Exploring the iceberg: Prospects of coordinated myosin V and actin assembly functions in transport processes

ABSTRACT

Spir actin nucleators and myosin V motor proteins were recently discovered to coexist in a protein complex. The direct interaction allows the coordinated activation of actin motor proteins and actin filament track generation at vesicle membranes. By now the cooperation of myosin V (MyoV) motors and Spir actin nucleation function has only been shown in the exocytic transport of Rab11 vesicles in metaphase mouse oocytes. Next to Rab11, myosin V motors however interact with a variety of Rab GTPases including Rab3, Rab8 and Rab10. As a common theme most of the MyoV interacting Rab GTPases function at different steps along the exocytic transport routes. We here summarize the different transport functions of class V myosins and provide as proof of principle data showing a colocalization of Spir actin nucleators and MyoVa at Rab8a vesicles. This suggests that besides Rab11/MyoV transport also the Rab8/MyoV and possibly other MyoV transport processes recruit Spir actin filament nucleation function.

Rab family GTPases as master regulators of intracellular transport

The polarized nature of higher order eukaryotic cells, including transmembrane adhesion and signaling receptors for setting up communicating cellular networks, requires a sophisticated protein transport machinery, which enables the directional delivery of proteins. Despite the enormous progress in molecular cell biologic methodology, including live cell imaging technologies and detailed information on the molecular components as provided by the genome projects, our knowledge of polarized intracellular transport processes is still incomplete. The study of Rab (Ras-like in brain) small GTPases as members of the Ras superfamily, which function as molecular switches in the regulation of membrane transport processes, is indispensable to disclose the secrets of directional transport. Besides the regulation of the Rab molecular switches by incoming signal transduction cascades, unravelling the exact composition of protein complexes organized by the Rab GTPases at vesicle and endosomal membranes will be essential for our understanding of cellular transport. So far, 65 members of the Rab family have been identified in the mammalian genomes, divided into 9 functional groups.^1,2 The large diversity of Rab proteins is mainly explained by gene duplications during evolution. Individual Rab GTPases are localized to specific intracellular membrane compartments,^1-6 where they mediate their functions in membrane transport by recruiting specific effector proteins, such as vesicle coat proteins, motor proteins, tethering factors and signaling proteins, toward membranes.² The targeting of Rab proteins to intracellular membranes is achieved by posttranslational modifications in which geranylgeranyl lipid residues are linked to C-terminal cysteine residues of newly translated Rab proteins.²

Rab / myosin V motor protein complexes

A major role of Rab GTPases is the assembly of motor protein complexes to provide forces for vesicle transport along cytoskeletal filaments. The microtubule network serves as tracks for fast and long-range transport toward distinct cellular regions. Actin filament based transport is in an order of a magnitude slower compared with microtubule dependent processes, but allows transport beyond the microtubule networks to reach outlying regions of the cell and to provide delivery to the desired subcellular locales.⁷ To achieve vesicle transport along cytoskeletal tracks, Rab GTPases exert their function by recruiting motor proteins toward the vesicle surface, including the microtubule based kinesin and dynein motors, and the actin based myosin motors to form cargo associated motor protein complexes.

The formation of such Rab GTPase / motor protein complexes is well described for the unconventional class V myosin actin motors which have been shown to associate with Rab GTPases at vesicle membranes in both, yeast and higher order eukaryotic cells. Class V myosins are conserved across almost all eukaryotic species.⁸ In contrast to the conventional myosin II, which is involved in muscle contraction, myosin V (MyoV) proteins are actin filament associated motor proteins which processively walk along F-actin tracks to move cargoes throughout the cell.⁹ Three myosin V isoforms exist in mammals: MyoVa, MyoVb and MyoVc (Fig. 1, 2).^9-12 MyoVa and MyoVb are expressed throughout the body,¹³ including the brain.^12,14-18 Myosin Vc is highly abundant in epithelial and glandular tissues, and is mainly expressed in epithelial cells.¹²

Figure 1.

