Journal: Scientific Reports

Article Title: Oxyresveratrol suppressed melanogenesis, dendrite formation, and melanosome transport in melanocytes via regulation of the MC1R/cAMP/MITF pathway

doi: 10.1038/s41598-025-05248-x

Figure Lengend Snippet: Oxyresveratrol inhibited the expression of small GTPases and kinesin involved in dendrite formation and melanosome transport. ( a – h ) The effects of oxyresveratrol on the expression of eight genes associated with dendrite development and melanosome transport in B16F10 cells. OXY-5, 5 µg/mL oxyresveratrol group. OXY-10, 10 µg/mL oxyresveratrol group. OXY-15, 15 µg/mL oxyresveratrol group. The mRNA expression levels of Cdc42 , Rab17 , Rab11b , Rac1 , Kif5b , Rab27a , Mlph , and Myo5a were normalized to β-actin gene ( Actb ) levels. N = 3, Student’s t test, * P < 0.05, ** P < 0.01, *** P < 0.001 vs. CK. ( i ) The effects of oxyresveratrol on the protein expression of CDC42, RAB17, RAB11B, RAC1, KIF5B, and MLPH in B16F10 cells. The levels of these proteins were normalized to GAPDH levels.

Article Snippet: For western blot assay, antibodies CDC42 (10155-1-AP, 1:1000), RAC1 (24072-1-AP, 1:1000), MLPH (10338-1-AP, 1:1000), KIF5B (21632-1-AP, 1:1000), RAB11B (67780-1-Ig, 1:1000) and TRP-2 (13095-1-AP, 1:1000) were purchased from proteintech (China), RAB17 (TA350622S, 1:1000) and MC1R (TA321478S, 1:1000) were purchased from OriGene (USA), PMEL/gp100 (ab137078, 1:1000) was purchased from Abcam (UK), and GAPDH (KC-5G5, 1:10000) was purchased from Aksomics (China).

Techniques: Expressing

Journal: PloS one

Article Title: FABP7 and HMGCS2 are novel protein markers for apocrine differentiation categorizing apocrine carcinoma of the breast.

doi: 10.1371/journal.pone.0112024

Figure Lengend Snippet: Figure 7. Immunohistochemical analysis of molecular apocrine markers derived from trancriptomics data within the apocrine cyst and carcinoma dataset. Representative staining of FFPE of apocrine cysts and IACs with antibodies against SLC2A10 (Glucose transporter type 10, sections A and B) - cytoplasmic immunoreactivity; SLC7A8 (L-type amino acid transporter 2, sections C and D) - mainly cytoplasmic and membranous immunereactivity; MLPH (Melanophilin, sections E and F) - cytoplasmic and luminal membranous immunoreactivity; FOXA1 (Hepatocyte nuclear factor 3-alpha, sections G and H) – nuclear staining; XBP1 (X-box-binding protein 1, sections I and J) – mainly nuclear and cytoplasmic immunereactivity; BLVRA (Biliverdin reductase A, sections K and L) - cytoplasmic immunoreactivity which combined with rare nuclear positivity; RHOB (Rho-related GTP-binding protein RhoB, sections M and N) - cytoplasmic immunoreactivity; TSC22D3 (TSC22 domain family protein 3, sections O and P) - mainly nuclear and cytoplasmic immunereactivity; ABCA12 (ATP-binding cassette sub-family A member 12, sections Q and R) - cytoplasmic immunoreactivity and SIDT1 (SID1 transmembrane family member 1, sections I and G) - cytoplasmic immunoreactivity. Magnification: x10. Representative areas are shown in higher magnification (x20) and examples of positive staining are indicated by red arrows. doi:10.1371/journal.pone.0112024.g007

Article Snippet: Antibodies The polyclonal antibody raised against synthetic peptides of human FABP7 (HPA028825), BLVRA (HPA042865), ABCA12 (HPA043194), SIDT1 (HPA035862), MLPH (HPA014685) were purchased from Atlas Antibodies and were used in IHC at a dilution of 1:200, 1:100, 1:500, 1:50 and 1:100 respectively in accordance to the manufacturer instructions.

Techniques: Immunohistochemical staining, Derivative Assay, Staining, Binding Assay

Journal: PloS one

Article Title: FABP7 and HMGCS2 are novel protein markers for apocrine differentiation categorizing apocrine carcinoma of the breast.

