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Vol. 18, Issue 10, 4074-4084, October 2007

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Talin Influences the Dynamics of the Myosin VII-Membrane Interaction

Shawn A. Galdeen^*, Stephen Stephens^*, David D. Thomas, and Margaret A. Titus^*

*Department of Genetics, Cell Biology, and Development and Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455

Submitted July 10, 2006; Revised July 3, 2007; Accepted July 25, 2007
Monitoring Editor: Carole Parent

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Myosin VII (M7) and talin are ancient and ubiquitous actin-binding proteins with conserved roles in adhesion. Talin serves to link membrane receptors to the underlying actin cytoskeleton and forms a complex with M7 in Dictyostelium. The levels of talinA are tightly linked to M7 levels in Dictyostelium. Cells lacking M7 exhibit an 80% decrease in steady-state levels of talinA, whereas increased levels of M7 result in concomitant increases in total talinA. In contrast, changes in talinA levels do not affect M7 levels. Immunoprecipitation reveals that talinA and M7 are associated with each other in membrane fractions. Fluorescence recovery after photobleaching experiments on green fluorescent protein (GFP)-M7 cells expressing different levels of the M7 and talinA show that changes in the overall amounts of these two proteins influences the dynamics of membrane-associated M7. The recovery of GFP-M7 on the membrane is faster in cells lacking talinA and limited in the presence of excess amounts of talinA and M7. These results establish that M7 stabilizes talinA in the cytosol and, in return, talinA regulates the residence time of M7 at the plasma membrane, suggesting that these two proteins are both part of the same dynamic adhesion complex on the plasma membrane.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Efficient cellular migration is critically dependent on timely regulation of cell–cell and cell–substrate contacts. The two general modes of movement used by eukaryotic cells can be defined by the nature of the adhesive contacts that cells make with the substrata. The widely studied fibroblastic mode of migration is characterized by relatively high-affinity focal contacts between the cell and substrata, whereas the amoeboid mode of migration made by lymphocytes and Dictyostelium uses lower affinity, broader contacts that permit rapid movement. The two modes of adhesion employ the same general molecular machinery: a transmembrane adhesion receptor(s) linked to the cortical actin cytoskeleton via one or more adaptor proteins, suggesting that the differences may arise from variations in the strength or duration of the adhesive complex. Talin plays a key role in both fibroblastic and amoeboid motility. Detailed studies in mammalian cells have established its role in activation of integrin binding to its ligand and tension sensing in focal contacts (Nayal et al., 2004) and it is essential for cell–substrate adhesion in amoeboid cells (Niewöhner et al., 1997).

Talin is present in a wide range of organisms, from amoebozoa to humans (Senetar and McCann, 2005), consistent with this protein having an important and highly conserved role in cellular adhesion. It is a large protein (230 kDa) comprised of an N-terminal FERM domain, a long central rod and a C-terminal I/LWEQ actin binding domain. FERM domains can be considered a signature module for proteins that have a role in cellular adhesion, including cytoskeletal linker proteins such as ezrin and moesin and regulatory proteins such as focal adhesion kinase. The talin FERM domain binds to NPxY motifs on the cytoplasmic tails of transmembrane adhesion receptors such as integrins, but can also bind directly to membrane phospholipids. In addition to actin and membrane receptors, talin interacts with vinculin and PIPKI gamma in mammalian cells (Nayal et al., 2004). Talin can dimerize and effectively cross-link actin filaments into networks and bundles, suggesting additional roles in the general organization of cortical actin (Goldmann et al., 1997; Zhang et al., 1996). Consistent with its widespread distribution and ability to bind to both adhesion receptors and the actin cytoskeleton, talin is found in a range of different adhesion structures in addition to focal and sites of amoeboid cell contact with surfaces. It is present in podosomes, invasive structures found in transformed cells and the immunological synapse where it has been shown to be important for stabilizing the LFA-1–dependent (lymphocyte function–associated antigen) interaction between T-cells and antigen-presenting cells (Marchisio et al., 1988; Monks et al., 1998; Simonson et al., 2006).

