Myo1c binds phosphoinositides through a putative pleckstrin homology domain

Myo1c is a member of the myosin superfamily that binds phosphatidylinositol-4,5-bisphosphate (PIP(2)), links the actin cytoskeleton to cellular membranes and plays roles in mechano-signal transduction and membrane trafficking. We located and characterized two distinct membrane binding sites within the regulatory and tail domains of this myosin. By sequence, secondary structure, and ab initio computational analyses, we identified a phosphoinositide binding site in the tail to be a putative pleckstrin homology (PH) domain. Point mutations of residues known to be essential for polyphosphoinositide binding in previously characterized PH domains inhibit myo1c binding to PIP(2) in vitro, disrupt in vivo membrane binding, and disrupt cellular localization. The extended sequence of this binding site is conserved within other myosin-I isoforms, suggesting they contain this putative PH domain. We also characterized a previously identified membrane binding site within the IQ motifs in the regulatory domain. This region is not phosphoinositide specific, but it binds anionic phospholipids in a calcium-dependent manner. However, this site is not essential for in vivo membrane binding.     

Myo1c facilitates G-actin transport to the leading edge of migrating endothelial cells

Addition of actin monomer (G-actin) to growing actin filaments (F-actin) at the leading edge generates force for cell locomotion. The polymerization reaction and its regulation have been studied in depth. However, the mechanism responsible for transport of G-actin substrate to the cell front is largely unknown; random diffusion, facilitated transport via myosin II contraction, local synthesis as a result of messenger ribonucleic acid localization, or F-actin turnover all might contribute. By tracking a photoactivatable, nonpolymerizable actin mutant, we show vectorial transport of G-actin in live migrating endothelial cells (ECs). Mass spectrometric analysis identified Myo1c, an unconventional F-actin-binding motor protein, as a major G-actin-interacting protein. The cargo-binding tail domain of Myo1c interacted with G-actin, and the motor domain was required for the transport. Local microinjection of Myo1c promoted G-actin accumulation and plasma membrane ruffling, and Myo1c knockdown confirmed its contribution to G-actin delivery to the leading edge and for cell motility. In addition, there is no obvious requirement for myosin II contractile-based transport of G-actin in ECs. Thus, Myo1c-facilitated G-actin transport might be a critical node for control of cell polarity and motility.     

Motor protein Myo1c is a podocyte protein that facilitates the transport of slit diaphragm protein Neph1 to the podocyte membrane

The podocyte proteins Neph1 and nephrin organize a signaling complex at the podocyte cell membrane that forms the structural framework for a functional glomerular filtration barrier. Mechanisms regulating the movement of these proteins to and from the membrane are currently unknown. This study identifies a novel interaction between Neph1 and the motor protein Myo1c, where Myo1c plays an active role in targeting Neph1 to the podocyte cell membrane. Using in vivo and in vitro experiments, we provide data supporting a direct interaction between Neph1 and Myo1c which is dynamic and actin dependent. Unlike wild-type Myo1c, the membrane localization of Neph1 was significantly reduced in podocytes expressing dominant negative Myo1c. In addition, Neph1 failed to localize at the podocyte cell membrane and cell junctions in Myo1c-depleted podocytes. We further demonstrate that similarly to Neph1, Myo1c also binds nephrin and reduces its localization at the podocyte cell membrane. A functional analysis of Myo1c knockdown cells showed defects in cell migration, as determined by a wound assay. In addition, the ability to form tight junctions was impaired in Myo1c knockdown cells, as determined by transepithelial electric resistance (TER) and bovine serum albumin (BSA) permeability assays. These results identify a novel Myo1c-dependent molecular mechanism that mediates the dynamic organization of Neph1 and nephrin at the slit diaphragm and is critical for podocyte function.     

Myo1c binding to submembrane actin mediates insulin-induced tethering of GLUT4 vesicles

