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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
Particles labeled with FM4-64 and Abp1p-GFP exhibit linear, retrograde movement. Mid-log phase wild-type haploid cells expressing Abp1p-GFP from the chromosomal locus were incubated with FM4-64 for 1 min at RT. Cells were washed with lactate medium to remove excess FM4-64, and imaged within 3 min after initial incubation with FM4-64. Two-color time-lapse imaging was performed as described for Fig. 1. Images shown are still frames from a time-lapse series showing Abp1p-GFP in the top row, FM4-64 in the middle row, and a merged image showing Abp1p-GFP in green and FM4-64 in red in the bottom row. The outline of the cell is shown in panels at t = 0 s. The bud, mother-bud neck, and part of the mother cell are shown. Arrowheads in the merged images mark an actin patch/endosome undergoing linear movement. Bar, 2 μm.
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fig3: Particles labeled with FM4-64 and Abp1p-GFP exhibit linear, retrograde movement. Mid-log phase wild-type haploid cells expressing Abp1p-GFP from the chromosomal locus were incubated with FM4-64 for 1 min at RT. Cells were washed with lactate medium to remove excess FM4-64, and imaged within 3 min after initial incubation with FM4-64. Two-color time-lapse imaging was performed as described for Fig. 1. Images shown are still frames from a time-lapse series showing Abp1p-GFP in the top row, FM4-64 in the middle row, and a merged image showing Abp1p-GFP in green and FM4-64 in red in the bottom row. The outline of the cell is shown in panels at t = 0 s. The bud, mother-bud neck, and part of the mother cell are shown. Arrowheads in the merged images mark an actin patch/endosome undergoing linear movement. Bar, 2 μm.

Mentions: Using simultaneous two-color time-lapse imaging in living yeast cells, we found that FM4-64 colocalized with motile Abp1p-GFP–labeled actin patches. In the representative example shown, a particle labeled with FM4-64 and Abp1p-GFP undergoes linear, retrograde movement for a distance of ∼2 μm over a 3.6-s period (Fig. 3)Figure 3.


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)

Particles labeled with FM4-64 and Abp1p-GFP exhibit linear, retrograde movement. Mid-log phase wild-type haploid cells expressing Abp1p-GFP from the chromosomal locus were incubated with FM4-64 for 1 min at RT. Cells were washed with lactate medium to remove excess FM4-64, and imaged within 3 min after initial incubation with FM4-64. Two-color time-lapse imaging was performed as described for Fig. 1. Images shown are still frames from a time-lapse series showing Abp1p-GFP in the top row, FM4-64 in the middle row, and a merged image showing Abp1p-GFP in green and FM4-64 in red in the bottom row. The outline of the cell is shown in panels at t = 0 s. The bud, mother-bud neck, and part of the mother cell are shown. Arrowheads in the merged images mark an actin patch/endosome undergoing linear movement. Bar, 2 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172478&req=5

fig3: Particles labeled with FM4-64 and Abp1p-GFP exhibit linear, retrograde movement. Mid-log phase wild-type haploid cells expressing Abp1p-GFP from the chromosomal locus were incubated with FM4-64 for 1 min at RT. Cells were washed with lactate medium to remove excess FM4-64, and imaged within 3 min after initial incubation with FM4-64. Two-color time-lapse imaging was performed as described for Fig. 1. Images shown are still frames from a time-lapse series showing Abp1p-GFP in the top row, FM4-64 in the middle row, and a merged image showing Abp1p-GFP in green and FM4-64 in red in the bottom row. The outline of the cell is shown in panels at t = 0 s. The bud, mother-bud neck, and part of the mother cell are shown. Arrowheads in the merged images mark an actin patch/endosome undergoing linear movement. Bar, 2 μm.
Mentions: Using simultaneous two-color time-lapse imaging in living yeast cells, we found that FM4-64 colocalized with motile Abp1p-GFP–labeled actin patches. In the representative example shown, a particle labeled with FM4-64 and Abp1p-GFP undergoes linear, retrograde movement for a distance of ∼2 μm over a 3.6-s period (Fig. 3)Figure 3.

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