The Spatial and Temporal Dynamics of Pleckstrin Homology Domain Binding at the Plasma Membrane Measured by Imaging Single Molecules in Live Mouse Myoblasts

Pleckstrin homology (PH) domains act to target proteins to the plasma membrane and intracellular vesicles by binding to specific phosphoinositol phospholipids. We have investigated the binding kinetics of PH domains found in the tail region of the molecular motor, myosin X. Using total internal reflection fluorescence microscopy, we observed binding and release of individual PH domains fused to green fluorescent protein at the plasma membrane of living cells. Individual spots of light corresponding to single fluorescently tagged molecules were imaged onto a sensitive camera system, and digital image processing was then used to identify each fluorophore and store its trajectory in time and space. The PH domains bound with an apparent on-rate of 0.03 μm-1 μm-2 s-1 and a detachment rate constant of 0.05 s-1. The average residency time of the domains at the plasma membrane was about 20s. We found very limited movement of the membrane-bound PH domains in the mouse myoblast cells that we studied. This implies that the PH domains must either be attached to the cytoskeleton or corralled in a lipid compartment. Localization of the PH domains together with their rapid detachment rate is probably important in controlling the response of myosin X to signaling events and in regulating its cellular function. Pleckstrin homology (PH) domains act to target proteins to the plasma membrane and intracellular vesicles by binding to specific phosphoinositol phospholipids. We have investigated the binding kinetics of PH domains found in the tail region of the molecular motor, myosin X. Using total internal reflection fluorescence microscopy, we observed binding and release of individual PH domains fused to green fluorescent protein at the plasma membrane of living cells. Individual spots of light corresponding to single fluorescently tagged molecules were imaged onto a sensitive camera system, and digital image processing was then used to identify each fluorophore and store its trajectory in time and space. The PH domains bound with an apparent on-rate of 0.03 μm-1 μm-2 s-1 and a detachment rate constant of 0.05 s-1. The average residency time of the domains at the plasma membrane was about 20s. We found very limited movement of the membrane-bound PH domains in the mouse myoblast cells that we studied. This implies that the PH domains must either be attached to the cytoskeleton or corralled in a lipid compartment. Localization of the PH domains together with their rapid detachment rate is probably important in controlling the response of myosin X to signaling events and in regulating its cellular function. PH domains are found in over 250 proteins in the human genome (1Lemmon M.A. Ferguson K.M. Biochem. Soc. Trans. 2001; 29: 377-384Crossref PubMed Scopus (87) Google Scholar) and are known to bind specific phosphoinositol phospholipids (PtdIns). 1The abbreviations used are: PtdIns, phosphatidylinositol; PH, pleckstrin homology; TIRFM, total internal reflection fluorescence microscopy; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-triphosphate; PtdIns(3,4)P2, phosphatidylinositol 3,4-biphosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-biphosphate; PtdIns(3,5)P2, phosphatidylinositol 3,5-biphosphate. The binding of PH domains to their target phospholipids is controlled by phosphorylation of the inositol moiety of the lipids. Because there are five available phosphorylation positions, this could give rise to 32 different PtdIns forms (for example, a 5-bit binary encoding). Although binding affinities of different PH domains have been measured in vitro and their localization has been observed in vivo, little is known regarding the membrane binding dynamics of these domains. Recently, several laboratories have developed optical methods to make wide field observations of individual fluorophores in aqueous solution and these techniques enable the dynamic behavior (e.g. kinetics of membrane binding and release, diffusion times, and trafficking) of proteins, ligands, and viruses (2Axelrod D. Lakowicz J.R. Topics inFluorescent Spectroscopy. 3. Plenum Press, New York1992: 289-342Google Scholar, 3Burghardt T.P. Axelrod D. Biophys. J. 1981; 33: 455-467Abstract Full Text PDF PubMed Scopus (147) Google Scholar, 4Cinelli R.A. Ferrari A. Pellegrini V. Tyagi M. Giacca M. Beltram F. Photochem. Photobiol. 2000; 71: 771-776Crossref PubMed Scopus (60) Google Scholar, 5Garcia-Parajo M.F. Segers-Nolten G.M. Veerman J.A. Greve J. van Hulst N.