BMP-2 regulates the expression of myosin Va via smad in melan-a melanocyte.

Myosin Va (Myo Va) is one of three protein complexes involved in melanosome transport. In this study, we identified BMP-2 as an up-regulator of Myo Va expression using 2-methyl-naphtho[1,2,3-de]quinolin-8-one (MNQO). Our results showed that MNQO reduced the mRNA and protein expression of Myo Va and BMP-2 in melanocytes. Knockdown of BMP-2 by siRNA also affected Myo Va mRNA and protein expression, confirming that MNQO regulates Myo Va through BMP-2. Furthermore, phosphorylation of Smad1/5/8 by BMP2 treatment confirmed that the BMP-2/Smad signaling pathway regulates Myo Va expression in Melan-a melanocytes. Smad-binding elements were found in the Myo Va promoter and phosphorylated Smad1/5/8 bind directly to the Myo Va promoter to activate Myo Va transcription and BMP-2 enhances this binding. These findings provide insight into a new role for BMP-2 in Melan-a melanocytes and a mechanism of regulation of Myo Va expression that may be beneficial in the treatment of albinism or hyperpigmentation disorders.

Myosin Va-dependent Transport of NMDA Receptors in Hippocampal Neurons.

N-methyl-D-aspartate receptor (NMDAR) trafficking is a key process in the regulation of synaptic efficacy and brain function. However, the molecular mechanism underlying the surface transport of NMDARs is largely unknown. Here we identified myosin Va (MyoVa) as the specific motor protein that traffics NMDARs in hippocampal neurons. We found that MyoVa associates with NMDARs through its cargo binding domain. This association was increased during NMDAR surface transport. Knockdown of MyoVa suppressed NMDAR transport. We further demonstrated that Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates NMDAR transport through its direct interaction with MyoVa. Furthermore, MyoVa employed Rab11 family-interacting protein 3 (Rab11/FIP3) as the adaptor proteins to couple themselves with NMDARs during their transport. Accordingly, the knockdown of FIP3 impairs hippocampal memory. Together, we conclude that in hippocampal neurons, MyoVa conducts active transport of NMDARs in a CaMKII-dependent manner.

The kinesin Kif21b binds myosin Va and mediates changes in actin dynamics underlying homeostatic synaptic downscaling.

Homeostatic synaptic plasticity adjusts the strength of synapses to restrain neuronal activity within a physiological range. Postsynaptic guanylate kinase-associated protein (GKAP) controls the bidirectional synaptic scaling of AMPA receptors (AMPARs); however, mechanisms by which chronic activity triggers cytoskeletal remodeling to downscale synaptic transmission are barely understood. Here, we report that the microtubule-dependent kinesin motor Kif21b binds GKAP and likewise is located in dendritic spines in a myosin Va- and neuronal-activity-dependent manner. Kif21b depletion unexpectedly alters actin dynamics in spines, and adaptation of actin turnover following chronic activity is lost in Kif21b-knockout neurons. Consistent with a role of the kinesin in regulating actin dynamics, Kif21b overexpression promotes actin polymerization. Moreover, Kif21b controls GKAP removal from spines and the decrease of GluA2-containing AMPARs from the neuronal surface, thereby inducing homeostatic synaptic downscaling. Our data highlight a critical role of Kif21b at the synaptic actin cytoskeleton underlying homeostatic scaling of neuronal firing.

Myosin Va, a Novel Interaction Partner of STXBP1, Is Required to Transport Syntaxin1A to the Plasma Membrane.