doi: 10.1371/journal.pone.0112024

Figure Lengend Snippet: Figure 8. Protein expression profile of transcriptome-derived apocrine markers among breast cancer subtypes. The diagram presents immunohistochemical profiles of four breast tumor subtypes, TNBC, Luminal A Luminal B and HER2, reacted with antibodies against ten transcriptome-derived apocrine markers: XBP1, TSC22D3, ABCA12, SIDT1, FOXA1, RHOB, BLVRA, SLC2A10, SLC7A8 and MLPH. The rows indicate the expression of particular protein in every core in breast cancer TMA (BRC1501, 1502 and 1503; Pantomics INC, USA): red box – positive staining, white box – negative staining grey box – parameter was not determined. Corresponding frequencies of positives are shown on the right side of the diagram. Samples were considered as positive if 10% or more of the cells showed a clear positive staining with the antibodies. IDC = invasive ductal carcinoma; TNBC = triple negative breast cancer. Tumors have been stratified as specified in Materials and Methods. doi:10.1371/journal.pone.0112024.g008

Article Snippet: Antibodies The polyclonal antibody raised against synthetic peptides of human FABP7 (HPA028825), BLVRA (HPA042865), ABCA12 (HPA043194), SIDT1 (HPA035862), MLPH (HPA014685) were purchased from Atlas Antibodies and were used in IHC at a dilution of 1:200, 1:100, 1:500, 1:50 and 1:100 respectively in accordance to the manufacturer instructions.

Techniques: Expressing, Derivative Assay, Immunohistochemical staining, Staining, Negative Staining

Journal: PloS one

Article Title: FABP7 and HMGCS2 are novel protein markers for apocrine differentiation categorizing apocrine carcinoma of the breast.

doi: 10.1371/journal.pone.0112024

Figure Lengend Snippet: Figure 9. MLPH is expressed by non-malignant apocrine cells but is lost in IACs. Representative images of FFPE sections immunostained with antibody against MLPH. (A) normal ducts, (B) benign apocrine cysts (mainly luminal membranous immunoreactivity), (C) sclerosing adenosis with apocrine differentiation, (D) IAC showing positive immunostaining in pseudo-glands structures (cytoplasmic and luminal membranous immunoreactivity) and (E) IAC with negative immunostaining. Magnification: x20. doi:10.1371/journal.pone.0112024.g009

Article Snippet: Antibodies The polyclonal antibody raised against synthetic peptides of human FABP7 (HPA028825), BLVRA (HPA042865), ABCA12 (HPA043194), SIDT1 (HPA035862), MLPH (HPA014685) were purchased from Atlas Antibodies and were used in IHC at a dilution of 1:200, 1:100, 1:500, 1:50 and 1:100 respectively in accordance to the manufacturer instructions.

Techniques: Immunostaining

Journal: BMC Medical Genetics

Article Title: Tissue-specific patterns of gene expression in the epithelium and stroma of normal colon in healthy individuals in an aspirin intervention trial

doi: 10.1186/s12881-015-0161-6

Figure Lengend Snippet: Real-Time PCR confirms differences in gene expression in colonic epithelium and stroma. Quantitative real-time RT-PCR was done on both biopsies (taken at two separate visits) from 10 study participants for four candidate genes. The histogram bars from the same individual are located next to each other, i.e., samples 1 and 2 are from the same person but taken at two separate visits. Total RNA was reverse transcribed and then amplified by PCR and normalized to β-glucuronidase (GUSB) expression. Blue bars -epithelial tissue. Red bars -stromal tissue. Upper panel shows expression of (i) ABCC3, an epithelial gene involved in drug and lipid metabolism and (ii) MLPH (melanophilin), an epithelial gene involved in protein binding and protein transport. Lower panel shows expression of (iii) ANXA1 (annexin A1), a stromal gene involved in cell migration and cell adhesion and (iv) ITGA8 (integrin α8), a stromal gene involved in extracellular matrix formation.

Article Snippet: Relative gene expression was examined by Taqman PCR using 2X Taqman Gene Expression Master Mix (Applied Biosystems, Foster City, California) and Taqman Gene Expression Assays for two epithelial genes (ABCC3, MLPH; assay identifiers Hs00358677_m1 and Hs00983106_m1) and two stromal genes (ITGα8 and ANXA1; assay identifiers Hs00943530_m1 and Hs00945401_m1).

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Quantitative RT-PCR, Reverse Transcription, Amplification, Expressing, Protein Binding, Migration

Journal: PLoS ONE

Article Title: Bifunctional effects of O-methylated flavones from Scutellaria baicalensis Georgi on melanocytes: Inhibition of melanin production and intracellular melanosome transport

doi: 10.1371/journal.pone.0171513

Figure Lengend Snippet: B16F10 cells were cultured for 3 days with wogonin at a concentration of 50 μM ( A ) or the indicated concentrations ( B–C ). ( A ) Nuclei (blue) and F-actin (magenta) were analyzed by confocal microscopy. Bright field images show the melanosome (black pigment) distribution. Scale bar = 10 μm. ( B ) MLPH, Rab27A, and myosin Va levels were determined by immunoblotting with anti-MLPH, Rab27A, and myosin Va antibodies, respectively. ( C ) The expression of Mlph mRNA was quantified using qPCR.

