Kinetic signatures of myosin-5B, the motor involved in microvillus inclusion disease.

Myosin-5B is a ubiquitous molecular motor that transports cargo vesicles of the endomembrane system in intracellular recycling pathways. Myosin-5B malfunction causes the congenital enteropathy microvillus inclusion disease, underlining its importance in cellular homeostasis. Here we describe the interaction of myosin-5B with F-actin, nucleotides, and the pyrazolopyrimidine compound myoVin-1. We show that single-headed myosin-5B is an intermediate duty ratio motor with a kinetic ATPase cycle that is rate-limited by the release of phosphate. The presence of a second head generates strain and gating in the myosin-5B dimer that alters the kinetic signature by reducing the actin-activated ADP release rate to become rate-limiting. This kinetic transition into a high-duty ratio motor is a prerequisite for the proposed transport function of myosin-5B in cellular recycling pathways. Moreover, we show that the small molecule compound myoVin-1 inhibits the enzymatic and functional activity of myosin-5B in vitro Partial inhibition of the actin-activated steady-state ATPase activity and sliding velocity suggests that caution should be used when probing the effect of myoVin-1 on myosin-5-dependent transport processes in cells.

Downregulation of myosin VI reduced cell growth and increased apoptosis in human colorectal cancer.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide, with the mortality increasing steadily over the last decade. Myosin VI (MYO6) expression is found to be elevated in some types of human carcinoma cell types, suggesting that it may be a sensitive biomarker for the diagnosis and follow-up. In this study, we first used the Oncomine database to explore the expression of MYO6 in CRC tissues, and then constructed a plasmid of RNA interference targeting MYO6 gene. After transfection of lentivirus targeting MYO6 into SW1116 cells, cell viability and proliferation were measured with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assay. Cell cycle distribution was assayed by flow cytometry and apoptosis was evaluated by Annexin V. MYO6 expression was detected by quantitative real-time polymerase chain reaction and western blot analysis. It was found that MYO6 mRNA was upregulated in CRC tissues using data mining of public Oncomine microarray datasets. Depletion of MYO6 significantly inhibited cell proliferation and colony formation. In addition, knockdown of MYO6 slightly arrested cell cycle in G0/G1 phase, but remarkably increased the proportion of the sub-G1 phase of cell with the increase of apoptotic cells. These results suggest that MYO6 may promote cell growth and may be used as a potential target for anticancer therapy of CRC.

Peroxynitrite induces degradation of myosin heavy chain via p38 MAPK and muscle-specific E3 ubiquitin ligases in C2 skeletal myotubes.

Oxidative stress and inflammation play an important role in the catabolism of skeletal muscles. Recently, cigarette smoke (CS) was shown to stimulate muscle catabolism by activation of p38 MAPK and up-regulation of the muscle-specific E3 ubiquitin ligases (E3s) atrogin-1 and MuRF1 which are over-expressed during muscle atrophy. Peroxynitrite (ONOO-), an oxidative ingredient of CS, also produced during oxidative stress and inflammation, was previously shown to induce ubiquitination and degradation of muscle proteins. To investigate the involvement of p38 MAPK and the muscle-specific E3s in ONOO--induced muscle catabolism, C2 myotubes, differentiated from a myoblast cell line, were exposed to ONOO- (25 µM) in a time-dependent manner. Following exposure, degradation of myosin heavy chain (MyHC) and actin, activation of p38 MAPK, and levels of atrogin-1 and MuRF1 were studied by Western blotting. Peak phosphorylation of p38 MAPK was observed at 1 h of ONOO- exposure. ONOO- caused a significant increase in the levels of atrogin-1 and MuRF1. In accordance, a significant decrease in MyHC levels was observed in a time-dependent manner. These findings support previous studies in which the catabolic effects of ONOO- were shown. In addition, ONOO- was demonstrated to induce degradation of muscle proteins by activation of p38 MAPK and up-regulation of the muscle-specific E3s atrogin-1 and MuRF1.

Phosphorylation of the kinase domain regulates autophosphorylation of myosin IIIA and its translocation in microvilli.