Syntaxin-binding protein 1 (STXBP1, also known as Munc18-1) regulates exocytosis as a chaperone protein of Syntaxin1A. The haploinsufficiency of STXBP1 causes early infantile-onset developmental and epileptic encephalopathy, known as STXBP1 encephalopathy. Previously, we reported impaired cellular localization of Syntaxin1A in induced pluripotent stem cell-derived neurons from an STXBP1 encephalopathy patient harboring a nonsense mutation. However, the molecular mechanism of abnormal Syntaxin1A localization in the haploinsufficiency of STXBP1 remains unknown. This study aimed to identify the novel interacting partner of STXBP1 involved in transporting Syntaxin1A to the plasma membrane. Affinity purification coupled with mass spectrometry analysis identified a motor protein Myosin Va as a potential binding partner of STXBP1. Co-immunoprecipitation analysis of the synaptosomal fraction from the mouse and tag-fused recombinant proteins revealed that the STXBP1 short splice variant (STXBP1S) interacted with Myosin Va in addition to Syntaxin1A. These proteins colocalized at the tip of the growth cone and axons in primary cultured hippocampal neurons. Furthermore, RNAi-mediated gene silencing in Neuro2a cells showed that STXBP1 and Myosin Va were required for membrane trafficking of Syntaxin1A. In conclusion, this study proposes a potential role of STXBP1 in the trafficking of the presynaptic protein Syntaxin1A to the plasma membrane in conjunction with Myosin Va.

Molecular machinery regulating organelle dynamics during axon growth and guidance.

Axon growth and guidance in the developing nervous system rely on intracellular membrane dynamics that involve endosome maturation and transport, as well as its regulated tethering to the endoplasmic reticulum (ER). Recent studies have identified several key molecules, such as protrudin, which plays a dynamic role at membrane contact sites between the ER and endosomes/lysosomes, and myosin Va, which acts as a sensor for ER-derived Ca2+ that triggers peri-ER membrane export. These molecules form different types of multiprotein complexes at the interface of organelles and, in response to their surrounding microenvironments, such as Ca2+ concentrations and lipid contents, regulate the directional movement of endosomal vesicles in extending axons. Here, we review the molecular mechanisms underlying membrane dynamics and inter-organelle interactions during neuronal morphogenesis.

Myosin Va: Capturing cAMP for synaptic plasticity.

The plus-end directed actin-dependent motor protein, myosin Va, is of particular relevance for outward vesicular protein trafficking and for restraining specific cargo vesicles within the actin cortex. The latter is a preferred site of cAMP production, and the specificity of cAMP signaling is largely mediated through the formation of microdomains that spatially couple localized metabotropic receptor activity and cAMP production to selected effectors and downstream targets. This review summarizes the core literature on the role of myosin Va for the creation of such a cAMP microdomain at the mammalian nerve-muscle synapse that serves the activity-dependent recycling of nicotinic acetylcholine receptors (nAChRs)-a principal ligand-gated ion channel which is imperative for voluntary muscle contraction. It is discussed that i) the nerve-muscle synapse is a site with a unique actin-dependent microstructure, ii) myosin Va and protein kinase A regulatory subunit Iα as well as nAChR and its constitutive binding partner, rapsyn, colocalize in endocytic/recycling vesicles near the postsynaptic membrane, and iii) impairment of myosin Va or displacement of protein kinase A regulatory subunit Iα leads to the loss of nAChR stability. Regulation of this signaling process and underlying basic pieces of machinery were covered in previous articles, to which the present review refers.

The Effects of Patchouli Alcohol on Diarrhea-Predominant Irritable Bowel Syndrome are Correlated with Phenotypic Plasticity in Myenteric Neurons and the Targeted Regulation of Myosin Va.

Patchouli alcohol (PA) has been widely used for the treatment of diarrhea-predominant irritable bowel syndrome (IBS-D) in traditional Chinese medicine, and the related mechanism remains to be fully understood. Our previous study has indicated that PA significantly reduced visceral sensitivity and defecation area in IBS-D rats. In this study, we prepared an IBS-D rat model and observed the dynamic intestinal motility and colonic longitudinal muscle and myenteric plexus (LMMP) neurons, as well as their subtypes at D14, D21, and D28. After PA administration, we observed the effects on the changes in intestinal motility, colonic LMMP neurons, and LMMP Myosin Va in IBS-D rats and their co-localization with inhibitory neurotransmitter-related proteins. The results indicated that PA treatment could alleviate IBS-D symptoms, regulate the abnormal expression of LMMP neurons, increase Myosin Va expression, up-regulate co-localization levels of Myosin Va with neuronal nitric oxide synthase (nNOS), and promote co-localization levels of Myosin Va with vasoactive intestinal polypeptide (VIP). In conclusion, this study demonstrated the neuropathic alterations in the colon of chronic restraint stress-induced IBS-D rat model. PA reversed the neuropathological alteration by affecting the transport process of nNOS and VIP vesicles via Myosin Va and the function of LMMP inhibitory neurons, and these effects were related to the mechanism of enteric nervous system (ENS) remodeling.

