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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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Assembly, movement, and disassembly of Sac6p-GFP. Mid-log phase wild-type haploid cells expressing Sac6p-GFP from the chromosomal locus. FM4-64 staining to detect endosomes in A was performed as described in Fig. 1. FM4-64 treatment to stain internal compartments in B was performed as described in Fig. 4. Two-color time-lapse imaging was performed as for Fig. 1. Images shown are still frames from time-lapse series showing Sac6p-GFP–labeled actin patches in green, FM4-64 in red, and sites of colocalization in yellow. The outline of each cell is shown in both panels at t = 0 s. In A, arrowheads indicate the coassembly of a Sac6p-containing actin patch and an FM4-64 labeled endosome. Arrow in A points to the beginning of a linear, retrograde movement of a Sac6p- and FM4-64-containing structure. In B, an arrow points to a Sac6p-labeled actin patch that undergoes linear, retrograde movement and subsequent disassembly at the FM4-64–labeled internal compartment. Bar, 2 μm.
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fig7: Assembly, movement, and disassembly of Sac6p-GFP. Mid-log phase wild-type haploid cells expressing Sac6p-GFP from the chromosomal locus. FM4-64 staining to detect endosomes in A was performed as described in Fig. 1. FM4-64 treatment to stain internal compartments in B was performed as described in Fig. 4. Two-color time-lapse imaging was performed as for Fig. 1. Images shown are still frames from time-lapse series showing Sac6p-GFP–labeled actin patches in green, FM4-64 in red, and sites of colocalization in yellow. The outline of each cell is shown in both panels at t = 0 s. In A, arrowheads indicate the coassembly of a Sac6p-containing actin patch and an FM4-64 labeled endosome. Arrow in A points to the beginning of a linear, retrograde movement of a Sac6p- and FM4-64-containing structure. In B, an arrow points to a Sac6p-labeled actin patch that undergoes linear, retrograde movement and subsequent disassembly at the FM4-64–labeled internal compartment. Bar, 2 μm.

Mentions: Abp1p-GFP and Sac6p-HcRed assemble at the same punctate structures in living yeast. Wild-type haploid cells expressing Abp1p-GFP and Sac6p-HcRed from the chromosomal loci were grown to mid-log phase in lactate medium and 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 Abp1p-GFP–labeled actin patches in the top row, Sac6p-HcRed–labeled actin patches in the middle row, and a merged image showing Abp1p-GFP in green and Sac6p-HcRed in red in the bottom row. Arrowheads indicate the point of emergence of fluorescent signal in the top and middle rows. The outline of the cell is shown in the top panel at t = 0 s. Bar, 2 μm.


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)

Assembly, movement, and disassembly of Sac6p-GFP. Mid-log phase wild-type haploid cells expressing Sac6p-GFP from the chromosomal locus. FM4-64 staining to detect endosomes in A was performed as described in Fig. 1. FM4-64 treatment to stain internal compartments in B was performed as described in Fig. 4. Two-color time-lapse imaging was performed as for Fig. 1. Images shown are still frames from time-lapse series showing Sac6p-GFP–labeled actin patches in green, FM4-64 in red, and sites of colocalization in yellow. The outline of each cell is shown in both panels at t = 0 s. In A, arrowheads indicate the coassembly of a Sac6p-containing actin patch and an FM4-64 labeled endosome. Arrow in A points to the beginning of a linear, retrograde movement of a Sac6p- and FM4-64-containing structure. In B, an arrow points to a Sac6p-labeled actin patch that undergoes linear, retrograde movement and subsequent disassembly at the FM4-64–labeled internal compartment. Bar, 2 μm.
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Related In: Results  -  Collection

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fig7: Assembly, movement, and disassembly of Sac6p-GFP. Mid-log phase wild-type haploid cells expressing Sac6p-GFP from the chromosomal locus. FM4-64 staining to detect endosomes in A was performed as described in Fig. 1. FM4-64 treatment to stain internal compartments in B was performed as described in Fig. 4. Two-color time-lapse imaging was performed as for Fig. 1. Images shown are still frames from time-lapse series showing Sac6p-GFP–labeled actin patches in green, FM4-64 in red, and sites of colocalization in yellow. The outline of each cell is shown in both panels at t = 0 s. In A, arrowheads indicate the coassembly of a Sac6p-containing actin patch and an FM4-64 labeled endosome. Arrow in A points to the beginning of a linear, retrograde movement of a Sac6p- and FM4-64-containing structure. In B, an arrow points to a Sac6p-labeled actin patch that undergoes linear, retrograde movement and subsequent disassembly at the FM4-64–labeled internal compartment. Bar, 2 μm.
Mentions: Abp1p-GFP and Sac6p-HcRed assemble at the same punctate structures in living yeast. Wild-type haploid cells expressing Abp1p-GFP and Sac6p-HcRed from the chromosomal loci were grown to mid-log phase in lactate medium and 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 Abp1p-GFP–labeled actin patches in the top row, Sac6p-HcRed–labeled actin patches in the middle row, and a merged image showing Abp1p-GFP in green and Sac6p-HcRed in red in the bottom row. Arrowheads indicate the point of emergence of fluorescent signal in the top and middle rows. The outline of the cell is shown in the top panel at t = 0 s. Bar, 2 μm.

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