Molecular basis of dynamic relocalization of Dictyostelium myosin IB

Abstract

Class I myosins have a single heavy chain comprising an N-terminal motor domain with actin-activated ATPase activity and a C-terminal globular tail with a basic region that binds to acidic phospholipids. These myosins contribute to the formation of actin-rich protrusions such as pseudopodia, but regulation of the dynamic localization to these structures is not understood. Previously, we found that Acanthamoeba myosin IC binds to acidic phospholipids in vitro through a short sequence of basic and hydrophobic amino acids, BH site, based on the charge density of the phospholipids. The tail of Dictyostelium myosin IB (DMIB) also contains a BH site. We now report that the BH site is essential for DMIB binding to the plasma membrane and describe the molecular basis of the dynamic relocalization of DMIB in live cells. Endogenous DMIB is localized uniformly on the plasma membrane of resting cells, at active protrusions and cell-cell contacts of randomly moving cells, and at the front of motile polarized cells. The BH site is required for association of DMIB with the plasma membrane at all stages where it colocalizes with phosphoinositide bisphosphate/phosphoinositide trisphosphate (PIP(2)/PIP(3)). The charge-based specificity of the BH site allows for in vivo specificity of DMIB for PIP(2)/PIP(3) similar to the PH domain-based specificity of other class I myosins. However, DMIB-head is required for relocalization of DMIB to the front of migrating cells. Motor activity is not essential, but the actin binding site in the head is important. Thus, dynamic relocalization of DMIB is determined principally by the local PIP(2)/PIP(3) concentration in the plasma membrane and cytoplasmic F-actin.

FIGURE 1.

Colocalization of endogenous DMIB and PIP₂/PIP₃ in fixed AX3 cells. AX3 cells were fixed and endogenous DMIB, and PIP₂/PIP₃ were visualized with antibodies against DMIB (red) and PIP₂/PIP₃ (green). F-actin was stained with phalloidin Alexa Fluor 633 (blue). In nonpolarized cells, DMIB colocalizes with PIP₂/PIP₃ in random cell protrusions (A). In polarized cells, DMIB and PIP₂/PIP₃ colocalize at the cell front (B). In chemotaxing cells, DMIB and PIP₂/PIP₃ colocalize at the front of the leading cell and in the engulfing mouth of the following cell (C). Actin colocalizes with DMIB and PIP₂/PIP₃ at random cell protrusions (D) and at the front of polarized cells (E). Arrows mark the sites of colocalization of DMIB with PIP₂/PIP₃ (A–C) and with F-actin (D and E). Scale bars, 10 μm. DIC, differential interference control microscopy.

FIGURE 2.

Localization of expressed GFP-DMIB in live cells. AX3 cells were cotransfected with DMIB and ABD-120 fused to GFP (green) and RFP (red), respectively. Live cells images are shown. DMIB is localized uniformly on the plasma membrane of freshly plated cells (A), in pseudopods (B), cups (C), and at cell-cell contacts (D) of randomly moving cells. DMIB is mostly diffuse in cells starved for about 4 h (E) and is localized to the cell front in elongated polarized cells (F). In all cases except E, DMIB colocalizes with F-actin. Arrows mark sites of DMIB and F-actin colocalization. Scale bar, 10 μm. See also supplemental Movie S1.

FIGURE 3.

Schematic representation of DMIB and its mutants expressed in Dictyostelium cells. The major regions that were deleted are labeled (S = SH3). The positions of residues mutated within the head and the position of BH site are indicated. The point mutations in the head in full-length DMIB were: S332A (DMIB-S332A), which results in motor-dead myosin, and E407K (DMIB-E407K), which results in severely reduced binding to F-actin. Mutations of the BH sites in full-length DMIB, dGPQSH3, and the tail were: deletion of entire BH site (dBH), substitution of 5 basic residues within the BH site with Ala (BH-Ala), and point mutation I810D. See supplemental Fig. S2 for more detailed description of DMIB-I810D.

FIGURE 4.

BH site is necessary for localization of DMIB to the plasma membrane. Constructs are identified as described in Fig. 3 and “Results.” A, localization of GFP-labeled DMIB, DMIB deletion constructs, PH-PLCδ, and PH-CRAC in AX2 myoB⁻ cells newly plated in nonnutrient buffer. B, localization of mutants with BH site deleted (dBH) or mutated (BH-Ala, I810D). Left panels in A and B show GFP images, and right panels show differential interference contrast microscopy images. Scale bar, 10 μm.

FIGURE 5.

