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. 2017 Jun 13;114(24):E4714-E4723.
doi: 10.1073/pnas.1619473114. Epub 2017 May 30.

Myosin Va's adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

Angela Oberhofer  1 , Peter Spieler  1 , Yuliya Rosenfeld  1 , Willi L Stepp  1 , Augustine Cleetus  1 , Alistair N Hume  2 , Felix Mueller-Planitz  3 , Zeynep Ökten  4   5
Affiliations

Myosin Va's adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

Angela Oberhofer et al. Proc Natl Acad Sci U S A. .

Abstract

Pigment organelles, or melanosomes, are transported by kinesin, dynein, and myosin motors. As such, melanosome transport is an excellent model system to study the functional relationship between the microtubule- and actin-based transport systems. In mammalian melanocytes, it is well known that the Rab27a/melanophilin/myosin Va complex mediates actin-based transport in vivo. However, pathways that regulate the overall directionality of melanosomes on the actin/microtubule networks have not yet been delineated. Here, we investigated the role of PKA-dependent phosphorylation on the activity of the actin-based Rab27a/melanophilin/myosin Va transport complex in vitro. We found that melanophilin, specifically its C-terminal actin-binding domain (ABD), is a target of PKA. Notably, in vitro phosphorylation of the ABD closely recapitulated the previously described in vivo phosphorylation pattern. Unexpectedly, we found that phosphorylation of the ABD affected neither the interaction of the complex with actin nor its movement along actin tracks. Surprisingly, the phosphorylation state of melanophilin was instead important for reversible association with microtubules in vitro. Dephosphorylated melanophilin preferred binding to microtubules even in the presence of actin, whereas phosphorylated melanophilin associated with actin. Indeed, when actin and microtubules were present simultaneously, melanophilin's phosphorylation state enforced track selection of the Rab27a/melanophilin/myosin Va transport complex. Collectively, our results unmasked the regulatory dominance of the melanophilin adaptor protein over its associated motor and offer an unexpected mechanism by which filaments of the cytoskeletal network compete for the moving organelles to accomplish directional transport on the cytoskeleton in vivo.

