Myosin VA Research Articles

MYO5A overexpression promotes invasion and correlates with low lymphocyte infiltration in head and neck squamous carcinoma

Head and neck squamous carcinoma (HNSC) poses a significant public health challenge due to its substantial morbidity. Nevertheless, despite advances in current treatments, the prognosis for HNSC remains unsatisfactory. To address this, single-cell RNA sequencing (RNA-seq) and bulk RNA-seq data combined with in vitro studies were conducted to examine the role of MYO5A (Myosin VA) in HNSC. Our investigation revealed an overexpression of MYO5A in HNSC that promotes HNSC migration in vitro. Remarkably, knockdown of MYO5A suppressed vimentin expression. Furthermore, analyzing the TCGA database evidenced that MYO5A is a risk factor for human papillomavirus positive (HPV+) HNSC (HR = 0.81, P < 0.001). In high MYO5A expression HNSC, there was a low count of tumor infiltrating lymphocytes (TIL), including activated CD4+ T cells, CD8+ T cells, and B cells. Of note, CD4+ T cells and B cells were positively associated with improved HPV+ HNSC outcomes. Correlation analysis demonstrated a decreased level of immunostimulators in high MYO5A-expressing HNSC. Collectively, these findings suggest that MYO5A may promote HNSC migration through vimentin and involve itself in the process of immune infiltration in HNSC, advancing the understanding of the mechanisms and treatment of HNSC.

Circ-FNDC3B Functions as an Oncogenic Factor in Esophageal Squamous Cell Carcinoma via Upregulating MYO5A by Absorbing miR-136-5p and miR-370-3p.

Circular RNAs (circRNAs) are a class of key regulators in cancers via regulating gene levels by acting as sponges of miRNAs. This study was devoted to explore the functional mechanism of circRNA fibronectin type III domain-containing protein 3B (circ-FNDC3B) in esophageal squamous cell carcinoma (ESCC). RNA levels were examined via reverse transcription-quantitative polymerase chain reaction assay. Cell viability detection was performed using Cell Counting Kit-8 assay. The proliferation ability was determined through colony formation assay and EDU assay. Flow cytometry was applied for analysis of apoptosis. Invasion ability was assessed via transwell assay. Target binding was analyzed by dual-luciferase reporter assay. The protein expression was measured using western blot. In vivo research was conducted via xenograft model in mice. Circ-FNDC3B exhibited significant upregulation in ESCC tissues and cells. Downregulation of circ-FNDC3B inhibited ESCC cell proliferation and invasion but accelerated cell apoptosis. Circ-FNDC3B interacted with miR-136-5p or miR-370-3p. The function of circ-FNDC3B was achieved by sponging miR-136-5p or miR-370-3p. Myosin VA (MYO5A) acted as a downstream target of miR-136-5p or miR-370-3p. MYO5A reversed miR-136-5p/miR-370-3p-induced tumor inhibition in ESCC cells. Circ-FNDC3B targeted miR-136-5p or miR-370-3p to affect MYO5A expression. Circ-FNDC3B knockdown reduced tumor growth in vivo by inhibiting miR-136-5p or miR-370-3p-mediated MYO5A expression. These findings demonstrated that circ-FNDC3B contributed to malignant progression of ESCC cells via miR-136-5p/MYO5A or miR-370-3p/MYO5A axis.

A mutation in the switch 1 region of myosin VA alters the load-dependence of phosphate rebinding

The load-dependence of reversible product release in myosins is key to understanding how it converts chemical energy into mechanical force. Following the powerstroke and phosphate (Pi) release, the rebinding of Pi to the actomyosin⋅ADP complex is highly load-dependent, but the underlying mechanisms/structures involved remain unclear. Therefore, we introduced a mutation (S217A) into the switch I region of the active site to examine its impact on the load dependence of Pi rebinding to single-headed myosin Va (MV) in a mini-ensemble (∼10 heads) three-bead laser trap assay at 100 uM ATP.

