Different splice variants of filamin-B affect myogenesis, subcellular distribution, and determine binding to integrin β subunits

Integrins connect the extracellular matrix with the cell interior, and transduce signals through interactions of their cytoplasmic tails with cytoskeletal and signaling proteins. Using the yeast two-hybrid system, we isolated a novel splice variant (filamin-Bvar-1) of the filamentous actin cross-linking protein, filamin-B, that interacts with the cytoplasmic domain of the integrin β1A and β1D subunits. RT-PCR analysis showed weak, but wide, expression of filamin-Bvar-1 and a similar splice variant of filamin-A (filamin-Avar-1) in human tissues. Furthermore, alternative splice variants of filamin-B and filamin-C, from which the flexible hinge-1 region is deleted (ΔH1), were induced during in vitro differentiation of C2C12 mouse myoblasts. We show that both filamin-Avar-1 and filamin-Bvar-1 bind more strongly than their wild-type isoforms to different integrin β subunits. The mere presence of the high-affinity binding site for β1A is not sufficient for targeting the filamin-Bvar-1 construct to focal contacts. Interestingly, the simultaneous deletion of the H1 region is required for the localization of filamin-B at the tips of actin stress fibers. When expressed in C2C12 cells, filamin-Bvar-1(ΔH1) accelerates their differentiation into myotubes. Furthermore, filamin-B variants lacking the H1 region induce the formation of thinner myotubes than those in cells containing variants with this region. These findings suggest that specific combinations of filamin mRNA splicing events modulate the organization of the actin cytoskeleton and the binding affinity for integrins

Kidney failure in mice lacking the tetraspanin CD151

The tetraspanin CD151 is a cell-surface molecule known for its strong lateral interaction with the laminin-binding integrin α3β1. Patients with a nonsense mutation in CD151 display end-stage kidney failure associated with regional skin blistering and sensorineural deafness, and mice lacking the integrin α3 subunit die neonatally because of severe abnormalities in the lung and kidney epithelia. We report the generation of Cd151-null mice that recapitulate the renal pathology of human patients, i.e., with age they develop massive proteinuria caused by focal glomerulosclerosis, disorganization of the glomerular basement membrane, and tubular cystic dilation. However, neither skin integrity nor hearing ability are impaired in the Cd151-null mice. Furthermore, we generated podocyte-specific conditional knockout mice for the integrin α3 subunit that show renal defects similar to those in the Cd151 knockout mice. Our results support the hypothesis that CD151 plays a key role in strengthening α3β1-mediated adhesion in podocytes

Kindlin-1 Regulates Integrin Dynamics and Adhesion Turnover

<div><p>Loss-of-function mutations in the gene encoding the integrin co-activator kindlin-1 cause Kindler syndrome. We report a novel kindlin-1-deficient keratinocyte cell line derived from a Kindler syndrome patient. Despite the expression of kindlin-2, the patient’s cells display several hallmarks related to reduced function of β1 integrins, including abnormal cell morphology, cell adhesion, cell spreading, focal adhesion assembly, and cell migration. Defective cell adhesion was aggravated by kindlin-2 depletion, indicating that kindlin-2 can compensate to a certain extent for the loss of kindlin-1. Intriguingly, β1 at the cell-surface was aberrantly glycosylated in the patient’s cells, and its expression was considerably reduced, both in cells <i>in vitro</i> and in the patient’s epidermis. Reconstitution with wild-type kindlin-1 but not with a β1-binding defective mutant restored the aberrant β1 expression and glycosylation, and normalized cell morphology, adhesion, spreading, and migration. Furthermore, the expression of wild-type kindlin-1, but not of the integrin-binding-defective mutant, increased the stability of integrin-mediated cell-matrix adhesions and enhanced the redistribution of internalized integrins to the cell surface. Thus, these data uncover a role for kindlin-1 in the regulation of integrin trafficking and adhesion turnover.</p></div

Re-expression of kindlin-1 in KS cells.

<p><b>A</b>) Western blot showing expression of eGFP-kindlin-1 in KS and KSK cells. <b>B</b>) Morphology of KS and KSK cells. Bar, 20 µm. <b>C</b>) Proliferation of KS and KSK cells. Shown are the averages ±SEM from 3 independent experiments. <b>D</b>) eGFP-kindlin-1 (green), FAs (blue) and F-actin (red) in KSK cells. Bar, 5 µm. <b>E</b>) Cell adhesion to Col-1 and Ln-332 in KS and KSK cells. Bars represent averages ±SEM of 3 independent experiments. AU, arbitrary units. <b>F</b>) Number of KS and KSK cells spread on Ln-332 and Col-1. Shown are the average values ±SEM from ∼500 cells out of a representative experiment. <b>G</b>) Surface area of KS and KSK cells on Ln-332 and Col-1. Shown are the averages ±SEM from ∼500 cells of a representative experiment. <b>H</b>) Rose-plots depicting migration tracks of KS and KSK cells generated by time-lapse video microscopy. <b>I</b>) Quantification of the velocity of cell migration (average ±SEM from ∼300 cells out of 3 experiments). <b>J</b>) FACS histograms of NHK and KS cells showing β1 cell-surface expression (left) and quantification (average ±SEM from 3 independent experiments) (right). AU, arbitrary units.</p

Regulation of β1 expression and cell spreading by kindlin-1 require the F3 domain.