Keywords: intracellular transport; melanophilin; myosin Va; transport regulation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The Mlph subunit of the tripartite complex is specifically phosphorylated by PKA. (A) Schematic illustration of the MyoVa-dependent tripartite transport complex on the melanosome surface. The Rab27a GTPase resides in the melanosome membrane and recruits Mlph in a GTP-dependent manner. Mlph in turn recruits the MyoVa motor to form the tripartite transport complex. (B) Domain structure of the adaptor protein Mlph. The Rab27a-binding domain (Rab27a-BD) is located at Mlph’s N terminus (42, 43). MyoVa binds to Mlph’s middle domain with its globular tail domain and the melanocyte-specific alternatively spliced exon F (MyoVa-BD) (15, 65, 66). The C terminus of Mlph harbors an ABD (23, 24). (C) Tripartite complex reconstituted with 6×His-SNAP–tagged Rab27aS-H, FLAG-tagged MyoVaF, and full-length (lane I) or C-terminally truncated (lanes II and III) FLAG-tagged MlphF purified by Ni-NTA affinity purification. As a control for nonspecific binding, FLAG-tagged MyoVaF was also subjected to Ni-NTA affinity purification (lane IV) (Materials and Methods for details). MW, molecular mass marker. (D) The individually expressed full-length subunits of the tripartite complex along with C-terminally truncated Mlph and MyoVa HMM constructs were treated with PKA and radiolabeled ATP. Autoradiography showed specific phosphorylation of Mlph. Deletion of the C terminus of Mlph significantly decreased the phosphorylation levels. A FLAG-mock purification was included to control for unspecific phosphorylation.
Fig. S1.
Fig. S1.
Alignment of Mlph ABDs from mouse (Mm), human (Hs), Fukomys (Fd), dog (Cl), sheep (Oa), and Xenopus (Xt). Sequence alignment of selected Mlph ABDs revealed numerous conserved serine/threonine residues that represent potential PKA targets. Serine and threonine residues that were found to be phosphorylated in vivo are in bold. Boxes indicate conserved phosphorylatable serine residues. Numbers represent the residue numbers according to MmMlph. Asterisks, colons, and dots indicate positions that are fully, partially, or weakly conserved, respectively.
Fig. S2.
Fig. S2.
Mlph is the sole phosphorylation target of the reconstituted tripartite complex. (A) Coomassie-stained SDS/PAGE image of the reconstituted Rab27a/Mlph/MyoVa transport complex that was subjected to in vitro phosphorylation. (B) PKA specifically phosphorylated Mlph in the presence of Rab27a and MyoVa (autoradiograph).
Fig. 2.
Fig. 2.
Mlph’s phosphorylation state does not interfere substantially with actin binding. (A) Actin decoration experiments were performed with surface-immobilized and Atto488-labeled actin filaments (red). Filaments were incubated with the complex formed between Mlph and Alexa Fluor 647-labeled Rab27a (green). Dephosphorylated (Dephos; Left) and the phosphorylated (Phos; Right) Mlph decorated actin filaments similarly well. Removal of the C-terminal ABD of Mlph (Rab27a/Mlph ΔABD) abolished this interaction regardless of Mlph’s phosphorylation state. (B) The dephosphorylated, Alexa Fluor 488-labeled Rab27a/Mlph complex was mixed in equal amounts with the phosphorylated, Alexa Fluor 647-labeled Rab27a/Mlph complex and was incubated with surface-attached, Atto565-labeled actin filaments. The quantification of the actin-associated fluorescence signals from the respective PKA- and phosphatase-treated Rab27a/Mlph complexes showed that the phosphorylation state of Mlph did not substantially interfere with actin binding. Error bars represent SD. (Scale bars: 3 µm.)
Fig. S3.
Fig. S3.
Mlph’s phosphorylation state does not interfere substantially with actin binding. Dephosphorylated Alexa Fluor 647-labeled Rab27a/Mlph complex (cyan) was mixed in equal amounts with phosphorylated, Alexa 488-labeled Rab27a/Mlph complex (green) and was incubated with surface-attached, Atto565-labeled actin filaments (red; dye swap control for Fig. 2B). The quantification showed results identical to those demonstrated in Fig. 2B: Phosphorylation of Mlph does not alter the interaction between Mlph and actin filaments. Error bars represent SD. (Scale bar: 3 µm.)
Fig. 3.
Fig. 3.
Transport parameters of the tripartite complex on surface-attached actin filaments in single-molecule TIRF assays. The tripartite complex assembled with Mlph that lacked its ABD (A) and the complexes assembled with phosphorylated (B) and dephosphorylated (C) Mlph all moved at consistent velocities. The absence of ABD (A) or the phosphorylation state of the Mlph (B vs. C) did not interfere with the velocities and run lengths of the respective complexes. The majority of the complexes displayed a single-step photobleaching of the SNAP-tagged Rab27a subunit as shown in A–C, Right, demonstrating that the transport parameters are derived from single molecules of Rab27a (also see Fig. S4).