The role of talin in the establishment of and signaling from focal contacts is well-known but is role in cell–substrate contact in amoeboid cells remains poorly understood. The ability of amoeboid cells to move relatively fast is the result of their making low-affinity, transient contacts with surfaces, suggesting either that talin association with integrins is not accompanied by activation of these receptors or that talin may interact with a different class of receptors altogether. Studies of talin function in lower eukaryotes reveals its essential role in the adhesion of amoeboid cells. For example, the social amoeba Dictyostelium discoideum expresses two talin homologues, talinA and talinB, both of which contribute to adhesion (Niewöhner et al., 1997; Tsujioka et al., 2004). TalinA, in particular, has been shown directly to have a critical role in both cell–cell and cell–substrate adhesion. Mutations in talinA result in defects in cellular adhesion, phagocytosis, and cytokinesis. This is similar to what has been found when talin function is disrupted in the higher eukaryotes (Nuckolls et al., 1992; Niewöhner et al., 1997; Priddle et al., 1998; Brown et al., 2002), consistent with a fundamental conservation of talin function in mediating cellular adhesion.

Myosin VII (M7) is the major talinA-binding partner in Dictyostelium (Tuxworth et al., 2005). The class VII myosins are also a family of highly conserved cytoskeletal proteins with roles in adhesion in different organisms (Tuxworth et al., 2001; El-Amraoui and Petit, 2005; Richards and Cavalier-Smith, 2005). Dictyostelium mutants lacking M7 exhibit defects in substrate adhesion and phagocytosis (Titus, 1999; Tuxworth et al., 2001), phenotypes similar to those of the talinA null mutant. Both proteins are found at the leading edge of migrating cells and in filopodia but they localize to the plasma membrane independently of one another (Kreitmeier et al., 1995; Tuxworth et al., 2001, 2005). M7 and talinA appear to be found in an exclusive complex with each other in the cytosol, suggesting that they operate as a complex to generate optimal adhesion during migration upon recruitment to the membrane. The relationship between these two conserved adhesion proteins was further investigated to better understand their potentially shared and individual contributions to adhesion.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Strains, Cell Growth, and Maintenance
All Dictyostelium strains were maintained using standard methods (Sussman, 1987). Cells were grown on tissue culture plates in HL5 growth medium supplemented with 10,000 U/ml penicillin G (Fisher Scientific, Pittsburgh, PA) and 10 µg/ml streptomycin sulfate (Sigma Chemical Co., St. Louis, MO). The M7 null strain HTD17–1 (in Ax3; Tuxworth et al., 2001), and talinA null strain HG1666 (in Ax2; Niewöhner et al., 1997) were periodically selected in HL5 supplemented with 10 µg/ml Blasticidin S (ICN Biomedicals, Costa Mesa, CA). Transformants expressing green fluorescent protien (GFP)-M7 or GFP-M7 wild-type and mutant tails or tail fragments (see below) in the wild-type Ax2, M7, or talinA null mutant backgrounds were generated as previously described (Tuxworth et al., 2001, 2005) and maintained in HL5 supplemented with 10 µg/ml G418 (Fisher Chemical, Fairlawn, NJ).

GFP-M7 Expression Plasmids
The extrachromosomal GFP fusion expression plasmids for full-length M7 (pDTi112), full-length tail (pDTi35), FERM1/FERM2 tail (pDTi114), and Pro1 region (pDTi183) have been described previously (Tuxworth et al., 2005). GFP fusions of the tail lacking both MyTH4/FERM regions (MFMF; nucleotides 2759–3795 and 4994–5968; amino acids 809-1154 plus 1602–1878), the coil-Pro1 region alone (cP; nucleotides 2759–3795; amino acids 809-1154), the SH3-Pro2 region alone (SP2; nucleotides 5192–5968; amino acids 1620–1878), combined Pro1-Pro2 regions (Pro1–2; nucleotides 3041–3795 and 4994–5968; amino acids 903-1154 plus 1554–1878) and combined Pro1-SH3 regions (PS; nucleotides 3041–3795 and 4994–5371; amino acids 903-1154 plus 1554–1679) of the M7 tail were generated by a combination of PCR (PCR), overlapping PCR, and TOPO-TA cloning (Invitrogen, Carlsbad, CA) using either Ax2 genomic DNA or available genomic clones as templates. All PCR-derived clones were verified by sequencing, and the inserts were ligated in-frame to pTX-GFP, an extrachromosomal expression plasmid that carries the Neo gene, conferring G418 resistance (Levi et al., 2000) resulting in pDTi179 (MFMF), pDTi201 (cP), pDTi203 (SP2), pDTi204 (Pro1–2), and pDTi206 (PS) expression plasmids. Each GFP-expression plasmid was transformed into the appropriate strain and transformants selected for by growth in 10 µg/ml G418, screened for fluorescence, and then analyzed for expression of GFP fusions by Western blotting.

