Disruption in the tropomodulin1 (Tmod1) gene compromises cardiomyocyte development in murine embryonic stem cells by arresting myofibril maturation

Tropomodulins (Tmods) comprise a family of capping proteins for actin filament pointed ends. To decipher the significance of Tmod1 functions during de novo myofibrillogenesis, we generated Tmod1 null embryonic stem (ES) cells and studied their differentiation into cardiomyocytes. Strikingly, in vitro cardiomyocyte differentiation of wild type (WT) ES cells faithfully recapitulates in vivo cardiomyocyte differentiation, allowing us to evaluate the phenotypes of Tmod1 knockout (KO) myofibrils irrespective of embryonic lethality of Tmod1 KO mice. Immunofluorescence and electron microscopy studies revealed that Tmod1 null cardiac myocytes were round, morphologically immature, and contained underdeveloped myofibrils that were shorter, narrower, and had fewer thin filaments than those in WT cells. Unexpectedly, clear gaps in the staining pattern for F-actin at the H-zone were detected in most KO cells, indicating the presence of filaments at uniform lengths. This indicates that additional mechanisms other than capping proteins are responsible for thin filament length maintenance in cardiac myocytes. Also unexpectedly, approximately 40% of the KO cardiac myocytes exhibited contractile activity. Our data indicate that differentiating ES cells are a powerful system to investigate the functional properties of contractile proteins and that Tmod1 functions are critical for late stages of myofibrillogenesis, and for the maturation of myofibrils.     

Myosin flares and actin leptomeres as myofibril assembly/disassembly intermediates in sonic muscle fibers

The sonic muscle of type 1 male midshipman fish produces loud and enduring mating calls. Each sonic muscle fiber contains a tubular contractile apparatus with radially arranged myofibrillar plates encased in a desmin-rich cytoskeleton that is anchored to broad Z bands (~1.2 μm wide). Immunomicroscopy has revealed patches of myosin-rich “flares” emanating from the contractile tubes into the peripheral sarcoplasm along the length of the fibers. These flares contain swirls of thick filaments devoid of associated thin filaments. In other regions of the sarcoplasm at the inner surface of the sarcolemma and near Z bands, abundant ladder-like leptomeres occur with rungs every 160 nm. Leptomeres consist of dense arrays of filaments (~4 nm) with a structure that resembles myofibrillar Z band structure. We propose that flares and leptomeres are distinct filamentous arrays representing site-specific processing of myofibrillar components during the assembly and disassembly of the sarcomere. Recent reports that myosin assembles into filamentous aggregates before incorporating into the A band in the skeletal muscles of vertebrates and Caenorhabditis elegans suggest that sonic fibers utilize a similar pathway. Thus, sonic muscle fibers, with their tubular design and abundant sarcoplasmic space, may provide an attractive muscle model to identify myofibrillar intermediates by structural and molecular techniques. This work was supported by the Intramural Research Program of the NIAMS, NIH, HHS (KW).     

Myofibrillogenesis in the first cardiomyocytes formed from isolated quail precardiac mesoderm

De novo assembly of myofibrils was investigated in explants of precardiac mesoderm from quail embryos to address a controversy about different models of myofibrillogenesis. The sequential expression of sarcomeric components was visualized in double- and triple-stained explants before, during, and just after the first cardiomyocytes began to beat. In explants from stage 6 embryos, cultured for 10 h, ectoderm, endoderm, and the precardiac mesoderm displayed arrays of stress fibers with alternating bands of the nonmuscle isoforms of alpha-actinin and myosin IIB. With increasing time in culture, mesoderm cells contained fibrils composed of actin, nonmuscle myosin IIB, and sarcomeric alpha-actinin. Several hours later, before beating occurred, both nonmuscle and muscle myosin II localized in some of the fibrils in the cells. Concentrations of muscle myosin began as thin bundles, dispersed in the cytoplasm, often overlapping one another, and progressed to small, aligned A-band-sized aggregates. The amount of nonmuscle myosin decreased dramatically when Z-bands formed, the muscle myosin became organized into A-bands, and the cells began beating. The sequential changes in protein composition of the fibrils in the developing muscle cells supports the model of myofibrillogenesis in which assembly begins with premyofibrils and progresses through nascent myofibrils to mature myofibrils.     