GLUT4-containing vesicles cycle between the plasma membrane and intracellular compartments. Insulin promotes GLUT4 exocytosis by regulating GLUT4 vesicle arrival at the cell periphery and its subsequent tethering, docking, and fusion with the plasma membrane. The molecular machinery involved in GLUT4 vesicle tethering is unknown. We show here that Myo1c, an actin-based motor protein that associates with membranes and actin filaments, is required for insulin-induced vesicle tethering in muscle cells. Myo1c was found to associate with both mobile and tethered GLUT4 vesicles and to be required for vesicle capture in the total internal reflection fluorescence (TIRF) zone beneath the plasma membrane. Myo1c knockdown or overexpression of an actin binding–deficient Myo1c mutant abolished insulin-induced vesicle immobilization, increased GLUT4 vesicle velocity in the TIRF zone, and prevented their externalization. Conversely, Myo1c overexpression immobilized GLUT4 vesicles in the TIRF zone and promoted insulin-induced GLUT4 exposure to the extracellular milieu. Myo1c also contributed to insulin-dependent actin filament remodeling. Thus we propose that interaction of vesicular Myo1c with cortical actin filaments is required for insulin-mediated tethering of GLUT4 vesicles and for efficient GLUT4 surface delivery in muscle cells.     

The transmission of nuclear pore complexes to daughter cells requires a cytoplasmic pool of Nsp1

Nuclear pore complexes (NPCs) are essential protein assemblies that span the nuclear envelope and establish nuclear–cytoplasmic compartmentalization. We have investigated mechanisms that control NPC number in mother and daughter cells during the asymmetric division of budding yeast. By simultaneously tracking existing NPCs and newly synthesized NPC protomers (nups) through anaphase, we uncovered a pool of the central channel nup Nsp1 that is actively targeted to the bud in association with endoplasmic reticulum. Bud targeting required an intact actin cytoskeleton and the class V myosin, Myo2. Selective inhibition of cytoplasmic Nsp1 or inactivation of Myo2 reduced the inheritance of NPCs in daughter cells, leading to a daughter-specific loss of viability. Our data are consistent with a model in which Nsp1 releases a barrier that otherwise prevents NPC passage through the bud neck. It further supports the finding that NPC inheritance, not de novo NPC assembly, is primarily responsible for controlling NPC number in daughter cells.     

Motor domain-dependent localization of myo1b (myr-1)

Myosin-I is the single-headed, membrane binding member of the myosin superfamily that plays a role in membrane dynamics and transport [1-6]. Its molecular functions and its mechanism of regulation are not known. In mammalian cells, myosin-I is excluded from specific microfilament populations, indicating that its localization is tightly regulated. Identifying the mechanism of this localization, and the specific actin populations with which myosin-I interacts, is crucial to understanding the molecular functions of this motor. eGFP chimeras of myo1b [7] were imaged in live and fixed NRK cells. Ratio-imaging microscopy shows that myo1b-eGFP concentrates within dynamic areas of the actin cytoskeleton, most notably in membrane ruffles. Myo1b-eGFP does not associate with stable actin bundles or stress fibers. Truncation mutants consisting of the motor or tail domains show a partially overlapping cytoplasmic localization with full-length myo1b, but do not concentrate in membrane ruffles. A chimera consisting of the light chain and tail domains of myo1b and the motor domain from nonmuscle myosin-IIb (nmMIIb) concentrates on actin filaments in ruffles as well as to stress fibers. In vitro motility assays show that the exclusion of myo1b from certain actin filament populations is due to the regulation of the actomyosin interaction by tropomyosin. Therefore, we conclude that tropomyosin and spatially regulated actin polymerization play important roles in regulating the function and localization of myo1b.     

T cell glycolipid-enriched membrane domains are constitutively assembled as membrane patches that translocate to immune synapses

In T cells, glycolipid-enriched membrane (GEM) domains, or lipid rafts, are assembled into immune synapses in response to Ag presentation. However, the properties of T cell GEM domains in the absence of stimulatory signals, such as their size and distribution in the plasma membrane, are less clear. To address this question, we used confocal microscopy to measure GEM domains in unstimulated T cells expressing a GEM-targeted green fluorescent protein molecule. Our experiments showed that the GEM domains were assembled into membrane patches that were micrometers in size, as evidenced by a specific enrichment of GEM-associated molecules and resistance of the patches to extraction by Triton X-100. However, treatment of cells with latrunculin B disrupted the patching of the GEM domains and their resistance to Triton X-100. Similarly, the patches were coenriched with F-actin, and actin occurred in the detergent-resistant GEM fraction of T cells. Live-cell imaging showed that the patches were mobile and underwent translocation in the plasma membrane to immune synapses in stimulated T cells. Targeting of GEM domains to immune synapses was found to be actin-dependent, and required phosphatidylinositol 3-kinase activity and myosin motor proteins. We conclude from our results that T cell GEM domains are constitutively assembled by the actin cytoskeleton into micrometer-sized membrane patches, and that GEM domains and the GEM-enriched patches can function as a vehicle for targeting molecules to immune synapses.     