Suo Quan Wan ameliorates bladder overactivity and regulates neurotransmission via regulating Myosin Va protein expression.

BACKGROUND: Ancient prescriptions of Suo Quan Wan (SQW) have therapeutic effects on diabetic bladder dysfunction. However, the underlying mechanism remains unclear. Here, we hypothesized that SQW ameliorates bladder overactivity and regulates neurotransmission via regulating Myosin Va protein expression. METHODS: After diabetic rats were induced by streptozotocin (65 mg/kg), the model of diabetic bladder dysfunction was established by detecting fasting blood glucose, urodynamic test, in vitro muscle strip experiments, and histological examination. One week after induction, SQW was given to observe the therapeutic effect. The expression levels of Myosin Va in control, Model, SQW L and SQW H groups were detected by RT-qPCR, RNAscope and immunofluorescence assay. The expression levels of ChAT, SP, nNOS and VIP proteins were observed by immunofluorescence assay. After knockdown and overexpression of Myosin Va, the expression changes of ChAT, SP, nNOS and VIP and the regulatory role of SQW were observed. RESULTS: STZ-induced DM rats had significantly higher serum glucose levels and lower body weight. Compared with the diabetic rats, SQW treatment significantly improved urination function with decreased residual volume (RV), bladder compliance (BC), non-voiding contractions (NVCs), and increased voided efficiency (VE). In addition, contractile responses of muscle strips to electrical-field stimulation (EFS), carbachol (CCh), KCl were significantly lower in the SQW H and SQW L groups than those in the model group. RT-qPCR found that the expression of Myosin Va in the bladder tissue or bladder neurons in model group was significantly increased compared with the control group, and SQW treatment significantly decreased the levels of Myosin Va. In DM rats, ChAT and SP expression were significantly increased, while nNOS and VIP expression were significantly decreased, and SQW improved this phenomenon. Interestingly, SQW ameliorated the abnormal expression of ChAT, SP, nNOS and VIP caused by myosin Va knockdown, and Myosin Va overexpression results are consistent with these. CONCLUSIONS: SQW ameliorates overactive bladder and regulate neurotransmission via regulating Myosin Va mRNA and protein expression.

Human Cytomegalovirus Nuclear Egress Complex Subunit, UL53, Associates with Capsids and Myosin Va, but Is Not Important for Capsid Localization towards the Nuclear Periphery.

After herpesviruses encapsidate their genomes in replication compartments (RCs) within the nuclear interior, capsids migrate to the inner nuclear membrane (INM) for nuclear egress. For human cytomegalovirus (HCMV), capsid migration depends at least in part on nuclear myosin Va. It has been reported for certain herpesviruses that the nucleoplasmic subunit of the viral nuclear egress complex (NEC) is important for this migration. To address whether this is true for HCMV, we used mass spectrometry and multiple other methods to investigate associations among the HCMV NEC nucleoplasmic subunit, UL53, myosin Va, major capsid protein, and/or capsids. We also generated complementing cells to derive and test HCMV mutants null for UL53 or the INM NEC subunit, UL50, for their importance for these associations and, using electron microscopy, for intranuclear distribution of capsids. We found modest associations among the proteins tested, which were enhanced in the absence of UL50. However, we found no role for UL53 in the interactions of myosin Va with capsids or the percentage of capsids outside RC-like inclusions in the nucleus. Thus, UL53 associates somewhat with myosin Va and capsids, but, contrary to reports regarding its homologs in other herpesviruses, is not important for migration of capsids towards the INM.

Cargo properties play a critical role in myosin Va-driven cargo transport along actin filaments.