Quantitative Immunoblotting
A total of 3 x 10⁶ cells of each strain were centrifuged at 2700 x g and resuspended in 0.1 ml ULSB (6 M urea, 4% SDS, 20% glycerol, 125 mM Tris, pH 7.5). Samples of 5, 7.5, and 10 µl were adjusted to 15 µl with ULSB and loaded on 6% SDS-PAGE gels. Electrophoresis, transfer to PVDF membrane (Millipore, Bedford, MA), and immunodetection was then performed. Immunodetection of the class I myosin myoB was used as a loading control. Raw data were plotted and linear regression analysis was performed; all R² values for the analysis exceeded 0.9. Rabbit serum containing antibodies directed against the N-terminal portion of the M7 heavy-chain tail region (UMN87; Tuxworth et al., 2005) was used at 1:1000 for immunoblotting. A rabbit polyclonal antibody specific for the heavy chain of the class I myosin, myoB, was also used at 1:1000 (Novak et al., 1995). A mouse mAb specific for the N-terminus of Dictyostelium talinA, mAb 341 (Niewöhner et al., 1997) was initially a generous gift of Dr. Günther Gerisch (MPI, Martinsreid, Germany) and subsequently obtained from Developmental Studies Hybridoma Bank (Iowa City, IA). The antibody was used without dilution. All of the primary antibodies, with the exception of talinA, were diluted in 0.1% casein in phosphate-buffered saline at pH 7.4 (Bio-Rad, Hercules, CA). Washed blots were incubated with Alexa fluor 680– or 800–conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (Molecular Probes, Eugene, OR) and detected and quantified with an Odyssey infared imaging system (LI-COR Biosciences, Lincoln, NE).

Kinetics of talinA Digestion
A total of 1 x 10⁸ cells of wild-type strain Ax2 and M7 null strain HTD17 were washed twice and resuspended in 500 µl MES buffer (2 mM MgSO₄, 0.2 mM CaCl₂, 20 mM MES, pH 6.8). Cells were lysed on ice with 1% Triton X-100 (Anatrace, Maumee, OH) and then 50 µl removed and mixed with 50 µl ULSB at the indicated time points. These samples were heated at 100°C for 3 min, triplicate samples of increasing volumes were adjusted to 10 µl with additional ULSB and loaded on 6% SDS-PAGE gels as described above. Electrophoresis, transfer to PVDF membrane, and immunodetection was then performed. The data were plotted using Origin software (Rockware, Golden, CO).

RNA Isolation and cDNA Preparation
A total of 1 x 10⁷ cells of wild-type strain Ax2 and M7 null strain HTD17 were resuspended in 1 ml of TRIZOL reagent (Invitrogen) and RNA isolated according to manufacturer's instructions. RNA samples were treated with DNAse I (Ambion, Austin, TX), quantified, and overall quality examined on a 1% agarose MOPS-formaldehyde gel. cDNA was prepared from 5 µg total RNA with Superscript III reverse transcriptase mix (Invitrogen) using a 20-base poly-dT primer. No RT reactions were performed to control for contaminating genomic DNA.

Quantitative PCR
Quantitative PCR was performed with the Roche LightCycler real-time PCR system (Roche Diagnostics, Indianapolis, IN) with Roche FastStart DNA Master mix (SYBR Green I). Optimal annealing temperatures to reduce secondary products were determined for each reaction by temperature gradient PCR, and melting temperature analysis was performed on real-time reactions to ensure that secondary products were not present. Relative amounts of sample RT product were standardized to H7, a control gene (Singleton et al., 1988). Annealing was performed for 5 s at 47°C, and extension time was 14 s. Primers for talA were talA3: 5'-CCATGGTTGCTGCAACAATCGTAGATGC-3' (nucleotides 7092–7114) and talA4: 5'-CTCGAGTTAATTTTTATTATAATTTTGTTTTCTTG-3' (nucleotides 7648–7676); primers for H7 were H7S: 5'-ACGTTCAAACTAAATACGGAGCTGGT-3' (nucleotides 5–30) and H7AS: 5'-TTTGAGTGGTTTGCCAATTTCTTTT-3' (nucleotides 288–312).

