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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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Visualization of the disassembly of Abp1p-GFP at FM4-64–labeled internal compartments by 3D reconstruction combined with time-lapse imaging. Mid-log phase wild-type haploid cells expressing Abp1p-GFP were stained with FM4-64 as for Fig. 4. Cells were analyzed by simultaneous two-color imaging and 3D reconstruction combined with time-lapse imaging. Simultaneous two-color imaging was performed as for Fig. 1. Z-sections were obtained at 0.4-μm increments. The time interval between each successive set of z-sections is 1.6 s. The still frames shown are z-sections at focal planes that show sites of disassembly of Abp1p-GFP (top) as the particle interacts with an FM4-64–labeled internal compartment (bottom) at different time-points during the disassembly process. The cell shown is a large-budded cell. The structures of interest are in the mother cell and are pseudocolored as in Fig. 2. Optical sections taken above and below the plane of Abp1p-GFP–labeled particle disassembly indicate that the loss of Abp1p-GFP signal is due to disassembly, and not to movement of the particle out of the plane of imaging. Bar, 2 μm.
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fig5: Visualization of the disassembly of Abp1p-GFP at FM4-64–labeled internal compartments by 3D reconstruction combined with time-lapse imaging. Mid-log phase wild-type haploid cells expressing Abp1p-GFP were stained with FM4-64 as for Fig. 4. Cells were analyzed by simultaneous two-color imaging and 3D reconstruction combined with time-lapse imaging. Simultaneous two-color imaging was performed as for Fig. 1. Z-sections were obtained at 0.4-μm increments. The time interval between each successive set of z-sections is 1.6 s. The still frames shown are z-sections at focal planes that show sites of disassembly of Abp1p-GFP (top) as the particle interacts with an FM4-64–labeled internal compartment (bottom) at different time-points during the disassembly process. The cell shown is a large-budded cell. The structures of interest are in the mother cell and are pseudocolored as in Fig. 2. Optical sections taken above and below the plane of Abp1p-GFP–labeled particle disassembly indicate that the loss of Abp1p-GFP signal is due to disassembly, and not to movement of the particle out of the plane of imaging. Bar, 2 μm.

Mentions: Abp1p-GFP disassembles after actin patches interact with FM4-64–labeled internal compartments. Mid-log phase wild-type haploid cells expressing Abp1p-GFP from the chromosomal locus were stained with FM4-64 for 2 min at RT. Cells were washed with lactate medium to remove excess FM4-64 and imaged 7 min after initial incubation of cells with FM4-64. Under these conditions, FM4-64 stains the endosomal sorting compartment (Holthuis et al., 1998). 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–labeled actin patches in the top row, FM4-64–labeled internal compartments 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 the top panel at t = 0 s. Arrows mark an Abp1p-GFP–labeled actin patch that undergoes retrograde movement and interacts with an FM4-64–labeled internal compartment, indicated by arrowheads. 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)

Visualization of the disassembly of Abp1p-GFP at FM4-64–labeled internal compartments by 3D reconstruction combined with time-lapse imaging. Mid-log phase wild-type haploid cells expressing Abp1p-GFP were stained with FM4-64 as for Fig. 4. Cells were analyzed by simultaneous two-color imaging and 3D reconstruction combined with time-lapse imaging. Simultaneous two-color imaging was performed as for Fig. 1. Z-sections were obtained at 0.4-μm increments. The time interval between each successive set of z-sections is 1.6 s. The still frames shown are z-sections at focal planes that show sites of disassembly of Abp1p-GFP (top) as the particle interacts with an FM4-64–labeled internal compartment (bottom) at different time-points during the disassembly process. The cell shown is a large-budded cell. The structures of interest are in the mother cell and are pseudocolored as in Fig. 2. Optical sections taken above and below the plane of Abp1p-GFP–labeled particle disassembly indicate that the loss of Abp1p-GFP signal is due to disassembly, and not to movement of the particle out of the plane of imaging. Bar, 2 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172478&req=5

fig5: Visualization of the disassembly of Abp1p-GFP at FM4-64–labeled internal compartments by 3D reconstruction combined with time-lapse imaging. Mid-log phase wild-type haploid cells expressing Abp1p-GFP were stained with FM4-64 as for Fig. 4. Cells were analyzed by simultaneous two-color imaging and 3D reconstruction combined with time-lapse imaging. Simultaneous two-color imaging was performed as for Fig. 1. Z-sections were obtained at 0.4-μm increments. The time interval between each successive set of z-sections is 1.6 s. The still frames shown are z-sections at focal planes that show sites of disassembly of Abp1p-GFP (top) as the particle interacts with an FM4-64–labeled internal compartment (bottom) at different time-points during the disassembly process. The cell shown is a large-budded cell. The structures of interest are in the mother cell and are pseudocolored as in Fig. 2. Optical sections taken above and below the plane of Abp1p-GFP–labeled particle disassembly indicate that the loss of Abp1p-GFP signal is due to disassembly, and not to movement of the particle out of the plane of imaging. Bar, 2 μm.
Mentions: Abp1p-GFP disassembles after actin patches interact with FM4-64–labeled internal compartments. Mid-log phase wild-type haploid cells expressing Abp1p-GFP from the chromosomal locus were stained with FM4-64 for 2 min at RT. Cells were washed with lactate medium to remove excess FM4-64 and imaged 7 min after initial incubation of cells with FM4-64. Under these conditions, FM4-64 stains the endosomal sorting compartment (Holthuis et al., 1998). 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–labeled actin patches in the top row, FM4-64–labeled internal compartments 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 the top panel at t = 0 s. Arrows mark an Abp1p-GFP–labeled actin patch that undergoes retrograde movement and interacts with an FM4-64–labeled internal compartment, indicated by arrowheads. 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