Myosin Va interacts with the exosomal protein spermine synthase

Abstract

Myosin Va (MyoVa) is an actin-based molecular motor that plays key roles in the final stages of secretory pathways, including neurotransmitter release. Several studies have addressed how MyoVa coordinates the trafficking of secretory vesicles, but why this molecular motor is found in exosomes is still unclear. In this work, using a yeast two-hybrid screening system, we identified the direct interaction between the globular tail domain (GTD) of MyoVa and four protein components of exosomes: the WD repeat-containing protein 48 (WDR48), the cold shock domain-containing protein E1 (CSDE1), the tandem C2 domain-containing protein 1 (TC2N), and the enzyme spermine synthase (SMS). The interaction between the GTD of MyoVa and SMS was further validated in vitro and displayed a K_d in the low micromolar range (3.5 ± 0.5 µM). SMS localized together with MyoVa in cytoplasmic vesicles of breast cancer MCF-7 and neuroblastoma SH-SY5Y cell lines, known to produce exosomes. Moreover, MYO5A knockdown decreased the expression of SMS gene and rendered the distribution of SMS protein diffuse, supporting a role for MyoVa in SMS expression and targeting.

Keywords: cellular localization; exocytosis; myosins; protein-protein interactions; trafficking; transcription.

Figure 1. Yeast two-hybrid and in vitro assays reveal SMS as a novel binding partner of MyoVa-GTD

(A) Pairwise two-hybrid assays showing the activation of four reporter genes (QDO/X/A selective medium) in yeast cells co-transformed with pGADT7-prey (or empty pGADT7 as a control) plasmids and pGBKT7-GTD constructs (EE = phosphomimetic and AA = non-phosphorylated) showing that the identified interactions are not specific to the phosphomimetic mutant. The first line represents the negative control assays, where each clone was tested against the empty pGBKT7 vector (DBD). (B) Schematic representation of the domain architecture of the new-found partners. The red bar indicates the prey boundaries found in the yeast two-hybrid screening: SMS^90–366 = catalytic domain of SMS (NCBI accession number: EAW98985); WDR48^451–677 = DUF3337 domain of the WDR48 (NCBI accession number EAW64548); TC2N^277–490 = contains the second C2 domain of the tandem C2 domains nuclear protein (TC2N; NCBI accession number: EAW81464); CSDE1^692–798 = contains the SUZ-C domain of the cold shock domain-containing protein E1, isoform 4 (CSDE1; NCBI accession number: NP_001007554). Figures were made using SMART [19] and IBS [41]. (C) MST assays showing that MyoVa-GTD binds to SMS with a K_d in the low micromolar range. F_norm = normalized fluorescence. Data are presented as mean ± SD (error bars) from triplicates.

Figure 2. MyoVa and SMS localize together in discrete puncta at the cytoplasm

Immunocytochemistry showing the colocalization of MyoVa (green; anti-MyoVa-Medial tail) and SMS (red) labeling in MCF-7 (A-C) and SH-SY5Y (D-F) cell lines. The overlap of the fluorescent signals is shown in yellow (overlay). (B,E) Linescan analysis of two representative colocalization sites. (C, F) Pearson’s correlation coefficients support the partial colocalization of MyoVa (green) and SMS (red) labeling. The correlation is based on the average of 16 and 18 independent cells, for MCF-7 and SH-SY5Y cells, respectively, with standard deviations shown. The correlation between MyoVa (rotated 180°) and SMS channels was used as negative control (random colocalization). Nucleus were stained with DAPI (Blue). Scale bar: 10 µm (field), 1 µm (zoom). **** P≤0.001 from two-tailed Wilcoxon’s non-parametric rank test in C (because one of the populations were not normally distributed) and two-tailed paired t test in F).

Figure 3. MYO5A knockdown affects SMS distribution and expression in MCF-7 cells

(A) Immunocytochemistry showing that the puncta distribution of SMS (siControl) became diffuse in cells treated with siMYO5A. Nucleus were stained with DAPI (Blue). Scale bar: 10 µm. (B) qPCR showing that the siMYO5A treatment was effective to reduce MYO5A mRNA expression. (C) The qPCR showing the relative mRNA expression of SMS in cells treated with siControl or siMYO5A and in (D) MYO5A-null fibroblasts (FO^−/−) and the normal control (RO). The qPCR analysis of expression of the marker for neuronal differentiation NeuN (E), MYO5A (F), and SMS (G) in normal and differentiated neuronal SH-SY5Y cells. * P≤0.05; ** P≤0.01; *** P≤0.001, **** P≤0.0001. Paired t test, two-tailed.