Immunoprecipitation
Total cellular membranes and cytosol were prepared as described previously (Senda et al., 2001). Total membranes were diluted to the volume of the cytosolic fraction with immunoprecipitation lysis buffer (ILB; 25 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 10 mM Mg-ATP) and 1% Triton X-100 (Anatrace) and protease inhibitors (PIs; 1 mM Pefablock, 100 µM TLCK (N-p-tosyl-L-lysine chloromethyl ketone), 100 µM TPCK (N-tosyl-L- phenylalanine chloromethyl ketone), 333 µM E64, and 0.4 µM ALLN) were added to both fractions. To remove proteins that nonspecifically bind the beads, both fractions were incubated sequentially with three separate solutions of protein A Sepharose Fast Flow beads (Amersham Biosciences, Piscataway, NJ). For each incubation, fractions were added to 100 µl of protein A beads that had been washed twice with ILB (25 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 10 mM Mg-ATP). The sample was gently mixed at 4°C for 1 h, and then the beads were pelleted by centrifugation at low speed for 3 min. The resulting supernatant was collected for the next incubation with beads. A rabbit polyclonal anti-GFP antibody (Molecular Probes) was added to the precleared supernatant, at a final concentration of 12 µg/ml, and the sample was incubated with gentle mixing at 4°C for 1–12 h. The antibody-supernatant solution was then added to 100 µl of protein A beads that had been washed with ILB, and the slurry was incubated with gentle mixing at 4°C for 6 h. The beads were then collected by gentle centrifugation, and the supernatant was saved for analysis. The bead complex was then washed several times with ILB containing PIs, and the final bead pellet was carefully drained of all excess liquid. A total of 30 µl of ULSB was added to the beads, and the samples were heated at 100°C for 3 min and then applied to a 6% SDS-PAGE gel. The gels were then either transferred to PVDF membrane (Millipore) for immunoblotting or stained with silver.

Phagocytosis and Adhesion Assays
A modified small-scale version of the standard phagocytosis assay (Vogel et al., 1980; Tuxworth et al., 2001) using 1-µm fluorescent latex beads (Polysciences, Warrington, PA) was performed. In brief, a total of 1 x 10⁶ cells in a 900 µl volume were shaken in phosphate buffer (16.6 mM phosphate, pH 6.1) at 150 rpm in each well of a 24-well plate at room temperature for 1 h. A 200-fold excess of 1.0-µm fluorescent latex beads in 100 µl was then added; this point defined as time = 0, and 75 µl of cell suspension was removed at various times and added immediately to 5 ml of ice-cold stop solution (0.04% sodium azide in phosphate buffer). After sample collection, cell suspensions were spun at 1140 x g for 10 min, and all but 200 µl of supernatant was removed. Cells were resuspended in the remaining liquid and quantified using a FACSCalibar flow cytometer (Benton-Dickson, Franklin Lakes, NJ).

Bead binding was measured using a slightly modified bead adhesion assay (Tuxworth et al., 2001). Equal numbers of axenically growing wild-type, M7 null, or cP-expressing cells were seeded at subconfluent density for 30 min onto glass coverslips at 20°C. The coverslips were then transferred to 4°C for 30 min. Latex beads of 4.0-µm diameter were washed once and diluted to a density of 6.0 x 10⁶ particles/ml in ice-cold HL5. After the cells were chilled, the media covering them was replaced with 300 µl bead suspension and incubated at 4°C for 15 min. Cells were then fixed for 10 min by replacement of the bead suspension with 300 µl picric acid fixative (Humbel and Biegelmann, 1992). No wash was necessary to remove nonadherent particles. The cells with adhered beads were visualized using a 63x DIC (differential interference contrast) objective mounted on a Zeiss Axiovert microscope (Carl Zeiss MicroImaging, Thornwood, NY). At least nine areas were selected at random in each experiment, and every cell in the field of view was analyzed. A modified plate adhesion assay was performed as described (Fey et al., 2002; Bukharova et al., 2005). In brief, a total of 2.8 x 10⁶ cells total were seeded into each well of a six-well plate and allowed to attach for 15 min at room temperature. The growth medium was replaced with 2 ml of starvation buffer, and the samples were incubated for another 1 h before placing on a shaker at 150 rpm and counting the number of detached cells over time using a Coulter counter (Beckman Coulter, Fullerton, CA).