Article Snippet: First-strand cDNA was synthesized with 1 μg of total RNA using a PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan) per the manufacturer’s instructions. mRNA expression levels of target genes were measured using an Applied Biosystems 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) and the following TaqMan Gene Expression Assays: Mitf (assay ID Mm00434954_m1); Mlph (assay ID Mm01286059_m1); and Gapdh (assay ID Mm99999915_g1).

Techniques: Cell Culture, Concentration Assay, Confocal Microscopy, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Bifunctional effects of O-methylated flavones from Scutellaria baicalensis Georgi on melanocytes: Inhibition of melanin production and intracellular melanosome transport

doi: 10.1371/journal.pone.0171513

Figure Lengend Snippet: B16F10 cells were cultured for 2 days with or without 50 μM wogonin. Following replacement of the medium, B16F10 cells were cultured for an additional 24 h with or without wogonin (50 μM) in combination with MG132 (120 nM) or leupeptin (20 μM). ( A–B ) MLPH levels were determined by immunoblotting with an anti-MLPH antibody. ( C ) Bright field images show the melanosome distribution. Scale bar = 50 μm.

Article Snippet: First-strand cDNA was synthesized with 1 μg of total RNA using a PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan) per the manufacturer’s instructions. mRNA expression levels of target genes were measured using an Applied Biosystems 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) and the following TaqMan Gene Expression Assays: Mitf (assay ID Mm00434954_m1); Mlph (assay ID Mm01286059_m1); and Gapdh (assay ID Mm99999915_g1).

Techniques: Cell Culture, Western Blot

Journal: PLoS ONE

Article Title: Bifunctional effects of O-methylated flavones from Scutellaria baicalensis Georgi on melanocytes: Inhibition of melanin production and intracellular melanosome transport

doi: 10.1371/journal.pone.0171513

Figure Lengend Snippet: B16F10 cells were cultured for 3 days with or without 50 μM wogonin. Following replacement of wogonin-free medium, B16F10 cells were cultured for an additional 24 h. ( A ) Bright field images show the melanosome distribution. Scale bar = 50 μm. ( B ) The MLPH level was determined by immunoblotting with an anti-MLPH antibody.

Article Snippet: First-strand cDNA was synthesized with 1 μg of total RNA using a PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan) per the manufacturer’s instructions. mRNA expression levels of target genes were measured using an Applied Biosystems 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) and the following TaqMan Gene Expression Assays: Mitf (assay ID Mm00434954_m1); Mlph (assay ID Mm01286059_m1); and Gapdh (assay ID Mm99999915_g1).

Techniques: Cell Culture, Western Blot

Journal: PLoS ONE

Article Title: Bifunctional effects of O-methylated flavones from Scutellaria baicalensis Georgi on melanocytes: Inhibition of melanin production and intracellular melanosome transport

doi: 10.1371/journal.pone.0171513

Figure Lengend Snippet: B16F10 cells were cultured for 3 days with 50 μM wogonin, wogonoside, or norwogonin. ( A ) Bright field images show the melanosome distribution. Scale bar = 10 μm. ( B ) The results are expressed as the percentage of cells showing perinuclear melanosome aggregation. ( C ) MLPH levels were determined by immunoblotting with an anti-MLPH antibody.

Article Snippet: First-strand cDNA was synthesized with 1 μg of total RNA using a PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan) per the manufacturer’s instructions. mRNA expression levels of target genes were measured using an Applied Biosystems 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) and the following TaqMan Gene Expression Assays: Mitf (assay ID Mm00434954_m1); Mlph (assay ID Mm01286059_m1); and Gapdh (assay ID Mm99999915_g1).

Techniques: Cell Culture, Western Blot

Journal: PLoS ONE

Article Title: Bifunctional effects of O-methylated flavones from Scutellaria baicalensis Georgi on melanocytes: Inhibition of melanin production and intracellular melanosome transport

doi: 10.1371/journal.pone.0171513

Figure Lengend Snippet: ( A ) The structures of three mono- O -methylated flavones. ( B–C ) B16F10 cells were cultured for 3 days with 50 μM wogonin or each of the two wogonin analogs. The results are expressed as the percentage of cells showing perinuclear melanosome aggregation. MLPH levels were determined by immunoblotting with an anti-MLPH antibody. ( D ) B16F10 cells were cultured for 24 h with 50 μM of each flavone. The melanin content in each culture was quantified.

Article Snippet: First-strand cDNA was synthesized with 1 μg of total RNA using a PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan) per the manufacturer’s instructions. mRNA expression levels of target genes were measured using an Applied Biosystems 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) and the following TaqMan Gene Expression Assays: Mitf (assay ID Mm00434954_m1); Mlph (assay ID Mm01286059_m1); and Gapdh (assay ID Mm99999915_g1).

