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.

Keywords: Rab GTPases; Spir; actin nucleation; formin; myosin V; myosin actin motor proteins; vesicle transport.

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.

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.

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.