A two-photon FRAP analysis of the cytoskeleton dynamics in the microvilli of intestinal cells

The molecular structure of the brush-border of enterocytes has been investigated since the 1980s, but the dynamics of this highly specialized subcellular domain have been difficult to study due to its small size. To perform a detailed analysis of the dynamics of cytoskeleton proteins in this domain, we developed two-photon fluorescence recovery after photobleaching and a theoretical framework for data analysis. With this method, fast dynamics of proteins in the microvilli of the brush border of epithelial intestinal cells can be measured on the millisecond timescale in volumes smaller than 1 microm3. Two major proteins of the cytoskeleton of the microvilli, actin and myosin 1a (Myo1a; formerly named brush border myosin I), are mobile in the brush-border of Caco-2 cells, an enterocyte-like cellular model. However, the mobility of actin is very different from that of Myo1a and they appear to be unrelated (diffusion coefficient of 15 microm2 s(-1) with a mobile fraction of 60% for actin, and 4 microm2 s(-1) with a mobile fraction of 90% for Myo1a). Furthermore, we show for the first time, in vivo, that the dynamics of Myo1a in microvilli reflect its motor activity.     

The Class V Myosin Myo2p Is Required for Fus2p Transport and Actin Polarization during the Yeast Mating Response

Mating yeast cells remove their cell walls and fuse their plasma membranes in a spatially restricted cell contact region. Cell wall removal is dependent on Fus2p, an amphiphysin-associated Rho-GEF homolog. As mating cells polarize, Fus2p-GFP localizes to the tip of the mating projection, where cell fusion will occur, and to cytoplasmic puncta, which show rapid movement toward the tip. Movement requires polymerized actin, whereas tip localization is dependent on both actin and a membrane protein, Fus1p. Here, we show that Fus2p-GFP movement is specifically dependent on Myo2p, a type V myosin, and not on Myo4p, another type V myosin, or Myo3p and Myo5p, type I myosins. Fus2p-GFP tip localization and actin polarization in shmoos are also dependent on Myo2p. A temperature-sensitive tropomyosin mutation and Myo2p alleles that specifically disrupt vesicle binding caused rapid loss of actin patch organization, indicating that transport is required to maintain actin polarity. Mutant shmoos lost actin polarity more rapidly than mitotic cells, suggesting that the maintenance of cell polarity in shmoos is more sensitive to perturbation. The different velocities, differential sensitivity to mutation and lack of colocalization suggest that Fus2p and Sec4p, another Myo2p cargo associated with exocytotic vesicles, reside predominantly on different cellular organelles.     

The class V myosin motor protein, Myo2, plays a major role in mitochondrial motility in Saccharomyces cerevisiae

The actin cytoskeleton is essential for polarized, bud-directed movement of cellular membranes in Saccharomyces cerevisiae and thus ensures accurate inheritance of organelles during cell division. Also, mitochondrial distribution and inheritance depend on the actin cytoskeleton, though the precise molecular mechanisms are unknown. Here, we establish the class V myosin motor protein, Myo2, as an important mediator of mitochondrial motility in budding yeast. We found that mutants with abnormal expression levels of Myo2 or its associated light chain, Mlc1, exhibit aberrant mitochondrial morphology and loss of mitochondrial DNA. Specific mutations in the globular tail of Myo2 lead to aggregation of mitochondria in the mother cell. Isolated mitochondria lacking functional Myo2 are severely impaired in their capacity to bind to actin filaments in vitro. Time-resolved fluorescence microscopy revealed a block of bud-directed anterograde mitochondrial movement in cargo binding-defective myo2 mutant cells. We conclude that Myo2 plays an important and direct role for mitochondrial motility and inheritance in budding yeast.     

