Actin‐dependent, Retrograde Motility of Surface‐attached Beads and Aggregating Pigment Granules in Dissociated Teleost Retinal Pigment Epithelial Cells

Basciano, Paul; King-Smith, Christina

doi:10.1034/j.1600-0749.2002.01078.x

Cited by 7 publications

(14 citation statements)

References 36 publications

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“…The mean rate of melanosome movement observed in the Nitella assay was about 2 m/min. This rate is comparable to rates of melanosome motility in apical projections of dissociated, cultured RPE cells, which ranges from 1-2 m/min [King-Smith et al, 2001;Basciano and King-Smith, 2002]. The very rapid velocities observed in the Nitella assay were possibly from endogenous motors that were not inactivated by the NEM pre-treatment.…”

Section: Discussionmentioning

confidence: 63%

Actin‐dependent motility of melanosomes from fish retinal pigment epithelial (RPE) cells investigated using in vitro motility assays

McNeil

Tacelosky

Basciano

et al. 2004

Cell Motil. Cytoskeleton

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Melanosomes (pigment granules) within retinal pigment epithelial (RPE) cells of fish and amphibians undergo massive migrations in response to light conditions to control light flux to the retina. Previous research has shown that melanosome motility within apical projections of dissociated fish RPE cells requires an intact actin cytoskeleton, but the mechanisms and motors involved in melanosome transport in RPE have not been identified. Two in vitro motility assays, the Nitella assay and the sliding filament assay, were used to characterize actin-dependent motor activity of RPE melanosomes. Melanosomes applied to dissected filets of the Characean alga, Nitella, moved along actin cables at a mean rate of 2 microm/min, similar to the rate of melanosome motility in dissociated, cultured RPE cells. Path lengths of motile melanosomes ranged from 9 to 37 microm. Melanosome motility in the sliding filament assay was much more variable, ranging from 0.4-33 microm/min; 70% of velocities ranged from 1-15 microm/min. Latex beads coated with skeletal muscle myosin II and added to Nitella filets moved in the same direction as RPE melanosomes, indicating that the motility is barbed-end directed. Immunoblotting using antibodies against myosin VIIa and rab27a revealed that both proteins are enriched on melanosome membranes, suggesting that they could play a role in melanosome transport within apical projections of fish RPE.

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Section: Discussionmentioning

confidence: 63%

Actin‐dependent motility of melanosomes from fish retinal pigment epithelial (RPE) cells investigated using in vitro motility assays

McNeil

Tacelosky

Basciano

et al. 2004

Cell Motil. Cytoskeleton

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“…In contrast, the unidirectional, coordinated motility of melanosomes during aggregation is hypothesized to occur by passive association of melanosomes to actin filaments undergoing retrograde flow. A possible link to retrograde flow is suggested by the smooth, coordinated motility of melanosomes during aggregation, at rates similar to those reported for retrograde actin flow [King-Smith et al, 1997;Basciano and King-Smith, 2002]. This hypothesis requires that melanosomes attach to a ''linker'' protein or protein complex that passively attaches them to actin filaments.…”

Section: Introductionmentioning

confidence: 88%

Myosin II and Rho kinase activity are required for melanosome aggregation in fish retinal pigment epithelial cells

Barsoum¹,

King-Smith²

2007

Cell Motil. Cytoskeleton

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In the retinal pigment epithelium (RPE) of fish, melanosomes (pigment granules) migrate long distances through the cell body into apical projections in the light, and aggregate back into the cell body in the dark. RPE cells can be isolated from the eye, dissociated, and cultured as single cells in vitro. Treatment of isolated RPE cells with cAMP or the phosphatase inhibitor, okadaic acid (OA), stimulates melanosome aggregation, while cAMP or OA washout in the presence of dopamine triggers dispersion. Previous studies have shown that actin filaments are both necessary and sufficient for aggregation and dispersion of melanosomes within apical projections of isolated RPE. The role of myosin II in melanosome motility was investigated using the myosin II inhibitor, blebbistatin, and a specific rho kinase (ROCK) inhibitor, H-1152. Blebbistatin and H-1152 partially blocked melanosome aggregation triggered by cAMP in dissociated, isolated RPE cells and isolated sheets of RPE. In contrast, neither drug affected melanosome dispersion. In cells exposed to either blebbistatin or H-1152, then triggered to aggregate using OA, melanosome aggregation was completely inhibited. These results demonstrate that (1) melanosome aggregation and dispersion occur through different, actin-dependent mechanisms; (2) myosin II and ROCK activity are required for full melanosome aggregation, but not dispersion; (3) partial aggregation that occurred despite myosin II or ROCK inhibition suggests a second component of aggregation that is dependent on cAMP signaling, but independent of ROCK and myosin II.