Techniques: Methylation, Cell Culture, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: The Mlph subunit of the tripartite complex is specifically phosphorylated by PKA. (A) Schematic illustration of the MyoVa-dependent tripartite transport complex on the melanosome surface. The Rab27a GTPase resides in the melanosome membrane and recruits Mlph in a GTP-dependent manner. Mlph in turn recruits the MyoVa motor to form the tripartite transport complex. (B) Domain structure of the adaptor protein Mlph. The Rab27a-binding domain (Rab27a-BD) is located at Mlph’s N terminus (42, 43). MyoVa binds to Mlph’s middle domain with its globular tail domain and the melanocyte-specific alternatively spliced exon F (MyoVa-BD) (15, 65, 66). The C terminus of Mlph harbors an ABD (23, 24). (C) Tripartite complex reconstituted with 6×His-SNAP–tagged Rab27aS-H, FLAG-tagged MyoVaF, and full-length (lane I) or C-terminally truncated (lanes II and III) FLAG-tagged MlphF purified by Ni-NTA affinity purification. As a control for nonspecific binding, FLAG-tagged MyoVaF was also subjected to Ni-NTA affinity purification (lane IV) (Materials and Methods for details). MW, molecular mass marker. (D) The individually expressed full-length subunits of the tripartite complex along with C-terminally truncated Mlph and MyoVa HMM constructs were treated with PKA and radiolabeled ATP. Autoradiography showed specific phosphorylation of Mlph. Deletion of the C terminus of Mlph significantly decreased the phosphorylation levels. A FLAG-mock purification was included to control for unspecific phosphorylation.

Article Snippet: Four point-mutated and N-terminally FLAG-tagged Mlph constructs mimicking the dephosphorylated state by substituting serine or threonine with alanine residues were synthesized (GenScript).

Techniques: Binding Assay, Purification, Affinity Purification, Marker, Construct, Autoradiography

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: Alignment of Mlph ABDs from mouse (Mm), human (Hs), Fukomys (Fd), dog (Cl), sheep (Oa), and Xenopus (Xt). Sequence alignment of selected Mlph ABDs revealed numerous conserved serine/threonine residues that represent potential PKA targets. Serine and threonine residues that were found to be phosphorylated in vivo are in bold. Boxes indicate conserved phosphorylatable serine residues. Numbers represent the residue numbers according to MmMlph. Asterisks, colons, and dots indicate positions that are fully, partially, or weakly conserved, respectively.

Article Snippet: Four point-mutated and N-terminally FLAG-tagged Mlph constructs mimicking the dephosphorylated state by substituting serine or threonine with alanine residues were synthesized (GenScript).

Techniques: Sequencing, In Vivo

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: Mlph’s phosphorylation state does not interfere substantially with actin binding. (A) Actin decoration experiments were performed with surface-immobilized and Atto488-labeled actin filaments (red). Filaments were incubated with the complex formed between Mlph and Alexa Fluor 647-labeled Rab27a (green). Dephosphorylated (Dephos; Left) and the phosphorylated (Phos; Right) Mlph decorated actin filaments similarly well. Removal of the C-terminal ABD of Mlph (Rab27a/Mlph ΔABD) abolished this interaction regardless of Mlph’s phosphorylation state. (B) The dephosphorylated, Alexa Fluor 488-labeled Rab27a/Mlph complex was mixed in equal amounts with the phosphorylated, Alexa Fluor 647-labeled Rab27a/Mlph complex and was incubated with surface-attached, Atto565-labeled actin filaments. The quantification of the actin-associated fluorescence signals from the respective PKA- and phosphatase-treated Rab27a/Mlph complexes showed that the phosphorylation state of Mlph did not substantially interfere with actin binding. Error bars represent SD. (Scale bars: 3 µm.)

Article Snippet: Four point-mutated and N-terminally FLAG-tagged Mlph constructs mimicking the dephosphorylated state by substituting serine or threonine with alanine residues were synthesized (GenScript).

Techniques: Binding Assay, Labeling, Incubation, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: Mlph’s phosphorylation state does not interfere substantially with actin binding. Dephosphorylated Alexa Fluor 647-labeled Rab27a/Mlph complex (cyan) was mixed in equal amounts with phosphorylated, Alexa 488-labeled Rab27a/Mlph complex (green) and was incubated with surface-attached, Atto565-labeled actin filaments (red; dye swap control for Fig. 2B). The quantification showed results identical to those demonstrated in Fig. 2B: Phosphorylation of Mlph does not alter the interaction between Mlph and actin filaments. Error bars represent SD. (Scale bar: 3 µm.)

Article Snippet: Four point-mutated and N-terminally FLAG-tagged Mlph constructs mimicking the dephosphorylated state by substituting serine or threonine with alanine residues were synthesized (GenScript).