Myosin-1A targets to microvilli using multiple membrane binding motifs in the tail homology 1 (TH1) domain

One of the most abundant components of the enterocyte brush border is the actin-based monomeric motor, myosin-1a (Myo1a). Within brush border microvilli, Myo1a carries out a number of critical functions at the interface between membrane and actin cytoskeleton. Proper physiological function of Myo1a depends on its ability to bind to microvillar membrane, an interaction mediated by a C-terminal tail homology 1 (TH1) domain. However, little is known about the mechanistic details of the Myo1a-TH1/membrane interaction. Structure-function analysis of Myo1a-TH1 targeting in epithelial cells revealed that an N-terminal motif conserved among class I myosins and a C-terminal motif unique to Myo1a-TH1 are both required for steady state microvillar enrichment. Purified Myo1a bound to liposomes composed of phosphatidylserine and phosphoinositol 4,5-bisphosphate, with moderate affinity in a charge-dependent manner. Additionally, peptides of the N- and C-terminal regions required for targeting were able to compete with Myo1a for binding to highly charged liposomes in vitro. Single molecule total internal reflection fluorescence microscopy showed that these motifs are also necessary for slowing the membrane detachment rate in cells. Finally, Myo1a-TH1 co-localized with both lactadherin-C2 (a phosphatidylserine-binding protein) and PLCδ1-PH (a phosphoinositol 4,5-bisphosphate-binding protein) in microvilli, but only lactaderin-C2 expression reduced brush border targeting of Myo1a-TH1. Together, our results suggest that Myo1a targeting to microvilli is driven by membrane binding potential that is distributed throughout TH1 rather than localized to a single motif. These data highlight the diversity of mechanisms that enable different class I myosins to target membranes in distinct biological contexts.     

The myosin phosphatase targeting protein (MYPT) family: a regulated mechanism for achieving substrate specificity of the catalytic subunit of protein phosphatase type 1δ

The mammalian MYPT family consists of the products of five genes, denoted MYPT1, MYPT2, MBS85, MYPT3 and TIMAP, which function as targeting and regulatory subunits to confer substrate specificity and subcellular localization on the catalytic subunit of type 1δ protein serine/threonine phosphatase (PP1cδ). Family members share several conserved domains, including an RVxF motif for PP1c binding and several ankyrin repeats that mediate protein–protein interactions. MYPT1, MYPT2 and MBS85 contain C-terminal leucine zipper domains involved in dimerization and protein–protein interaction, whereas MYPT3 and TIMAP are targeted to membranes via a C-terminal prenylation site. All family members are regulated by phosphorylation at multiple sites by various protein kinases; for example, Rho-associated kinase phosphorylates MYPT1, MYPT2 and MBS85, resulting in inhibition of phosphatase activity and Ca²⁺ sensitization of smooth muscle contraction. A great deal is known about MYPT1, the myosin targeting subunit of myosin light chain phosphatase, in terms of its role in the regulation of smooth muscle contraction and, to a lesser extent, non-muscle motile processes. MYPT2 appears to be the key myosin targeting subunit of myosin light chain phosphatase in cardiac and skeletal muscles. MBS85 most closely resembles MYPT2, but little is known about its physiological function. Little is also known about the physiological role of MYPT3, although it is likely to target myosin light chain phosphatase to membranes and thereby achieve specificity for substrates involved in regulation of the actin cytoskeleton. MYPT3 is regulated by phosphorylation by cAMP-dependent protein kinase. TIMAP appears to target PP1cδ to the plasma membrane of endothelial cells where it serves to dephosphorylate proteins involved in regulation of the actin cytoskeleton and thereby control endothelial barrier function. With such a wide range of regulatory targets, MYPT family members have been implicated in diverse pathological events, including hypertension, Parkinson’s disease and cancer.     

Myo3A,one of two class III myosin genes expressed in vertebrate retina,is localized to the calycal processes of rod and cone photoreceptors and is expressed in the sacculus

The striped bass has two retina-expressed class III myosin genes, each composed of a kinase, motor, and tail domain. We report the cloning, sequence analysis, and expression patterns of the long (Myo3A) and short (Myo3B) class III myosins, as well as cellular localization and biochemical characterization of the long isoform, Myo3A. Myo3A (209 kDa) is expressed in the retina, brain, testis, and sacculus, and Myo3B (155 kDa) is expressed in the retina, intestine, and testis. The tails of these two isoforms contain two highly conserved domains, 3THDI and 3THDII. Whereas Myo3B has three IQ motifs, Myo3A has nine IQ motifs, four in its neck and five in its tail domain. Myo3A localizes to actin filament bundles of photoreceptors and is concentrated in the calycal processes. An anti-Myo3A antibody decorates the actin cytoskeleton of rod inner/outer segments, and this labeling is reduced by the presence of ATP. The ATP-sensitive actin association is a feature characteristic of myosin motors. The numerous IQ motifs may play a structural or signaling role in the Myo3A, and its localization to calycal processes indicates that this myosin mediates a local function at this site in vertebrate photoreceptors.     