A new unique form of microRNA from human heart,microRNA-499c,promotes myofibril formation and rescues cardiac development in mutant axolotl embryos

Background

A recessive mutation “c” in the Mexican axolotl, Ambystoma mexicanum, results in the failure of normal heart development. In homozygous recessive embryos, the hearts do not have organized myofibrils and fail to beat. In our previous studies, we identified a noncoding Myofibril-Inducing RNA (MIR) from axolotls which promotes myofibril formation and rescues heart development.

Results

We randomly cloned RNAs from fetal human heart. RNA from clone #291 promoted myofibril formation and induced heart development of mutant axolotls in organ culture. This RNA induced expression of cardiac markers in mutant hearts: tropomyosin, troponin and α-syntrophin. This cloned RNA matches in partial sequence alignment to human microRNA-499a and b, although it differs in length. We have concluded that this cloned RNA is unique in its length, but is still related to the microRNA-499 family. We have named this unique RNA, microRNA-499c. Thus, we will refer to this RNA derived from clone #291 as microRNA-499c throughout the rest of the paper.

Conclusions

This new form, microRNA-499c, plays an important role in cardiac development.     

Myosin 1G (Myo1G) is a haematopoietic specific myosin that localises to the plasma membrane and regulates cell elasticity

Immune cells navigate through different environments where they experience different mechanical forces. Responses to external forces are determined by the mechanical properties of a cell and they depend to a large extent on the actin-rich cell cortex. We report here that Myo1G, a previously uncharacterised member of class I myosins, is expressed specifically in haematopoietic tissues and cells. It is associated with the plasma membrane. This association is dependent on a conserved PH-domain-like myosin I tail homology motif and the head domain. However, the head domain does not need to be a functional motor. Knockdown of Myo1G in Jurkat cells decreased cell elasticity significantly. We propose that Myo1G regulates cell elasticity by deformations of the actin network at the cell cortex.

Structured summary

MINT-7307273: MYO1G (uniprotkb:B0I1T2) and Actin (uniprotkb:P60709) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT-7307283: TfR (uniprotkb:P02786) and MYO1G (uniprotkb:B0I1T2) colocalize (MI:0403) by cosedimentation through density gradients (MI:0029)     

A centrosomal inclusion (striped nebulous body) in pinealocytes of the golden hamster

Summary The postnatal maturation of regions of the epididymis and intragonadal segment of the deferens duct was studied in the rat by light-and transmission electron microscopy. Maturation of the genital duct starts in the distal cauda epididymidis and ductus deferens after one week of life, and one week later, in the more cranial segments of the epididymis. Epithelial principal cells and peritubular contractile cells are structurally mature 35 days after birth. The synchronous changes of these cells indicate that the same factors control their postnatal maturation. The epithelial principal cells obtain an endocytotic apparatus and long stereocilia, whereas peritubular cells acquire contractile features. These changes are associated with a progressive increase in the immunoreaction for smooth muscle actin in both cell types. Smooth muscle myosin is detected in the apical region of the epithelial cells and the peritubular cell cytoplasm by day one of postnatal development. The differentiation of contractile cells in the wall is accompanied by progressive organization of the pericellular matrix into a continuous basement membrane. Although fibronectin is visible at birth, it is gradually removed from the tubule wall.     

