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Microfilament-dependent movement of the ß3 integrin subunit within focal contacts of endothelial cells¹

Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois, USA; and
^* Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, North Carolina, USA

²Correspondence: Department of Cell and Molecular Biology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611, USA. E-mail: j-jones3{at}nwu.edu

SPECIFIC AIMS

The goal of our study was to characterize the mobility of focal contacts and their constituents in endothelial cells since these matrix adhesion sites play a crucial role in angiogenesis during development, tissue remodeling, and cancer. We focused our analyses on ß3 subunit-containing integrin heterodimers, since these are major matrix receptors expressed by endothelial cells, and tested an hypothesis that integrins are mobile within the confines of each focal contact in endothelial cells.

PRINCIPAL FINDINGS

1. Green fluorescent protein-tagged ß3 integrin and actinin-1 preferentially incorporate into focal contact-like structures in endothelial cells
Green fluorescent protein-tagged ß3 integrin (GFP-ß3) and actinin-1 (GFP-actinin-1) incorporate into focal contacts of human endothelial cells. These tagged focal contacts show migration at the rate of 0.1 µm/min, primarily toward the nuclear region of the cell. On occasion, focal contact movement was observed toward or along the cell surface.

2. Integrin subunits show dynamic properties within the context of an individual focal contact whereas actinin-1 exchanges readily and rapidly with a cytoplasmic pool of protein
We analyzed ß3 integrin and actinin-1 dynamics in focal contacts using fluorescence recovery after photobleaching (FRAP). When an entire GFP-ß3 integrin-containing focal contact is bleached, recovery of a detectable signal takes at least 30 min and recovery remains incomplete at 60 min (Fig. 1 A–D). In contrast, FRAP of GFP-actinin-1 labeled focal contacts occurs rapidly in live transformed human bone marrow endothelial cells (TrHBMECs) and is complete within 4 min (Fig. 1E-H ). When a narrow stripe of fluorescence is bleached across an individual, moving focal contact containing GFP-ß3 integrin, recovery is complete within 16 min (Fig. 2 A–C). Remarkably, FRAP occurs within 1 min after bleaching of a narrow band of fluorescence across a GFP-actinin-1-labeled focal contact (Fig. 2J-L ).

View larger version (82K): [in this window] [in a new window]   Figure 1. GFP-ß3 integrin and GFP-actinin-1 dynamics in focal contacts using FRAP. Images of cells expressing either GFP-ß3 integrin (A–D) or GFP-actinin-1 (E–H) are shown before bleaching (A, E), immediately after bleaching (B, F), and after treatment (C, D, G, H). A–D) One focal contact was bleached (arrow); E–H) three distinct focal contacts were bleached (region marked by arrow). Note that even at 60 min there is minimal recovery in signal in the bleached focal contact in panel D. Signal is restored in as little as 4 min into focal contacts shown in panel H. Bar, 2 µm.

View larger version (70K): [in this window] [in a new window]   Figure 2. FRAP of partially bleached focal contacts in TrHBMECs. Images of cells expressing either GFP-ß3 integrin (A–I) or GFP-actinin-1 (J–L) are shown before bleaching (A, D, G, J), immediately after bleaching (B, E, H, K), and at the indicated times after treatment (C, F, I, L). A narrow stripe across one or more focal contacts was bleached in the cells. The cell in panels D–F was incubated in medium containing 0.05% sodium azide and 50 mM 2-deoxy-D-glucose whereas the cell in panels G–I was incubated in medium supplemented with 0.1 µM cytochalasin D. Recovery of signal is inhibited in panels F, I vs. panel C. There is very rapid recovery of the bleached area in panel L. The inset in panel C shows an overlay of colorized images of the focal contacts shown in panels A–C taken immediately after (blue) and 8 (green) and 16 (red) min after photobleaching. Note that the focal contacts show movement as indicated by the rainbow color effect. C) Bar = 2 µm; inset: bar = 1 µm.

3. A molecular motor associated with the actin cytoskeleton is involved in integrin dynamics in focal contacts
We next analyzed the energy requirements for recovery after photobleaching of focal contacts containing GFP-actinin-1 and GFP-ß3 integrin. To deplete energy stores, TrHBMECs were incubated in medium containing a combination of sodium azide and 2-deoxy-D-glucose for 15–30 min. FRAP of a bleached stripe across a GFP-ß3 integrin-containing focal contact and a fully bleached GFP-actinin-1-labeled focal contact is inhibited under these conditions (Fig. 2D-F ).

