1. Examining the Effects of VAP on Vinculin
Melissa Palma, Elke S. Nelson, and Dr. Kris DeMali
Department of Biochemistry
University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA
ABSTRACT
Sites where cells adhere to the extracellular matrix are rich in integrins, the major cell surface adhesion receptors. Integrins are coupled to the actin cytoskeleton by a series of actin-binding proteins including vinculin. Maintenance of a connection to the underlying actin cytoskeleton is critical for integrin function; interruptions in this linkage contribute to the formation and progression of numerous diseased states including cancer, leukocyte adhesion deficiency, heart disease, and some muscular dystrophies. In spite of the critical role of integrins, effective targets for manipulating integrin function are still lacking. We have recently identified a short vinculin activating peptide (VAP) that increases integrin-mediated adhesion to fibronectin or collagen. The increased adhesion is both vinculin- and integrin-dependent. An analysis of the vinculin activating peptide reveals that it is comprised of three vinculin binding sites. Using a series of truncation mutants, we have mapped the residues sufficient for binding vinculin. We find that any one of the three binding sites in VAP is sufficient for vinculin binding and co-localization with vinculin in focal adhesions. We are investigating the mechanism by which the vinculin activating peptide increases adhesion, with an emphasis on which residues in vinculin are required for VAP to exert its effects, and how these effects regulate integrin activity. These studies provide novel insight into how vinculin activation regulates integrin function and will open up novel therapeutic possibilities.
VAP Fragments Containing Vinculin Binding Sites (VBS)
Schematic of the GFP-tagged VAP constructs and their Vinculin Binding Sites (VBS). M21 cells were stably transfected with GFP-tagged VAP fragment constructs.
1. GFP
2. GFP
3. GFP (VAP)
4. GFP
5. GFP
6. GFP
7. GFP
8. GFP
9. GFP
VAP Peptide Nomenclature:
Vinculin, An Important Modulator of Cell Adhesion, Is Regulated by Conformational changes
(A) Upon loss of its intramolecular head-tail interaction, vinculin transitions from its closed, inactive conformation to its open, active conformation. (B) In its open conformation, vinculin binds many proteins. D5 D4 D3 D2 D1
Proline -
rich a
actinin
Proline-rich
VAP
Head
Tail A. B.
VAP Elevates Cell Adhesion to the Extracellular Matrix (ECM)
        Fibronectin           No Fibronectin Fibronectin
VBS+VBS2 +VBS3
VBS2
VBS+VBS2
VBS2
VAP Increases Cell Adhesion to the Extracellular Matrix (ECM)
VBS+VBS)
VBS
Western Blot
Biological Processes for Which Adhesion Through Integrins is Critical
VBS2+VBS3
VBS1+VBS2
VBS2
VBS2
VBS1
M21 cells expressing GFP-VAP truncations were incubated on fibronectin-coated 24-well plates (10 µg/ml) for 2 hours at 37 °C. The wells were washed gently, stained, and the number of adherent cells per field of view were counted using phase microscopy. The results from 20 fields of view were averaged, and the averages from several independent experiments were plotted.
Muscular Dystrophy
ECM a b X
Vinculin
Dystrophin
Dystrophin-associated
Complex
Integrins
Sarcolemma
Tumor
Tumor cell invasion
Basement
Membrane
Blood vessel
APC
T cell
Killer T-cell Activation A. B. C.
Vinculin (Loading Control)
A Single VBS Is Sufficient To Increase Adhesion to the ECM
M21 cells expressing GFP-VAP truncations were incubated on fibronectin-coated 24-well plates (10 µg/ml) for 2 hours at 37 °C. The wells were washed gently, stained, and the number of adherent cells per field of view were counted using phase microscopy. The results from 20 fields of view were averaged, and the averages from several independent experiments were plotted.
Vinculin
VAP Enhances Integrin-Mediated Adhesion
GFP
VAP
GFP-VAP D. A.
GFP + anti-b1
VAP + anti-b1
+ anti-β1 B.
% Cells Adhered * C.
[Fibronectin] in µg/mL
(A-C) HeLa cells expressing GFP or GFP-VAP (VAP) were plated on surfaces coated with fibronectin. Panel A represents typical fields of view for the indicated conditions. (B and C) The numbers of adherent cells from ten representative fields were averaged, and the averages from three independent experiments are shown expressed as a percentage of the total cells +/- the standard error of the mean. Samples were pre-incubated with a function-blocking antibody (anti-β1) against 1-integrins (6S6). (B) Graphs depicting the percentage cells that adhered to 10 µg/mL fibronectin or (C) fibronectin concentrations ranging from 1-50 µg/mL. (D) Western blot analysis of total cell lysates with antibodies against vinculin and GFP confirm expression of the indicated proteins. Bar=100 µm. p values of <0.001 are indicated by an asterisk (*).
GFP-VAP Proteins
M21 cells stably transfected with GFP-tagged VAP truncation mutants were lysed. Western blots were run on a SDS-PAGE gel and blotted with antibodies against vinculin and GFP.
Any VBS is Sufficient to Co-localize with Vinculin in Focal Adhesions
The potential for therapeutic enhancement of integrin-mediated adhesion. (A) Proper cell adhesion is essential to maintain tissue organization and prevent tumor cell invasion and metastasis. (B) In Duchenne’s muscular dystrophy patients integrins can play a compensatory role in restoring muscle integrity. (C) The integrin-based immune synapse is crucial for appropriate T-cell signaling and the generation of an immune response.
Integrins are the Major Transmembrane Adhesion Receptors in Cell Matrix Adhesions/ Focal Adhesions
Actin stress fibers “terminate” at focal adhesions
Talin
Vinculin
Actin
Integrins a b
ECM
Paxillin
Focal adhesions are enriched in integrins, the major cell surface adhesion receptor, that are comprised of heterodimers of α- and -subunits. Upon binding ECM ligands, the cytoplasmic integrin β1-tail binds numerous cytoskeletal proteins including vinculin.
VBS1+VBS2 +VBS3
VBS2
VBS1+VBS2
VBS2
CONCLUSIONS
The Vinculin Activating Peptide (VAP) increases adhesion.
VAP fragments containing a combination of VBS can be stably expressed in M21 cells .
Only a single VBS is required for VAP to increase adhesion to the ECM.
VBS1+VBS2
VBS1
M21s expressing the indicated VAP peptides were fixed, permeabilized, stained with antibodies against vinculin, and visualized using immunofluorescence. Images show 63x magnification of GFP and Texas Red. Areas of yellow indicate co-localization of vinculin within focal adhesions.