Photobleaching
Growth-phase cells were seeded into a custom stainless steel chamber (Tuxworth et al., 2001) at a density of 0.5–2 x 10⁶ cell/ml in starvation buffer (16.6 mM phosphate, pH 6.1) for 1–2 h before each experiment. The chamber was inverted, and cells were visualized on an inverted Nikon TE-200 microscope equipped with a 63x 1.4 NA TIRF (total internal reflection fluorescence) objective and a 20 mW argon laser (Melles Griot, Carlsbad, CA) with 488-nm and 514-nm lines. A 488-nm filter was used to remove the 514-nm line, and all experiments were performed at full power, with measured laser intensity at the focal plane of 1.2–1.5 mW. Single pulses of 10 ms were applied to cells and changes in fluorescence intensity in the bleached area (an 0.5-µm spot) monitored with respect to time after background subtraction. Images were captured every 100 ms using a Cascade 2 Digital camera (Photometrics, Tucson, AZ), and all data were collected at 14 bits gray depth. The data were fit with curves using Origin graphical analysis software (Originlab, Northhampton, MA). The time course of recovery was fitted to the following equation for a single exponential rise to maximum:

where A is the amplitude of the exponential, k is the rate constant, t is time (in milliseconds), and t₀ is the time of the bleach. For double exponential rises to maximum, the following equation was used for fitting:

where A and B are respective amplitudes and k₁ and k₂ are rate constants. The half-time for recovery (t_1/2) for a given rate constant k was calculated using the following equation:

The fractional recovery was determined using the following:

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
TalinA Protein Levels Are Directly Correlated to M7 Tail Expression
TalinA and M7 form a complex in the Dictyostelium cytosol (Tuxworth et al., 2005) and, consistent with an intimate relationship between these two proteins, there is an observed decrease in the overall levels of talinA in the absence of M7 (Gebbie et al., 2004; Tuxworth et al., 2005). The extent of talinA decrease in the M7 null mutant was investigated as a first step toward understanding the dependence of talinA levels on M7. Quantitative immunoblotting of talinA and M7 using whole cell lysates from wild-type and M7 null mutants was carried out and the levels of each protein relative to myoB, a class I myosin that served as a loading control and then compared (Figure 1A). Cells lacking M7 have a significantly reduced amount of talinA, down to 17% of wild-type levels (Figure 1A, Table 1).

View larger version (67K): [in this window] [in a new window]   Figure 1. The level of M7 expression affect TalinA levels. The levels of M7 and talinA, relative to the loading control myoB, were determined by quantitative Western blotting of total cell lysates. The antibody used to probe each set of blots in indicated on the left. (A) Comparison of talinA levels in wild-type Ax2 and M7 null cells. Numbers above each lane indicate sample volumes loaded (in µl). (B) TalinA levels in cell lines expressing different levels of either GFP-tagged full-length M7 (GFP-M7), or tail in wild-type or M7 null cells or in talinA null cells. Note that the top panel shows immunoblotting with the M7 antibody.

View larger version (30K): [in this window] [in a new window]   Figure 2. TalinA degradation occurs faster in M7 null cell lysates than in wild-type lysates. (A) Representative Western blots showing changes in talinA levels in wild-type Ax2 and M7 null detergent lysates incubated at 4°C for up to 1 h. (B) Time course of talinA degradation in total cell lysates. The levels in each sample were standardized to time = 0 (100%). Data points from seven separate experiments were plotted together and exponential decay regression analysis was performed for wild-type Ax2 (dashed line) and M7 null (solid line) samples. , levels of talinA in wild-type Ax2 cells (WT); , levels in M7 null cells.

A Minimal Portion of the M7 Tail Responsible for talinA Stabilization
Prevention of talinA turnover by M7 could occur by masking of protease-sensitive sites, either by directly binding to these sites or through steric blocking by adjacent M7 domains. Protection of talinA could alternatively be accomplished by M7 binding, inducing a conformational change in talinA that stabilizes the protein. A series of cell lines were generated to identify the region of the M7 tail that confers proteolytic protection and gain information concerning the prevention of talinA degradation by M7.