Fig. S4.
Fig. S4.
Bleaching step analysis of the Rab27a/Mlph ΔABD complex labeled on the Rab27a subunit dependent on the laser intensity. For each laser intensity, 112 frames with a frame rate of 223 ms were acquired and analyzed according to the procedure described in Materials and Methods. With decreasing laser intensity, the zero-step population increased strongly relative to the one-step population, as is consistent with photobleaching but not dissociation of the complex from the surface.
Fig. 4.
Fig. 4.
Mlph interacts with microtubules in a phosphorylation-dependent manner. In microtubule decoration experiments Atto488-labeled microtubules (red) were incubated with the Alexa Fluor 647-labeled Rab27a/Mlph complex (green). Decoration of microtubules was strictly dependent on the phosphorylation state of Mlph. The fluorescent background from the phosphorylated and dephosphorylated Rab27a/Mlph complex seen in the green channel was comparable, indicating similar amounts of protein. (Scale bar: 3 µm.)
Fig. 5.
Fig. 5.
Mlph interacts with microtubules via its ABD, and mutations in candidate phosphorylation sites of Mlph prevent PKA-induced dissociation of Mlph from microtubules. (A) Microtubule decoration experiments were performed as in Fig. 4. Removal of the ABD (Rab27a/Mlph ΔABD) abolished the interaction of Mlph with microtubules. (B) PKA-treated Mlph mutants T443A/S445A/T446A, S491/498A, and S544/546/547A all decorated microtubules, but the Dephos control mutant (T392A/S393/396/398/399A/T400A/S401A) did not. (Scale bars: 3 μm.)
Fig. S5.
Fig. S5.
Mutated serine and threonine residues in conserved regions within the ABD of Mlph. Serine and threonine residues in three conserved regions (Fig. S1) were mutated to alanines to mimic the dephosphorylated state. Mutated residues are shown in bold letters. A serine- and threonine-rich stretch outside the ABD was mutated as a negative control.
Fig. S6.
Fig. S6.
Dephosphorylated wild-type Rab27a/Mlph decorates microtubules in vitro, but phosphorylated wild-type Rab27a/Mlph does not. The Rab27a/Mlph complex (green) reconstituted with wild-type Mlph was purified side by side with the Rab27a/Mlph complex containing the point mutations in Mlph’s ABD (see Fig. 5) and used as a control. Consistent with Fig. 4, the dephosphorylated complex decorated microtubules (red), but the phosphorylated Rab27a/Mlph complex failed to decorate microtubules. (Scale bar: 3 μm.)
Fig. S7.
Fig. S7.
Mutations in candidate phosphorylation sites of Mlph do not interfere with F-actin binding. Decoration of fluorescently labeled F-actin (red) with the constructs used in Fig. 5 shows that the interaction of all Rab27a/Mlph complexes containing the respective point mutations (green) with F-actin was similar to the interaction of the wild-type complex with F-actin. (Scale bar: 3 µm.)
Fig. 6.
Fig. 6.
Phosphorylation of S491/498A is the main contributor to phosphorylation-dependent binding of Mlph to microtubules. (A) Nonphosphorylatable alanine mutations in three predicted phosphorylation sites reduced PKA-dependent phosphorylation. In vitro phosphorylation assays (Lower: autoradiograph; Upper: corresponding Coomassie-stained SDS/PAGE as loading control) with wild-type Mlph and its mutants showed that the S491/498A mutant suppressed the PKA-dependent phosphorylation of Mlph more efficiently (26%) than the wild-type (100%) or mutants containing weaker and less conserved PKA consensus sites (76% and 57%). (B) Microtubule cosedimentation assays were performed side by side with the truncated Mlph that lacked its ABD (I, ΔABD), phosphorylated wild-type Mlph (II, 1–590 Phos), dephosphorylated wild-type Mlph (III, 1–590 Dephos) along with phosphorylated Mlph that carried the respective nonphosphorylatable alanine mutations (IV–VI). The total reaction (T), supernatant (SN), and pellet (P) were analyzed with SDS/PAGE. As expected from the results shown in Figs. 4 and 5, Mlph that lacked its ABD and the phosphorylated wild-type Mlph failed to display a pronounced interaction with the microtubules (N/D and 4%, I and II, respectively). Also, in line with results from Fig. 5, the dephosphorylated wild-type Mlph and all rescue mutants displayed significant pelleting with microtubules [27%, 17%, 8%, and 9% (III–VI), respectively]. The mutant S491/498A containing the strong and most conserved PKA consensus site S498 also demonstrated the most pronounced effect of rescue (IV). Percentages are obtained from two independent assays ± SD. N/D, not determinable.
Fig. S8.
Fig. S8.
Cosedimentation assay of microtubules (2 µM) with a reduced amount (3 µM) of dephosphorylated wild-type Mlph (1-590 Dephos). Forty-seven percent of the total amount of Mlph protein pelleted with microtubules, noticeably more than in Fig. 6, where excess Mlph (5 µM) was used and only 27% pelleted. These observations suggested that binding sites for Mlph on microtubules become limiting when Mlph exceeds the concentration of microtubules.