Functions of class V myosin motor proteins in intracellular transport processes. The figure summarizes the interactions of MyoV actin motors with Rab GTPases and indicates the distinct vesicular, endosomal and organell localizations of the Rab/MyoV complexes. Endocytic events are drawn in yellow, exocytic events in green. In general, Rab GTPases are crucial for exocytosis from post-Golgi vesicles (e.g., Rab3A) and by recycling pathways (e.g., Rab11), but also for endocytic uptake (not shown here). Rab8 is involved in recycling processes via macropinocytosis, a tubular membrane network and exocytic vesicles, e.g., for recycling of the transferrin receptor. Rab10 is localized to the same tubular network and might thus function in a similar way. A number of Rab GTPases are critically involved in different steps of melanosome biogenesis, maturation and transport (Rab32/Rab38, Rab7a, Rab8a, Rab27a), arising from the early endosome (EE) and the recycling endosome (RE), and in the release of melanin for subsequent endocytic uptake by keratinocytes (Rab11b). Rab GTPases are also involved in intra-organelle trafficking (e.g., Rab6). Not much is known about the role of Rab GTPases in mitochondria function, but a body of evidence exists for MyoV proteins mediating mitochondria dynamics, including fission and motility. EE, early endosome; LE/MVB, late endosome/multi-vesicular body; RE, recycling endosome; ER, endoplasmatic reticulum; TGN, trans-Golgi network; GLUT4, glucose transporter type 4; Dm-MyoV, Drosophila melanogaster MyoV.

Myosin V actin motor proteins functionally interact with a multitude of Rab family GTPases and are thus involved in diverse cellular functions (Fig. 1). So far, a body of direct and indirect interactions of myosin Va motors and Rab proteins has been revealed, including Rab3A,¹⁹ Rab3b, Rab3c, Rab3d, Rab6a, Rab6a’, Rab6b,²⁰ Rab8a, Rab10, Rab11a,¹³ Rab11b, Rab14, Rab11c (or Rab25),²⁰ Rab27a,²¹ Rab27b²² and Rab39b.²⁰ Rab3A as well as Rab27b form complexes with myosin Va to enable exocytosis of synaptic vesicles for synaptic transmission,^19,22 and also Rab39b is a neuron specific Rab GTPase involved in neurite transport.²⁰ Myosin Vb directly interacts with Rab8a, Rab10 and Rab11a.^13,23,24 Myosin Vc directly interacts with Rab10,¹³ and interactions with Rab GTPases have been shown to drive melanosome biogenesis and maturation as it is associated with Rab38 and the closely related Rab32,²⁵ Rab7a at immature melanosomes, and Rab8a at mature melanosomes (Fig. 1).^26,27

The MyoV heavy chain is composed of the N-terminal motor domain which binds to actin filaments and harbours the actin dependent ATPase activity for force generation (Fig. 2). Six IQ motifs bind calmodulin light chains and an elongated coiled-coil region is required for heavy chain dimerization. The very C-terminal globular tail domain (GTD) is considered as the cargo binding domain (Fig. 2A).¹⁸ Both, the coiled-coil regions and the globular tail domain bind distinct Rab GTPases (Fig. 2B). The globular tail domain of myosin Va interacts with Rab3 family GTPases^19,20 and with Rab39b.²⁰ MyoVa and MyoVb GTDs interact with Rab11 family GTPases.^13,20 A short sequence within the coiled-coil regions of myosin Va mediates interactions with Rab6 family GTPases and Rab14.²⁰ Alternatively spliced exons within the myosin V C-terminal coiled-coil regions specifically determine the interaction of the motor proteins with several Rab GTPases (Fig. 2B).¹³ The MYO5A gene encodes the 6 alternatively spliced exons A, B, C, D, E and F, whereas MYO5B has only 5 as it misses exon F sequences. Particularly, the 3 exons B, D and F undergo alternative splicing in myosin Va (Fig. 2B).^11,28,29 In the process of melanosome transport, exon F supports the association of myosin Va with melanophilin (MLPH) to form a functional complex with Rab27a (see below) (Fig. 2B, Fig. 3A). Exon B plays a role in MyoVa interaction with dynein light chain 2 (DLC2), which, however, is not conserved for exon B of MyoVb.^30,31 Exon D is required for MyoVa binding to Rab8a and Rab10. MyoVb exon D is also essential for Rab10 interaction, but, in contrast to MyoVa exon D, which also interacts with Rab8a, the MyoVb alternate exon D sequences inhibit binding to Rab8a.¹³ MyoVc proteins contain regions within their coiled-coil domains that resemble the sequences of exon D, exon E and exon F, which are also critical for binding to distinct Rab GTPases. (Fig. 2B)^13,25 MyoVc binding to Rab8a and Rab10 depends on the exon D- and exon E-like regions.^13,25 Two Rab GTPases which are essential for melanosome biogenesis, Rab38 and the related Rab32, have partially overlapping binding regions with Rab38 binding to exon E-like and exon F-like regions and Rab32 binding to the exon F-like region (Fig. 1, Fig. 2B).²⁵