The talinA binding site on M7 resides in the N-terminal region of the tail (Tuxworth et al., 2005), a 253-amino acid proline-rich region (Pro1) located between the coil domain and the first MyTH4/FERM repeat (Figure 3A). Quantitative Western blotting of M7 null cells expressing GFP-tagged Pro1 reveals that talinA levels are not restored (Figure 3, Table 2). A series of different GFP-tagged M7 tail fragments were then expressed in the M7 null strain to identify the minimal region necessary for effective talinA stabilization. Expression of a tail lacking both MyTH4/FERM domains was capable of restoring near wild-type levels of talinA (Table 2). The N-terminal region of the tail comprising both the region of predicted coil and Pro1 (cP) or Pro1 fused to the centrally located SH3 and Pro2 domains (Pro1–2) restored almost wild-type levels of talinA (Table 2, Figure 3B). However, the SH3-Pro2 domain alone does not stabilize talinA. These results suggest that steric hindrance is responsible for talinA stabilization and that the precise sequence required is somewhat promiscuous but requires the minimal talinA-binding region, Pro1 (Figure 3, Table 2).

View larger version (47K): [in this window] [in a new window]   Figure 3. Identification of the region of the M7 tail that stabilizes TalinA levels. (A) Schematic illustration of full-length M7 and expressed tail fragments indicating major protein domains. Note that all of the fragments were fused to GFP. The region of predicted coiled-coil is labeled "C," and the MyTH4 domains are indicated by the dark gray box labeled "M." The two proline-rich regions are labeled "Pro1" and "Pro2," and the SH3 domain is represented by the light gray box labeled "S." (B) Immunodetection of M7, talinA, and myoB levels in tail deletion constructs. The antibody used to detect the M7 tail fragments is indicated at the bottom of each set of blots.

View larger version (23K): [in this window] [in a new window]   Figure 4. Restoration of wild-type talinA levels in M7 null cells does not rescue the M7 null mutant phenotype. (A) The uptake of 1-µm fluorescent latex beads was assayed. Cells were incubated with a 200-fold excess of beads, samples were taken at the times indicated, and uptake was measured by flow cytometry. Error bars, SD from three experiments. , wild-type Ax2; , M7 null; , M7 tail in M7 null. (B) The uptake of 1-µm fluorescent latex beads by M7 null cells expressing the cP fragment is shown. Error bars, SD from three experiments. , wild-type Ax2; , M7 null; , cP in M7 null. (C) Binding of 4-µm latex beads at 4°C. The number of beads attached per cell was measured for wild-type (WT; black), M7 null (gray), or cP in M7 null (light gray) cells. The average number of beads/cell was 0.421 (WT; n = 95), 0.09 (M7 null; n = 220), and 0.05 (cP in M7 null; n = 365). (D) A representative assay showing the percentage of cells detached from a six-well plate after shaking at 150 rpm. Shown are the results for wild-type Ax2 (), M7 null (), or cP in M7 null () cells.

TalinA Modulates M7 Membrane Dynamics
The similar adhesion defect of the M7 and talinA null mutants suggests that M7 and talinA may be associated with each other at the plasma membrane where their interaction could be required for proper function of an adhesion complex. The existence of a M7-talinA complex in membrane fractions was investigated using an immunoprecipitation (IP) approach. Cytosol and total membrane fractions were isolated from GFP-M7–expressing cells using a sucrose step gradient. The membrane fraction was solubilized with Triton X-100, and the M7 was immunoprecipitated from both fractions using an anti-GFP antibody. Two high-molecular-weight bands are present in both the cytosol and membrane IP pellets, one at the predicted 300 kDa size of GFP-M7 and a second band migrating slightly below that, at the 260 kDa size of talinA. Immunoblotting with either a M7 or talinA antibody confirms that the higher molecular weight band is M7 and the lower one is talinA (Figure 5). Additional IP experiments using a variety of detergents to solubilize the membrane fraction were performed and the same results obtained: talinA is the only protein that coprecipitates with GFP-M7 (Stephens and Titus, unpublished observations). Taken together with the finding that GFP-M7 is localized only to the plasma membrane of cells (Tuxworth et al., 2001), this result suggests the existence of a plasma membrane–associated M7-talinA complex.

View larger version (29K): [in this window] [in a new window]   Figure 5. M7 and talinA coprecipitate from isolated total membranes. Detergent lysates of a total membranes fraction from cells expressing GFP-M7 were incubated with a GFP-specific antibody, and the bound protein precipitated with protein A beads. The immunoprecipitated pellets were analyzed on a 6% SDS-PAGE gel stained with silver or by immunoblotting with specific M7 and talinA antibodies (shown at the bottom of the panel). The positions of known molecular mass standards, in kDa, are indicated to the left of the gel.

