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

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.melanophilin | myosin Va | intracellular transport | transport regulation

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“…89 Similarly, the actin-binding domain of the adapter protein Melanophilin could be phosphorylated and change MYO5A's preference from actin to microtubules. 90 This evidence also adds a layer of complexity that not only the motor domain but also the adapters could dictate crosstalk between these transport systems. Despite these exciting findings, the subject of filament switching still requires further investigation and validation in other cell systems.…”

“…One study bridged this gap where it modeled MYO5A gliding over microtubules, suggesting a mechanism that allows the switching of microtubule and actin cargoes . Similarly, the actin‐binding domain of the adapter protein Melanophilin could be phosphorylated and change MYO5A's preference from actin to microtubules . This evidence also adds a layer of complexity that not only the motor domain but also the adapters could dictate crosstalk between these transport systems.…”

The road to lysosome‐related organelles: Insights from Hermansky‐Pudlak syndrome and other rare diseases

“…This results in hypopigmentation of the skin and hair in GS1, 2 and 3 patients and corresponding mouse models . Interestingly, MLPH that has not been phosphorylated by protein kinase A can bind to microtubules in vitro and relocate the associated MYOVA and RAB27A from actin filaments to microtubules; this might provide a mechanism for protein kinase A‐dependent regulation of melanosome localization in vivo . MLPH function in RAB27A‐dependent organelle motility appears to be limited to skin melanocytes, and hence GS3 patients display pigment dilution in the skin and hair, but not in the eye .…”

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“…DrMlph-b, DrMlph-bX2, DrMyoVa, XlRab27a, XtMlph, XtMlphΔFBD, and XlMyoVa were described 322 earlier (13,28). DrMlph-b-TPQP was synthesized with residues 551 to 554 changed to TPQP.…”

“…Protein expression, purification, fluorescent labeling, and dephosphorylation/phosphorylation 324 and reconstitution of tripartite complexes 325 All procedures were performed as described in (13,28 Rab27a/Mlph/MyoVa complexes were flowed into these chambers and reconstitution assays were 336 carried out with 4 mM ATP as described previously (28). Movies were acquired at room temperature 337 with the microscope setup described earlier (28). were for 1 hour at 27°C or 4°C for nocodazole conditions, respectively.…”