Molecular mechanism of the E99K mutation in cardiac actin (ACTC Gene) that causes apical hypertrophy in man and mouse

We generated a transgenic mouse model expressing the apical hypertrophic cardiomyopathy-causing mutation ACTC E99K at 50% of total heart actin and compared it with actin from patients carrying the same mutation. The actin mutation caused a higher Ca(2+) sensitivity in reconstituted thin filaments measured by in vitro motility assay (2.3-fold for mice and 1.3-fold for humans) and in skinned papillary muscle. The mutation also abolished the change in Ca(2+) sensitivity normally linked to troponin I phosphorylation. MyBP-C and troponin I phosphorylation levels were the same as controls in transgenic mice and human carrier heart samples. ACTC E99K mice exhibited a high death rate between 28 and 45 days (48% females and 22% males). At 21 weeks, the hearts of the male survivors had enlarged atria, increased interstitial fibrosis, and sarcomere disarray. MRI showed hypertrophy, predominantly at the apex of the heart. End-diastolic volume and end-diastolic pressure were increased, and relaxation rates were reduced compared with nontransgenic littermates. End-systolic pressures and volumes were unaltered. ECG abnormalities were present, and the contractile response to β-adrenergic stimulation was much reduced. Older mice (29-week-old females and 38-week-old males) developed dilated cardiomyopathy with increased end-systolic volume and continuing increased end-diastolic pressure and slower contraction and relaxation rates. ECG showed atrial flutter and frequent atrial ectopic beats at rest in some ACTC E99K mice. We propose that the ACTC E99K mutation causes higher myofibrillar Ca(2+) sensitivity that is responsible for the sudden cardiac death, apical hypertrophy, and subsequent development of heart failure in humans and mice.     

Central role of mitofusin 2 in autophagosome-lysosome fusion in cardiomyocytes

In the heart, autophagy has been implicated in cardioprotection and ischemia-reperfusion tolerance, and the dysregulation of autophagy is associated with the development of heart failure. Mitochondrial dynamic proteins are profoundly involved in autophagic processes, especially the initiation and formation of autophagosomes, but it is not clear whether they play any role in cardiac autophagy. We previously reported that mitofusin 2 (MFN2), a mitochondrial outer membrane protein, serves as a major determinant of cardiomyocyte apoptosis mediated by oxidative stress. Here, we reveal a novel and essential role of MFN2 in mediating cardiac autophagy. We found that specific deletion of MFN2 in cardiomyocytes caused extensive accumulation of autophagosomes. In particular, the fusion of autophagosomes with lysosomes, a critical step in autophagic degradation, was markedly retarded without altering the formation of autophagosomes and lysosomes in response to ischemia-reperfusion stress. Importantly, MFN2 co-immunoprecipitated with RAB7 in the heart, and starvation further increased it. Knockdown of MFN2 by shRNA prevented, whereas re-expression of MFN2 restored, the autophagosome-lysosome fusion in neonatal cardiomyocytes. Hearts from cardiac-specific MFN2 knock-out mice had abnormal mitochondrial and cellular metabolism and were vulnerable to ischemia-reperfusion challenge. Our study defined a novel and essential role of MFN2 in the cardiac autophagic process by mediating the maturation of autophagy at the phase of autophagosome-lysosome fusion; deficiency of MFN2 caused multiple molecular and functional defects that undermined cardiac reserve and gradually led to cardiac vulnerability and dysfunction.     

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.     

Role of zinc metallothionein-3 (ZnMt3) in epidermal growth factor (EGF)-induced c-Abl protein activation and actin polymerization in cultured astrocytes

Recent evidence indicates that zinc plays a major role in neurochemistry. Of the many zinc-binding proteins, metallothionein-3 (Mt3) is regarded as one of the major regulators of cellular zinc in the brain. However, biological functions of Mt3 are not yet well characterized. Recently, we found that lysosomal dysfunction in metallothionein-3 (Mt3)-null astrocytes involves down-regulation of c-Abl. In this study, we investigated the role of Mt3 in c-Abl activation and actin polymerization in cultured astrocytes following treatment with epidermal growth factor (EGF). Compared with wild-type (WT) astrocytes, Mt3-null cells exhibited a substantial reduction in the activation of c-Abl upon treatment with EGF. Consistent with previous studies, activation of c-Abl by EGF induced dissociation of c-Abl from F-actin. Mt3 added to astrocytic cell lysates bound F-actin, augmented F-actin polymerization, and promoted the dissociation of c-Abl from F-actin, suggesting a possible role for Mt3 in this process. Conversely, Mt3-deficient astrocytes showed significantly reduced dissociation of c-Abl from F-actin following EGF treatment. Experiments using various peptide fragments of Mt3 showed that a fragment containing the N-terminal TCPCP motif (peptide 1) is sufficient for this effect. Removal of zinc from Mt3 or pep1 with tetrakis(2-pyridylmethyl)ethylenediamine abrogated the effect of Mt3 on the association of c-Abl and F-actin, indicating that zinc binding is necessary for this action. These results suggest that ZnMt3 in cultured astrocytes may be a normal component of c-Abl activation in EGF receptor signaling. Hence, modulation of Mt3 levels or distribution may prove to be a useful strategy for controlling cytoskeletal mobilization following EGF stimulation in brain cells.     