View larger version (22K): [in this window] [in a new window]   Figure 6. Recovery of GFP-M7 fluorescence at the leading edge. The overall recovery levels for each cells line and representative images and curves of a FRAP analysis of GFP-M7 membrane dynamics are presented. (A) Comparison of the percent recovery of fluorescence in either the cytosol or at the membrane in wild-type Ax2 (WT), M7 null (M7), and talinA (talA) null cells. The number of samples ranged from 8 to 15 per cell line, and the SD is shown. (B) FRAP of GFP-M7 expressed in M7 null cells. Shown are selected images demonstrating recovery of fluorescence at the leading edge before (pre) and at select times after the bleach (left). A time course of fluorescence recovery at the leading edge of the same cell is presented. The initial value for the unbleached leading edge is set to 1.0 (dashed line), and the value immediately after bleaching is set to 0. The arrow indicates the time of the bleach. The light gray line is the calculated curve that fits the experimental data.

View larger version (37K): [in this window] [in a new window]   Figure 7. TalinA influences GFP-M7 membrane dynamics. The membrane dynamics of GFP-M7 expressed in wild-type cells and talinA null cells was analyzed by FRAP. (A) An example of the FRAP of GFP-M7 expressed in talinA null cell. (B) Representative FRAP experiment of wild-type cells expressing GFP-M7 showing selected images that illustrate the minimal recovery after bleach (left). Quantitative analysis of the time course of fluorescence recovery at the leading edge of the same cell is presented. The initial value for the unbleached leading edge is set to 1.0, and the value immediately after bleaching is set to 0. The arrow indicates the time of the bleach and the dashed line 1.0. The light gray line is the calculated curve that fits the experimental data.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The interaction of migrating cells with the substrate must be finely tuned to achieve optimal translocation. Talin is a key player in adhesion in multicellular organisms where it has critical roles in linking integrins to the actin cytoskeleton in migrating cells, acting as both a tension sensor and a mediator of "inside-out" signaling (Nayal et al., 2004). Talin is an ancient and ubiquitous protein (Senetar and McCann, 2005) and in simpler systems such as Dictyostelium, it also has a significant role in cell–substrate adhesion (Niewöhner et al., 1997). Here it is shown that talinA influences the membrane dynamics of another conserved adhesion protein, M7, in Dictyostelium. These two proteins are associated with each other both in the cytosol and on the membrane, as revealed by coIP experiments (Tuxworth et al., 2005; Figure 5). FRAP analysis of cells expressing various levels of M7 and talinA reveals that the lifetime of GFP-M7 on the membrane is significantly increased when an excess of M7 and talinA is present (Figures 6A and 7B, Table 3). This decreased recovery rate is unlikely to be due simply to an increase in the levels of GFP-M7 as recovery is actually faster in talinA cells that have endogenous levels of M7 plus ectopically expressed GFP-M7. Thus, decreased exchange correlates with increased amounts of the talinA/M7 complex.

Talin regulates adhesion protein complex dynamics at the plasma membrane. In mammalian cells, talin1 is specifically cleaved by calpain 2 at a site C-terminal to the FERM domain, and mutations in the site result in the increased lifetime of talin in focal adhesion complexes in cultured cells, indicating that this cleavage releases talin1 from focal complexes (Franco et al., 2004). The persistence of talin1 in adhesion complexes is accompanied by reduction in the turnover of adhesion complex components such as zyxin (Huang et al., 2003). The observation that M7 turnover at the plasma membrane is slowed in cells that have increased levels of the talinA is consistent with a conserved role for talins in controlling the dynamics of adhesion complex components in both simple and complex organisms. It will be interesting to determine if the mechanism of controlling talinA dynamics is shared with other systems or if the requirement for calpain cleavage is found only in higher eukaryotes. Although the molecular details of how talin controls the dynamics of cellular adhesion may depend on the system or cell type, it seems likely that the basic mechanism will be conserved.

The role of talin in clustering and activating adhesion receptors in a number of cell types has been well described (Nayal et al., 2004), yet little is known about control of the cytosolic pool of available talin. The work described here reveals that steady-state levels of this critical adhesion protein are tightly regulated by its association with M7, its major binding partner in Dictyostelium. It interesting to note that there is no detectable free talinA in the Dictyostelium cytosol of wild-type cells—all of it appears to be present in a complex with M7 (Tuxworth et al., 2005). In addition to determining the overall levels of talinA, it is possible that binding to M7 in the cytosol may prevent potentially deleterious effects of free talin. For example, uncontrolled binding of the I/LWEQ region to actin may cause general disruption of the cytoskeleton (Weber et al., 2002). The association with M7 in the cytosol could be required to maintain talinA in the "off" state to prevent this possibility. Alternatively, keeping talinA together in a complex with M7 may coordinate the recruitment of these two functionally related linker proteins to the membrane as a means of efficiently initiating adhesion and subsequently recruiting the additional, necessary adhesion complex components.