High-resolution experiments revealed that a single myosin-Va motor can transport micron-sized cargo on actin filaments in a stepwise manner. However, intracellular cargo transport is mediated through the dense actin meshwork by a team of myosin Va motors. The mechanism of how motors interact mechanically to bring about efficient cargo transport is still poorly understood. This study describes a stochastic model where a quantitative understanding of the collective behaviors of myosin Va motors is developed based on cargo stiffness. To understand how cargo properties affect the overall cargo transport, we have designed a model in which two myosin Va motors were coupled by wormlike chain tethers with persistence length ranging from 10 to 80 nm and contour length from 100 to 200 nm, and predicted distributions of velocity, run length, and tether force. Our analysis showed that these parameters are sensitive to both the contour and persistence length of cargo. While the velocity of two couple motors is decreased compared to a single motor (from 531 ± 251 nm/s to as low as 318 ± 287 nm/s), the run length (716 ± 563 nm for a single motor) decreased for short, rigid tethers (to as low as 377 ± 187 µm) and increased for long, flexible tethers (to as high as 1.74 ± 1.50 µm). The sensitivity of processive properties to tether rigidity (persistence length) was greatest for short tethers, which caused the motors to exhibit close, yet anti-cooperative coordination. Motors coupled by longer tethers stepped more independently regardless of tether rigidity. Therefore, the properties of the cargo or linkage must play an essential role in motor-motor communication and cargo transport.

Diabetic bladder dysfunction in T2D KK-Ay mice and its changes in the level of relevant gene expression.

OBJECTIVE: Diabetic bladder dysfunction (DBD) is one of the most common and bothersome complications of diabetes mellitus (DM). The purpose of the present study is to investigate DBD in KK-Ay mice, and to identify the expression of relative genes. METHOD: Totally twenty-seven KK-Ay mice and thirty C57BL/6 J mice, respectively, were randomly divided into 12-, 18-, and 25-week old groups. The weight, water intake, voided volume, the frequency of micturition, fasting blood glucose (FBG), oral glucose tolerance test (OGTT) were measured at varying time points. Maximum bladder volume (MBC), residual volume (RV), bladder compliance (BC), micturition efficiency (VE) and maximum micturition pressure (MVP) were assessed by urodynamic test, and contractile responses to α, ß-methylene ATP, KCl, electrical-field stimulation, carbachol were performed by detrusor smooth muscle strips contractility test. The bladders were stained with hematoxylin and eosin (H&E) and Masson's trichrome to determine bladder wall thickness. Additionally, the mRNA expression of Myosin Va, SLC17A9, P2X1, M3 and M2 were then verified by qRT-PCR. RESULT: The weight, water intake, voided volumes, micturition frequency, FBG, the blood glucose AUC0-2h of KK-Ay mice were significantly increased at three time points. MBC, RV and BC were significantly increased; VE was significantly lower at the age of 18 and 25 weeks in KK-Ay mice; MVP was significantly increased at the age of 25 weeks in KK-Ay mice. In DSM strips contractility test, the amplitude of the spontaneous activity in KK-Ay mice significant increased at 12 weeks and 18 weeks, while both the amplitude and frequency were significantly decreased at the age of 25 weeks. The level of Myosin Va, SLC17A9 and M3 receptor significantly decreased in KK-Ay mice at 12 weeks, while Myosin Va markedly increased at 18 weeks; P2X1 and M2 receptors of KK-Ay mice was significantly increased at all three time points. CONCLUSION: Taken together, this study demonstrates that KK-Ay mice can be a proper model to investigate DBD whose transformation from compensatory state to decompensated state may ascribe to the time-dependent alternations of Myosin Va, SLC17A9, P2X1, M3 and M2 expression levels.

Calcium triggers the dissociation of myosin-Va from ribosomes in ribonucleoprotein complexes.