Fumonisin B(1) alters sphingolipid metabolism and tumor necrosis factor alpha expression in heart and lung of mice

Fumonisin B(1) (FB(1)), produced by Fusarium verticillioides, is a common contaminant in foods and feeds. Increase in tissue free sphingoid bases resulting from the inhibition of ceramide synthase is a biomarker of fumonisin exposure. Tumor necrosis factor alpha (TNFalpha) is induced in liver in response to FB(1) treatment. This study determined whether fumonisin B(1) caused increases in free sphingoid bases and altered the expression of TNFalpha in heart and lung, organs that are not targets of FB(1) toxicity, of male and female mice treated with 5-daily subcutaneous injection of 2.25 mg/kg FB(1). A significant increase in free sphingoid bases was observed in both heart and lung of FB(1)-exposed mice. The magnitude of increases in free sphingoid bases in both organs of female mice was much higher than that in males. The expression of TNFalpha was increased by FB(1) treatment in the lung of male mice and in the heart of female mice, whereas the expression of interferon gamma was unaltered. Results suggest that both sphingolipid accumulation and TNFalpha induction are observed in the tissues of mice that are not associated with FB(1) toxicity.     

In vitro cleavage of eIF4GI but not eIF4GII by HIV-1 protease and its effects on translation in the rabbit reticulocyte lysate system

The vertebrate muscle Z-band organizes and tethers antiparallel actin filaments in adjacent sarcomeres and hence propagates the tension generated by the actomyosin interaction during muscular contraction. The axial width of the Z-band varies with fibre and muscle type: fast twitch muscles have narrow (approximately 30-50 nm) Z-bands, while slow-twitch and cardiac muscles have wide (approximately 100-140 nm) Z-bands. In electron micrographs of longitudinal sections of fast fibres like those found in fish body white muscle, the Z-band appears as a characteristic zigzag layer of density connecting the mutually offset actin filament arrays in adjacent sarcomeres. Wide Z-bands in slow fibres such as the one studied here (bovine neck muscle) show a stack of three or four zigzag layers. The variable Z-band width incorporating variable numbers of zigzag layers presumably relates to the different mechanical properties of the respective muscles. Three-dimensional reconstructions of Z-bands reveal that individual zigzag layers are often composed of more than one set of protein bridges, called Z-links, probably alpha-actinin, between oppositely oriented actin filaments. Fast muscle Z-bands comprise two or three layers of Z-links. Here we have applied Fourier reconstruction methods to obtain clear three-dimensional density maps of the Z-bands in beef muscle. The bovine slow muscle investigated here reveals a Z-band comprising six sets of Z-links, which, due to their shape and the way their projected densities overlap, appear in longitudinal sections as either three or four zigzag layers, depending on the lattice view. There has been great interest recently in the suggestion that Z-band variability with fibre type may be due to differences in the repetitive region (tandem Z-repeats) in the Z-band part of titin (also called connectin). We discuss this in the context of our results and present a systematic classification of Z-band types according to the numbers of Z-links and titin Z-repeats.     