Natural Citrus flavanone 5-demethylnobiletin stimulates melanogenesis through the activation of cAMP/CREB pathway in B16F10 cells

Background5-demethylnobiletin is a natural polymethoxyflavone which is isolated from the extract of citrus fruits peels. It exhibits a broad spectrum of biological activities such as anti-cancer, anti-inflammatory, cardiovascular protective and neuroprotective effects, however, its effect in melanogenesis remains uninvestigated. PurposeMelanin synthesis is a very important biological process in curing disease such as vitiligo with depigmentation on the skin. In the current work, we aim to confirm the bioactivity and mechanism of 5-demethylnobiletin in stimulating melanogenesis. Study DesignTo confirm the mechanistic role of 5-demethylnobiletin in enhancing melanogenesis, its effect on the activity of tyrosinase, together with the level of microphthalmia-associated transcription factor (MITF), Trp-1, Trp-2, melanocyte-specific marker protein PMEL17, Rab27a, Melanophilin and Myosin VA were studied in B16F10 melanoma cells. MethodsMultiple biological assays on melanogenesis-associated proteins such as melanin content detection, tyrosinase activity colorimetric assay, qPCR, western blot analysis, dual-luciferase reporter assay, cAMP activity assay and Fontana-Masson ammoniacal silver staining were used to confirm the role of 5-demethylnobiletin in stimulating melanin synthesis and the transportation of melanosomes. ResultsAs confirmed by multiple biological assays, 5-demethylnobiletin is found to stimulate dendrite structure formation in cells, melanin synthesis and the transportation of melanosomes, via inducing the phosphorylation of cAMP response element-binding protein (CREB) and increasing the intracellular levels of cAMP in vitro through the PKA-dependent pathway. ConclusionThe findings suggested that 5-demethylnobiletin may be considered as a potential natural product candidate for patients with pigment disorder.

Rhamnazin suppresses melanosome transport by promoting the ubiquitin-mediated proteasomal degradation of melanophilin

BackgroundMelanosomes are intracellularly transported from the perinuclear region to the cell periphery and then to neighboring keratinocytes. We recently reported that the flavonoid rhamnazin suppresses melanosomal transport within pigment cells, yet the action mechanism remained unclear. ObjectiveOur aim was to elucidate how rhamnazin influences the intracellular transport of melanosomes. MethodsA melanosome distribution assay and immunostaining were performed using B16F10 mouse melanoma cells and normal human epidermal melanocytes, respectively. Expression levels of melanosome transport-related proteins, including melanophilin (MLPH), RAB27A, and myosin VA (MYO5A), were analyzed by immunoblotting. Ubiquitinated MLPH was detected using a commercial ubiquitin detection kit. To investigate the interaction between rhamnazin and MLPH, we prepared rhamnazin conjugated with magnetic FG beads. ResultsImmunoblotting analysis revealed that rhamnazin specifically reduces the expression of MLPH but not RAB27A or MYO5A proteins. The ubiquitin detection assay, which made use of a proteasome inhibitor, showed that MLPH accumulated as a polyubiquitinated protein after treatment with rhamnazin. We speculated that the affinity of rhamnazin for the components of the melanosome transport-related tripartite complex may alter the stability of the formation of the tripartite assembly. By using affinity-based techniques with B16F10 whole cell lysates or recombinant MLPH and RAB27A proteins, we revealed the interaction of rhamnazin with the components of the tripartite complex. ConclusionWe found that rhamnazin inhibits intracellular transport of melanosomes through proteasomal degradation of MLPH. Our results suggest that topical application of rhamnazin may provide a new approach for treating skin pigmentation disorders.

MYO5A Frameshift Variant in a Miniature Dachshund with Coat Color Dilution and Neurological Defects Resembling Human Griscelli Syndrome Type 1.

A 1-month-old, female, smooth-haired miniature Dachshund with dilute color and neurological defects was investigated. The aim of this study was to characterize the clinical signs, histopathological changes and underlying genetic defect. The puppy had visible coat color dilution and was unable to hold its head on its own or to remain in a stable prone position for an extended period. Histopathological examination revealed an accumulation of clumped melanin and deposition of accumulated keratin within the hair follicles, accompanied by dermal pigmentary incontinence. These dermatological changes were compatible with the histopathology described in dogs with an MLPH-related dilute coat color. We sequenced the genome of the affected dog and compared the data to 795 control genomes. MYO5A, coding for myosin VA, was investigated as the top functional candidate gene. This search revealed a private homozygous frameshift variant in MYO5A, XM_022412522.1:c.4973_4974insA, predicted to truncate 269 amino acids (13.8%) of the wild type myosin VA protein, XP_022268230.1:p.(Asn1658Lysfs*28). The genotypes of the index family showed the expected co-segregation with the phenotype and the mutant allele was absent from 142 additionally genotyped, unrelated Dachshund dogs. MYO5A loss of function variants cause Griscelli type 1 syndrome in humans, lavender foal in horses and the phenotype of the dilute mouse mutant. Based on the available data, together with current knowledge on other species, we propose the identified MYO5A frameshift insertion as a candidate causative variant for the observed dermatological and neurological signs in the investigated dog.