The sorting of RNAs to specific regions of the cell for local translation represents an important mechanism directing protein distribution and cell compartmentalization. While significant progress has been made in understanding the mechanisms underlying the transport and localization of mRNAs, the mechanisms governing ribosome mobilization are less well understood. Ribosomes present in the cytoplasm of multiple cell types can form ribonucleoprotein complexes that also contain myosin-Va (Myo5a), a processive, actin-dependent molecular motor. Here, we report that Myo5a can be disassociated from ribosomes when ribonucleoprotein complexes are exposed to calcium, both in vitro and in vivo. We suggest that Myo5a may act as a molecular switch able to anchor or release ribosomes from the actin cytoskeleton in response to intracellular signaling.

Suo Quan Wan Protects Mouse From Early Diabetic Bladder Dysfunction by Mediating Motor Protein Myosin Va and Transporter Protein SLC17A9.

Objective: To investigate the effects of Suo Quan Wan (SQW), a traditional Chinese herbal formula, on the overactive bladder (OAB) of type 2 diabetes mellitus (T2DM) mouse models, particularly on its function of mediating the gene and protein expression levels of myosin Va and SLC17A9. Materials and Methods: After 4 weeks high-fat diet (HFD) feeding, C57BL/6J mice were injected with streptozotocin (100 mg/kg) for four times. After 3 weeks, the diabetic mice were treated with SQW for another 3 weeks. Voided stain on paper assay, fasting blood glucose (FBG) test, and oral glucose tolerance test (OGTT) were conducted. Urodynamic test, tension test [α,ß-methylene ATP, electrical-field stimulation (EFS), KCl, and carbachol] and histomorphometry were also performed. Western blot analysis and qPCR assays were used to quantify the expression levels of myosin Va and SLC17A9. Results: The diabetic mice exhibited decreased weight but increased water intake, urine production, FBG, and OGTT. No significant changes were observed after 3 weeks SQW treatment. Urodynamic test indicated that the non-voiding contraction (NVC) frequency, maximum bladder capacity (MBC), residual volume (RV), and bladder compliance (BC) were remarkably increased in the diabetic mice, whereas the voided efficiency (VE) was decreased as a feature of overactivity. Compared with the model mice, SQW treatment significantly improved urodynamic urination with decreased NVC, MBC, RV, and BC, and increased VE. Histomorphometry results showed that the bladder wall of the diabetic mice thickened, and SQW effectively attenuated the pathological alterations. The contract responses of bladder strips to all stimulators were higher in the DSM strips of diabetic mice, whereas SQW treatment markedly decreased the contraction response for all stimuli. Moreover, the protein and gene expression levels of myosin Va and SLC17A9 were up-regulated in the bladders of diabetic mice, but SQW treatment restored such alterations. Conclusion: T2DM mice exhibited the early phase of diabetic bladder dysfunction (DBD) characterized by OAB and bladder dysfunction. SQW can improve the bladder storage and micturition of DBD mice by mediating the protein and gene expression levels of myosin Va and SLC17A9 in the bladder, instead of improving the blood glucose level.

Time-dependent functional, morphological, and molecular changes in diabetic bladder dysfunction in streptozotocin-induced diabetic mice.

AIM: Diabetic bladder dysfunction (DBD) is one of the most common and bothersome complications of diabetes mellitus (DM). This study aimed to investigate the functional, structural, and molecular changes of the bladder at 0, 3, 6, 9, and 12 weeks after DM induction by streptozotocin (STZ) in male C57BL/6 mice. METHODS: Male C57BL/6J mice were injected with STZ (130 mg/kg). Then, diabetic general characteristics, cystometry test, histomorphometry, and contractile responses to α, ß-methylene ATP, KCl, electrical-field stimulation, carbachol were performed at 0, 3, 6, 9, and 12 weeks after induction. Finally, protein and messenger RNA (mRNA) expressions of myosin Va and SLC17A9 were quantified. RESULTS: DM mice exhibited lower body weight, voiding efficiency and higher water intake, urine production, fasting blood glucose, oral glucose tolerance test, bladder wall thickness, maximum bladder capacity, residual volume, bladder compliance. In particular, nonvoiding contractions has increased more than five times at 6 weeks. And the amplitudes of spontaneous activity, contractile responses to all stimulus was about two times higher at 6 weeks but cut almost in half at 12 weeks. The protein and mRNA expressions of myosin Va and SLC17A9 were about two times higher at 6 weeks, but myosin Va was reverted nearly 40% while SLC17A9 is still higher at 12 weeks. CONCLUSIONS: DBD transitioned from a compensated state to a decompensated state in STZ-induced DM mice at 9 to 12 weeks after DM induction. Our molecular data suggest that the transition may be closely related to the alterations of myosin Va and SLC17A9 expression levels in the bladder with time.

Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits.

The alpha1 (α1) subunit of the sodium/potassium ATPase (i.e., Na+/K+-ATPase α1), the prototypical sodium pump, is expressed in each eukaryotic cell. They pump out three sodium ions in exchange for two extracellular potassium ions to establish a cellular electrochemical gradient important for firing of neuronal and cardiac action potentials. We hypothesized that myosin (myo or myh) motor proteins might interact with Na+/K+-ATPase α1 subunits in order for them to play an important role in the transport and trafficking of sodium pump. To this end immunoassays were performed to determine whether class II non-muscle myosins (i.e., NMHC-IIA/myh9, NMHC-IIB/myh10 or NMHC-IIC/myh14), myosin Va (myoVa) and myosin VI (myoVI) would interact with Na+/K+-ATPase α1 subunits. Immunoprecipitation of myh9, myh10, myh14, myoVa and myoVI from rat brain tissues led to the co-immunoprecipitation of Na+/K+-ATPase α1 subunits expressed there. Heterologous expression studies using HEK293 cells indicated that recombinant myh9, myh10, myh14 and myoVI interact with Na+/K+-ATPase α1 subunits expressed in HEK293 cells. Additional results indicated that loss of tail regions in recombinant myh9, myh10, myh14 and myoVI did not affect their interaction with Na+/K+-ATPase α1 subunits. However, recombinant myh9, myh10 and myh14 mutants having reduced or no actin binding ability, as a result of loss of their actin binding sites, displayed greatly reduced or null interaction with Na+/K+-ATPase α1 subunits. These results suggested the involvement of the actin binding site, but not tail regions, of NMHC-IIs in their interaction with Na+/K+-ATPase α1 subunits. Overall these results suggest a role for these diverse myosins in the trafficking and transport of sodium pump in neuronal and non-neuronal tissues.

Myosin Va from Eriocheir sinensis: cDNA cloning, expression and involvement in growth and development.

Myosin Va, a member of the myosin superfamily, has been widely identified associated with processes of cellular motility, which include neurotransmitter release and synaptic plasticity during neurodevelopment. However, the function of myosin Va in the growth and development of crustaceans has not yet been reported. In this study, a full-length cDNA of myosin Va (named as EsMyoVa) was cloned from the Chinese mitten crab, Eriocheir sinensis, and the expression patterns were detected in different tissues and larval developmental stages. The full-length cDNA of EsMyoVa was 6037 bp in length. Real time quantitative reverse transcription PCR (qRT-PCR) analysis showed that EsMyoVa transcript has a wide tissue distribution pattern and is expressed in zoeae, megalopa, juvenile crab stages and adults. In order to further study the function of this gene, we used RNAi technology in the muscle, hepatopancreas, gill, and gonad. After double-stranded RNA (dsRNA) injection, the expression level of EsMyoVa was significantly decreased in all tissues in both sexes and the gene knockdown effects of dsRNA persisted for at least 6 days. Subsequently, the role of EsMyoVa was revealed by silencing the transcript through one month injections of Myosin Va dsRNA. Crabs with reduced levels of EsMyoVa transcripts displayed a dramatic slowing in growth rate and considerably higher mortality compared to control groups, which indicated that this gene had important role of regulating growth and development.

Creation of a myosin Va-TAP-tagged mouse and identification of potential myosin Va-interacting proteins in the cerebellum.