Regulation of the microfilament system in normal and polyoma virus transformed cultured (BHK) cells

The content and state of actin in baby hamster kidney (BHK) cells before and after transformation with polyoma virus were examined by deoxyribonuclease assay and gel electrophoresis followed by dye elution. The actin content of the transformed cells, relative to total cell protein, was lower than that of the normal cells by 30-50%. In both the normal and transformed cells the greater part of the total actin was found on lysis to be in the monomeric state. Cytoplasmic and membrane fractions of the two cell lines were, in qualitative terms, very similar in their protein compositions. The plasma membrane isolated from the transformed cells was richer in actin than that from the untransformed, and both membrane fractions contained proteins corresponding to myosin, filamin and alpha-actinin on SDS-polyacrylamide gels. The cell extract from both the normal and transformed lines formed an actin-based gel on incubation at 30 degrees C, although the amount of the cross-linked actin was much smaller in the latter. This was a consequence not only of the lower concentration of total actin in the cell, but also, presumably, of a gross relative deficiency in the concentration or activity of filament cross-linking protein(s) in the cytoplasm. Thus, small aliquots of cytoplasmic fractions from transformed cells, when added to an excess of exogenous F-actin, were able to cross-link the filaments to a much smaller extent than those from the normal cells. A similar range of proteins was found to be associated with the actin gels formed from both cell extracts. One conspicuous difference was that a species migrating in SDS-gel electrophoresis as a doublet with a subunit molecular weight of about 58,000, and tentatively identified as intermediate filament protein, was replaced in the transformed cells by a single band. Filament cross-linking activity of the cytoplasmic fractions was enhanced by addition of Triton extracts of crude membranes, although the latter were not capable of cross-linking exogenous F-actin on their own. The effect of Triton extracts was much greater in the case of membranes from the transformed cells. The cytoplasmic fractions of BHK cells contain capping protein(s) and/or complexes of such proteins with actin; these reveal themselves by the propensity of the extracts to nucleate polymerization of exogenous G-actin. This activity was more abundant in transformed cells, despite their lower actin content. Their membranes were also more effective in nucleating G-actin polymerization, indicating the presence of a greater number of filament ends.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of myosin II to chromosome arms and spindle fibers in PtK1 cells: a possible role for an actomyosin system in mitosis

Summary. The enzymes of importance in moving chromosomes are called motor proteins and include dynein, kinesin, and possibly myosin II. These three molecules are all included in the category of ATPases, in that they have the ability to convert chemical energy into mechanical energy. Both dynein and kinesin have been documented as molecules that “walk” along microtubules in the mitotic spindle, carrying cargo such as chromosomes. Myosin II, analogous to the muscle contraction system, transiently interacts along actin filaments and associates with kinetochore microtubules. In this paper we present evidence that a third ATPase, myosin II, may act as a “thruster” to propel chromosomes during the mitotic process. Double-label immunocytochemistry to actin and myosin II shows that myosin II is localized on chromosome arms at the beginning of mitosis and remains localized to the chromosomes throughout mitosis. Specific staining of myosin II is relegated to the outside of chromosomes with the highest density of staining occurring between the spindle poles and the chromosomes. This specific localization could account for the movement of chromosomes during mitosis, since they segregate towards the spindle poles, along kinetochore microtubules containing actin filaments, after aligning at the equatorial region of the cell at metaphase. We conclude from this study that there is an actomyosin system present in the mitotic spindle and that myosin is attached to chromosome arms and may act as a thruster in moving chromosomes during the mitotic process. Correspondence and reprints: Department of Biological Sciences, University of Denver, 2190 E Iliff Avenue, Denver, CO 80208, U.S.A.     