Techniques: Binding Assay, Labeling, Incubation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: Transport parameters of the tripartite complex on surface-attached actin filaments in single-molecule TIRF assays. The tripartite complex assembled with Mlph that lacked its ABD (A) and the complexes assembled with phosphorylated (B) and dephosphorylated (C) Mlph all moved at consistent velocities. The absence of ABD (A) or the phosphorylation state of the Mlph (B vs. C) did not interfere with the velocities and run lengths of the respective complexes. The majority of the complexes displayed a single-step photobleaching of the SNAP-tagged Rab27a subunit as shown in A–C, Right, demonstrating that the transport parameters are derived from single molecules of Rab27a (also see Fig. S4).

Article Snippet: Four point-mutated and N-terminally FLAG-tagged Mlph constructs mimicking the dephosphorylated state by substituting serine or threonine with alanine residues were synthesized (GenScript).

Techniques: Derivative Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: Mlph interacts with microtubules in a phosphorylation-dependent manner. In microtubule decoration experiments Atto488-labeled microtubules (red) were incubated with the Alexa Fluor 647-labeled Rab27a/Mlph complex (green). Decoration of microtubules was strictly dependent on the phosphorylation state of Mlph. The fluorescent background from the phosphorylated and dephosphorylated Rab27a/Mlph complex seen in the green channel was comparable, indicating similar amounts of protein. (Scale bar: 3 µm.)

Article Snippet: Four point-mutated and N-terminally FLAG-tagged Mlph constructs mimicking the dephosphorylated state by substituting serine or threonine with alanine residues were synthesized (GenScript).

Techniques: Labeling, Incubation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: Mutated serine and threonine residues in conserved regions within the ABD of Mlph. Serine and threonine residues in three conserved regions (Fig. S1) were mutated to alanines to mimic the dephosphorylated state. Mutated residues are shown in bold letters. A serine- and threonine-rich stretch outside the ABD was mutated as a negative control.

Article Snippet: Four point-mutated and N-terminally FLAG-tagged Mlph constructs mimicking the dephosphorylated state by substituting serine or threonine with alanine residues were synthesized (GenScript).

Techniques: Negative Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: Dephosphorylated wild-type Rab27a/Mlph decorates microtubules in vitro, but phosphorylated wild-type Rab27a/Mlph does not. The Rab27a/Mlph complex (green) reconstituted with wild-type Mlph was purified side by side with the Rab27a/Mlph complex containing the point mutations in Mlph’s ABD (see Fig. 5) and used as a control. Consistent with Fig. 4, the dephosphorylated complex decorated microtubules (red), but the phosphorylated Rab27a/Mlph complex failed to decorate microtubules. (Scale bar: 3 μm.)

Article Snippet: Four point-mutated and N-terminally FLAG-tagged Mlph constructs mimicking the dephosphorylated state by substituting serine or threonine with alanine residues were synthesized (GenScript).

Techniques: In Vitro, Purification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: Phosphorylation of S491/498A is the main contributor to phosphorylation-dependent binding of Mlph to microtubules. (A) Nonphosphorylatable alanine mutations in three predicted phosphorylation sites reduced PKA-dependent phosphorylation. In vitro phosphorylation assays (Lower: autoradiograph; Upper: corresponding Coomassie-stained SDS/PAGE as loading control) with wild-type Mlph and its mutants showed that the S491/498A mutant suppressed the PKA-dependent phosphorylation of Mlph more efficiently (26%) than the wild-type (100%) or mutants containing weaker and less conserved PKA consensus sites (76% and 57%). (B) Microtubule cosedimentation assays were performed side by side with the truncated Mlph that lacked its ABD (I, ΔABD), phosphorylated wild-type Mlph (II, 1–590 Phos), dephosphorylated wild-type Mlph (III, 1–590 Dephos) along with phosphorylated Mlph that carried the respective nonphosphorylatable alanine mutations (IV–VI). The total reaction (T), supernatant (SN), and pellet (P) were analyzed with SDS/PAGE. As expected from the results shown in Figs. 4 and ​and5,5, Mlph that lacked its ABD and the phosphorylated wild-type Mlph failed to display a pronounced interaction with the microtubules (N/D and 4%, I and II, respectively). Also, in line with results from Fig. 5, the dephosphorylated wild-type Mlph and all rescue mutants displayed significant pelleting with microtubules [27%, 17%, 8%, and 9% (III–VI), respectively]. The mutant S491/498A containing the strong and most conserved PKA consensus site S498 also demonstrated the most pronounced effect of rescue (IV). Percentages are obtained from two independent assays ± SD. N/D, not determinable.

Article Snippet: Four point-mutated and N-terminally FLAG-tagged Mlph constructs mimicking the dephosphorylated state by substituting serine or threonine with alanine residues were synthesized (GenScript).

Techniques: Binding Assay, In Vitro, Autoradiography, Staining, SDS Page, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: Cosedimentation assay of microtubules (2 µM) with a reduced amount (3 µM) of dephosphorylated wild-type Mlph (1-590 Dephos). Forty-seven percent of the total amount of Mlph protein pelleted with microtubules, noticeably more than in Fig. 6, where excess Mlph (5 µM) was used and only 27% pelleted. These observations suggested that binding sites for Mlph on microtubules become limiting when Mlph exceeds the concentration of microtubules.

