PTEN hopping on the cell membrane is regulated via a positively-charged C2 domain

Abstract

PTEN, a tumor suppressor that is frequently mutated in a wide spectrum of cancers, exerts PI(3,4,5)P3 phosphatase activities that are regulated by its dynamic shuttling between the membrane and cytoplasm. Direct observation of PTEN in the interfacial environment can offer quantitative information about the shuttling dynamics, but remains elusive. Here we show that positively charged residues located in the cα2 helix of the C2 domain are necessary for the membrane localization of PTEN via stable electrostatic interactions in Dictyostelium discoideum. Single-molecule imaging analyses revealed that PTEN molecules moved distances much larger than expected had they been caused by lateral diffusion, a phenomenon we call "hopping." Our novel single-particle tracking analysis method found that the cα2 helix aids in regulating the hopping and stable-binding states. The dynamically established membrane localization of PTEN was revealed to be essential for developmental processes and clarified a fundamental regulation mechanism of the protein quantity and activity on the plasma membrane.

Figure 1. Phenotype of wild-type PTEN and PTEN_i mutants (i = 1,2,…,7).

(A) Human PTEN crystal structure (residues 14–351) . PTEN_i mutants had different positive charges in the cα2 helix. Regions colored in green and blue show the C2 domain and phosphatase domain, respectively. The red, yellow, and orange regions show the cα2 helix, T1 loop, and CBR3 loop, respectively. The PBM at the N-terminus (residues 1–13) and 24 residues in the C2 domain (residues 282–312) are not shown. The upper side of the structure faces the membrane. (B) Fluorescence images of Dictyostelium discoideum cells expressing wild-type PTEN or PTEN mutants. PTEN was labeled with TMR via HaloTag (PTEN-Halo-TMR). Images were captured by confocal microscopy. Scale bar, 5 µm. (C) The ratio of the plasma membrane and cytoplasm fluorescence intensities. (D) Images of fruiting bodies formed by wild-type cells or pten-null cells expressing wild-type PTEN or PTEN mutants. Scale bar, 500 µm (E) The diameter of the sorus in the fruiting bodies. Data are mean +/− SD.

Figure 2. Single-molecule imaging of wild-type PTEN and PTEN_i mutants.

(A) Images of cells expressing wild-type PTEN or PTEN_i mutants labeled with TMR captured under TIRFM. Scale bar, 5 µm. (B) The numbers of PTEN-Halo-TMR and PTEN_i-Halo-TMR molecules that remained bound to the membrane are plotted against time after membrane association. Lines are three-component exponential fits (Eq. 1). The cumulative plots were obtained from 16,088 molecules in 8 cells (wild-type PTEN), 12,164 molecules in 8 cells (PTEN₁), 9,022 molecules in 7 cells (PTEN₂), 11,386 molecules in 8 cells (PTEN₃), 12,406 molecules in 8 cells (PTEN₄), 11,079 molecules in 8 cells (PTEN₅), 10,822 molecules in 7 cells (PTEN₆) and 20,683 molecules in 8 cells (PTEN₇). (C, D) Dissociation constants k _1–3 (C) and frequencies A _1–3/k _1–3 (D) of PTEN_i mutants obtained from the fitting in (B). Estimated parameters are shown in Table 2. (E) Frequency of slow, moderate and fast diffusion mobility states, a_i, from the displacement distribution analysis in Fig. S3.

Figure 3. PTEN molecules hopping on the cell membrane.

(A, B) Hopping occurred between the second and third frames. (C, D) Hopping occurred within the second frame. (E, F) Blinking. (A, C, E) Images of single molecules. (B, D, F) Fluorescence intensity profiles along the long axis of the rectangles shown in A, C and E. (G) Cloud-like fluorescence (dotted rectangle at t = 66 msec) of hopping molecules. Numbers in the upper right of each panel are time in milliseconds. Scale bar, 1 µm. (H) Trajectories of molecules showing hopping, blinking and lateral diffusion. Thick lines show hopping displacements. Colors in the trajectories indicate the moments of hopping shown in (B) and (D).

Figure 4. The analysis method for hopping molecules proposed in this study.

(A) Two coordinate systems are introduced. In the global coordinate system, O(X,Y,T), the time origin is the initial frame and the spatial origin is the lower-left corner of the image. In the coordinate system about the j-th molecule, o_j(x_j,y_j,t_j), the time origin is the vanished time and the spatial origin is the vanished position of the molecule. (B) Membrane-associating molecules are classified into two types: those rebinding to the membrane after hopping (hopping molecule) and those recruited from the cytoplasm (recruited molecule). (C) The spatial distribution of wild-type PTEN trajectories (blue) observed in a single cell. Scale bar, 5 µm. (D) The number of molecules appeared in a single cell after excitation of the fluorophore. Time interval, 1.666 sec. (E) Schematics of the simulation of hopping molecules. (F) Results of the rebinding probability estimated from simulated molecules. Data are mean +/− SD.

Figure 5. Rebinding probability analysis.

(A) Rebinding probabilities of wild-type PTEN, PTEN mutants and Latrunculin A treated PTEN₄ mutant. The probability that a molecule rebinds to the membrane between 0.45 and 2 µm from the vanished position and within 133 msec after the vanished time was calculated using Eq. 9. * and ** indicate p<0.05 and p<0. 01 obtained by Student's t-test, respectively. (B) Spatial distribution of rebinding probabilities of wild-type PTEN (orange) and PTEN₄ (blue). The spatial distributions were obtained between 0 and 33 msec (upper) and 33 and 66 msec (lower) after spots vanished (right panels). The bin range is 0.05 µm. Typical images of hopping molecules that rebind between 0 and 33 msec (upper) and 33 and 66 msec (lower) are shown (left panels). The minimum limit of the analysis is 0.45 µm from the previous position (white dotted circles). Trajectories before hopping are shown in yellow. (C) Temporal changes in rebinding probability of wild-type PTEN (upper) and PTEN₄ (lower). Data are mean +/− SD. (D) Typical images showing a succession of jumps and the corresponding trajectory (yellow). Numbers are time in milliseconds. Scale bar, 1 µm. (E) Hopping lifetimes of wild-type PTEN, PTEN₄ and PTEN₇.

Figure 6. Effect of actin filaments on PTEN hopping.

(A) Hopping lifetimes of PTEN₄ in the absence (blue) and presence (red) of Latrunculin A. (B) Spatial distribution of the rebinding probability of PTEN₄ in the absence (blue) and presence (red) of Latrunculin A. (C) Temporal changes in the rebinding probability of PTEN₄ in the absence (blue) and presence (red) of Latrunculin A. Data are mean +/− SD.

Figure 7. A “search-and-stabilization” model describes the role of the cα2 helix in the PTEN-membrane interaction.

PTEN mainly adopts three states on the membrane: a stabilized state (S ₁), two cα2-related states (S ₂, S ₃) plus a hopping state. The cα2 helix is involved in suppressing the membrane dissociation of PTEN by directly regulating the latter two states. When the state changes from S ₂ or S ₃ to S ₁, the membrane interaction becomes stabilized and the substrate PI(3,4,5)P₃ is more accessible. In the S ₂ and S ₃ states, PTEN exhibits membrane dissociation at a faster rate than in the S ₁ state. Of the dissociating molecules, 8% rebind to the membrane after hopping, which offers a chance for PTEN to search for the substrate again.