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Live cell imaging of the assembly, disassembly, and actin cable-dependent movement of endosomes and actin patches in the budding yeast, Saccharomyces cerevisiae.

Huckaba TM, Gay AC, Pantalena LF, Yang HC, Pon LA - J. Cell Biol. (2004)

Bottom Line: An Arp2/3 complex mutation decreases the frequency of cortical, nonlinear actin patch movements, but has no effect on the velocity of linear, retrograde actin patch movement.Moreover, actin patches require actin cables for retrograde movements and colocalize with actin cables as they undergo retrograde movement.Our studies support a mechanism whereby actin cables serve as "conveyor belts" for retrograde movement and delivery of actin patches/endosomes to FM4-64-labeled internal compartments.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.

ABSTRACT
Using FM4-64 to label endosomes and Abp1p-GFP or Sac6p-GFP to label actin patches, we find that (1) endosomes colocalize with actin patches as they assemble at the bud cortex; (2) endosomes colocalize with actin patches as they undergo linear, retrograde movement from buds toward mother cells; and (3) actin patches interact with and disassemble at FM4-64-labeled internal compartments. We also show that retrograde flow of actin cables mediates retrograde actin patch movement. An Arp2/3 complex mutation decreases the frequency of cortical, nonlinear actin patch movements, but has no effect on the velocity of linear, retrograde actin patch movement. Rather, linear actin patch movement occurs at the same velocity and direction as the movement of actin cables. Moreover, actin patches require actin cables for retrograde movements and colocalize with actin cables as they undergo retrograde movement. Our studies support a mechanism whereby actin cables serve as "conveyor belts" for retrograde movement and delivery of actin patches/endosomes to FM4-64-labeled internal compartments.

Show MeSH
FM4-64 and Abp1p-GFP assemble at the same punctate structures in living yeast. Mid-log phase wild-type haploid cells expressing Abp1p-GFP from the chromosomal locus were incubated with FM4-64 for 30 s at RT. Cells were washed with lactate medium to remove excess FM4-64 and analyzed by time-lapse fluorescence imaging within 2 min after initial incubation with FM4-64. Under these staining and imaging conditions, FM4-64 localizes to sites of endocytosis and endosomes. Cells were imaged in a single, cortical focal plane at RT using an optical beam splitter that allows for simultaneous imaging of Abp1p-GFP and FM4-64 (see Materials and methods). Images are still frames from a time-lapse series showing Abp1p-GFP in the left column, FM4-64 in the middle column, and a merged image showing Abp1p-GFP (green) and FM4-64 (red) in the right column. Outline of the cell is shown at t = 0 s. Arrowheads in merged images mark the site of FM4-64 and Abp1p-GFP accumulation. Arrows indicate the first time-points in which a signal is detectable for Abp1p-GFP (left) and FM4-64 (middle). Bar, 2 μm.
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fig1: FM4-64 and Abp1p-GFP assemble at the same punctate structures in living yeast. Mid-log phase wild-type haploid cells expressing Abp1p-GFP from the chromosomal locus were incubated with FM4-64 for 30 s at RT. Cells were washed with lactate medium to remove excess FM4-64 and analyzed by time-lapse fluorescence imaging within 2 min after initial incubation with FM4-64. Under these staining and imaging conditions, FM4-64 localizes to sites of endocytosis and endosomes. Cells were imaged in a single, cortical focal plane at RT using an optical beam splitter that allows for simultaneous imaging of Abp1p-GFP and FM4-64 (see Materials and methods). Images are still frames from a time-lapse series showing Abp1p-GFP in the left column, FM4-64 in the middle column, and a merged image showing Abp1p-GFP (green) and FM4-64 (red) in the right column. Outline of the cell is shown at t = 0 s. Arrowheads in merged images mark the site of FM4-64 and Abp1p-GFP accumulation. Arrows indicate the first time-points in which a signal is detectable for Abp1p-GFP (left) and FM4-64 (middle). Bar, 2 μm.

Mentions: We found that FM4-64 and Abp1p-GFP assemble into the same punctate structures in living yeast. In the example shown, Abp1p-GFP appears as a weakly fluorescent spot at the mother-bud neck of a yeast cell (Fig. 1)Figure 1.