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7237-7242Crossref PubMed Scopus (152) Google Scholar, 6Sund S.E. Axelrod D. Biophys. J. 2000; 79: 1655-1669Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 7Thompson N.L. Burghardt T.P. Axelrod D. Biophys. J. 1981; 33: 435-454Abstract Full Text PDF PubMed Scopus (240) Google Scholar) to be measured simply by making steady-state observations. However, one of the main problems in imaging single fluorophores within a living cell is the high autofluorescence that overlaps the emission spectrum of the enhanced (S65T mutant) green fluorescent protein (eGFP) used in many such studies (8Harms G.S. Cognet L. Lommerse P.H.M. Blab G.A. Schmidt T. Biophys. J. 2001; 80: 2396-2408Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). To overcome this problem, we exploited total internal reflection fluorescence microscopy (TIRFM) to observe single fluorophores in living cells. TIRFM is a wide-field method (e.g. illuminates the entire cross-section of a specimen) that relies on near-field excitation (by the evanescent wave); thus, only a thin layer of the cell that lies close to the glasswater interface is illuminated (9Mashanov G.I. Tacon D. Knight A.E. Peckham M. Molloy J.E. Methods. 2003; 29: 142-152Crossref PubMed Scopus (113) Google Scholar). This technique enables the activity of individual molecules to be observed directly allowing membrane binding dynamics of single molecules to be measured by steady-state observation. We are interested in the behavior of myosin X, which is a dimeric molecular motor consisting of a highly conserved motor domain that interacts with actin to produce force and movement and a modular tail region that is thought to bind to membranes via its PH domains. Here, we studied the three tandem PH domains present in its tail region (10Berg J.S. Derfler B.H. Pennisi C.M. Corey D.P. Cheney R.E. J. Cell Sci. 2000; 113: 3439-3451Crossref PubMed Google Scholar). The first domain is split, such that the second domain is inserted into a loop in the first domain. The tail domain of myosin X fused to eGFP localizes to the actin rich cortex (11Berg J.S. Cheney R.E. Nat. Cell Biol. 2002; 4: 246-250Crossref PubMed Scopus (275) Google Scholar), and of the three identified tail domains, only the PH domains show any membrane localization (12Yonezawa S. Yoshizaki N. Sano M. Hanai A. Masaki S. Takizawa T. Kageyama T. Moriyama A. Dev. Growth Differ. 2003; 45: 175-185Crossref PubMed Scopus (26) Google Scholar). Sequence homology mapping suggests that all three domains bind to phosphoinositol lipids with a phosphate at the 3′ position such as PtdIns(3,4,5)P3 or PtdIns(3,4)P2 following phosphatidylinositol 3-kinase phosphorylation of PtdIns(4,5)P2 upon cell stimulation (13Isakoff S.J. Cardozo T. Andreev J. Li Z. Ferguson K.M. Abagyan R. Lemmon M.A. Aronheim A. Skolnik E.Y. EMBO J. 1998; 17: 5374-5387Crossref PubMed Scopus (286) Google Scholar). Phospholipid blot overlay assays showed that the PH domains in fact bind to PtdIns(3,4,5)P3, PtdIns(3,5)P2, and with lower affinity to PtdIns(4,5)P2 in vitro. 2D. Tacon and M. Peckham, unpublished findings. In vivo, the PH domains are recruited to the plasma membrane, areas of dynamic ruffling activity, and intracellular vesicles in live cells. In this study, the binding kinetics of single molecules of eGFP-PH123 at the plasma membrane of live cells was investigated using TIRFM. Time-lapse TIRFM was employed to visualize individual fluorophores for long periods (>500 s), and this enabled data from up to 1000 individual fluorophores to be collected from each cell specimen. By handling the data obtained from each individual molecule separately, the power of the single molecule approach is retained, whereas sufficiently large data sets could be collected to ensure that the results are statistically valid. Steady-state observation of single molecules enabled their binding kinetics and spatial dynamics to be measured without perturbing the cell. Furthermore, the use of a novel time-lapse illumination method to systematically change the illumination intensity enabled the effects of photobleaching to be obviated. Cloning—The full-length myosin X cDNA was amplified from mouse myoblasts by long reverse transcription-PCR using primers based on the mouse sequence (GenBank™ accession number AJ249706) (14Yonezawa S. Kimura A. Koshiba S. Masaki S. Ono T. Hanai A. Sonta S. Kageyama T. Takahashi T. Moriyama A. Biochem. Biophys. Res. Commun. 