Myosin'S Powerstroke Occurs with Phosphate Still in the Active Site

A fundamental property of myosin is its ability to harness the power of ATP hydrolysis to generate force. A key unanswered question is the timing of the force-generating powerstroke relative to phosphate (Pi) -release from its active site. We examined the ability of single-headed myosin Va (MV) construct to bind to actin and generate a powerstroke in a single molecule laser trap assay in the presence of elevated Pi. We examined both WT MV and the impact of a mutation (S217A) in the conserved switch I region of the active site which slows actin-activated Pi-release. Upon binding to actin WT myosin rapidly (>500/s) generated a 7±0.5 nm powerstroke. In the presence of 30mM Pi, no decrease in the size (7±0.4 nm) or delay in the rate of the development of the powerstroke (>500/s) was observed, however the average duration of strong actomyosin binding (ton) was reduced in the presence of Pi (240±20 vs 113± ms, p<0.05). The reduced ton is consistent with Pi rebinding to the active site and accelerating detachment from actin. Therefore, this suggests myosin generates the powerstroke with Pi still in the active site. S217A generated a powerstroke with a similar rate (>500/s) and size (9±0.9 nm) to the WT, but ton was shorter, but not significantly different, than WT (143±20 ms, p=0.22 and 62±5 ms, p<0.05 for S217A in the absence and presence of Pi, respectively). Since the S217A mutant slows actin-activated Pi-release from the active site our results fit best to a model in which the powerstroke occurs with Pi still in the active site. Together these findings suggest that actin-binding, and not the release of Pi from the active site, trigger the conformational changes that lead to the powerstroke, and thus force-generation by myosin.

A thirteen‑gene set efficiently predicts the prognosis of glioblastoma.

Glioblastoma multiforme (GBM) is the most common type of brain cancer; it usually recurs and patients have a short survival time. The present study aimed to construct a gene expression classifier and to screen key genes associated with GBM prognosis. GSE7696 microarray data set included samples from 10 recurrent GBM tissues, 70 primary GBM tissues and 4 normal brain tissues. Seed genes were identified by the ‘survival’ package in R and subjected to pathway enrichment analysis. Prognostic genes were selected from the seed genes using the ‘rbsurv’ package in R, unsupervised hierarchical clustering, survival analysis and enrichment analysis. Multivariate survival analysis was performed for the prognostic genes, and the GBM data set from The Cancer Genome Atlas database was utilized to validate the prognostic genes. Of the 1,785 seed genes analyzed, 13 prognostic feature genes, including collagen type XXVIII α1 chain (COL28A1), PDS5 cohesin-associated factor A (PDS5A), zinc-finger DHHC-type containing 2 (ZDHHC2), zinc-finger protein 24 (ZNF24), myosin VA (MYO5A) and myeloid/lymphoid or mixed-lineage leukemia translocated to 4 (MLLT4), were identified. These genes performed well on sample classification and prognostic risk differentiation, and six pathways, including adherens junction, cyclic adenosine 3′,5′-monophosphate signaling and Ras signaling pathways, were enriched for these feature genes. The high-risk group was slightly older compared with the low-risk group. The validation data set confirmed the prognostic value of the 13 feature genes for GBM; of these, COL28A1, PDS5A, ZDHHC2, ZNF24, MYO5A and MLLT4 may be crucial. These results may aid the understanding of the pathogenesis of GBM and provide important clues for the development of novel diagnostic markers or therapeutic targets.