Fig. 7.
Fig. 7.
Dephosphorylation is sufficient to relocate Mlph from actin to microtubules efficiently. Surface-immobilized and Atto565-labeled actin filaments (red; Upper) and Atto488-labeled microtubules (red; Lower) were incubated with phosphorylated (A) and dephosphorylated (B) complex formed between Mlph and Alexa Fluor 647-labeled Rab27a (green). (A) The phosphorylated Rab27a/Mlph complex largely ignored microtubules (MT) and associated with actin filaments (17 ± 4% vs. 83 ± 4%). (B) Upon dephosphorylation, the behavior of the Rab27a/Mlph complex was reversed, and the microtubule binding clearly dominated (76 ± 4%) over the actin binding (24 ± 4%). Error bars represent SD. (Scale bar: 3 µm.)
Fig. S9.
Fig. S9.
Dephosphorylation is sufficient to relocate Mlph from actin to microtubules efficiently. Alternative analysis of decoration assays with phosphorylated (Left) or dephosphorylated (Right) Rab27a/Mlph complex on both microtubules and actin. The Mlph intensity bound to each filament type was normalized to the total length of this filament type present in the channel. Error bars represent SD. a.u., arbitrary units.
Fig. S10.
Fig. S10.
Dephosphorylated Rab27a/Mlph/MyoVa complexes (green) in the presence (Left) or absence (Right) of ATP on surface-attached networks of actin (blue) and microtubules (red) in vitro. (Scale bar: 10 µm.)
Fig. 8.
Fig. 8.
Phosphorylation state of Mlph’s ABD dictates the directionality of switching at the actin–microtubule intersections. The movement of the tripartite complex on actin and microtubules is represented by single- and double-headed black arrows, respectively. Cyan arrows depict switching from actin to microtubules; red arrows indicate switching from microtubules to actin. (A and B) The tripartite complex reconstituted with dephosphorylated Mlph displayed a significantly higher probability of switching from actin to microtubules at the interfilament intersections (A). Although 32.3% of complexes switched from actin to microtubules (67.7% continued directional movement on actin; A), the propensity of switching from actin to microtubules was abolished when the tripartite complex was assembled with phosphorylated Mlph (B). The phosphorylated complex completely ignored the interfilament intersections (0% switching) and continued its directional movement on the actin (100%; B). Conversely, dephosphorylated Mlph significantly suppressed the probability of the complex switching from microtubules to actin (26.1%; A) compared with the complex built with phosphorylated Mlph (100%; B). Indeed, the phosphorylated complex rarely interacted with the microtubules, substantially decreasing the probability of switching events from microtubules to actin (Movie S1). (C and D) In contrast, tripartite complexes assembled with dephosphorylated Mlph ΔABD (C) and phosphorylated Mlph ΔABD (D) displayed similar probabilities of switching between the two filament types, confirming that phosphorylation outside Mlph’s ABD does not interfere with the switching behavior of the tripartite complex. N indicates the number of events for each switching direction.
Fig. S11.
Fig. S11.
Examples of switching events at the actin/microtubule intersections. (A) Example of a tripartite complex (green) that diffused on a microtubule (red) and switched to an intersecting actin filament (blue) (Movie S3). (B) Example of a tripartite complex that moved directionally on the actin filament and switched to an intersecting microtubule (Movie S4). (Scale bars: 3 µm.)
Fig. 9.
Fig. 9.
Proposed model for regulating the affinities of the moving organelles on the microtubule and actin networks in vivo. In our mechanistic dissection, we unmasked the regulatory dominance of the adaptor protein Mlph over its associated motor. Even though MyoVa is an actin-associated motor, the dephosphorylation of Mlph’s ABD was sufficient to redirect the MyoVa from directional movement on the actin network to microtubules for diffusive movement. Consequently, the phosphorylation state of Mlph’s ABD regulated the probability of directional switching of MyoVa between the microtubule and actin networks. Based on these findings, we propose that Mlph serves to bias the transport of organelles on the microtubule or actin networks in vivo. (A) Specifically, phosphorylation of Mlph’s ABD promotes MyoVa-dependent motility on the actin network by suppressing the affinity of the tripartite complex toward microtubules. (B) To reverse this process, Mlph is dephosphorylated to increase the affinity of the tripartite complex for the microtubule network.
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