Figure 2.

The mammalian class V actin motor proteins. (A) Mammalian MyoV proteins contain an N-terminal motor domain (also called head) which binds to actin filaments and mediates the actin dependent ATPase activity. Six IQ motifs each bind calmodulin light chains and are also referred to as neck and forming the lever arm required for forward movement. The C-terminal tail is divided into a coiled-coil region which is periodically interrupted and required for heavy chain dimerization, and the very C-terminal globular tail domain (GTD) which is the cargo binding domain by binding to specific membrane receptors, and a major protein interaction site. (B) Schematic representation of GTPase binding sites and alternatively spliced exons of mammalian MyoVa and MyoVb proteins and the corresponding regions in MyoVc. Rab3 and Rab11 family GTPases and Rab39b bind to the globular tail domain of MyoVa and also the MyoVb GTD binds Rab11. A binding site for Rab6 and Rab14 is present within the coiled-coil region of MyoVa. Alternatively spliced exons are located within the coiled-coil regions of the MyoV-tail (exons A, B, C, D, E and F for MyoVa, and exons A, B, C, D and E for MyoVb). Three exons are particularly subjected to alternative splicing in MyoVa: exons B, D and F (drawn in red). Exon B mediates interaction with dynein light chain 2 (DLC2). Exon D is essential for MyoVa interaction with Rab8a and Rab10. The melanocyte specific exon F is required for efficient interaction with melanophilin (MLPH). Only 2 exons in particular undergo alternative splicing in MyoVb: exons B and D (drawn in red). A specific function for exon B has not been demonstrated so far. Exon D mediates MyoVb interaction with Rab10, similar to MyoVa, but, in contrast, inhibits its interaction with Rab8a, which binds to the same region in absence of exon D. There is no exon F present in MyoVb. The MyoVc protein does not contain alternatively spliced exons per se, but exon-like regions are present which resemble the sequences of MyoVa/b exons D, E and F and which are required for Rab GTPase interactions. Rab8a and Rab10 bind to exon D- and exon E-like regions. Rab38 binding needs presence of exon E- and exon F-like regions and Rab32 binding depends on exon F-like regions. Numbers on the protein domains indicate amino acids for mouse (Mm) MyoVa, human (Hs) MyoVb and human (Hs) MyoVc; aa, amino acids; IQ, isoleucine/glutamine; GTD, globular tail domain; aa, amino acids.

Figure 3.

