recording of an HFF cell expressing GFP-tensin, before, during and after incubation with 150 μM H-7. Images were recorded at 2-min intervals starting 24 h after transfection. Time is indicated by the time bar, and the timing of drug addition and removal is marked by arrows. Scale bar represents 10 μm. (MOV 4148 kb)

Movie 5

Time-lapse recording of an HFF cell expressing GFP-tensin, before, during and after incubation with 100 μM ML-7. Images were recorded at 2-min intervals starting 24 h after transfection. Time is indicated by the time bar, and the timing of drug addition and removal is marked by arrows. Scale bar represents 10 μm. (MOV 4376 kb)

Movie 6

Time-lapse recording of two HFF cells expressing GFP-tensin, before, during and after incubation with 100 μM (left cell) or 300 μM (right cell) Y-27632. Images were recorded at 2-min intervals starting 24 h after transfection. Time is indicated by the time bar, and the timing of drug addition and removal is marked by arrows. Scale bar represents 10 μm. (MOV 4189 kb)

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Zamir, E., Katz, M., Posen, Y. et al. Dynamics and segregation of cell–matrix adhesions in cultured fibroblasts. Nat Cell Biol 2, 191–196 (2000). https://doi.org/10.1038/35008607

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