<p><b>A</b>) Schematic representation of full-length kindlin-1 (top) and kindlin-1^del581 (bottom). <b>B</b>) Expression of full-length eGFP-kindlin-1 and eGFP-kindlin-1^del581 in KSK and KSK^del581 cells. <b>C</b>) Expression of precursor β1 (110 kDa) and mature β1 (130 kDa) in KS, KSK, and KSK^del581 cells. Expression of mature β1 was quantified by densitometry, normalized to actin, and expressed relative to the expression in KSK cells. Shown are the values acquired from a representative blot. <b>D</b>) Immunoprecipitated β1 was treated with neuraminidase (NANase) and analyzed by Western blotting. <b>E</b>) FACS histograms (left) and averages ±SEM quantified from 3 independent experiments (right) of β1 cell-surface expression in KS, KSK, KSK^del581, and NHK, expressed relative to that in KS. <b>F</b>) Phase-contrast images of KS, KSK, KSK^del581 and NHK on Col-1 (left), and average surface area ±SEM of KS, KSK, KSK^del581 and NHK cells (quantified from ∼250 cells from a representative experiment) (right). Bar, 10 µm. <b>G</b>) Subcellular distribution of eGFP-kindlin-1 and eGFP-kindlin-1^del581 (green). FAs (blue), F-actin (red). Bar, 5 µm.</p

Abnormalities in KS cells.

<p><b>A</b>) Schematic representation of the <i>KIND1</i> gene (top), indicating the position of the c.1161delA mutation, and kindlin-1 protein (bottom). Exons are represented by boxes, introns are not to scale. <b>B</b>) Western blot showing the expression of kindlin-1 and kindlin-2 in NHK and KS cells. <b>C</b>) Phase/contrast images of NHK and KS cells. Bar, 20 µm. <b>D</b>) Adhesion of KS cells to Col-1 and Ln-332, expressed relative to that of NHK. Shown are the averages ±SEM from 3 independent experiments. <b>E</b>) Cell spreading of NHK and KS cells on Col-1. Shown are the averages ±SEM from 3 independent experiments. <b>F</b>) Rose-plots depicting migration tracks of NHK and KS cells. <b>G</b>) Quantification of the velocity of cell migration (Bars represent averages ±SEM from ∼250 cells out of 3 experiments). <b>H</b>) Confocal images of FAs, visualized using an antibody against P(Y) (green), and F-actin (red). Scale bar, 10 µm.</p

Decreased integrin expression in the absence of kindlin-1.

<p><b>A</b>) FACS histograms (top) and quantification (bottom; average ±SEM from 3 independent experiments) of NHK and KS cells showing cell-surface expression of β1 (left) and active β1 (right), as measured by 9EG7 staining. AU, arbitrary units. <b>B</b>) Western blot showing the precursor β1 (110 kDa) and the mature form of β1 (130 kDa) in NHK and KS cells. <b>C</b>) Expression of β1 (green) and Ln-332 (red) in the skin of an unaffected individual (normal) and the KS patient. The upper border of the epidermis is indicated with a white line. Bar, 50 µm. d; dermis, e; epidermis. <b>D</b>) Depletion of kindlin-2 causes detachment of KS cells. The numbers above the blot indicate the normalized kindlin-2 expression in the remaining (attached) cells, relative to that in untreated cells. Bar, 20 µm. <b>E</b>) Expression of kindlin-1 (top) and kindlin-2 (bottom) in the skin. Bar, 50 µm. d; dermis, e; epidermis.</p

Kindlin-1 interaction with β1 regulates integrin trafficking.

<p>Cell-surface β1 integrins on KS, KSK, and KSK^del581 cells were labelled with DyLight 649-conjugated K-20 at 4°C (top panel), after which they were allowed to internalize in serum-free medium at 37°C for 2 hrs (middle panel). Recycling of the internal pool was induced with 20% FCS for 7.5 min (bottom panel). Cells were then fixed and processed for confocal microscopy. β1 is pseudo-colored green, nuclei were counterstained with DAPI (pseudocolored red). Arrows indicate delivery of recycled β1 to adhesions. Percentages of cells with recycled integrins are shown (from ∼120 cells out of 3 independent experiments). Bar, 10 µm.</p

Kindlin-1 targeting to adhesions and adhesion stability depend on the F3 domain.

<p><b>A</b>) Stills from a TIRF movie, showing the dynamics of mCherry-vinculin in KS cells. <b>B</b>) Dynamics of mCherry-vinculin (top), and eGFP-kindlin-1 (bottom) in KSK cells. <b>C</b>) Dynamics of mCherry-vinculin (top), and eGFP-kindlin-1 (bottom) in KSK^del581 cells. Look-up table ‘fire’ was used to enhance visibility of adhesions. Shown are images at 0, 7.5, 15, 22.5, and 30 min. Boxed regions are enlarged. Arrows indicate retraction fibers. Bar, 10 µm.</p