Elucidating the Role of Mineralocorticoid Receptors in Skeletal Muscle as a Potential Therapeutic Target for Duchenne Muscular Dystrophy

Our lab identified a potential new treatment for Duchenne Muscular Dystrophy using the mineralocorticoid receptor (MR) antagonist spironolactone and ACE inhibitor lisinopril. Drug studies using dystrophic Het (utrn+/-;mdx) mice showed a dramatic improvement in both respiratory and limb muscle force and a reduction of ongoing muscle damage, in addition to preventing cardiomyopathy. We show MR is present in both skeletal muscle tissue and myogenic cultures, supporting a direct affect by MR antagonists on skeletal muscle. Global analysis of gene expression between treated and untreated dystrophic mice identified potential molecular targets to unravel the drugs' mode of action. Preliminary microarray data comparing quadriceps muscle from lisinopril and spironolactone treated het mice to untreated controls revealed changes in the expression of several gene targets with known roles in striated muscle, Gene Name Gene Symbol Fold-change (Treated/Control) Role in Striated Muscle ankyrin repeat domain 1 Ankrd1 -2.15634 Marker of skeletal muscle pathological remodeling tight junction protein 2 Tjp2 -2.02372 Important molecular target in vascular remodeling myosin VA Myo5a -2.12497 Involved in maturation of NMJs in regenerating muscle mid1 interacting protein 1 Mid1ip1 -2.48061 Potential biomarker of skeletal myopathy myosin binding protein H Mybph 2.059236 Muscle remodeling in response to injury thyroid hormone responsive SPOT14 homolog Thrsp 2.138901 Modulator of fatty acid synthesis which have been confirmed using immunofluorescence and western blot analysis. Myogenic cultures treated with MR agonists and antagonists are being used to test whether these potential downstream targets are affected in a cell autologous manner in skeletal muscle and represent bona fide MR gene targets. NIH 1 R01 NS082868; T32 NS077984

Bone Morphogenetic Protein Signaling Suppresses Wound-Induced Skin Repair by Inhibiting Keratinocyte Proliferation and Migration

Bone morphogenetic protein (BMP) signalling plays a key role in the control of skin development and postnatal remodelling by regulating keratinocyte proliferation, differentiation and apoptosis. To study the role of BMPs in wound-induced epidermal repair, we used transgenic mice overexpressing the BMP downstream component Smad1 under the control of a K14 promoter as an in vivo model, as well as ex vivo and in vitro assays. K14-caSmad1 mice exhibited retarded wound healing associated with significant inhibition of proliferation and increased apoptosis in healing wound epithelium. Furthermore, microarray and qRT-PCR analyses revealed decreased expression of a number of cytoskeletal/cell motility-associated genes including wound-associated keratins (Krt16, Krt17) and Myo5a, in the epidermis of K14-caSmad1 mice versus wild-type controls during wound healing. BMP treatment significantly inhibited keratinocyte migration ex vivo, and primary keratinocytes of K14-caSmad1 mice showed retarded migration compared to wild-type controls. Finally, siRNA-mediated silencing of Bmpr-1B in primary mouse keratinocytes accelerated cell migration and was associated with increased expression of Krt16, Krt17 and Myo5a compared to controls. Thus, this study demonstrates that BMPs inhibit keratinocyte proliferation, cytoskeletal organization and migration in regenerating skin epithelium during wound healing, and raises a possibility for using BMP antagonists for the management of chronic wounds.

A frameshift mutation in the melanophilin gene causes the dilute coat colour in rabbit (Oryctolagus cuniculus) breeds

In rabbit, the dilute locus is determined by a recessive mutated allele (d) that causes the dilution of both eumelanic and pheomelanic pigmentations. In mice, similar phenotypes are determined by mutations in the myosin VA, Rab27a and melanophilin (MLPH) genes. In this study, we investigated the rabbit MLPH gene and showed that a mutation in this gene appears responsible for the dilute coat colour in this species. Checkered Giant F1 families segregating for black and grey (diluted or blue) coat colour were first genotyped for a complex indel in intron 1 of the MLPH gene that was completely associated with the coat colour phenotype (θ = 0.00; LOD = 4.82). Then, we sequenced 6357 bp of the MLPH gene in 18 rabbits of different coat colours, including blue animals. A total of 165 polymorphisms were identified: 137 were in non-coding regions and 28 were in coding exons. One of them was a frameshift deletion in exon 5. Genotyping the half-sib families confirmed the complete cosegregation of this mutation with the blue coat colour. The mutation was analysed in 198 rabbits of 23 breeds. All Blue Vienna and all other blue/grey/ash rabbits in other breeds (Californian, Castor Rex, Checkered Giant, English Spot, Fairy Marburg and Fairy Pearly) were homozygous for this deletion. The identification of MLPH as the responsible gene for the dilute locus in rabbit provides a natural animal model for human Griscelli syndrome type 3 and a new mutant to study the role of this gene on pigmentation.