2000; 271: 526-533Crossref PubMed Scopus (24) Google Scholar). It was then sequenced in its entirety to check for sequence fidelity. A fragment containing all three PH domains (eGFP-PH123, nucleotides 3615–4500) was subcloned into either peGFPN1 or peGFPC1 (Clontech), such that eGFP was fused either to the N terminus with a 15 amino acid linker or the C terminus with a 6 amino acid linker. In the experiments reported here, we used exclusively the C-terminal constructs. These were transiently expressed in COS-7 cells, and the cell lysates were immunoblotted using an anti-GFP antibody (AbCam Ltd., Cambridge, United Kingdom) to confirm that the expressed proteins were of the predicted size (data not shown). In the experiments described here, we used eGFP-PH123 in which eGFP was fused to the C terminus; however, we found that N-terminal fusions gave similar cellular localization. Cell Culture, Transfection, and Expression Levels—Mouse myoblasts isolated from the “immortal mouse” (H2kb-tsA58) were cultured as described previously (15Peckham M. Miller G. Wells C. Zicha D. Dunn G.A. J. Cell Sci. 2001; 114: 1367-1377PubMed Google Scholar). They were transfected with the eGFP constructs using FuGENE 6 (Roche Diagnostics) or Genejuice (Novagen) following the recommended protocol. After 24 h, the cells were plated onto glass coverslips and allowed to settle. 8 h later, the medium was replaced with Hanks' balanced salt solution containing 20 mm HEPES (pH 7.4) without serum and the coverslip was assembled into a small chamber for viewing the cells on the inverted microscope (see below). Approximately, 10% of the cells had a suitable level of expression for TIRF measurements following this transfection procedure, such that single molecules could be visualized as single spots. The final concentration of eGFP fusion protein in cells used in the TIRF experiments was in the nanomolar range (i.e. ∼1 molecule/μm3; 5000 molecules/cell). This was confirmed by independent measurements using confocal microscopy where fluorescence of a range of concentrations of eGFP was compared with that of live transfected cells. From the calibration curve, we estimated the concentration to be around 10 nm (9Mashanov G.I. Tacon D. Knight A.E. Peckham M. Molloy J.E. Methods. 2003; 29: 142-152Crossref PubMed Scopus (113) Google Scholar). Under equivalent conditions in neutrophils, the concentration of target lipid, PtdIns(3,4,5)P3, in the plasma membrane was ∼50 nm (local concentration of 5 μm) (16Stephens L.R. Hughes K.T. Irvine R.F. Nature. 1991; 351: 33-39Crossref PubMed Scopus (408) Google Scholar). Objective Type TIRF Microscopy—The imaging apparatus was based around an inverted microscope (Axiovert S100, Zeiss) with a high numerical aperture objective lens (Fluor ×100, 1.45NA, Zeiss). An argon ion laser (532-AP-A01, Melles Griot) was used to illuminate the cells using a custom optical arrangement to produce evanescent field excitation (TIRFM) (9Mashanov G.I. Tacon D. Knight A.E. Peckham M. Molloy J.E. Methods. 2003; 29: 142-152Crossref PubMed Scopus (113) Google Scholar). Illumination was controlled using a mechanical shutter system and neutral density filter. A small (∼2.5 mm diameter) mirror placed close to the back aperture of the objective lens directed the laser beam to the periphery of the lens so that light the at an the for total internal A second mirror on the of the back aperture the beam of the microscope system and onto a used for (see G.I. Tacon D. Knight A.E. Peckham M. Molloy J.E. Methods. 2003; 29: 142-152Crossref PubMed Scopus (113) Google for a of were by a digital camera United Kingdom) and to the of a for transfected with PH domains were plated onto glass coverslips and in a viewing The chamber of a with a The coverslip with cells was to the lower of the and the chamber was then with medium to the from The was then with a second coverslip to the enabled to be eGFP in the single fluorophore TIRFM system, eGFP was controlled were using a glass microscope which of coverslip were 15 mm using of United A coverslip was onto the coverslip to the mm of coverslip from either The was to light the was of a solution (pH 7.4) containing 5 was to the for 5 This was and the was using serum of a solution containing 20 eGFP then was for 5 and eGFP was using an of were then using the TIRFM The eGFP attached to the glass at a density of molecule eGFP molecules were on glass as isolated spots of a The and of individual fluorophores together with their rapid photobleaching were used to the system for studies using live cells. cell studies were using the and camera were used as the of the experiments were at of the data was using a custom single fluorophore (9Mashanov G.I. Tacon D. Knight A.E. Peckham M. Molloy J.E. Methods. 