Motor activity of myosin III is regulated by autophosphorylation. To investigate the role of the kinase activity on the transporter function of myosin IIIA (Myo3A), we identified the phosphorylation sites of kinase domain (KD), which is responsible for the regulation of kinase activity and thus motor function. Using mass spectrometry, we identified six phosphorylation sites in the KD, which are highly conserved among class III myosins and Ste20-related misshapen (Msn) kinases. Two predominant sites, Thr¹84 and Thr¹88, in KD are important for phosphorylation of the KD as well as the motor domain, which regulates the affinity for actin. In the Caco2 cells, the full-length human Myo3A (hMyo3AFull) markedly enlarged the microvilli, although it did not show discrete localization within the microvilli. On the other hand, hMyo3AFull(T184A) and hMyo3AFull(T188A) both showed clear localization at the microvilli tips. Our results suggest that Myo3A induces large actin bundle formation to form microvilli, and phosphorylation of KD at Thr¹84 and Thr¹88 is critical for the kinase activity of Myo3A, and regulation of Myo3A translocation to the tip of microvilli. Retinal extracts potently dephosphorylate both KD and motor domain without IQ motifs (MDIQo), which was inhibited by okadaic acid (OA) with nanomolar range and by tautomycetin (TMC) with micromolar range. The results suggest that Myo3A phosphatase is protein phosphatase type 2A (PP2A). Supporting this result, recombinant PP2Ac potently dephosphorylates both KD and MDIQo. We propose that the phosphorylation-dephosphorylation mechanism plays an essential role in mediating the transport and actin bundle formation and stability functions of hMyo3A.

Overexpression of myosin is associated with the development of uterine myoma.

AIM: Myosin is involved in cell contraction and motility, but it is unclear whether it is involved in cell proliferation in uterine myoma. In this study therefore we aimed to explore the role of myosin in uterine myoma. MATERIAL AND METHODS: Immunohistochemistry and real-time polymerase chain reaction were used to determine the expression of myosin light chain (MLC), myosin heavy chain (MHC) and myosin light chain kinase (MLCK) in patient uterine myoma and adjacent smooth muscle tissue. Human uterine fibroid cells were isolated and cultured in vitro, myosin heavy chain 11 (MHC subtype expressed in uterine fibroid cells) was knocked down by RNA interference to reduce the expression of myosin, then cell proliferation was determined by the methyl thiazol tetrazolium bromide method. To explore the possible mechanism of reduced cell proliferation after myosin heavy chain 11 knockdown, the downstream proteins collagen I, insulin-like growth factor-1, fibronectin and proteoglycans were analyzed. RESULTS: Expression of MLC, MHC, MLCK and p-MLCK in uterine myoma cells was significantly higher than in adjacent smooth muscle cells. After knockdown of MHC, smooth muscle cell proliferation decreased, and the production of collagen I, insulin-like growth factor-1 and fibronectin was also reduced, but proteoglycans did not show any significant change. CONCLUSION: Myosin is overexpressed in uterine myoma, and the overexpression of myosin is associated with both uterine contraction and tumor development of uterine myoma.

Actin and myosin contribute to mammalian mitochondrial DNA maintenance.

Mitochondrial DNA maintenance and segregation are dependent on the actin cytoskeleton in budding yeast. We found two cytoskeletal proteins among six proteins tightly associated with rat liver mitochondrial DNA: non-muscle myosin heavy chain IIA and ß-actin. In human cells, transient gene silencing of MYH9 (encoding non-muscle myosin heavy chain IIA), or the closely related MYH10 gene (encoding non-muscle myosin heavy chain IIB), altered the topology and increased the copy number of mitochondrial DNA; and the latter effect was enhanced when both genes were targeted simultaneously. In contrast, genetic ablation of non-muscle myosin IIB was associated with a 60% decrease in mitochondrial DNA copy number in mouse embryonic fibroblasts, compared to control cells. Gene silencing of ß-actin also affected mitochondrial DNA copy number and organization. Protease-protection experiments and iodixanol gradient analysis suggest some ß-actin and non-muscle myosin heavy chain IIA reside within human mitochondria and confirm that they are associated with mitochondrial DNA. Collectively, these results strongly implicate the actomyosin cytoskeleton in mammalian mitochondrial DNA maintenance.

Myosin Va cooperates with PKA RIalpha to mediate maintenance of the endplate in vivo.

