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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.

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Colocalization of actin patches and actin cables during retrograde movement. Wild-type haploid cells expressing Abp1p-HcRed and Abp140p-GFP from the chromosomal loci were grown to mid-log phase in lactate medium and were imaged in a single cortical focal plane using simultaneous two-color imaging as for Fig. 1. Images shown are still frames from a time-lapse series showing Abp140p-GFP–labeled actin cables in the top row, Abp1p-HcRed–labeled actin patches in the middle row, and a merged image showing Abp140p-GFP in green and Abp1p-HcRed in red in the bottom row. The cell shown is an unbudded cell in which the presumptive bud site is at the top of the cell. Arrows in the merged images mark an Abp1p-HcRed–labeled actin patch that undergoes linear, retrograde movement along an Abp140p-GFP–labeled actin cable. Bar, 2 μm.
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fig9: Colocalization of actin patches and actin cables during retrograde movement. Wild-type haploid cells expressing Abp1p-HcRed and Abp140p-GFP from the chromosomal loci were grown to mid-log phase in lactate medium and were imaged in a single cortical focal plane using simultaneous two-color imaging as for Fig. 1. Images shown are still frames from a time-lapse series showing Abp140p-GFP–labeled actin cables in the top row, Abp1p-HcRed–labeled actin patches in the middle row, and a merged image showing Abp140p-GFP in green and Abp1p-HcRed in red in the bottom row. The cell shown is an unbudded cell in which the presumptive bud site is at the top of the cell. Arrows in the merged images mark an Abp1p-HcRed–labeled actin patch that undergoes linear, retrograde movement along an Abp140p-GFP–labeled actin cable. Bar, 2 μm.

Mentions: Finally, we found that Abp1p-HcRed–labeled actin patches undergoing linear, retrograde movement localize to Abp140p-GFP–labeled actin cables (Fig. 9Figure 9.


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)

Colocalization of actin patches and actin cables during retrograde movement. Wild-type haploid cells expressing Abp1p-HcRed and Abp140p-GFP from the chromosomal loci were grown to mid-log phase in lactate medium and were imaged in a single cortical focal plane using simultaneous two-color imaging as for Fig. 1. Images shown are still frames from a time-lapse series showing Abp140p-GFP–labeled actin cables in the top row, Abp1p-HcRed–labeled actin patches in the middle row, and a merged image showing Abp140p-GFP in green and Abp1p-HcRed in red in the bottom row. The cell shown is an unbudded cell in which the presumptive bud site is at the top of the cell. Arrows in the merged images mark an Abp1p-HcRed–labeled actin patch that undergoes linear, retrograde movement along an Abp140p-GFP–labeled actin cable. Bar, 2 μm.
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Related In: Results  -  Collection

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fig9: Colocalization of actin patches and actin cables during retrograde movement. Wild-type haploid cells expressing Abp1p-HcRed and Abp140p-GFP from the chromosomal loci were grown to mid-log phase in lactate medium and were imaged in a single cortical focal plane using simultaneous two-color imaging as for Fig. 1. Images shown are still frames from a time-lapse series showing Abp140p-GFP–labeled actin cables in the top row, Abp1p-HcRed–labeled actin patches in the middle row, and a merged image showing Abp140p-GFP in green and Abp1p-HcRed in red in the bottom row. The cell shown is an unbudded cell in which the presumptive bud site is at the top of the cell. Arrows in the merged images mark an Abp1p-HcRed–labeled actin patch that undergoes linear, retrograde movement along an Abp140p-GFP–labeled actin cable. Bar, 2 μm.
Mentions: Finally, we found that Abp1p-HcRed–labeled actin patches undergoing linear, retrograde movement localize to Abp140p-GFP–labeled actin cables (Fig. 9Figure 9.

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