2003; 29: 142-152Crossref PubMed Scopus (113) Google Scholar). data sets obtained from eGFP molecules attached to the coverslip via antibody showed of single We these the the fluorescent spots of light had intensity of emission for a single single and of the fluorophore photobleaching directly to laser excitation on these we developed a that spatial and on to identify individual fluorophores (see below). the first of were in time to for the change in intensity of rapid and of single spots that the and of intensity change and which had the size were then to a second of The second a of the intensity and in intensity were the first the final of the the trajectory in and time for each fluorophore was by the of each fluorescent The an that could be the data sets and which all of the regarding the individual time for each fluorophore and the average using TIRFM were by the fact that fluorophores very to and from the evanescent excitation region and binding at the plasma membrane is close to the coverslip This that spots of light and then at the membrane for several either or the molecule In either the of light The time of system is to by rapid diffusion of the excitation field and In the average diffusion time for a small protein in (e.g. diffusion from an evanescent field of is by Because photobleaching is directly to laser whereas is independent of illumination photobleaching be from by systematically the average laser To time-lapse illumination was In the laser was on for or or on for the 5 The illumination was over the range from to This approach the that of the camera system and the TIRF the of illumination of eGFP in eGFP attached to coverslips via anti-GFP could be identified as isolated spots of light The spatial intensity by individual fluorophores to an with a by the imaging Full at be close to 250 nm optical numerical aperture of the imaging system, of for aqueous system gave a measured at of nm This was by image by the of intensity of individual fluorophores time showed an constant level of fluorescence with level by were collected so that the in intensity to was (i.e. of the intensity The of measured for many different fluorophores is from the of an individual (e.g. that the average be within The large in individual probably from the of the eGFP on the light was used to and in its in which the of of individual fluorophores photobleaching were show that the rate of of fluorophores to photobleaching was with a that in a on the illumination intensity (see eGFP-PH123 in TIRFM was used to image eGFP-PH123 expressed in living cells at very expression Individual spots of light were observed that had the as experiments using eGFP bound to a glass via antibody (see the cells were illuminated at high laser the average fluorescence of the cell The time of photobleaching was similar to the average of the individual fluorescent spots in and The from this is that of the observed fluorophores are probably the observation the average illumination intensity was so that the cells were illuminated for only in any the fluorescence intensity of the cell then high for many of a and and by only over the entire Under these fluorescent spots and at the membrane for many and the average of the spots was and as long as of eGFP-PH123 that from the membrane photobleaching from the excitation field so that camera system could not directly this from Illumination power was systematically as eGFP bound to an and the average of the fluorophores photobleaching was found to upon illumination However, a of the rate constant for the of the spots of the average laser power showed a be the of rate photobleaching and detachment and be to illumination and that these are at high average laser the be whereas at power be This for eGFP-PH123 in living cells showed that at 0.05 s-1 In eGFP molecules attached via to glass had an detachment to at laser power a single (i.e. for photobleaching and The in the the and or not the is suitable and In the we give the to the of the to the The that a single is an of Because size is observations for as a the be of the as in where number of and The apparent binding rate of eGFP-PH123 at the plasma membrane was estimated by the (i.e. the of a fluorescent within a of membrane or rate in a upon the observation size (i.e. of the of rate were obtained using a 5 We found that the apparent binding of eGFP-PH123 at the plasma membrane was μm-2 s-1 The apparent binding upon the concentration of eGFP-PH123 in the the density of binding the phosphoinositol and the concentration of any PH domains. The expression level of eGFP-PH123 was found to be nm by the fluorescence measured in the of cells to that of eGFP in solution by confocal microscopy (9Mashanov G.I. Tacon D. Knight A.E. Peckham M. Molloy J.E. Methods. 