DMIB head is required for dissociation of DMIB from the plasma membrane, and BH site is required for DMIB localization to cell-cell contacts in randomly moving cells. A, within about 2 h, DMIB (supplemental Movie S2) relocates to cell protrusions whereas Tail remains uniformly concentrated on the plasma membrane (supplemental Movie S3). B, DMIB, dGPQSH3 (supplemental Movie S4), DMIB-S332A, and DMIB-E407K are enriched at random cell-cell contacts, where PIP₂ (PH-PLCδ) also concentrates, and are absent from other regions of the plasma membrane. Tail remains relatively uniformly associated with the plasma membrane and DMIB-BH-Ala, and Head+IQ remains diffused in the cytoplasm. Scale bars, 10 μm.

FIGURE 6.

DMIB head is required for relocation of DMIB to front of polarized cells. Images of live starved elongated cells expressing proteins as marked at the top of the panels are shown. DMIB and Head+IQ show the same diffuse localization at the front as does motor-dead DMIB mutant (DMIB-S332A) and DMIB minus GPQ and SH3 domains (dGPQSH3). Tail and PIP₂ (as monitored with PH-PLCδ) localize mostly uniformly on the plasma membrane with enrichment at the rear. DMIB point mutant with weakened actin binding (DMIB-E407K) localizes on plasma membrane with strong enrichment at the rear but shows stronger cytoplasmic presence than does Tail. Arrows mark the direction of cell movement. Scale bar, 10 μm. See also supplemental Movies S5–S7.

FIGURE 7.

Localization of DMIB in streaming cells. Images of live streaming cells expressing proteins as marked at the top of the panels are shown. Expressed DMIB colocalizes with PIP₃ (PH-CRAC) at the engulfing mouth of cells in chemotaxing streams as does motor-dead DMIB-S332A and DMIB missing the GPQ and SH3 domains (dGPQSH3). Head+IQ and mutant missing BH site (dGPQSHdBH) are fully cytoplasmic. Expressed Tail and PIP₂ (PH-PLCδ) remain mostly uniformly distributed on the plasma membrane. DMIB point mutant with weakened actin binding (DMIB-E470K) also localizes primarily to the plasma membrane but has a higher cytoplasmic component than does Tail. See also supplemental Movies S8–S11.

FIGURE 8.

Comparison of cytoplasmic and membrane-associated fractions of DMIB, Tail, DMIB-N154A, and dGPQSH3. A and B, fluorescence images of freshly plated Dictyostelium cells expressing DMIB or Tail were scanned. Examples of individual linear cross-scans for DMIB (A) and Tail (B) are shown. Each cell was scanned twice across two different lines. Scans were normalized for each cell taking the average maximum fluorescence intensity of membrane peaks as 100%. The ratios of the maximum fluorescence intensity on the plasma membrane to the average fluorescence intensity in the cytoplasm for single cells shown in A and B were 1.7 and 3.6, respectively, as indicated at the sides of the panels. C, ratio of the maximum fluorescence intensity on the plasma membrane to the average fluorescence intensity in the cytoplasm in five experiments. Each pair of bars represents a separate experiment, and the number of cells scanned in each experiment is indicated on the top of bars. D, average of fluorescence ratios for DMIB, dGPQSH3, DMIB-E407K, and Tail. The average fluorescence ratios are indicated at the tops of the bars. The number of scanned cells and independent experiments were as follow: 35 cells from 5 experiments for DMIB, 22 cells from 3 experiments for dGPQSH3, 30 cells from 3 experiments for DMIB-E407K, and 40 cells from 5 experiments for Tail. Error bars, S.E.

FIGURE 9.

Localization of DMIB-N154A. Aa, freshly plated cells. Ab, cells starved for 2 h. Ac, cells starved for 8 h. B, more detail of DMIB N154A distribution in cells represented by cell in Ab. C, more detail of DMIB N154A distribution in cells represented by cell in Ac. Arrows point to the sites of DMIB N154A location. Scale bars, 10 μm.

FIGURE 10.

Relocation of DMIB to plasma membrane in cells treated with LatA. AX3 cells cotransfected with DMIB and F-actin probe ABD-120 were starved for 4 h and treated with 7.5 μm LatA. A, in cells before treatment DMIB is mostly diffused and cortical actin is present. B and C, after 20-min exposure to LatA, cortical F-actin is absent, and DMIB reappears on the plasma membrane, alone (arrowheads) or accompanied by F-actin patches (arrows). Scale bar, 10 μm.

FIGURE 11.

Localization of dGPQSH3 in PI3K1–5⁻,PTEN⁻ cells. Freshly plated cells (A) and cells starved for 6 h (B) are shown. Arrows indicate the direction of cell movement. Scale bars, 10 μm.