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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 assembly of FM4-64 and Abp1p-GFP by 3D reconstruction combined with time-lapse imaging. Mid-log phase wild-type haploid cells expressing Abp1p-GFP from their chromosomal locus were incubated with FM4-64 as for Fig. 1. 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 incorporation of Abp1p-GFP (top) and FM4-64 (bottom) at different time-points during the assembly process. The cell shown is unbudded and has polarized toward the presumptive bud site in the top of the cell. The structure of interest is pseudocolored according to the strength of the fluorescent signal, where yellow corresponds to greatest intensity, orange corresponds to medium intensity, and red corresponds to low intensity. Abp1p and FM4-64 accumulate in an intermediate focal plane in the second column and are not present either above or below the plane of appearance in the preceding column. Thus, the structures are indeed assembling, rather than moving into the focal plane. Bar, 2 μm.
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fig2: Visualization of the assembly of FM4-64 and Abp1p-GFP by 3D reconstruction combined with time-lapse imaging. Mid-log phase wild-type haploid cells expressing Abp1p-GFP from their chromosomal locus were incubated with FM4-64 as for Fig. 1. 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 incorporation of Abp1p-GFP (top) and FM4-64 (bottom) at different time-points during the assembly process. The cell shown is unbudded and has polarized toward the presumptive bud site in the top of the cell. The structure of interest is pseudocolored according to the strength of the fluorescent signal, where yellow corresponds to greatest intensity, orange corresponds to medium intensity, and red corresponds to low intensity. Abp1p and FM4-64 accumulate in an intermediate focal plane in the second column and are not present either above or below the plane of appearance in the preceding column. Thus, the structures are indeed assembling, rather than moving into the focal plane. Bar, 2 μm.

Mentions: 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.


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 assembly of FM4-64 and Abp1p-GFP by 3D reconstruction combined with time-lapse imaging. Mid-log phase wild-type haploid cells expressing Abp1p-GFP from their chromosomal locus were incubated with FM4-64 as for Fig. 1. 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 incorporation of Abp1p-GFP (top) and FM4-64 (bottom) at different time-points during the assembly process. The cell shown is unbudded and has polarized toward the presumptive bud site in the top of the cell. The structure of interest is pseudocolored according to the strength of the fluorescent signal, where yellow corresponds to greatest intensity, orange corresponds to medium intensity, and red corresponds to low intensity. Abp1p and FM4-64 accumulate in an intermediate focal plane in the second column and are not present either above or below the plane of appearance in the preceding column. Thus, the structures are indeed assembling, rather than moving into the focal plane. Bar, 2 μm.
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Related In: Results  -  Collection

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fig2: Visualization of the assembly of FM4-64 and Abp1p-GFP by 3D reconstruction combined with time-lapse imaging. Mid-log phase wild-type haploid cells expressing Abp1p-GFP from their chromosomal locus were incubated with FM4-64 as for Fig. 1. 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 incorporation of Abp1p-GFP (top) and FM4-64 (bottom) at different time-points during the assembly process. The cell shown is unbudded and has polarized toward the presumptive bud site in the top of the cell. The structure of interest is pseudocolored according to the strength of the fluorescent signal, where yellow corresponds to greatest intensity, orange corresponds to medium intensity, and red corresponds to low intensity. Abp1p and FM4-64 accumulate in an intermediate focal plane in the second column and are not present either above or below the plane of appearance in the preceding column. Thus, the structures are indeed assembling, rather than moving into the focal plane. Bar, 2 μm.
Mentions: 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.

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