Identification and analysis of single nucleotide polymorphisms in the myosin VA (MYO5A) gene and its exclusion as the causative gene of the dilute coat colour locus in rabbit

Classical genetic studies have identified different coat colour loci in rabbit and comparative analyses have established corresponding loci across species. In particular, the rabbit dilute locus is determined by a recessive coat colour mutation that modifies the basic colours influenced by the agouti and extension mutations. In mice, similar phenotypic effects are determined by a similarly named locus. This locus encodes the myosin VA ( Myo5a ) gene, whose protein product is an unconventional myosin that plays an essential role in melanosome transport in the melanocytes. We selected the same gene as a strong candidate for explaining the dilute coat colour in rabbit. To this end, 1399 bp were re-sequenced, spanning 4 exons out of 41 exons and a portion of intronic regions of the rabbit MYO5A gene to identify polymorphisms that could be useful to confirm or exclude this gene as causative of the rabbit dilute locus. Nine polymorphisms were identified, one of which was used to follow the segregation of the blue and black colours in a Checkered Giant F1 family. The single nucleotide polymorphism (SNP) analysed did not co-segregate with the two colours. These results excluded the MYO5A gene as determinant of the dilute locus in rabbit. The two alleles of this SNP were also present in several other breeds with different coat colours, further indicating that this marker is not associated with the dilute mutation in rabbits. Other candidates should be investigated to identify the causative gene of this locus in rabbit.

Ethnic and Mouse Strain Differences in Central Corneal Thickness and Association with Pigmentation Phenotype

The cornea is a transparent structure that permits the refraction of light into the eye. Evidence from a range of studies indicates that central corneal thickness (CCT) is strongly genetically determined. Support for a genetic component comes from data showing significant variation in CCT between different human ethnic groups. Interestingly, these studies also appear to show that skin pigmentation may influence CCT. To validate these observations, we undertook the first analysis of CCT in an oculocutaneous albinism (OCA) and Ugandan cohort, populations with distinct skin pigmentation phenotypes. There was a significant difference in the mean CCT of the OCA, Ugandan and Australian-Caucasian cohorts (Ugandan: 517.3±37 µm; Caucasian: 539.7±32.8 µm, OCA: 563.3±37.2 µm; p<0.001). A meta-analysis of 53 studies investigating the CCT of different ethnic groups was then performed and demonstrated that darker skin pigmentation is associated with a thinner CCT (p<0.001). To further verify these observations, we measured CCT in 13 different inbred mouse strains and found a significant difference between the albino and pigmented strains (p = 0.008). Specific mutations within the melanin synthesis pathway were then investigated in mice for an association with CCT. Significant differences between mutant and wild type strains were seen with the nonagouti (p<0.001), myosin VA (p<0.001), tyrosinase (p = 0.025) and tyrosinase related protein (p = 0.001) genes. These findings provide support for our hypothesis that pigmentation is associated with CCT and identifies pigment-related genes as candidates for developmental determination of a non-pigmented structure.