Article Snippet: Four point-mutated and N-terminally FLAG-tagged Mlph constructs mimicking the dephosphorylated state by substituting serine or threonine with alanine residues were synthesized (GenScript).

Techniques: Binding Assay, Concentration Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: Dephosphorylation is sufficient to relocate Mlph from actin to microtubules efficiently. Surface-immobilized and Atto565-labeled actin filaments (red; Upper) and Atto488-labeled microtubules (red; Lower) were incubated with phosphorylated (A) and dephosphorylated (B) complex formed between Mlph and Alexa Fluor 647-labeled Rab27a (green). (A) The phosphorylated Rab27a/Mlph complex largely ignored microtubules (MT) and associated with actin filaments (17 ± 4% vs. 83 ± 4%). (B) Upon dephosphorylation, the behavior of the Rab27a/Mlph complex was reversed, and the microtubule binding clearly dominated (76 ± 4%) over the actin binding (24 ± 4%). Error bars represent SD. (Scale bar: 3 µm.)

Article Snippet: Four point-mutated and N-terminally FLAG-tagged Mlph constructs mimicking the dephosphorylated state by substituting serine or threonine with alanine residues were synthesized (GenScript).

Techniques: De-Phosphorylation Assay, Labeling, Incubation, Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: Dephosphorylation is sufficient to relocate Mlph from actin to microtubules efficiently. Alternative analysis of decoration assays with phosphorylated (Left) or dephosphorylated (Right) Rab27a/Mlph complex on both microtubules and actin. The Mlph intensity bound to each filament type was normalized to the total length of this filament type present in the channel. Error bars represent SD. a.u., arbitrary units.

Article Snippet: Four point-mutated and N-terminally FLAG-tagged Mlph constructs mimicking the dephosphorylated state by substituting serine or threonine with alanine residues were synthesized (GenScript).

Techniques: De-Phosphorylation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: Dephosphorylated Rab27a/Mlph/MyoVa complexes (green) in the presence (Left) or absence (Right) of ATP on surface-attached networks of actin (blue) and microtubules (red) in vitro. (Scale bar: 10 µm.)

Article Snippet: Four point-mutated and N-terminally FLAG-tagged Mlph constructs mimicking the dephosphorylated state by substituting serine or threonine with alanine residues were synthesized (GenScript).

Techniques: In Vitro

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: Phosphorylation state of Mlph’s ABD dictates the directionality of switching at the actin–microtubule intersections. The movement of the tripartite complex on actin and microtubules is represented by single- and double-headed black arrows, respectively. Cyan arrows depict switching from actin to microtubules; red arrows indicate switching from microtubules to actin. (A and B) The tripartite complex reconstituted with dephosphorylated Mlph displayed a significantly higher probability of switching from actin to microtubules at the interfilament intersections (A). Although 32.3% of complexes switched from actin to microtubules (67.7% continued directional movement on actin; A), the propensity of switching from actin to microtubules was abolished when the tripartite complex was assembled with phosphorylated Mlph (B). The phosphorylated complex completely ignored the interfilament intersections (0% switching) and continued its directional movement on the actin (100%; B). Conversely, dephosphorylated Mlph significantly suppressed the probability of the complex switching from microtubules to actin (26.1%; A) compared with the complex built with phosphorylated Mlph (100%; B). Indeed, the phosphorylated complex rarely interacted with the microtubules, substantially decreasing the probability of switching events from microtubules to actin (Movie S1). (C and D) In contrast, tripartite complexes assembled with dephosphorylated Mlph ΔABD (C) and phosphorylated Mlph ΔABD (D) displayed similar probabilities of switching between the two filament types, confirming that phosphorylation outside Mlph’s ABD does not interfere with the switching behavior of the tripartite complex. N indicates the number of events for each switching direction.

Article Snippet: Four point-mutated and N-terminally FLAG-tagged Mlph constructs mimicking the dephosphorylated state by substituting serine or threonine with alanine residues were synthesized (GenScript).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: Proposed model for regulating the affinities of the moving organelles on the microtubule and actin networks in vivo. In our mechanistic dissection, we unmasked the regulatory dominance of the adaptor protein Mlph over its associated motor. Even though MyoVa is an actin-associated motor, the dephosphorylation of Mlph’s ABD was sufficient to redirect the MyoVa from directional movement on the actin network to microtubules for diffusive movement. Consequently, the phosphorylation state of Mlph’s ABD regulated the probability of directional switching of MyoVa between the microtubule and actin networks. Based on these findings, we propose that Mlph serves to bias the transport of organelles on the microtubule or actin networks in vivo. (A) Specifically, phosphorylation of Mlph’s ABD promotes MyoVa-dependent motility on the actin network by suppressing the affinity of the tripartite complex toward microtubules. (B) To reverse this process, Mlph is dephosphorylated to increase the affinity of the tripartite complex for the microtubule network.

Article Snippet: Four point-mutated and N-terminally FLAG-tagged Mlph constructs mimicking the dephosphorylated state by substituting serine or threonine with alanine residues were synthesized (GenScript).

Techniques: In Vivo, Dissection, De-Phosphorylation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: The Mlph subunit of the tripartite complex is specifically phosphorylated by PKA. (A) Schematic illustration of the MyoVa-dependent tripartite transport complex on the melanosome surface. The Rab27a GTPase resides in the melanosome membrane and recruits Mlph in a GTP-dependent manner. Mlph in turn recruits the MyoVa motor to form the tripartite transport complex. (B) Domain structure of the adaptor protein Mlph. The Rab27a-binding domain (Rab27a-BD) is located at Mlph’s N terminus (42, 43). MyoVa binds to Mlph’s middle domain with its globular tail domain and the melanocyte-specific alternatively spliced exon F (MyoVa-BD) (15, 65, 66). The C terminus of Mlph harbors an ABD (23, 24). (C) Tripartite complex reconstituted with 6×His-SNAP–tagged Rab27aS-H, FLAG-tagged MyoVaF, and full-length (lane I) or C-terminally truncated (lanes II and III) FLAG-tagged MlphF purified by Ni-NTA affinity purification. As a control for nonspecific binding, FLAG-tagged MyoVaF was also subjected to Ni-NTA affinity purification (lane IV) (Materials and Methods for details). MW, molecular mass marker. (D) The individually expressed full-length subunits of the tripartite complex along with C-terminally truncated Mlph and MyoVa HMM constructs were treated with PKA and radiolabeled ATP. Autoradiography showed specific phosphorylation of Mlph. Deletion of the C terminus of Mlph significantly decreased the phosphorylation levels. A FLAG-mock purification was included to control for unspecific phosphorylation.

Article Snippet: Rab27a (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_023635.4","term_id":"146141108","term_text":"NM_023635.4"}} NM_023635.4 ; used for ) and the constitutively active Rab27a-Q78L [used for all TIRF assays ( 42 )] with a C-terminal SNAP-tag followed by a 6×His-tag, Mlph (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_053015.2","term_id":"87080830","term_text":"NM_053015.2"}} NM_053015.2 ) with an N-terminal FLAG-tag, and MyoVa (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_010864.1","term_id":"6754783","term_text":"NM_010864.1"}} NM_010864.1 ) with an N-terminal FLAG-tag were commercially synthesized (GenScript).

Techniques: Membrane, Binding Assay, Purification, Affinity Purification, Control, Marker, Construct, Autoradiography, Phospho-proteomics

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: Mlph is the sole phosphorylation target of the reconstituted tripartite complex. (A) Coomassie-stained SDS/PAGE image of the reconstituted Rab27a/Mlph/MyoVa transport complex that was subjected to in vitro phosphorylation. (B) PKA specifically phosphorylated Mlph in the presence of Rab27a and MyoVa (autoradiograph).

Article Snippet: Rab27a (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_023635.4","term_id":"146141108","term_text":"NM_023635.4"}} NM_023635.4 ; used for ) and the constitutively active Rab27a-Q78L [used for all TIRF assays ( 42 )] with a C-terminal SNAP-tag followed by a 6×His-tag, Mlph (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_053015.2","term_id":"87080830","term_text":"NM_053015.2"}} NM_053015.2 ) with an N-terminal FLAG-tag, and MyoVa (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_010864.1","term_id":"6754783","term_text":"NM_010864.1"}} NM_010864.1 ) with an N-terminal FLAG-tag were commercially synthesized (GenScript).

Techniques: Phospho-proteomics, Staining, SDS Page, In Vitro, Autoradiography

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: Mlph’s phosphorylation state does not interfere substantially with actin binding. (A) Actin decoration experiments were performed with surface-immobilized and Atto488-labeled actin filaments (red). Filaments were incubated with the complex formed between Mlph and Alexa Fluor 647-labeled Rab27a (green). Dephosphorylated (Dephos; Left) and the phosphorylated (Phos; Right) Mlph decorated actin filaments similarly well. Removal of the C-terminal ABD of Mlph (Rab27a/Mlph ΔABD) abolished this interaction regardless of Mlph’s phosphorylation state. (B) The dephosphorylated, Alexa Fluor 488-labeled Rab27a/Mlph complex was mixed in equal amounts with the phosphorylated, Alexa Fluor 647-labeled Rab27a/Mlph complex and was incubated with surface-attached, Atto565-labeled actin filaments. The quantification of the actin-associated fluorescence signals from the respective PKA- and phosphatase-treated Rab27a/Mlph complexes showed that the phosphorylation state of Mlph did not substantially interfere with actin binding. Error bars represent SD. (Scale bars: 3 µm.)

Article Snippet: Rab27a (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_023635.4","term_id":"146141108","term_text":"NM_023635.4"}} NM_023635.4 ; used for ) and the constitutively active Rab27a-Q78L [used for all TIRF assays ( 42 )] with a C-terminal SNAP-tag followed by a 6×His-tag, Mlph (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_053015.2","term_id":"87080830","term_text":"NM_053015.2"}} NM_053015.2 ) with an N-terminal FLAG-tag, and MyoVa (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_010864.1","term_id":"6754783","term_text":"NM_010864.1"}} NM_010864.1 ) with an N-terminal FLAG-tag were commercially synthesized (GenScript).

Techniques: Phospho-proteomics, Binding Assay, Labeling, Incubation, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: Transport parameters of the tripartite complex on surface-attached actin filaments in single-molecule TIRF assays. The tripartite complex assembled with Mlph that lacked its ABD (A) and the complexes assembled with phosphorylated (B) and dephosphorylated (C) Mlph all moved at consistent velocities. The absence of ABD (A) or the phosphorylation state of the Mlph (B vs. C) did not interfere with the velocities and run lengths of the respective complexes. The majority of the complexes displayed a single-step photobleaching of the SNAP-tagged Rab27a subunit as shown in A–C, Right, demonstrating that the transport parameters are derived from single molecules of Rab27a (also see Fig. S4).

Article Snippet: Rab27a (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_023635.4","term_id":"146141108","term_text":"NM_023635.4"}} NM_023635.4 ; used for ) and the constitutively active Rab27a-Q78L [used for all TIRF assays ( 42 )] with a C-terminal SNAP-tag followed by a 6×His-tag, Mlph (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_053015.2","term_id":"87080830","term_text":"NM_053015.2"}} NM_053015.2 ) with an N-terminal FLAG-tag, and MyoVa (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_010864.1","term_id":"6754783","term_text":"NM_010864.1"}} NM_010864.1 ) with an N-terminal FLAG-tag were commercially synthesized (GenScript).

Techniques: Phospho-proteomics, Derivative Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: Dephosphorylated Rab27a/Mlph/MyoVa complexes (green) in the presence (Left) or absence (Right) of ATP on surface-attached networks of actin (blue) and microtubules (red) in vitro. (Scale bar: 10 µm.)

Article Snippet: Rab27a (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_023635.4","term_id":"146141108","term_text":"NM_023635.4"}} NM_023635.4 ; used for ) and the constitutively active Rab27a-Q78L [used for all TIRF assays ( 42 )] with a C-terminal SNAP-tag followed by a 6×His-tag, Mlph (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_053015.2","term_id":"87080830","term_text":"NM_053015.2"}} NM_053015.2 ) with an N-terminal FLAG-tag, and MyoVa (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_010864.1","term_id":"6754783","term_text":"NM_010864.1"}} NM_010864.1 ) with an N-terminal FLAG-tag were commercially synthesized (GenScript).

Techniques: In Vitro

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

doi: 10.1073/pnas.1619473114

Figure Lengend Snippet: Proposed model for regulating the affinities of the moving organelles on the microtubule and actin networks in vivo. In our mechanistic dissection, we unmasked the regulatory dominance of the adaptor protein Mlph over its associated motor. Even though MyoVa is an actin-associated motor, the dephosphorylation of Mlph’s ABD was sufficient to redirect the MyoVa from directional movement on the actin network to microtubules for diffusive movement. Consequently, the phosphorylation state of Mlph’s ABD regulated the probability of directional switching of MyoVa between the microtubule and actin networks. Based on these findings, we propose that Mlph serves to bias the transport of organelles on the microtubule or actin networks in vivo. (A) Specifically, phosphorylation of Mlph’s ABD promotes MyoVa-dependent motility on the actin network by suppressing the affinity of the tripartite complex toward microtubules. (B) To reverse this process, Mlph is dephosphorylated to increase the affinity of the tripartite complex for the microtubule network.

Article Snippet: Rab27a (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_023635.4","term_id":"146141108","term_text":"NM_023635.4"}} NM_023635.4 ; used for ) and the constitutively active Rab27a-Q78L [used for all TIRF assays ( 42 )] with a C-terminal SNAP-tag followed by a 6×His-tag, Mlph (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_053015.2","term_id":"87080830","term_text":"NM_053015.2"}} NM_053015.2 ) with an N-terminal FLAG-tag, and MyoVa (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_010864.1","term_id":"6754783","term_text":"NM_010864.1"}} NM_010864.1 ) with an N-terminal FLAG-tag were commercially synthesized (GenScript).

Techniques: In Vivo, Dissection, De-Phosphorylation Assay, Phospho-proteomics