Rab/MyoV motor protein complexes at distinct vesicle membranes. (A) The tripartite Spir/MyoV/Rab11 complex. Both, MyoVa and MyoVb can form a tripartite complex with Rab11 and Spir proteins at vesicle membranes (left panel), which depends on the Spir/MyoV interaction mediated by the MyoV globular tail domain and the Spir GTBM. Formation of such complexes coordinates the Spir/FMN mediated actin nucleation activity and the Rab11/MyoV based force generation and is supposed at vesicle populations which require de novo actin nucleation for transport. The functional interplay of Spir, FMN-2, Rab11 and MyoVb proteins is fundamental for oocytic vesicle transport and oocyte maturation. The tripartite Rab27a/MLPH/MyoVa complex (middle panel) is critical to drive peripheral melanosome transport in melanocytes as the base for skin and hair pigmentation. Important, MLPH specifically interacts with the MyoVa isoform and has 2 contact sites, the GTBM (similar to Spir) and the exon-F binding domain (EFBD) binding to exon F encoded MyoVa sequences (light green). MyoVb forms a complex with Rab11 and the Rab11-family interacting protein Rab11-FIP2 at recycling endosomes to drive recycling endosome trafficking, such as the activity dependent insertion of AMPA receptor subunits into the postsynaptic densities of dendritic spines (right panel). Rab11-FIP2 contains a membrane binding C2 domain which might stabilize the complex at vesicle membranes. (B) Spir and MLPH proteins share similar domain organizations and functional units. Both proteins encode a MyoV/MyoVa interaction unit in their central regions (GTBM for Spir-2; GTBM and EFBD for MLPH). Both proteins express a membrane targeting unit at their C-terminus (Spir-2) and N-terminus (MLPH), respectively, consisting of a FYVE-type zinc-finger and a related Spir-box (SB in Spir-2) and the Spir-box related synaptotagmin-like protein homology H1 and H2 domains in MLPH. The MLPH-H1-FYVE-H2 cluster is essential for MLPH interaction with Rab27a.⁵⁷ Spir-2 encodes the actin nucleating KIND/WH2 domains at its N-terminus, and MLPH encodes an F-actin binding domain (ABD) at its C-terminus. KIND, kinase non-catalytic C-lobe domain; WH2, Wiskott-Aldrich syndrome protein homology 2; GTBM, globular tail domain binding motif; FYVE, after Fab1, YOTB/ZK632.12, VAC1, EEA1; ABD, actin binding domain.

Rab11 / MyoV dependent transport processes

One of the best studied Rab GTPase / MyoV motor protein complexes includes the Rab11 GTPase and MyoVa and MyoVb, respectively.^{13,20,24,32,33} Four Rab11 contact site residues which are conserved between MyoVa and MyoVb are substituted by nonhomologous residues within the MyoVc sequence,²⁴ which explains why MyoVc does not bind to Rab11.¹³ The Rab11 protein regulates exocytic and recycling transport processes of plasma membrane components^7,23 as it is localized to the trans-Golgi network (TGN) and post-Golgi vesicles to support secretory pathways,³⁴ and to the pericentriolar recycling endosome, thus participating in late recycling.³⁵ A multitude of cargoes has been described depending on Rab11 mediated transport, including the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and the epidermal growth factor (EGF) receptor. Therefore, Rab11 is involved in multiple cellular processes depending on secretory vesicle transport, such as neuritogenesis and oogenesis.^36,37

Coordination of actin filament assembly and motor protein activity in Rab11 transport