2003; 29: 142-152Crossref PubMed Scopus (113) Google Scholar). Using this of the concentration of the apparent second rate constant for is as μm-1 μm-2 s-1. for and experiments were using the first PH domains of myosin X and the domain fused to eGFP to the of each PH domain to the observed binding and release at the plasma of these constructs bound as as and there was a of to bound an illumination of was s-1 for and From the and is as s-1 that at power was of PH to the the position of fluorophores bound at the plasma membrane by the intensity obtained from the of three to a of and position of We found that the in position not with time close to the of position The (e.g. the of movement that one by was only and of the fluorescent spots in position by a one the in position to with such that where time and diffusion in The limited movement of the PH domains found in these experiments to be a of the mouse cell with which we to In experiments using human cells, and T. unpublished findings. we found that the fluorescently tagged PH domains (i.e. using and over many a observation of PH domains have been used previously to the cellular localization of lipids such as PtdIns(3,4,5)P3, PtdIns(4,5)P2, or Here, we have observed individual eGFP-PH123 molecules binding to the of the plasma Steady-state observations of single fluorophores to for binding and of these PH domains the living cell. three PH domains present in the tail of myosin X are for binding to Using a time-lapse method to systematically the laser excitation the of photobleaching and detachment or of eGFP-PH123 were The and apparent on-rate for this binding together with the rate of movement were from the single molecule data From lipid eGFP-PH123 is to bind to PtdIns(4,5)P2 and PtdIns(3,4,5)P3 at the plasma PtdIns(4,5)P2 in cells, whereas of PtdIns(3,4,5)P3, which is by rapid of from PtdIns(4,5)P2 in response to cell are probably in the conditions used studies D. Nat. Biol. 2000; PubMed Scopus Google Scholar, Miller J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, EMBO J. 2001; PubMed Scopus Google Scholar, J. Biol. 2002; PubMed Scopus Google Scholar) that PtdIns(4,5)P2 is to of the plasma membrane such as lipid membrane and of the membrane with (e.g. T. J. Cell Biol. 1998; PubMed Scopus Google Scholar). example, has been that PtdIns(4,5)P2 to actin to the cortex and PtdIns(3,4,5)P3 the region of cortex at which actin D. Nat. Biol. 2000; PubMed Scopus Google Scholar). Although the TIRFM excitation method used that we were to observe in which the cell and of the region of the evanescent the spots that we observe at the plasma membrane for eGFP-PH123 could to lipid or for at of the movement of eGFP-PH123 bound to plasma membrane were in that the fluorophores the of in position for the entire observation measurements T. A. J. Cell Biol. 2002; PubMed Scopus Google Scholar, M. S. Biophys. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, F. M. T. J. Cell Biol. 2000; PubMed Scopus Google Scholar) of the diffusion of fluorescently phospholipids range from to s-1. The diffusion for PtdIns(3,4,5)P3, measured using fused to was single molecules were not imaged in that F. M. T. J. Cell Biol. 2000; PubMed Scopus Google Scholar). The movement observed in the present be by the binding of PH domains either directly or to the actin cytoskeleton a second binding PtdIns(3,4,5)P3 phospholipids be PtdIns(4,5)P2 or the diffusion have been in The limited residency time of eGFP-PH123 molecules at the cell membrane is similar to F. M. T. J. Cell Biol. 2000; PubMed Scopus Google Scholar). This is probably important for dynamic of the cytoskeleton cell movement and and PtdIns(4,5)P2 is to and PtdIns(3,4,5)P3 is to such that membrane ruffling results from of vesicles from the into the plasma membrane Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). X has been to be important in and D. J.S. M. Cheney R.E. S. Nat. Cell Biol. 2002; 4: PubMed Scopus Google Scholar). The present TIRFM studies show that isolated domains bind at the plasma membrane and probably target myosin X to this in The here, together with implies that the PH domains of myosin X probably act to the motor to of actin at the cell The is for myosin X to to in of PtdIns(3,4,5)P3 or PtdIns(4,5)P2 following cell In experiments not reported here, we with a full-length myosin X that within the myosin X motor domain. of cells showed that this full-length molecule to the plasma membrane and to not to the of This that a molecule the one used in the and bind to we used the sequenced full-length myosin X of R. of and found that in mouse myoblasts this molecule as reported for cell (i.e. by to the of the Although we that the motor domain is to actin in the of its is that the tail in the PH domains are for myosin X to (11Berg J.S. Cheney R.E. Nat. Cell Biol. 2002; 4: 246-250Crossref PubMed Scopus (275) Google Scholar) and be important for of It is that the the techniques and methods of data be to In the the of different PH domains with different fluorescent enable the of signaling to be observed directly living cells.