MYO1A (brush border myosin I) dynamics in the brush border of LLC-PK1-CL4 cells

The kidney epithelial cell line, LLC-PK1-CL4 (CL4), forms a well ordered brush border (BB) on its apical surface. CL4 cells were used to examine the dynamics of MYO1A (M1A; formerly BB myosin I) within the BB using GFP-tagged MIA (GFP-M1A), MIA motor domain (GFP-MDIQ), and tail domain (GFP-Tail). GFP-beta-actin (GFP-Actin) was used to assess actin dynamics within the BB. GFP-M1A, GFP-Tail, but not GFP-MDIQ localized to the BB, indicating that the tail is sufficient for apical targeting of M1A. GFP-Actin targeted to all the actin domains of the cell including the BB. Fluorescence recovery after photobleaching analysis revealed that GFP-M1A and GFP-Tail turnover in the BB is rapid, approximately 80% complete in <1 min. As expected for an actin-based motor, ATP depletion resulted in significant inhibition of GFP-M1A turnover yet had little effect on GFP-Tail exchange. Rapid turnover of GFP-M1A and GFP-Tail was not due to actin turnover as GFP-Actin turnover in the BB was much slower. These results indicate that the BB population of M1A turns over rapidly, while its head and tail domains interact transiently with the core actin and plasma membrane, respectively. This rapidly exchanging pool of M1A envelops an actin core bundle that, by comparison, is static in structure.     

The novel adaptor protein, Mti1p, and Vrp1p, a homolog of Wiskott-Aldrich syndrome protein-interacting protein (WIP), may antagonistically regulate type I myosins in Saccharomyces cerevisiae

Type I myosins in yeast, Myo3p and Myo5p (Myo3/5p), are involved in the reorganization of the actin cytoskeleton. The SH3 domain of Myo5p regulates the polymerization of actin through interactions with both Las17p, a homolog of mammalian Wiskott-Aldrich syndrome protein (WASP), and Vrp1p, a homolog of WASP-interacting protein (WIP). Vrp1p is required for both the localization of Myo5p to cortical patch-like structures and the ATP-independent interaction between the Myo5p tail region and actin filaments. We have identified and characterized a new adaptor protein, Mti1p (Myosin tail region-interacting protein), which interacts with the SH3 domains of Myo3/5p. Mti1p co-immunoprecipitated with Myo5p and Mti1p-GFP co-localized with cortical actin patches. A null mutation of MTI1 exhibited synthetic lethal phenotypes with mutations in SAC6 and SLA2, which encode actin-bundling and cortical actin-binding proteins, respectively. Although the mti1 null mutation alone did not display any obvious phenotype, it suppressed vrp1 mutation phenotypes, including temperature-sensitive growth, abnormally large cell morphology, defects in endocytosis and salt-sensitive growth. These results suggest that Mti1p and Vrp1p antagonistically regulate type I myosin functions.     

The Src Homology Domain 3 (SH3) of a Yeast Type I Myosin, Myo5p, Binds to Verprolin and Is Required for Targeting to Sites of Actin Polarization

The budding yeast contains two type I myosins, Myo3p and Myo5p, with redundant functions. Deletion of both myosins results in growth defects, loss of actin polarity and polarized cell surface growth, and accumulation of intracellular membranes. Expression of myc-tagged Myo5p in myo3Δ myo5Δ cells fully restores wild-type characteristics. Myo5p is localized as punctate, cortical structures enriched at sites of polarized cell growth. We find that latrunculin-A–induced depolymerization of F-actin results in loss of Myo5p patches. Moreover, incubation of yeast cells at 37°C results in transient depolarization of both Myo5p patches and the actin cytoskeleton. Mutant Myo5 proteins with deletions in nonmotor domains were expressed in myo3Δ myo5Δ cells and the resulting strains were analyzed for Myo5p function. Deletion of the tail homology 2 (TH2) domain, previously implicated in ATP-insensitive actin binding, has no detectable effect on Myo5p function. In contrast, myo3Δ myo5Δ cells expressing mutant Myo5 proteins with deletions of the src homology domain 3 (SH3) or both TH2 and SH3 domains display defects including Myo5p patch depolarization, actin disorganization, and phenotypes associated with actin dysfunction. These findings support a role for the SH3 domain in Myo5p localization and function in budding yeast. The proline-rich protein verprolin (Vrp1p) binds to the SH3 domain of Myo3p or Myo5p in two-hybrid tests, coimmunoprecipitates with Myo5p, and colocalizes with Myo5p. Immunolocalization of the myc-tagged SH3 domain of Myo5p reveals diffuse cytoplasmic staining. Thus, the SH3 domain of Myo5p contributes to but is not sufficient for localization of Myo5p either to patches or to sites of polarized cell growth. Consistent with this, Myo5p patches assemble but do not localize to sites of polarized cell surface growth in a VRP1 deletion mutant. Our studies support a multistep model for Myo5p targeting in yeast. The first step, assembly of Myo5p patches, is dependent upon F-actin, and the second step, polarization of actin patches, requiresVrp1p and the SH3 domain of Myo5p.     