The actin-based motor myosin Va transports numerous cargos, including the smooth endoplasmic reticulum (SER) in cerebellar Purkinje neurons (PNs) and melanosomes in melanocytes. Identifying proteins that interact with this myosin is key to understanding its cellular functions. Toward that end, we used recombineering to insert via homologous recombination a tandem affinity purification (TAP) tag composed of the immunoglobulin G-binding domain of protein A, a tobacco etch virus cleavage site, and a FLAG tag into the mouse MYO5A locus immediately after the initiation codon. Importantly, we provide evidence that the TAP-tagged version of myosin Va (TAP-MyoVa) functions normally in terms of SER transport in PNs and melanosome positioning in melanocytes. Given this and other evidence that TAP-MyoVa is fully functional, we purified it together with associated proteins directly from juvenile mouse cerebella and subjected the samples to mass spectroscopic analyses. As expected, known myosin Va-binding partners like dynein light chain were identified. Importantly, numerous novel interacting proteins were also tentatively identified, including guanine nucleotide-binding protein G(o) subunit alpha (Gnao1), a biomarker for schizophrenia. Consistently, an antibody to Gnao1 immunoprecipitates myosin Va, and Gnao1's localization to PN dendritic spines depends on myosin Va. The mouse model created here should facilitate the identification of novel myosin Va-binding partners, which in turn should advance our understanding of the roles played by this important myosin in vivo.

Myosin Va plays essential roles in maintaining normal mitosis, enhancing tumor cell motility and viability.

Myosin Va, a member of Class V myosin, functions in organelle motility, spindle formation, nuclear morphogenesis and cell motility. The purpose of this study is to explore the expression and localization of myosin Va in testicular cancer and prostate cancer, and its specific roles in tumor progression including cell division, migration and proliferation. We detected myosin Va in testicular and prostate tumor tissues using sqRT-PCR, western blot, and immunofluorescence. Tumor samples showed an increased expression of myosin Va, abnormal actin and myosin Va distribution. Immunofluorescence images during the cell cycle showed that myosin Va tended to gather at cytoplasm during anaphase but co-localized with nucleus during other phases, suggesting the roles of myosin Va in disassembly of spindle microtubule, movement of chromosomes and normal cytokinesis. In addition, multi-nucleation and aberrant nuclear morphology were observed in myosin Va-knockdown cells. Wounding assay and CCK-8-based cell counting were conducted to explore myosin Va roles in cell migration, viability and proliferation. Our results suggest that myosin Va plays essential roles in maintaining normal mitosis, enhancing tumor cell motility and viability, and these properties are the hallmark of tumor progression and metastasis development. Therefore, an increased understanding of myosin Va expression and function will assist in the development of future oncodiagnosis and -therapy.

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

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.

KIFC1 and myosin Va: two motors for acrosomal biogenesis and nuclear shaping during spermiogenesis of Portunus trituberculatus.

To investigate the molecular mechanisms underlying the spermiogenesis of the swimming crab Portunus trituberculatus, full lengths of motor proteins KIFC1 and myosin Va were cloned by rapid-amplification of cDNA ends from P. trituberculatus testes cDNA, and their respective probes and specific antibodies were used to track their localization during sperm maturation. Antisense probes were designed from the gene sequences and used to detect the mRNA levels of each gene. According to the results of fluorescence in situ hybridization (FISH), the transcription of kifc1 and myosin Va began at the mid-stage of spermatids, with the kifc1 mRNA being most active at the location where the acrosome cap was formed and the myosin Va was more concentrated in the acrosome complex. Immunofluorescence results showed that KIFC1 and myosin Va were highly expressed in each stage of spermigenesis. In the early spermatids, they were randomly dispersed in the cytoplasm together with cytoskeletons. At the mid-stage, the motors were gathered above one side of the nucleus where the acrosome would later form. In the late spermatids and mature sperm, the KIFC1 was closely distributed in the perinuclear region, indicating its role in nucleus deformation. Myosin Va was distributed in the acrosome complex until sperm maturity. This suggests myosin Va's potential role in material transportation during acrosome formation and maturation. The above results provide a preliminary illustration of the essential roles of KIFC1 and myosin Va in the spermiogenesis of the swimming crab P. trituberculatus.