In metaphase oocytes, the MyoVb motor mediates the transport of Rab11 vesicles toward the oocyte cortex.³⁸ Here, the actin nucleators Spir-1 and Spir-2, and the formin-2 (FMN-2) of formin subfamily formins (FMNs) are localized to Rab11/MyoVb vesicles to form an actin filament meshwork which provides the tracks for Rab11 vesicle transport.³⁹ Spir proteins belong to the class of Wiskott-Aldrich syndrome protein homology 2 (WH2) domain containing actin nucleation factors and form a functional actin nucleator complex with formin family formins at intracellular vesicle membranes.⁴⁰ The 2 mammalian Spir proteins, Spir-1 and Spir-2, are modular proteins that share high similarity.⁴¹ The N-terminal KIND (kinase non-catalytic C-lobe domain) mediates interaction with FMN proteins.^42-44 A cluster of 4 WH2 domains binds actin monomers to form an actin nucleus.⁴⁵ Efficient actin nucleation, however, requires the interaction with FMN subgroup formins.^39,46,47 The C-terminal Spir-box and FYVE-type zinc-finger mediate targeting of Spir proteins to negatively charged membranes.⁴⁸ In somatic cells, the functional correlation of Spir actin nucleators and Rab11 GTPases in exocytic and recycling transport has been described a decade ago.⁴⁹ Spir proteins colocalize with Rab11 on membrane surfaces of the trans-Golgi network, post-Golgi vesicles and the recycling endosome, the major locales of Rab11 in eukaryotic cells.^34,35,50 Besides that, a direct physical interaction of both proteins in vitro and in vivo has never been observed and, thus, it remained unclear how the Spir/FMN actin nucleation function is recruited to Rab11/MyoVb vesicles. The mechanism for how the Spir/FMN driven actin filament generation and the Rab11/MyoV motor activity are coordinated was revealed recently.³³ Myosin V actin motor proteins were found to directly interact with Spir proteins. The interaction is mediated by the myosin V globular tail domain and a newly identified highly conserved sequence motif within the central Spir linker region (globular tail domain binding motif, GTBM). The direct Spir/MyoV interaction further determines the vesicle identity as the MyoV protein links Spir and Rab11 into a tripartite membrane associated complex, constituting a Rab11- and Spir-positive vesicle population (Fig. 3A). The formation of a tripartite Rab11/MyoV/Spir protein complex at vesicle membranes is suggested to facilitate vesicle transport by combining de novo actin filament nucleation and myosin dependent force generation. Considering the cooperative function of those proteins in mouse oocytes,^38,39 it seems obvious that the Spir/FMN actin nucleator complex generates actin filaments at the vesicle surface which might then be used by the Rab11/MyoV motor complex to generate forces to transport oocytic vesicles. However, there are still a lot of open questions regarding the actin/myosin force generation, the dynamics and quantities of the complex formation, and the role of additional proteins that might join the complex, and further investigations are demanded.

The actin filament binding protein melanophilin and the actin filament nucleator Spir share a similar MyoV interaction mode

The Spir/MyoV/Rab11 complex resembles in several aspects the very well-studied tripartite Rab27a/melanophilin/MyoVa complex, which functions in melanosome transport for skin and hair pigmentation (Fig. 3A).^21,24,51-57 Melanophilin (MLPH) connects the myosin Va actin motor with the melanosome associated Rab27a GTPase. In contrast to Spir proteins, MLPH specifically interacts with the MyoVa isoform and encodes 2 distinct MyoVa binding sites, the GTBM, similar to Spir, and the exon-F binding domain (EFBD) which binds to exon F encoded MyoVa sequences (Fig. 3B).⁵⁸ Defects of either of the complex partners underlie the manifestation of the human Griscelli syndrome (GS; reviewed in refs. 59, 60). Depending on the affected gene, there are 3 types of disease presentation all of which have in common a skin and hair hypopigmentation. Mutations in the MYO5A gene induce GS type 1 which further includes severe neurologic impairments and developmental delay. Defects in the RAB27A gene induce GS type 2 leading to deficits of the immune system and increased infection susceptibility. Mutations in the MLPH gene induce GS type 3 which only presents the partial albinism.

Interestingly, MLPH and Spir proteins share a similar domain organization (Fig. 3B). Both proteins encode a FYVE-type zinc-finger and related flanking regions for membrane targeting and MyoVa/MyoV interaction sequences. The proteins differ in the way they interact with actin. Whereas Spir proteins interact with G-actin and function as actin nucleation factors, MLPH is an actin filament binding protein. Based on the structural similarity, a combined action of the Spir actin nucleators and MLPH actin filament binding activities in MyoVa transport processes seems likely, and the coordination of membrane associated Rab GTPases, myosin V actin motors and actin assembly or actin binding proteins might be a common mechanism underlying actin driven vesicle transport.

A targeting mechanism of the MyoV motors toward Rab11 vesicles, which is independent of actin nucleation and actin filament binding factors has also been described. The Rab11-family interacting protein Rab11-FIP2, which interacts with both, MyoV motor proteins (MyoVa and MyoVb) and the Rab11 GTPase,^32,61-63 hereby was suggested to form a tripartite complex with MyoV and Rab11 (Fig. 3A). The complex, however, still awaits experimental confirmation. Rab11-FIP2 contains a membrane binding C2 domain which is thought to stabilize the complex at endosomal membranes.^64,65 The whole complex is suggested to consist of a MyoVb dimer binding 2 Rab11-FIP2 proteins which in turn bind 2 Rab11 proteins. As the MyoVb dimer by itself is also able to bind 2 Rab11 proteins by its globular tail domains, there might be 4 Rab11 proteins in total contributing to the complex.⁷