Myosin 1E interacts with synaptojanin-1 and dynamin and is involved in endocytosis

Myosin 1E is one of two "long-tailed" human Class I myosins that contain an SH3 domain within the tail region. SH3 domains of yeast and amoeboid myosins I interact with activators of the Arp2/3 complex, an important regulator of actin polymerization. No binding partners for the SH3 domains of myosins I have been identified in higher eukaryotes. In the current study, we show that two proteins with prominent functions in endocytosis, synaptojanin-1 and dynamin, bind to the SH3 domain of human Myo1E. Myosin 1E co-localizes with clathrin- and dynamin-containing puncta at the plasma membrane and this co-localization requires an intact SH3 domain. Expression of Myo1E tail, which acts in a dominant-negative manner, inhibits endocytosis of transferrin. Our findings suggest that myosin 1E may contribute to receptor-mediated endocytosis.     

Myosin surface loop 4 modulates inhibition of actomyosin 1b ATPase activity by tropomyosin

Structural studies of the class I myosin, MyoE, led to the predictions that loop 4, a surface loop near the actin-binding region that is longer in class I myosins than in other myosin subclasses, might limit binding of myosins I to actin when actin-binding proteins, like tropomyosin, are present, and might account for the exclusion of myosin I from stress fibers. To test these hypotheses, mutant molecules of the related mammalian class I myosin, Myo1b, in which loop 4 was truncated (from an amino acid sequence of RMNGLDES to NGLD) or replaced with the shorter and distinct loop 4 found in Dictyostelium myosin II (GAGEGA), were expressed in vitro and their interaction with actin and with actin-tropomyosin was tested. Saturating amounts of expressed fibroblast tropomyosin-2 resulted in a decrease in the maximum actin-activated Mg2+-ATPase activity of wild-type Myo1b but had little or no effect on the actin-activated Mg2+-ATPase activity of the two mutants. In motility assays, few actin filaments bound tightly to Myo1b-WT-coated cover slips when tropomyosin-2 was present, whereas actin filaments both bound and were translocated by Myo1b-NGLD or Myo1b-GAGEGA in both the presence and absence of tropomyosin-2. When expressed in mammalian cells, like the wild type, the mutant myosins were largely excluded from tropomyosin-containing actin filaments, indicating that in the cell additional factors besides loop 4 determine targeting of myosins I to specific subpopulations of actin filaments.     

A role for Vps1p, actin, and the Myo2p motor in peroxisome abundance and inheritance in Saccharomyces cerevisiae.

In vivo time-lapse microscopy reveals that the number of peroxisomes in Saccharomyces cerevisiae cells is fairly constant and that a subset of the organelles are targeted and segregated to the bud in a highly ordered, vectorial process. The dynamin-like protein Vps1p controls the number of peroxisomes, since in a vps1Delta mutant only one or two giant peroxisomes remain. Analogous to the function of other dynamin-related proteins, Vps1p may be involved in a membrane fission event that is required for the regulation of peroxisome abundance. We found that efficient segregation of peroxisomes from mother to bud is dependent on the actin cytoskeleton, and active movement of peroxisomes along actin filaments is driven by the class V myosin motor protein, Myo2p: (a) peroxisomal dynamics always paralleled the polarity of the actin cytoskeleton, (b) double labeling of peroxisomes and actin cables revealed a close association between both, (c) depolymerization of the actin cytoskeleton abolished all peroxisomal movements, and (d) in cells containing thermosensitive alleles of MYO2, all peroxisome movement immediately stopped at the nonpermissive temperature. In addition, time-lapse videos showing peroxisome movement in wild-type and vps1Delta cells suggest the existence of various levels of control involved in the partitioning of peroxisomes.