Myosin V motor proteins facilitate recycling of synaptic receptors, including AMPA and acetylcholine receptors, in central and peripheral synapses, respectively. To shed light on the regulation of receptor recycling, we employed in vivo imaging of mouse neuromuscular synapses. We found that myosin Va cooperates with PKA on the postsynapse to maintain size and integrity of the synapse; this cooperation also regulated the lifetime of acetylcholine receptors. Myosin Va and PKA colocalized in subsynaptic enrichments. These accumulations were crucial for synaptic integrity and proper cAMP signaling, and were dependent on AKAP function, myosin Va, and an intact actin cytoskeleton. The neuropeptide and cAMP agonist, calcitonin-gene related peptide, rescued fragmentation of synapses upon denervation. We hypothesize that neuronal ligands trigger local activation of PKA, which in turn controls synaptic integrity and turnover of receptors. To this end, myosin Va mediates correct positioning of PKA in a postsynaptic microdomain, presumably by tethering PKA to the actin cytoskeleton.

PRBC-derived plasma induces non-muscle myosin type IIA-mediated neutrophil migration and morphologic change.

CONTEXT: Neutrophils are the primary effector cells in the pathogenesis of transfusion-related acute lung injury or multiple organ failure after blood transfusion. OBJECTIVE: We aimed to investigate the effect of fresh (1 day preparation) and aged (42 day preparation) PRBC-derived plasma on neutrophil morphology, migration and phagocytosis. MATERIALS AND METHODS: We evaluated the production of reactive oxygen species (ROS) and the expression of non-muscle myosin heavy chain IIA (MYH9) in neutrophils treated with PRBC-derived plasma. We used western blots and antibody arrays to evaluate changes in signal transduction pathways in plasma-treated neutrophils. RESULTS: Aged PRBC-derived plasma elicited a stronger oxidative burst in neutrophils when compared with fresh PRBC-derived plasma (p < 0.05). Antibody arrays showed increased phosphorylation of NF-ĸB proteins (p105, p50 and Ikk) in aged PRBC-derived plasma-treated neutrophils. The expression of non-muscle myosin IIA (MYH9), a cytoskeleton protein involved in immune cell migration and morphological change, was also significantly upregulated in neutrophils treated with aged PRBC-derived plasma compared to fresh plasma (p < 0.05). Pretreatment of neutrophils with blebbistatin (a specific type II myosin inhibitor), ascorbic acid (an antioxidant), or staurosporine (a protein tyrosine kinase inhibitor), effectively abrogated the morphological changes, neutrophil migration, and phagocytosis induced by aged PRBC-derived plasma. CONCLUSION: Upregulation of MYH9 in neutrophils treated with aged PRBC-derived plasma and abrogation of neutrophil migration in blebbistatin-treated neutrophils suggested a functional role of MYH9 in the directional migration of immune cells. Our data help elucidate the cellular and molecular mechanisms of transfusion-related injury.

cGMP-dependent protein kinase Iα transfection inhibits hypoxia-induced migration, phenotype modulation and annexins A1 expression in human pulmonary artery smooth muscle cells.

Our previous work has demonstrated that the cellular phenotype changes of human pulmonary artery smooth muscle cells (PASMCs) play an important role during pulmonary vascular remodelling. However, little is known about the role of PASMCs phenotype modulation in the course of hypoxia-induced migration and its behind molecular mechanisms. In this study, we have shown that cGMP-dependent protein kinase (PKG) Iα transfection significantly attenuated the hypoxia-induced down-regulation of the expressions of SM-α-actin, MHC and calponin. Hypoxia-induced PASMC migration was also suppressed by PKGIα overexpression. Furthermore, this overexpression attenuated ANX A1 upregulation under hypoxic conditions. All those effects were reversed by a PKG inhibitor KT5823. Our data indicate that manipulating upstream entity e.g., PKGIa, may have a potential therapeutic value to prevent hypoxia-associated pulmonary arterial remodeling for pulmonary hypertension development.

DNA methyltransferase inhibitor CDA-II inhibits myogenic differentiation.

CDA-II (cell differentiation agent II), isolated from healthy human urine, is a DNA methyltransferase inhibitor. Previous studies indicated that CDA-II played important roles in the regulation of cell growth and certain differentiation processes. However, it has not been determined whether CDA-II affects skeletal myogenesis. In this study, we investigated effects of CDA-II treatment on skeletal muscle progenitor cell differentiation, migration and proliferation. We found that CDA-II blocked differentiation of murine myoblasts C2C12 in a dose-dependent manner. CDA-II repressed expression of muscle transcription factors, such as Myogenin and Mef2c, and structural proteins, such as myosin heavy chain (Myh3), light chain (Mylpf) and MCK. Moreover, CDA-II inhibited C1C12 cell migration and proliferation. Thus, our data provide the first evidence that CDA-II inhibits growth and differentiation of muscle progenitor cells, suggesting that the use of CDA-II might affect skeletal muscle functions.