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“…This movement likely represents the transport of virus-containing endocytic vesicles inside the cell, either directed by myosin motors on actin filaments (7) or propelled by actin filaments in a fashion similar to the actinbased motility of listeria (7,22,23). However, we cannot exclude the possibility that stage I involves an actin-dependent motion on the cell surface (24,25).…”

Section: Three-stage Active Transport Of Viruses In Cellsmentioning

confidence: 99%

Visualizing infection of individual influenza viruses

Lakadamyali

Rust

Babcock

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

673

645

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Influenza is a paradigm for understanding viral infections. As an opportunistic pathogen exploiting the cellular endocytic machinery for infection, influenza is also a valuable model system for exploring the cell's constitutive endocytic pathway. We have studied the transport, acidification, and fusion of single influenza viruses in living cells by using real-time fluorescence microscopy and have dissected individual stages of the viral entry pathway. The movement of individual viruses revealed a striking three-stage active transport process that preceded viral fusion with endosomes starting with an actin-dependent movement in the cell periphery, followed by a rapid, dynein-directed translocation to the perinuclear region, and finally an intermittent movement involving both plus-and minus-end-directed microtubule-based motilities in the perinuclear region. Surprisingly, the majority of viruses experience their initial acidification in the perinuclear region immediately following the dynein-directed rapid translocation step. This finding suggests a previously undescribed scenario of the endocytic pathway toward late endosomes: endosome maturation, including initial acidification, largely occurs in the perinuclear region.T he infection pathway of influenza is a complex, multistep process: viruses enter cells by receptor-mediated endocytosis, then move from endocytic vesicles to early endosomes and finally to late endosomes where the viruses fuse with the endosomal membrane to release viral genes (1-7), a pathway followed by many other medically important viruses (3, 4). Exploiting the cell's constitutive endocytic machinery for infection, influenza is an ideal probe for exploring the cell's endocytic pathway. However, despite intensive efforts in investigating influenza infection, many critical properties of the endocytosis and endocytic traffic of the virus remain elusive. Among these are important and general questions for cellular endocytosis: what are the transport mechanisms of endocytic compartments at different stages on the endocytic pathway, and how do these compartments and the enclosed endocytic cargo mature (6-8)?The difficulty in real-time imaging of the endocytic pathway presents one of the major hurdles in addressing the above questions. In this work, by tracking the behavior of single influenza viruses in real-time (9-12), we have dissected individual stages of the viral entry process, observed transient, previously unobserved steps in endocytic viral trafficking, and obtained a dynamic picture of the cell's endocytic pathway exploited by influenza. We have discovered that virus-bearing endosomes are transported in cells in three distinct stages, each with a distinct cytoskeleton-and motor-protein-dependent mechanism. We have revealed surprising endocytic acidification dynamics: the initial acidification of endocytic cargo mainly occurs in the perinuclear region, bringing into question the previous picture that early endosomes in the cell periphery are the early acidification sites for endocytic ca...

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Actin‐dependent, Retrograde Motility of Surface‐attached Beads and Aggregating Pigment Granules in Dissociated Teleost Retinal Pigment Epithelial Cells

Cited by 7 publications

References 36 publications

Actin‐dependent motility of melanosomes from fish retinal pigment epithelial (RPE) cells investigated using in vitro motility assays

Actin‐dependent motility of melanosomes from fish retinal pigment epithelial (RPE) cells investigated using in vitro motility assays

Myosin II and Rho kinase activity are required for melanosome aggregation in fish retinal pigment epithelial cells

Visualizing infection of individual influenza viruses

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