Spir-2 colocalizes with Rab8a and MyoVa at vesicle membranes

Besides Rab11, the Rab8 small GTPase plays a major role in exocytic and recycling transport. Rab8 is located at membrane vesicles, macropinosomes and tubular membrane structures which drive the recycling transport of, among others, β-1 integrin receptors and the transferrin receptor.⁶⁶ A fine-tuned interplay with the Arf (ADP-ribosylation factor) GTPase family member Arf6 enables macropinosome formation, subsequent tubulation and eventual vesicle formation for the recycling transport toward the plasma membrane. Rab8 has been described to interact with all 3 MyoV isoforms depending on the alternatively spliced exon D within the C-terminal coiled-coil regions of MyoVa and MyoVb,¹³ and the corresponding exon-like regions within the MyoVc sequence.²⁵ The presence of exon D is mandatory for MyoVa interaction with Rab8, but, in contrast, inhibits Rab8 interaction of MyoVb (Fig. 2B).¹³ As Spir proteins functionally interact with Rab11 in exocytic and recycling pathways,^38,39,49 it is intriguing to speculate about a Spir protein contribution to secretory and recycling transport depending on Rab GTPases other than Rab11, and consequently we aimed to investigate if Spir-2 proteins colocalize with MyoVa at Rab8a vesicles.

By means of fluorescence microscopy of HeLa cells transiently co-expressing an eGFP-tagged C-terminal MyoVa fragment encoding the coiled-coil and globular tail domain, but lacking exon D required for Rab8a interaction (GFP-MyoVa-CC-GTD), mStrawberry-tagged Rab8a (Straw-Rab8a) and Myc-epitope-tagged C-terminal Spir-2 fragments, encoding (Myc-Spir-2-GTBM-SB-FYVE) or lacking (Myc-Spir-2-SB-FYVE) the myosin V interaction motif, we here analyzed the potential formation of a tripartite Rab8a/MyoVa/Spir-2 complex depending on the direct Spir-2/MyoVa interaction (Fig. 4). Indeed, we observed a colocalization of Rab8a/MyoVa/Spir-2 at vesicle surfaces (Fig. 4A). As expected, the MyoVa-CC-GTD protein only very poorly colocalizes with Rab8a by itself as it misses exon D. The complex formation requires the ability of the Spir-2 protein to interact with MyoVa, as strong colocalization of Spir-2, MyoVa-CC-GTD and Rab8a was only obtained when using the Spir-2-GTBM-SB-FYVE protein (upper panel) and not using the Spir-2-SB-FYVE protein lacking the MyoV interaction sequence (lower panel). These results show that the Spir-2-GTBM-SB-FYVE protein is able to specifically target MyoVa-CC-GTD to Rab8a-positve vesicles. Important to note, both C-terminal Spir-2 fragments, regardless of their ability to interact with MyoV, strongly colocalize with Rab8a proteins at vesicle surfaces by themselves.

Figure 4.

Spir-2 colocalizes with Rab8a and MyoVa at vesicle surfaces. (A) HeLa cells were transiently transfected by lipofection. The cells were fixed 36 hours post-transfection and the localization of autofluorescent and immunostained proteins was determined by fluorescence microscopy (Leica AF6000LX microscope, 63x glycerol immersion objective). The localization of transiently co-expressed tagged MyoVa-CC-GTD (eGFP, eGFP-MyoVa-CC-GTD; green), Rab8a (mStrawberry, mStraw-Rab8a; red), and the Myc-epitope-tagged (Myc; cyan) C-terminal Spir-2 proteins encoding (Myc-Spir-2-GTBM-SB-FYVE) or lacking (Myc-Spir-2-SB-FYVE) the MyoV binding motif was analyzed by fluorescence microscopy. 3D-deconvoluted images indicate the localization of the proteins on vesicular structures. Only in presence of the MyoV binding Spir-2-GTBM-SB-FYVE (upper panel) MyoVa-CC-GTD is present at Rab8a-positive vesicles, which is not the case when Spir-2-SB-FYVE is co-expressed (lower panel) (merge). At least 5 cells were recorded for each condition and one representative cell is presented here. Scale bar represent 5 μm. (B) The colocalization of tagged proteins as described in (A) was quantified for the indicated co-expressions by determining the Pearson's correlation coefficient (PCC) as shown in a bar diagram. Each bar represents the mean PCC value for at least 5 cells analyzed. Error bars represent SEM. Statistical analysis was performed using Student's t-test to compare the mean PCC values of 2 co-expression conditions with a confidence interval of 95%. *, p < 0.05. (C) An overview of employed proteins is presented and the domains used for colocalization studies are highlighted.