The globular tail domain puts on the brake to stop the ATPase cycle of myosin Va.

Myosin Va is a well known processive motor involved in transport of organelles. A tail-inhibition model is generally accepted for the regulation of myosin Va: inhibited myosin Va is in a folded conformation such that the tail domain interacts with and inhibits myosin Va motor activity. Recent studies indicate that it is the C-terminal globular tail domain (GTD) that directly inhibits the motor activity of myosin Va. In the present study, we identified a conserved acidic residue in the motor domain (Asp-136) and two conserved basic residues in the GTD (Lys-1706 and Lys-1779) as critical residues for this regulation. Alanine mutations of these conserved charged residues not only abolished the inhibition of motor activity by the GTD but also prevented myosin Va from forming a folded conformation. We propose that Asp-136 forms ionic interactions with Lys-1706 and Lys-1779. This assignment locates the GTD-binding site in a pocket of the motor domain, formed by the N-terminal domain, converter, and the calmodulin in the first IQ motif. We propose that binding of the GTD to the motor domain prevents the movement of the converter/lever arm during ATP hydrolysis cycle, thus inhibiting the chemical cycle of the motor domain.

4H12, a Murine Monoclonal Antibody Directed against Myosin Heavy Chain-9 Expressed on Acinar Cell Carcinoma of Pancreas with Potential Therapeutic Application

Background: Pancreatic acinar cell carcinoma (PACC) is a rare type of pancreatic exocrine neoplasm that is frequently diagnosed at late stages with a high rate of metastasis. Identification of new biomarkers for PACC can improve our knowledge of its biology, early detection, or targeted therapy. In this study, hybridoma technology was used to generate mAbs against Faraz-ICR, a pancreatic acinar cell carcinoma cell line. Methods: Cell ELISA and flow cytometry were used for screening, and the 4H12 hybridoma clone was selected for further analysis. The 4H12 mAb was specific for myosin heavy chain-9 (MYH9) as determined by Immunoprecipitation, Western blot, and mass spectrometry. Results: This antibody reacted variably with other cancer cells, in comparison to Faraz-ICR cell. Besides, by immunohistochemical staining, the acinar cell tumor, which was the source of Faraz-ICR, showed high MYH9 expression. Among 21 pancreatic ductal adenocarcinoma cases, nine (42.8%) expressed MYH9 with low intensity, while 10 (47.8%) and 2 (9.5%) cases expressed MYH9 with moderate to strong intensities, respectively. The 4H12 mAb inhibited the proliferation of Faraz-ICR cells in a dose-dependent manner from 0.75 to 12.5 µg/ml concentrations (p < 0.0001 and p < 0.002). IC50 values were achieved at 12.09 ± 4.19 µg/ml and 7.74 ± 4.28 µg/ml after 24- and 48-h treatment, respectively. Conclusion: Our data suggest that the 4H12 mAb can serve as a tool for investigating the role of MYH9 pancreatic cancer biology and prognosis.

Effects of acetylation on dissociation and phosphorylation of actomyosin in postmortem ovine muscle during incubation at 4 °C in vitro.

This study aimed to assess the effects of acetylation levels on actomyosin disassociation and phosphorylation of lamb during incubation at 4 °C. Samples of whole proteins from lamb longissimus thoracis muscles were prepared and assigned into three treatments (high, middle and low acetylation groups). The results showed that deacetylation of myosin heavy chain and actin was inhibited by lysine deacetylase inhibitor trichostatin A and nicotinamide in this study. Phosphorylation levels of myosin heavy chain and actin were inhibited by their acetylation during incubation in vitro. Actomyosin disassociation degree in high acetylation group was significantly lower than that in middle and low acetylation groups (P < 0.05). The ATPase activity in high acetylation group was significantly higher than that in middle and low acetylation groups (P < 0.05). In conclusion, acetylation of myosin heavy chain and actin inhibited actomyosin dissociation by inhibiting their phosphorylation at 4 °C in vitro.

Megalin and nonmuscle myosin heavy chain IIA interact with the adaptor protein Disabled-2 in proximal tubule cells.

