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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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Retrograde movement of actin patches occurs with the same velocity as retrograde actin cable movement and requires actin cables. (A) The velocity of actin cable and patch movement. Wild-type cells expressing either Abp140p-GFP or Abp1p-GFP from the chromosomal loci were grown to mid-log phase in lactate medium. Cells were imaged by time-lapse fluorescence imaging and the velocities of linear, retrograde movements of actin cables (n = 41) and actin patches (n = 42) was determined as described in Materials and methods. (B) Destabilization of actin cables results in loss of linear, retrograde actin patch movement. Abp1p was tagged at its chromosomal locus with HcRed in wild-type cells and yeast bearing a deletion of the BNR1 gene and a temperature-sensitive mutation in the BNI1 gene (bni1-11 bnr1Δ). Cells were grown to mid-log phase in lactate medium. At t = 0, aliquots of the liquid culture were removed and either maintained at permissive temperature (RT) or incubated at restrictive temperature (35°C) for 2 min. Time-lapse imaging of Abp1p-HcRed–labeled actin patches was performed at 23 and 35°C. The frequency of linear retrograde actin patch movement was defined by the number of linear retrograde actin patch movements per mother cell in the 20-s imaging period (n = 67–108 cells). Linear retrograde movement was defined as a movement away from the bud neck in the mother cell over three consecutive time-points.
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fig8: Retrograde movement of actin patches occurs with the same velocity as retrograde actin cable movement and requires actin cables. (A) The velocity of actin cable and patch movement. Wild-type cells expressing either Abp140p-GFP or Abp1p-GFP from the chromosomal loci were grown to mid-log phase in lactate medium. Cells were imaged by time-lapse fluorescence imaging and the velocities of linear, retrograde movements of actin cables (n = 41) and actin patches (n = 42) was determined as described in Materials and methods. (B) Destabilization of actin cables results in loss of linear, retrograde actin patch movement. Abp1p was tagged at its chromosomal locus with HcRed in wild-type cells and yeast bearing a deletion of the BNR1 gene and a temperature-sensitive mutation in the BNI1 gene (bni1-11 bnr1Δ). Cells were grown to mid-log phase in lactate medium. At t = 0, aliquots of the liquid culture were removed and either maintained at permissive temperature (RT) or incubated at restrictive temperature (35°C) for 2 min. Time-lapse imaging of Abp1p-HcRed–labeled actin patches was performed at 23 and 35°C. The frequency of linear retrograde actin patch movement was defined by the number of linear retrograde actin patch movements per mother cell in the 20-s imaging period (n = 67–108 cells). Linear retrograde movement was defined as a movement away from the bud neck in the mother cell over three consecutive time-points.

Mentions: Previously, we showed that Abp140p-GFP, a GFP fusion protein containing the resident actin cable protein Abp140p, labels actin cables but has no obvious effect on cell growth, actin organization, or actin function. