scholarly journals In Vivo Role for Actin-regulating Kinases in Endocytosis and Yeast Epsin Phosphorylation

2001 ◽  
Vol 12 (11) ◽  
pp. 3668-3679 ◽  
Author(s):  
Hadiya A. Watson ◽  
M. Jamie T. V. Cope ◽  
Aaron Chris Groen ◽  
David G. Drubin ◽  
Beverly Wendland
Keyword(s):  
Consensus Sequence ◽  
Signaling Proteins ◽  
Wild Type ◽  
Cortical Actin ◽  
Growth Defects ◽  
Endocytic Protein ◽  
Mutant Phenotypes ◽  
Actin Localization ◽  
Synthetic Growth

The yeast actin-regulating kinases Ark1p and Prk1p are signaling proteins localized to cortical actin patches, which may be sites of endocytosis. Interactions between the endocytic proteins Pan1p and End3p may be regulated by Prk1p-dependent threonine phosphorylation of Pan1p within the consensus sequence [L/I]xxQxTG. We identified two Prk1p phosphorylation sites within the Pan1p-binding protein Ent1p, a yeast epsin homologue, and demonstrate Prk1p-dependent phosphorylation of both threonines. Converting both threonines to either glutamate or alanine mimics constitutively phosphorylated or dephosphorylated Ent1p, respectively. Synthetic growth defects were observed in a pan1–20 ENT1EE double mutant, suggesting that Ent1p phosphorylation negatively regulates the formation/activity of a Pan1p–Ent1p complex. Interestingly,pan1–20 ent2Δ but not pan1–20 ent1Δdouble mutants had improved growth and endocytosis over thepan1–20 mutant. We found that actin-regulating Ser/Thr kinase (ARK) mutants exhibit endocytic defects and that overexpressing either wild-type or alanine-substituted Ent1p partially suppressed phenotypes associated with loss of ARK kinases, including growth, endocytosis, and actin localization defects. Consistent with synthetic growth defects of pan1–20 ENT1EE cells, overexpressing glutamate-substituted Ent1p was deleterious to ARK mutants. Surprisingly, overexpressing the related Ent2p protein could not suppress ARK kinase mutant phenotypes. These results suggest that Ent1p and Ent2p are not completely redundant and may perform opposing functions in endocytosis. These data support the model that, as for clathrin-dependent recycling of synaptic vesicles, yeast endocytic protein phosphorylation inhibits endocytic functions.

scholarly journals Different Genetic Functions for the Rpd3(L) and Rpd3(S) Complexes Suggest Competition between NuA4 and Rpd3(S)

2008 ◽  
Vol 28 (14) ◽  
pp. 4445-4458 ◽  
Author(s):  
Debabrata Biswas ◽  
Shinya Takahata ◽  
David J. Stillman
Keyword(s):  
Transcriptional Activation ◽  
Chromatin Immunoprecipitation ◽  
Histone Acetyltransferase ◽  
Strand Break ◽  
Triple Mutant ◽  
Growth Defects ◽  
Mutant Phenotypes ◽  
Synthetic Growth ◽  
Synthetic Phenotype

ABSTRACT Rpd3(L) and Rpd3(S) are distinct multisubunit complexes containing the Rpd3 histone deacetylase. Disruption of the GCN5 histone acetyltransferase gene shows a strong synthetic phenotype when combined with either an sds3 mutation affecting only the Rpd3(L) complex or an rco1 mutation affecting only Rpd3(S). However, these synthetic growth defects are not seen in a gcn5 sds3 rco1 triple mutant, suggesting that the balance between Rpd3(L) and Rpd3(S) is critical in cells lacking Gcn5. Different genetic interactions are seen with mutations affecting the FACT chromatin reorganizing complex. An sds3 mutation affecting only Rpd3(L) has a synthetic defect with FACT mutants, while rco1 and eaf3 mutations affecting Rpd3(S) suppress FACT mutant phenotypes. Rpd3(L) therefore acts in concert with FACT, but Rpd3(S) opposes it. Combining FACT mutations with mutations in the Esa1 subunit of the NuA4 histone acetyltransferase results in synthetic growth defects, and these can be suppressed by an rco1 or set2 mutation. An rco1 mutation suppresses phenotypes caused by mutations in the ESA1 and ARP4 subunits of NuA4, while Rco1 overexpression exacerbates these defects. These results suggest a model in which NuA4 and Rpd3(S) compete. Chromatin immunoprecipitation experiments show that eliminating Rpd3(S) increases the amount of NuA4 binding to the ARG3 promoter during transcriptional activation and to the sites of DNA repair induced by a double-strand break. Our results suggest that the Rpd3(L) and Rpd3(S) complexes have distinct functions in vivo and that the relative amounts of the two forms alter the effectiveness of other chromatin-altering complexes, such as FACT and NuA4.

scholarly journals Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Mutational Analysis ◽  
Wild Type ◽  
Content Type ◽  
Growth Defects ◽  
C Terminus ◽  
K 12 ◽  
And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

A Bcr-Abl Mutant Lacking Direct Binding Sites for the Grb2, Cbl and CrkL Adapter Proteins Fails to Induce Leukemia in Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 718-718
Author(s):  
Kara J. Johnson ◽  
Ian J. Griswold ◽  
Amie Corbin ◽  
Michael W.N. Deininger ◽  
Brian J. Druker
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Cell Lines ◽  
Binding Sites ◽  
Signaling Proteins ◽  
Wild Type ◽  
Triple Mutant ◽  
Murine Bone Marrow ◽  
Direct Binding

Abstract The Bcr-Abl tyrosine kinase is detectable in greater than 95% of cases of chronic myelogenous leukemia (CML) and its kinase activity is required for induction of this disease. A number of signaling proteins are associated with and phosphorylated by Bcr-Abl. Proteins known to associate directly with Bcr-Abl include Grb2, c-Cbl, CrkL and p62Dok. Mutations of the direct binding sites for these proteins in Bcr-Abl abolish the direct interactions, but do not completely eliminate interactions, presumably due to the ability of many of these proteins to interact both directly and indirectly with Bcr-Abl. Individual mutations of the Grb2 and c-Cbl binding domains change the phenotype of disease induced in murine bone marrow transplantation assays from a myeloproliferative disorder to a B or T-lymphoid leukemia with prolonged latency. Thus, due to the complexity of Bcr-Abl signaling and a lack of a one to one correlation between direct binding sites, specific signaling proteins, and specific phenotypes, we created a triple mutant lacking the direct binding sites for Grb2, c-Cbl and CrkL. Stable myeloid cell lines were generated in the myeloid progenitor cell line, 32D, expressing the wild type and triple mutant forms of Bcr-Abl. Cell proliferation assays were performed in the presence and absence of WEHI (an IL-3 source) to assess growth factor requirements. Expression of the triple mutant in cell lines was able to confer growth factor dependence when expressed at levels comparable to wild type. Lysates from cell lines were analyzed by immunoprecipitation and immunoblotting and demonstrated that nearly all associations between Bcr-Abl and Grb2, c-Cbl, CrkL and p62Dok were eliminated in the triple mutant. Despite the lack of interaction, these proteins remained tyrosine phosphorylated at levels which correlate with Bcr-Abl expression. Phosphorylation was inhibited by treatment of cell lines with imatininb, indicating that the activity of Bcr-Abl is required for their phosphorylation, either directly or indirectly. Analysis of the activation of various signaling pathways (Akt, MAPK, MEK, Stat5), shows that only Stat5 remains phosphorylated in triple mutant cell lines. Despite inducing factor independent growth of 32D cells, the triple mutant was unable to induce the outgrowth of hematopoetic progenitors in B-cell lymphoid outgrowth assays. To test leukemogencity in vivo, murine bone marrow transplantation/transduction assays were also carried out using MSCV-MIGR1 vector constructs. The triple mutant failed to induce leukemia in the mice. In summary, a triple mutant of Bcr-Abl lacking the binding sites for Grb2, c-Cbl and CrkL is able to confer growth factor independence in cell lines. Although the associations of these proteins with Bcr-Abl are nearly eliminated, they are still tyrosine phosphorylated and this is dependent on the activity of Bcr-Abl. Despite its ability to transform cell lines, the triple mutant was not able to induce the outgrowth of hematopoetic progenitors in B-cell outgrowth assays, nor induce leukemia in mice. Although the phosphorylation of Stat5 correlates with factor independent growth, this is not sufficient to induce transformation in vivo suggesting that interactions with other signaling pathways disrupted in this triple mutant of Bcr-Abl are necessary. To our knowledge this is the only kinase active variant of Bcr-Abl that has failed to induce leukemia in vivo.

scholarly journals Quality Control of a Transcriptional Regulator by SUMO-Targeted Degradation

2009 ◽  
Vol 29 (7) ◽  
pp. 1694-1706 ◽  
Author(s):  
Zheng Wang ◽  
Gregory Prelich
Keyword(s):  
Quality Control ◽  
Protein Quality ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Physiological Importance ◽  
Sensitive Allele ◽  
Quality Control Mechanism ◽  
Mutant Phenotypes

ABSTRACT Slx5 and Slx8 are heterodimeric RING domain-containing proteins that possess SUMO-targeted ubiquitin ligase (STUbL) activity in vitro. Slx5-Slx8 and its orthologs are proposed to target SUMO conjugates for ubiquitin-mediated proteolysis, but the only in vivo substrate identified to date is mammalian PML, and the physiological importance of SUMO-targeted ubiquitylation remains largely unknown. We previously identified mutations in SLX5 and SLX8 by selecting for suppressors of a temperature-sensitive allele of MOT1, which encodes a regulator of TATA-binding protein. Here, we demonstrate that Mot1 is SUMOylated in vivo and that disrupting the Slx5-Slx8 pathway by mutation of the target lysines in Mot1, by deletion of SLX5 or the ubiquitin E2 UBC4, or by inhibition of the proteosome suppresses mot1-301 mutant phenotypes and increases the stability of the Mot1-301 protein. The Mot1-301 mutant protein is targeted for proteolysis by SUMOylation to a much greater extent than wild-type Mot1, suggesting a quality control mechanism. In support of this idea, growth of Saccharomyces cerevisiae in the presence of the arginine analog canavanine results in increased SUMOylation and Slx5-Slx8-mediated degradation of wild-type Mot1. These results therefore demonstrate that Mot1 is an in vivo STUbL target in yeast and suggest a role for SUMO-targeted degradation in protein quality control.

scholarly journals Deletion of the Dynein Heavy-Chain Gene DYN1 Leads to Aberrant Nuclear Positioning and Defective Hyphal Development in Candida albicans

2004 ◽  
Vol 3 (6) ◽  
pp. 1574-1588 ◽  
Author(s):  
R. Martin ◽  
A. Walther ◽  
J. Wendland
Keyword(s):  
Candida Albicans ◽  
Heavy Chain ◽  
Time Lapse ◽  
Yeast Cells ◽  
Chain Gene ◽  
Wild Type ◽  
Heavy Chain Gene ◽  
Mutant Strains ◽  
Mutant Phenotypes

ABSTRACT Cytoplasmic dynein is a microtubule-associated minus-end-directed motor protein. CaDYN1 encodes the single dynein heavy-chain gene of Candida albicans. The open reading frames of both alleles of CaDYN1 were completely deleted via a PCR-based approach. Cadyn1 mutants are viable but grow more slowly than the wild type. In vivo time-lapse microscopy was used to compare growth of wild-type (SC5314) and dyn1 mutant strains during yeast growth and after hyphal induction. During yeast-like growth, Cadyn1 strains formed chains of cells. Chromosomal TUB1-GFP and HHF1-GFP alleles were used both in wild-type and mutant strains to monitor the orientation of mitotic spindles and nuclear positioning in C. albicans. In vivo fluorescence time-lapse analyses with HHF1-GFP over several generations indicated defects in dyn1 cells in the realignment of spindles with the mother-daughter axis of yeast cells compared to that of the wild type. Mitosis in the dyn1 mutant, in contrast to that of wild-type yeast cells, was very frequently completed in the mother cells. Nevertheless, daughter nuclei were faithfully transported into the daughter cells, resulting in only a small number of multinucleate cells. Cadyn1 mutant strains responded to hypha-inducing media containing l-proline or serum with initial germ tube formation. Elongation of the hyphal tubes eventually came to a halt, and these tubes showed a defect in the tipward localization of nuclei. Using a heterozygous DYN1/dyn1 strain in which the remaining copy was controlled by the regulatable MAL2 promoter, we could switch between wild-type and mutant phenotypes depending on the carbon source, indicating that the observed mutant phenotypes were solely due to deletion of DYN1.

scholarly journals Candida albicans Vrp1 is required for polarized morphogenesis and interacts with Wal1 and Myo5

Microbiology ◽  
2010 ◽  
Vol 156 (10) ◽  
pp. 2962-2969 ◽  
Author(s):  
Nicole Borth ◽  
Andrea Walther ◽  
Patrick Reijnst ◽  
Sigyn Jorde ◽  
Yvonne Schaub ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Actin Cytoskeleton ◽  
Hyphal Growth ◽  
Potential Interaction ◽  
Wild Type ◽  
Terminal Part ◽  
Cortical Actin ◽  
Interacting Protein ◽  
Growth Defects ◽  
Interaction Sites

Recently, a link between endocytosis and hyphal morphogenesis has been identified in Candida albicans via the Wiskott–Aldrich syndrome gene homologue WAL1. To get a more detailed mechanistic understanding of this link we have investigated a potentially conserved interaction between Wal1 and the C. albicans WASP-interacting protein (WIP) homologue encoded by VRP1. Deletion of both alleles of VRP1 results in strong hyphal growth defects under serum inducing conditions but filamentation can be observed on Spider medium. Mutant vrp1 cells show a delay in endocytosis – measured as the uptake and delivery of the lipophilic dye FM4-64 into small endocytic vesicles – compared to the wild-type. Vacuolar morphology was found to be fragmented in a subset of cells and the cortical actin cytoskeleton was depolarized in vrp1 daughter cells. The morphology of the vrp1 null mutant could be complemented by reintegration of the wild-type VRP1 gene at the BUD3 locus. Using the yeast two-hybrid system we could demonstrate an interaction between the C-terminal part of Vrp1 and the N-terminal part of Wal1, which contains the WH1 domain. Furthermore, we found that Myo5 has several potential interaction sites on Vrp1. This suggests that a Wal1–Vrp1–Myo5 complex plays an important role in endocytosis and the polarized localization of the cortical actin cytoskeleton to promote polarized hyphal growth in C. albicans.

scholarly journals Pheromone-induced polarization is dependent on the Fus3p MAPK acting through the formin Bni1p

2004 ◽  
Vol 165 (1) ◽  
pp. 99-109 ◽  
Author(s):  
Dina Matheos ◽  
Metodi Metodiev ◽  
Eric Muller ◽  
David Stone ◽  
Mark D. Rose
Keyword(s):  
Cell Fusion ◽  
Cell Polarization ◽  
Yeast Cells ◽  
Cortical Actin ◽  
Cytoplasmic Microtubule ◽  
Multiple Phenotypes ◽  
Pheromone Signaling ◽  
Mutant Phenotypes

During mating, budding yeast cells reorient growth toward the highest concentration of pheromone. Bni1p, a formin homologue, is required for this polarized growth by facilitating cortical actin cable assembly. Fus3p, a pheromone-activated MAP kinase, is required for pheromone signaling and cell fusion. We show that Fus3p phosphorylates Bni1p in vitro, and phosphorylation of Bni1p in vivo during the pheromone response is dependent on Fus3p. fus3 mutants exhibited multiple phenotypes similar to bni1 mutants, including defects in actin and cell polarization, as well as Kar9p and cytoplasmic microtubule localization. Disruption of the interaction between Fus3p and the receptor-associated Gα subunit caused similar mutant phenotypes. After pheromone treatment, Bni1p-GFP and Spa2p failed to localize to the cortex of fus3 mutants, and cell wall growth became completely unpolarized. Bni1p overexpression suppressed the actin assembly, cell polarization, and cell fusion defects. These data suggest a model wherein activated Fus3p is recruited back to the cortex, where it activates Bni1p to promote polarization and cell fusion.

scholarly journals DAO1 catalyzes temporal and tissue-specific oxidative inactivation of auxin in Arabidopsis thaliana

2016 ◽  
Vol 113 (39) ◽  
pp. 11010-11015 ◽  
Author(s):  
Jun Zhang ◽  
Jinshan Ella Lin ◽  
Chinchu Harris ◽  
Fernanda Campos Mastrotti Pereira ◽  
Fan Wu ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Plant Growth ◽  
Expression Patterns ◽  
Loss Of Function ◽  
Wild Type ◽  
Iaa Oxidase ◽  
Oxidative Inactivation ◽  
Mutant Phenotypes

Tight homeostatic regulation of the phytohormone auxin [indole-3-acetic acid (IAA)] is essential to plant growth. Auxin biosynthetic pathways and the processes that inactivate auxin by conjugation to amino acids and sugars have been thoroughly characterized. However, the enzyme that catalyzes oxidation of IAA to its primary catabolite 2-oxindole-3-acetic acid (oxIAA) remains uncharacterized. Here, we show that DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1) catalyzes formation of oxIAA in vitro and in vivo and that this mechanism regulates auxin homeostasis and plant growth. Null dao1-1 mutants contain 95% less oxIAA compared with wild type, and complementation of dao1 restores wild-type oxIAA levels, indicating that DAO1 is the primary IAA oxidase in seedlings. Furthermore, dao1 loss of function plants have altered morphology, including larger cotyledons, increased lateral root density, delayed sepal opening, elongated pistils, and reduced fertility in the primary inflorescence stem. These phenotypes are tightly correlated with DAO1 spatiotemporal expression patterns as shown by DAO1pro:β-glucuronidase (GUS) activity and DAO1pro:YFP-DAO1 signals, and transformation with DAO1pro:YFP-DAO1 complemented the mutant phenotypes. The dominant dao1-2D mutant has increased oxIAA levels and decreased stature with shorter leaves and inflorescence stems, thus supporting DAO1 IAA oxidase function in vivo. A second isoform, DAO2, is very weakly expressed in seedling root apices. Together, these data confirm that IAA oxidation by DAO1 is the principal auxin catabolic process in Arabidopsis and that localized IAA oxidation plays a role in plant morphogenesis.

scholarly journals His20 Provides the Sole Functionally Significant Side Chain in the Essential TonB Transmembrane Domain

2007 ◽  
Vol 189 (7) ◽  
pp. 2825-2833 ◽  
Author(s):  
Ray A. Larsen ◽  
Gail E. Deckert ◽  
Kyle A. Kastead ◽  
Surendranathan Devanathan ◽  
Kimberly L. Keller ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
Protonmotive Force ◽  
Wild Type ◽  
Carboxy Terminus ◽  
Amino Terminal ◽  
In The Wild ◽  
Mutant Phenotypes

ABSTRACT The cytoplasmic membrane protein TonB couples the protonmotive force of the cytoplasmic membrane to active transport across the outer membrane of Escherichia coli. The uncleaved amino-terminal signal anchor transmembrane domain (TMD; residues 12 to 32) of TonB and the integral cytoplasmic membrane proteins ExbB and ExbD are essential to this process, with important interactions occurring among the several TMDs of all three proteins. Here, we show that, of all the residues in the TonB TMD, only His20 is essential for TonB activity. When alanyl residues replaced all TMD residues except Ser16 and His20, the resultant “all-Ala Ser16 His20” TMD TonB retained 90% of wild-type iron transport activity. Ser16Ala in the context of a wild-type TonB TMD was fully active. In contrast, His20Ala in the wild-type TMD was entirely inactive. In more mechanistically informative assays, the all-Ala Ser16 His20 TMD TonB unexpectedly failed to support formation of disulfide-linked dimers by TonB derivatives bearing Cys substitutions for the aromatic residues in the carboxy terminus. We hypothesize that, because ExbB/D apparently cannot efficiently down-regulate conformational changes at the TonB carboxy terminus through the all-Ala Ser16 His20 TMD, the TonB carboxy terminus might fold so rapidly that disulfide-linked dimers cannot be efficiently trapped. In formaldehyde cross-linking experiments, the all-Ala Ser16 His20 TMD also supported large numbers of apparently nonspecific contacts with unknown proteins. The all-Ala Ser16 His20 TMD TonB retained its dependence on ExbB/D. Together, these results suggest that a role for ExbB/D might be to control rapid and nonspecific folding that the unregulated TonB carboxy terminus otherwise undergoes. Such a model helps to reconcile the crystal/nuclear magnetic resonance structures of the TonB carboxy terminus with conformational changes and mutant phenotypes observed at the TonB carboxy terminus in vivo.

scholarly journals Visualization of Melanosome Dynamics within Wild-Type and Dilute Melanocytes Suggests a Paradigm for Myosin V Function In Vivo

1998 ◽  
Vol 143 (7) ◽  
pp. 1899-1918 ◽  
Author(s):  
Xufeng Wu ◽  
Blair Bowers ◽  
Kang Rao ◽  
Qin Wei ◽  
John A. Hammer
Keyword(s):  
Wild Type Mouse ◽  
Time Lapse ◽  
Tail Domain ◽  
Wild Type ◽  
Myosin V ◽  
Transport Models ◽  
Myosin Va ◽  
Cortical Shell ◽  
Mutant Phenotypes

Unlike wild-type mouse melanocytes, where melanosomes are concentrated in dendrites and dendritic tips, melanosomes in dilute (myosin Va−) melanocytes are concentrated in the cell center. Here we sought to define the role that myosin Va plays in melanosome transport and distribution. Actin filaments that comprise a cortical shell running the length of the dendrite were found to exhibit a random orientation, suggesting that myosin Va could drive the outward spreading of melanosomes by catalyzing random walks. In contrast to this mechanism, time lapse video microscopy revealed that melanosomes undergo rapid (∼1.5 μm/s) microtubule-dependent movements to the periphery and back again. This bidirectional traffic occurs in both wild-type and dilute melanocytes, but it is more obvious in dilute melanocytes because the only melanosomes in their periphery are those undergoing this movement. While providing an efficient means to transport melanosomes to the periphery, this component does not by itself result in their net accumulation there. These observations, together with previous studies showing extensive colocalization of myosin Va and melanosomes in the actin-rich periphery, suggest a mechanism in which a myosin Va–dependent interaction of melanosomes with F-actin in the periphery prevents these organelles from returning on microtubules to the cell center, causing their distal accumulation. This “capture” model is supported by the demonstration that (a) expression of the myosin Va tail domain within wild-type cells creates a dilute-like phenotype via a process involving initial colocalization of tail domains with melanosomes in the periphery, followed by an ∼120-min, microtubule-based redistribution of melanosomes to the cell center; (b) microtubule-dependent melanosome movement appears to be damped by myosin Va; (c) intermittent, microtubule-independent, ∼0.14 μm/s melanosome movements are seen only in wild-type melanocytes; and (d) these movements do not drive obvious spreading of melanosomes over 90 min. We conclude that long-range, bidirectional, microtubule-dependent melanosome movements, coupled with actomyosin Va–dependent capture of melanosomes in the periphery, is the predominant mechanism responsible for the centrifugal transport and peripheral accumulation of melanosomes in mouse melanocytes. This mechanism represents an alternative to straightforward transport models when interpreting other myosin V mutant phenotypes.

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