Megalin plays a critical role in the endocytosis of albumin and other filtered low-molecular-weight proteins. Here we studied the interaction between megalin and Disabled-2 (Dab2), an adaptor protein that binds to the cytoplasmic domain of megalin and appears to control its trafficking. We co-immunoprecipitated megalin and Dab2 from cultured proximal tubule cells and identified the proteins by liquid chromatography and tandem mass spectrometry. We found two proteins associated with the megalin/Dab2 complex, nonmuscle myosin heavy chain IIA (NMHC-IIA) and beta-actin. Subcellular fractionation followed by sucrose velocity gradient separation showed that megalin, Dab2, and NMHC-IIA existed as a complex in the same endosomal fractions. In vitro pull-down assays demonstrated that NMHC-IIA was bound to the carboxyl-terminal region of Dab2, but not to megalin's cytoplasmic domain. We then transfected COS-7 cells with plasmids that induced the expression of Dab2, NMHC-IIA, and the megalin minireceptor, a truncated form of megalin. Co-immunoprecipitation studies showed that the minireceptor and NMHC-IIA co-immunoprecipitated only with Dab2. Furthermore, the uptake of (125)I-lactoferrin, an endocytic ligand of megalin, by rat yolk sac-derived megalin-expressing L2 cells was inhibited by blebbistatin, a specific inhibitor of nonmuscle myosin II. Our study shows that NMHC-IIA is functionally linked to megalin by interaction with Dab2 and is likely involved in megalin-mediated endocytosis in proximal tubule cells.

MicroRNA-1 and MicroRNA-133 in spontaneous myocardial differentiation of mouse embryonic stem cells.

BACKGROUND: MicroRNAs (miRNAs) regulate various biological processes through inhibiting the translation of RNA transcripts. Although miRNA-1 (miR-1) and miRNA-133 (miR-133) are abundantly expressed in the adult heart and involved in cardiac hypertrophy, the roles of these miRNAs in spontaneous myocardial differentiation are unknown. METHODS AND RESULTS: The levels of miR-1 and miR-133 in mouse embryonic stem (ES) cells were increased during spontaneous differentiation by 2-dimensional culture, but reduced during forced myocardial differentiation by a histone deacetylase inhibitor, trichostatin A. The overexpression of miR-1 or miR-133 by lentiviral infection reduced the expression of a cardiac-specific gene, Nkx2.5, during differentiation of ES cells. In addition, miR-1 also inhibited alpha-myosin heavy chain expression. The results of luciferase assays revealed that miR-1 recognizes and targets the 3' untranslated region of cyclin-dependent kinase-9 (Cdk9) in ES cells. Overexpression of miR-1 decreased the protein amounts of Cdk9 without affecting the mRNA levels, indicating that miR-1 post-transcriptionally inhibits Cdk9 translation. CONCLUSIONS: miR-1 and miR-133 may play significant roles in the myocardial differentiation of mouse ES cells, and Cdk9 may be involved in this process as a target of miR-1.

Myosin VI Drives Clathrin-Mediated AMPA Receptor Endocytosis to Facilitate Cerebellar Long-Term Depression.

Myosin VI is an actin-based cytoskeletal motor implicated in various steps of membrane trafficking. Here, we investigated whether this myosin is crucial for synaptic function and plasticity in neurons. We find that myosin VI localizes at cerebellar parallel fiber to Purkinje cell synapses and that the myosin is indispensable for long-term depression of AMPA-receptor-mediated synaptic signal transmission at this synapse. Moreover, direct visualization of GluA2-containing AMPA receptors in Purkinje cells reveals that the myosin drives removal of AMPA receptors from the surface of dendritic spines in an activity-dependent manner. Co-immunoprecipitation and super-resolution microscopy indicate that specifically the interaction of myosin VI with the clathrin adaptor component α-adaptin is important during long-term depression. Together, these data suggest that myosin VI directly promotes clathrin-mediated endocytosis of AMPA receptors in Purkinje cells to mediate cerebellar long-term depression. Our results provide insights into myosin VI function and the molecular mechanisms underlying synaptic plasticity.

Myosin VI in the nucleus of neurosecretory PC12 cells: Stimulation-dependent nuclear translocation and interaction with nuclear proteins.