Effects of polyamines on apoptosis induced by simulated ischemia/reperfusion injury in cultured neonatal rat cardiomyocytes

We incubated neonatal Sprague-Dawley rat cardiomyocytes in primary culture in a medium simulating ischemia (consisting of hypoxia plus serum deprivation) for 2 h, then re-incubated them for 24 h in normal culture medium to establish a model of simulated ischemia/reperfusion (I/R) injury. Apoptotic cell death was measured by MTT assay, TUNEL staining and flow cytometry. Morphological alterations were assessed by transmission electron microscopy, the expression of caspases-3 and -9 and Bcl-2 and the release of cytochrome c by Western blotting, and the intracellular free-calcium concentration ([Ca2+]i) by laser confocal scanning microscopy. The results showed that pretreatment with 10 micromol/l spermine or spermidine significantly inhibited apoptosis in the I/R cells, suppressed the expression of caspases-3 and -9 and cytochrome c release, up-regulated Bcl-2 expression and decreased [Ca2+]i. However, pretreatment with 10 micromol/l putrescine had the opposite effects. Evidence for this "double-edged" effect of polyamines on apoptosis in I/R-injured cardiomyocytes is presented for the first time. It may suggest a novel pharmacological target for preventing and treating cardiac ischemia/reperfusion injury.     

SCCRO (DCUN1D1) promotes nuclear translocation and assembly of the neddylation E3 complex

SCCRO/DCUN1D1/DCN1 (squamous cell carcinoma-related oncogene/defective in cullin neddylation 1 domain containing 1/defective in cullin neddylation) serves as an accessory E3 in neddylation by binding to cullin and Ubc12 to allow efficient transfer of Nedd8. In this work we show that SCCRO has broader, pleiotropic effects that are essential for cullin neddylation in vivo. Reduced primary nuclear localization of Cul1 accompanying decreased neddylation and proliferation in SCCRO(-/-) mouse embryonic fibroblasts led us to investigate whether compartmentalization plays a regulatory role. Decreased nuclear localization, neddylation, and defective proliferation in SCCRO(-/-) mouse embryonic fibroblasts were rescued by transgenic expression of SCCRO. Expression of reciprocal SCCRO and Cul1-binding mutants confirmed the requirement for SCCRO in nuclear translocation and neddylation of cullins in vivo. Nuclear translocation of Cul1 by tagging with a nuclear localization sequence allowed neddylation independent of SCCRO, but at a lower level. We found that in the nucleus, SCCRO enhances recruitment of Ubc12 to Cul1 to promote neddylation. These findings suggest that SCCRO has an essential role in neddylation in vivo involving nuclear localization of neddylation components and recruitment and proper positioning of Ubc12.     

Analysis of cytoskeletal and motility proteins in the sea urchin genome assembly

The sea urchin embryo is a classical model system for studying the role of the cytoskeleton in such events as fertilization, mitosis, cleavage, cell migration and gastrulation. We have conducted an analysis of gene models derived from the Strongylocentrotus purpuratus genome assembly and have gathered strong evidence for the existence of multiple gene families encoding cytoskeletal proteins and their regulators in sea urchin. While many cytoskeletal genes have been cloned from sea urchin with sequences already existing in public databases, genome analysis reveals a significantly higher degree of diversity within certain gene families. Furthermore, genes are described corresponding to homologs of cytoskeletal proteins not previously documented in sea urchins. To illustrate the varying degree of sequence diversity that exists within cytoskeletal gene families, we conducted an analysis of genes encoding actins, specific actin-binding proteins, myosins, tubulins, kinesins, dyneins, specific microtubule-associated proteins, and intermediate filaments. We conducted ontological analysis of select genes to better understand the relatedness of urchin cytoskeletal genes to those of other deuterostomes. We analyzed developmental expression (EST) data to confirm the existence of select gene models and to understand their differential expression during various stages of early development.     

The transport of alpha(1A)-adrenergic receptor with 33-nm step size in live cells

We used the technique of single particle tracking (SPT) with high tempo-spatial resolution to efficiently explore the route and mechanism for the transport of alpha(1A)-adrenergic receptor (alpha(1A)-AR) in real time in living cells. We found that the initial transport of alpha(1A)-AR in cells depended on actin filaments with the velocity of 0.2 microm/s and exhibited discrete 33-nm steps. It was noted that the step size, the rate constant, and the velocities were in accordance with the character of single myosin in vitro, implying that while transporting each endosome myosins did not work in the "tug-of-war" mode and that they did not adopt the strategy to boost up transporting speed by working coordinately. These results provided insight into the mechanism of GPCR transport in vivo.