We studied the potential involvement of ligand and/or cytoskeleton in regulating mobility of GFP-ß3 integrin in assembled focal contacts using FRAP. Antibody LM609, which inhibits vß3 interaction with extracellular ligands, and Colcemid, a microtubule depolymerizing reagent, have no obvious effect on FRAP of GFP-ß3 integrin. Latrunculin B and cytochalasin D, both of which disrupt the microfilament cytoskeleton, inhibit the extent of recovery of a signal within a bleached stripe across a GFP-ß3 integrin labeled focal contact (Fig. 2G-I ).

The above data indicate that ß3 integrin mobility within the confines of a focal contact is ligand and microtubule independent but microfilament and energy dependent. This raises the possibility that a molecular motor associated with the actin cytoskeleton may be involved in integrin dynamics in focal contacts. An obvious candidate for such a motor is myosin. Immunofluorescence studies showed that myosin II is found associated with the microfilament network as well as at the sites of focal contacts. In FRAP studies, ML-7, a myosin light chain kinase inhibitor, reduces the rate of recovery of a stripe ablated across a GFP-ß3-labeled focal contact in TrHBMECs.

CONCLUSIONS AND SIGNIFICANCE

Despite evidence that focal contacts are dynamic in stationary cells, some reports have concluded that integrin receptors, actin, and actin-associated proteins show low mobility within an intact focal contact. It was our goal to test this long-standing idea by assessing the fate of the ß3 integrin subunit and actinin-1 in focal contacts in live stationary cells using FRAP technology.

Recovery of GFP-actinin-1 into fully bleached focal contacts in endothelial cells occurs in less than 4 min. Indeed, our data support the idea of a rapid, energy-dependent, exchange of actinin-1 between a soluble pool of actinin-1 and focal contacts. We also provide evidence that the ß3 integrin subunit is capable of moving in an energy-dependent manner within an individual focal contact (Fig. 3 ). Moreover, there is a net loss of fluorescence in the unbleached regions of a GFP-ß3 integrin-containing focal contact during recovery of a bleached stripe zone. This indicates that recovery of signal is as a result of the movement of protein from unbleached regions of focal contacts into a bleached zone. This was a surprise. It has been reported that integrins are immobile when assembled into a focal contact. Our data argue otherwise.

View larger version (40K): [in this window] [in a new window]   Figure 3. This schematic shows an integrin heterodimer within an individual focal contact and depicts its interaction with the actin-based microfilament cytoskeleton. Our data indicate that integrin heterodimers are mobile within the confines of each focal contact. Integrin heterodimers outlined by dotted lines represent receptors that have moved in the plane of the membrane. This movement is energy dependent and is likely driven by a molecular motor (MM). Actinin-1 shows exchange between a pool associated with integrin cytoplasmic tails and a soluble or possibly microfilament-bound pool.

The mobile fraction of labeled integrin in focal contacts is reduced by 60% in cells treated with drugs that perturb the microfilament cytoskeleton. This implies that the microfilament system does not merely position integrins but plays an ‘active’ role as a positive regulator of ß3 integrin mobility within a focal contact. FRAP is slowed in energy-depleted cells and is microfilament dependent, which raises the possibility that an actin-associated molecular motor is involved in the dynamic aspects of the ß3 integrin-containing heterodimers we have detailed. Our data concerning the localization of myosin II to focal contacts and the ability of the myosin light chain kinase inhibitor ML-7 to inhibit fluorescence recovery suggest that myosin is a good candidate for such a molecular motor. However, since ML-7 perturbs the actin cytoskeleton, we cannot yet rule out the possibility that the effects of this inhibitor on FRAP may be via a local disruption of the actin cytoskeleton.

In summary (Fig. 3) , our data reveal that certain cytoskeleton-associated proteins but also integrins are mobile within the confines of a focal contact. We hypothesize that such movement is crucial to focal contact function. Indeed, mobility of integrins in focal contacts would allow a cycling in receptor/ligand interactions as focal contacts move along the substratum-attached surface of cells even when they are stationary. We envision that precise regulation in integrin/ligand binding and modulation in integrin mobility by the cytoskeleton within a focal contact are necessary to permit focal contacts to be dynamic.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0878fje; to cite this article, use FASEB J. (April 10, 2002) 10.1096/fj.01-0878fje.

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