To quantify the colocalization experiments, we calculated the Pearson's correlation coefficient (PCC) for the colocalization of Rab8a with Spir-2 and MyoVa, respectively (Fig. 4B). A PCC value of 1 indicates a perfect colocalization of fluorescent intensities for all pixels, a value of 0 indicates a random overlap and a value of -1 indicates a mutually exclusive localization of the fluorescent proteins. As already revealed by microscopic images, we obtained high PCC values for the colocalization of Rab8a and both Spir-2 proteins used, in each of the experimental setups. The PCC for colocalization of Rab8a and MyoVa-CC-GTD was in contrast very low, both, in double expression conditions (Rab8a and MyoVa-CC-GTD) and triple expression conditions using the MyoV binding incompetent Spir-2 protein (Rab8a, MyoVa-CC-GTD, Spir-2-SB-FYVE). Importantly, the PCC for Rab8a and MyoVa-CC-GTD colocalization was significantly increased under triple expression conditions using the MyoV binding Spir-2 protein (Rab8a, MyoVa-CC-GTD and Spir-2-GTBM-SB-FYVE), reaching a similar range of the PCC as was observed for the Rab8a and Spir-2 colocalizations.

In summary, these data suggest the formation a complex by the vesicle associated Rab8a GTPase, the actin based myosin Va motor and the actin assembly factor Spir-2 at vesicle membranes. Our results are in accordance to what was already observed for Rab11, MyoVa/b and Spir-2. However, further investigations are necessary to analyze the underlying molecular mechanisms of complex formation, especially in respect to the observed colocalization of Spir-2 and Rab8a in the absence of MyoVa.

Multiple Rab/MyoV interactions suggest a multitude of multifunctional protein complexes

Considering the large diversity of Rab/MyoV protein interactions, their involvement in different cellular processes (Fig. 1) and the formation of protein complexes including Spir-2, MyoVa/b and Rab8a or Rab11, it is an intriguing idea that a functional Spir/MyoV/Rab-GTPase complex might exist in different compositions depending on the participating MyoV motor and Rab GTPase. A differential complex composition would allow targeting actin nucleation and myosin dependent force generation to specific vesicle populations to drive distinct vesicle transport processes that require de novo generation of actin filaments directly at vesicle surfaces.

In line with that, the small GTPase Rab3A has already been demonstrated to associate with the Spir-1 protein.⁶⁷ A functional interaction of both proteins is suggested in invadosomes of metastatic cells in which they might regulate the directed transport and the release of proteinases to drive degradation of the extracellular matrix and subsequent disruption of tissue and cellular barriers. Considering the reported direct interaction of Rab3A and myosin Va¹⁹ and the functional cooperation of Rab3A and Spir-1 in invadosome activity, it points toward the formation of a similar tripartite complex including a Rab family GTPase (Rab3A), a myosin V actin motor protein (MyoVa) and an actin nucleating protein (Spir-1), which finally decides on cell invasion and cancer metastasis.

Many open questions remain regarding the nature, function and the molecular dynamics for the formation of such multi-protein complexes. Deciphering these molecular and cellular aspects will be of fundamental importance for future studies and might be indispensable to understand intracellular transport processes as the base of cell structure and communication, but also in terms of disease manifestation and potential treatment targets.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by the German Research Foundation (DFG) under Grant DFG SPP 1464: KE 447/10–1, −2.