Live cell imaging of the assembly, disassembly, and actin cable-dependent movement of endosomes and actin patches in the budding yeast, Saccharomyces cerevisiae.

Huckaba TM, Gay AC, Pantalena LF, Yang HC, Pon LA - J. Cell Biol. (2004)

FM4-64 and Abp1p-GFP assemble at the same punctate structures in living yeast. Mid-log phase wild-type haploid cells expressing Abp1p-GFP from the chromosomal locus were incubated with FM4-64 for 30 s at RT. Cells were washed with lactate medium to remove excess FM4-64 and analyzed by time-lapse fluorescence imaging within 2 min after initial incubation with FM4-64. Under these staining and imaging conditions, FM4-64 localizes to sites of endocytosis and endosomes. Cells were imaged in a single, cortical focal plane at RT using an optical beam splitter that allows for simultaneous imaging of Abp1p-GFP and FM4-64 (see Materials and methods). Images are still frames from a time-lapse series showing Abp1p-GFP in the left column, FM4-64 in the middle column, and a merged image showing Abp1p-GFP (green) and FM4-64 (red) in the right column. Outline of the cell is shown at t = 0 s. Arrowheads in merged images mark the site of FM4-64 and Abp1p-GFP accumulation. Arrows indicate the first time-points in which a signal is detectable for Abp1p-GFP (left) and FM4-64 (middle). Bar, 2 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172478&req=5

fig1: FM4-64 and Abp1p-GFP assemble at the same punctate structures in living yeast. Mid-log phase wild-type haploid cells expressing Abp1p-GFP from the chromosomal locus were incubated with FM4-64 for 30 s at RT. Cells were washed with lactate medium to remove excess FM4-64 and analyzed by time-lapse fluorescence imaging within 2 min after initial incubation with FM4-64. Under these staining and imaging conditions, FM4-64 localizes to sites of endocytosis and endosomes. Cells were imaged in a single, cortical focal plane at RT using an optical beam splitter that allows for simultaneous imaging of Abp1p-GFP and FM4-64 (see Materials and methods). Images are still frames from a time-lapse series showing Abp1p-GFP in the left column, FM4-64 in the middle column, and a merged image showing Abp1p-GFP (green) and FM4-64 (red) in the right column. Outline of the cell is shown at t = 0 s. Arrowheads in merged images mark the site of FM4-64 and Abp1p-GFP accumulation. Arrows indicate the first time-points in which a signal is detectable for Abp1p-GFP (left) and FM4-64 (middle). Bar, 2 μm.
Mentions: We found that FM4-64 and Abp1p-GFP assemble into the same punctate structures in living yeast. In the example shown, Abp1p-GFP appears as a weakly fluorescent spot at the mother-bud neck of a yeast cell (Fig. 1)Figure 1.

Bottom Line: An Arp2/3 complex mutation decreases the frequency of cortical, nonlinear actin patch movements, but has no effect on the velocity of linear, retrograde actin patch movement.Moreover, actin patches require actin cables for retrograde movements and colocalize with actin cables as they undergo retrograde movement.Our studies support a mechanism whereby actin cables serve as "conveyor belts" for retrograde movement and delivery of actin patches/endosomes to FM4-64-labeled internal compartments.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.

ABSTRACT
Using FM4-64 to label endosomes and Abp1p-GFP or Sac6p-GFP to label actin patches, we find that (1) endosomes colocalize with actin patches as they assemble at the bud cortex; (2) endosomes colocalize with actin patches as they undergo linear, retrograde movement from buds toward mother cells; and (3) actin patches interact with and disassemble at FM4-64-labeled internal compartments. We also show that retrograde flow of actin cables mediates retrograde actin patch movement. An Arp2/3 complex mutation decreases the frequency of cortical, nonlinear actin patch movements, but has no effect on the velocity of linear, retrograde actin patch movement. Rather, linear actin patch movement occurs at the same velocity and direction as the movement of actin cables. Moreover, actin patches require actin cables for retrograde movements and colocalize with actin cables as they undergo retrograde movement. Our studies support a mechanism whereby actin cables serve as "conveyor belts" for retrograde movement and delivery of actin patches/endosomes to FM4-64-labeled internal compartments.

Show MeSH