Processive Runs of Full Length Myosin VA Are Interrupted by Pauses and Dwells

Full length myosin Va (FL-MyoVa) forms an inhibited, folded conformation at low salt, stabilized by interactions between the globular tails and the heads. High ionic strength disrupts this interaction, resulting in an extended, active processive motor. In vivo, it has been postulated that cargo binding disrupts the folded conformation and activates the motor. It is possible that splice variations in the tail (-B+D+F, melanocyte; +B-D-F, brain) could modify the ability of myosin Va to form the inhibited state. Two FL-MyoVa splice variants and an HMM-MyoVa, with biotin tags for Qdot labeling, were expressed in Sf9 cells. Sedimentation velocity experiments showed similar transitions from the folded-to-extended conformation for the two splice variants as a function of salt. TIRF microscopy was then used to observe processive runs on actin. The velocities of both FL-MyoVa splice variants were similar, and increased 270% (171-460nm/sec) with increasing KCl concentration (25-200mM). In contrast, the velocity of HMM-MyoVa increased by a more modest 50% (381-586nm/sec). The trajectories of the FL-MyoVa and HMM-MyoVa were also strikingly different. Both FL-MyoVa splice variants underwent processive runs that were interrupted by periods during which the motor dwelled at fixed points on the actin filament, presumably in the folded, inhibited state. At lower KCl concentration, FL-MyoVa dwelled approximately half of the total trajectory duration. Increasing ionic strength decreased duration of the dwells. HMM-MyoVa was fully active and maintained continuous processive movement at all KCl concentrations. The slower overall velocities for the FL-MyoVa splice variants, compared to HMM-MyoVa, results from inclusion of the dwell periods. We propose that during a processive run, a single FL-MyoVa can switch between an active and inhibited state without dissociating from actin, and that this phenomenon is independent of splice variations in the tail domain.

Myrip uses distinct domains in the cellular activation of myosin VA and myosin VIIA in melanosome transport

Myrip is a Rab27a and MyosinVIIa (MyoVIIa) linking protein that may regulate melanosome transport in the retinal pigment epithelium (RPE). Myrip also binds MyosinVa (MyoVa) in vitro however it is unclear whether this interaction is of sufficient affinity to be physiologically relevant. Here, we addressed the questions of whether Myrip interacts with MyoVa in cells and the molecular basis of cellular activation of MyoVa and MyoVIIa by Myrip. To answer these questions we used melanosome transport in skin melanocytes and RPE cells as read-outs of MyoVa and MyoVIIa activity. We found that Myrip recruits and activates MyoVa on skin melanosomes with similar efficiency to the established MyoVa activator Melanophilin (Mlph). Mutagenesis showed that a Myrip-Mlph conserved amphipathic helix (MMAH) is essential for MyoVa interaction while other Myrip regions, including the MyoVa exon F binding domain equivalent, play non-essential roles in this interaction. This suggests that, in contrast to Mlph, Myrip interacts with MyoVa lacking melanocyte-specific exon F. Parallel studies of RPE melanosome transport reveal that Myrip-specific inserts, but not the MMAH, are essential for MyoVIIa activation. We conclude that Myrip is a versatile Rab27a-associated myosin-activating protein that mediates cellular activation of MyoVa and MyoVIIa via non-overlapping domains.

Griscelli syndrome: a model system to study vesicular trafficking

Griscelli syndrome (GS) is a rare autosomal recessive disorder caused by mutations in either the myosin VA (GS1), RAB27A (GS2) or melanophilin (GS3) genes. The three GS subtypes are commonly characterized by pigment dilution of the skin and hair, due to defects involving melanosome transport in melanocytes. Here, we review how detailed studies concerning GS have contributed to a better understanding of the molecular mechanisms involved in vesicle transport and membrane trafficking processes. Additionally, we demonstrate that the identification and biological analysis of novel disease-causing mutations highlighted the functional importance of the RAB27A-MLPH-MYO5A tripartite complex in intracellular melanosome transport. As the small GTPase Rab27a is able to interact with multiple effectors, including Slp2-a and Myrip, we report on their presumed role in melanosome transport. Furthermore, we summarize data suggesting that RAB27B and RAB27A are functionally redundant and hereby provide further insight into the pathogenesis of GS2. Finally, we discuss how the gathered knowledge about the RAB27A-MLPH-MYO5A tripartite complex can be translated into a possible therapeutic application to reduce (hyper)pigmentation of the skin.