The observed differences in the dynamics of membrane-associated M7 in cells with different levels of M7 and talinA cannot be accounted for by proposing that talinA is simply required to tether M7 to the membrane. In talinA null cells, no gross change in the amount of M7 in total membrane fractions is observed and M7 localizes to the plasma membrane correctly (Tuxworth et al., 2005). A model that might account for both these observations as well as the FRAP results is presented in Figure 8. Membrane-binding sites for talinA and M7 could be physically associated with each other, bringing these into close proximity of each other as part of a large adhesion receptor complex. The existence of such a big complex might explain the failure to coprecipitate any protein other than talinA with M7 from membrane fractions treated with various detergents (Stephens, Galdeen, and Titus, unpublished observations). On binding to its own receptor, M7 would also be quite near to a talinA-binding site that its associated talinA could readily bind to. M7 binding to its receptor would not alter the affinity of M7 for talinA and, similarly, M7 binding to talinA would not change the affinity of M7 for its receptor. When M7 is released from its receptor, it can either bind to the nearby talinA or rebind to its receptor or diffuse away. Association with talinA might even increase the likelihood of M7 rebinding to a neighboring M7 receptor. Such a model could explain the observed changes in recovery rates in each cell line. For example, the presence of a large excess of M7 and talinA could result in occupation of all of the talinA and M7 membrane-binding sites, creating a relative abundance of M7 binding sites—both M7 receptors and talinA bound to neighboring receptors, and this slows the observed exchange of M7. Conversely, lack of talinA would not affect the ability of M7 to bind to the membrane, but its likelihood of its being retained at the membrane through combined interactions with the receptor and talinA would be reduced.

View larger version (18K): [in this window] [in a new window]   Figure 8. Speculative model of the dynamics of the M7-talinA association with the membrane. Schematic illustration of M7 (circled 7) and talinA (gray square) interactions with each other and with transmembrane receptors. The double curved lines indicate that plasma membrane. In the case of wild-type cells (top), M7 and talinA bind to each other (indicated by black line linking the two). They can also interact with their respective transmembrane adhesion receptors that are complexed together (joined by black oval). Note that the interaction between either talinA and/or M7 with adhesion receptors may be direct as illustrated or could occur via an adapter protein. Three binding states are illustrated: talinA only binds to its receptor, M7 binds only to its receptor, and M7 and talinA bind to their receptors that are adjacent to each other and at the same time they maintain an affinity for each other (dashed line). In talinA null cells (middle), M7 interacts with its membrane receptor. In cells expressing an excess of both M7 and talinA (bottom), there is an increased probably of M7 binding to both its receptor and talinA on the membrane, because it now has two binding sites at the membrane its residence time is increased.

	ACKNOWLEDGMENTS

 
The authors thank Dr. Günther Gerisch (Max-Planck Institut für Biochemie, Munich, Germany) for generously providing both the talinA null mutant strain HG1666 and Dictyostelium talinA hybridoma. The talinA mAb was obtained from the Developmental Studies Hybridoma Bank (DSHB; Iowa City, IA), which is supported by the National Institute of Child Health and Human Development. We thank Karen Jensen of the DSHB for her invaluable help with the talinA antibody. We also thank also Dr. Gaku Ashiba and Dr. E. Michael Ostap (University of Pennsylvania), Dr. John Cooper (Washington University), and Laura Breshears for stimulating discussions; Dr. Michael O'Connor for the use of a LI-COR fluorescent imaging workstation; and Casey Dorr for help with early real-time qPCR experiments. Finally, we thank to Seth Robia (Loyola University) and Ji Li, Mohac Tecman, Dave Kast, and other members of the Thomas lab for their invaluable assistance in initiating the FRAP experiments. This work was supported by a grant from the National Institutes of Health and a shared equipment grant from the Minnesota Medical Foundation for purchase of the CCD camera used in the FRAP experiments.

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-07-0586) on August 1, 2007.

Address correspondence to: Margaret A. Titus (titus004{at}umn.edu)

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