Myosin VI (MVI) is a unique actin-based motor protein moving towards the minus end of actin filaments, in the opposite direction than other known myosins. Besides well described functions of MVI in endocytosis and maintenance of Golgi apparatus, there are few reports showing its involvement in transcription. We previously demonstrated that in neurosecretory PC12 cells MVI was present in the cytoplasm and nucleus, and its depletion caused substantial inhibition of cell migration and proliferation. Here, we show an increase in nuclear localization of MVI upon cell stimulation, and identification of potential nuclear localization (NLS) and nuclear export (NES) signals within MVI heavy chain. These signals seem to be functional as the MVI nuclear presence was affected by the inhibitors of nuclear import (ivermectin) and export (leptomycin B). In nuclei of stimulated cells, MVI colocalized with active RNA polymerase II, BrUTP-containing transcription sites and transcription factor SP1 as well as SC35 and PML proteins, markers of nuclear speckles and PML bodies, respectively. Mass spectrometry analysis of samples of a GST-pull-down assay with the MVI tail domain as a "bait" identified several new potential MVI binding partners. Among them are proteins involved in transcription and post-transcriptional processes. We confirmed interaction of MVI with heterogeneous nuclear ribonucleoprotein U (hnRNPU) and nucleolin, proteins involved in pre-mRNA binding and transport, and nucleolar function, respectively. Our data provide an insight into mechanisms of involvement of MVI in nuclear processes via interaction with nuclear proteins and support a notion for important role(s) for MVI in gene expression.

Myosin-Va is required for preciliary vesicle transportation to the mother centriole during ciliogenesis.

Primary cilia play essential roles in signal transduction and development. The docking of preciliary vesicles at the distal appendages of a mother centriole is an initial/critical step of ciliogenesis, but the mechanisms are unclear. Here, we demonstrate that myosin-Va mediates the transportation of preciliary vesicles to the mother centriole and reveal the underlying mechanism. We also show that the myosin-Va-mediated transportation of preciliary vesicles is the earliest event that defines the onset of ciliogenesis. Depletion of myosin-Va significantly inhibits the attachment of preciliary vesicles to the distal appendages of the mother centriole and decreases cilia assembly. Myosin-Va functions upstream of EHD1- and Rab11-mediated ciliary vesicle formation. Importantly, dynein mediates myosin-Va-associated preciliary vesicle transportation to the pericentrosomal region along microtubules, while myosin-Va mediates preciliary vesicle transportation from the pericentrosomal region to the distal appendages of the mother centriole via the Arp2/3-associated branched actin network.

The Extract of Soybean Protein Increases Slow-Myosin Heavy Chain Expression in C2C12 Myotubes.

Skeletal muscle is composed of four types of fibers in mammals; oxidative slow-twitch type I, oxidative fast-twitch IIA, and glycolytic fast-twitch IIB and IIX/D. In this study using C2C12 myotubes, an extract of soybean protein significantly upregulated mRNA level of myosin heavy chain 7 (Myh7), the predominant isoform expressed in oxidative slow-twitch type I and downregulated mRNA levels of Myh4, the predominant isoform expressed in glycolytic fast-twitch IIB. Similarly, its hydrolysate prepared using digestive enzyme also significantly increased Myh7 expression. In contrast, no significant change was observed in Myh4 mRNA level after the hydrolysate treatment. These findings suggest that dietary intake of the soybean protein extract may increase oxidative slow-twitch fiber in skeletal muscle.

Proteomic investigation of the effects of preimplantation factor on human embryo implantation.

Despite the use of adjuvant therapies, the cumulative proportion of live births remains at ~40%. Accumulating data show that low pregnancy rates, even in the presence of high fertility rates, are due to implantation failure. The present study aimed to identify and construct a profile of proteins that react with preimplantation factor (PIF) and to provide an understanding into the molecular mechanisms by which PIF promotes trophoblast invasion. Cytoplasmic proteins were immunoprecipitated with biotin­labeled synthetic PIF or intralipid and scrambled PIF (PIFscr). The protein profiles were analyzed using isobaric tags for relative and absolute quantification coupled with mass spectrometry. Immunoprecipitation and western blot analyses were used to assess the interactions between PIF and myosin heavy chain 10 (MYH10) and heat shock protein family D1. Small interfering RNA­based silencing was performed to examine the function of MYH10. In the results of the present study, 21 proteins were identified with interactions with PIF. The immunoprecipitation and western blot analyses revealed an interaction between PIF and MYH10. Silencing of the expression of MYH10 in HEC­1­B cells significantly attenuated cell migration and invasion capacities. These data support the conclusion that MYH10­mediated cell migration and invasion act in conjunction with PIF to promote the trophoblast invasion procedure.