Melanosomes at a glance

Melanosomes, the pigment granules that provide tissues with colour and photoprotection, are the cellular site of synthesis, storage and transport of melanin pigments. They are synthesised in mammalian skin melanocytes, in choroidal melanocytes and retinal pigment epithelial (RPE) cells in the eye,

Interactions between SNP Alleles at Multiple Loci Contribute to Skin Color Differences between Caucasoid and Mongoloid Subjects

This study aimed to identify single nucleotide polymorphism (SNP) alleles at multiple loci associated with racial differences in skin color using SNP genotyping. A total of 122 Caucasians in Toledo, Ohio and 100 Mongoloids in Japan were genotyped for 20 SNPs in 7 candidate genes, encoding the Agouti signaling protein (ASIP), tyrosinase-related protein 1 (TYRP1), tyrosinase (TYR), melanocortin 1 receptor (MC1R), oculocutaneous albinism II (OCA2), microphthalmia-associated transcription factor (MITF), and myosin VA (MYO5A). Data were used to analyze associations between the 20 SNP alleles using linkage disequilibrium (LD). Combinations of SNP alleles were jointly tested under LD for associations with racial groups by performing a chi(2) test for independence. Results showed that SNP alleles at multiple loci can be considered the haplotype that contributes to significant differences between the two population groups and suggest a high probability of LD. Confirmation of these findings requires further study with other ethnic groups to analyze the associations between SNP alleles at multiple loci and skin color variation among races.

Separation of large mammalian ventricular myosin differing in ATPase activityThis paper is one of a selection of papers published in this Special Issue, entitled The Cellular and Molecular Basis of Cardiovascular Dysfunction, Dhalla 70th Birthday Tribute.

To investigate a possible heterogeneity of human ventricular myosin, papillary muscles of patients with valvular dysfunction were examined using a modified native gel electrophoresis. Myosin was separated into 2 components termed VA and VB, whereby the VA to VB proportion appeared to depend on the ventricular load. The proportion of the faster migrating band VA was correlated (P<0.05) with end-diastolic pressure and the aortic pressure-cardiac index product. The regression based on these variables accounted for 67% of the variation in VA (R2=0.67). The VA proportion was, however, not significantly correlated with cardiac norepinephrine concentration. The ATPase activity of the 2 components of myosin was assessed from the Ca3(PO4)2 precipitation by incubating the gel in the presence of ATP and CaCl2. The ATPase activity of VA was 60% of that of VB. The VA and VB forms were observed also in the cat (31.4% VA), dog (32.1% VA), pig (28.5% VA), wild pig (33.7% VA), and roe deer (30.5% VA). VA and VB were not detected in the rat exhibiting the 3 isoforms V1, V2, and V3, rabbit (100% V3), and hare (86% V1). The data demonstrate a heterogeneity of large mammalian ventricular myosin, whereby an increased cardiac load appeared to be associated with a higher myosin VA proportion that exhibited a reduced ATPase activity.

Myosin Va Mutation in Rats Is an Animal Model for the Human Hereditary Neurological Disease, Griscelli Syndrome Type 1

A spontaneous neurological mutation, dilute-opisthotonus (dop), was discovered in our breeding colony of Wistar rats. We found that the mutation affected the gene encoding Myosin Va (MyoVA), an actin-based molecular motor. Analysis of the myosin Va (Myo5a) gene of the dop genome showed the presence of a complex rearrangement consisting of a 306-bp inversion associated with 217-bp and 17-bp deletions. A 141-bp exon is skipped in the dop transcript, producing a dop cDNA with a 141 in-frame deletion in the sequences encoding the head region. Expression of the MyoVA protein is severely impaired in the brains of dop homozygous rats, suggesting they have a null mutation for Myo5a. In a morphological analysis of the cerebella of dop rats, we found an absence of smooth endoplasmic reticulum (SER) and of inositol 1,4,5-triphosphate (IP3) receptors in the dendritic spines of Purkinje cells (PC). The SER acts as an intracellular Ca(2+) store and IP3-mediated Ca(2+) signaling in dendritic spines plays a critical role in synaptic regulation. We therefore measured synaptic transmission and long-term depression (LTD), a form of synaptic plasticity underlying cerebellar motor learning, at PC synapses in the cerebella of dop rats. We found that synaptic transmission at the PC synapses is largely normal, whereas the LTD is deficient due to a decrease in IP3-mediated Ca(2+) release from the SER in the PC spines of the dop cerebella. These findings may account for the ataxic movements and clonic convulsions displayed by dop rats. They also contribute to our understanding of the neurological disease mechanisms of the human hereditary disease Griscelli syndrome type 1, which is caused by mutation of the Myo5a gene.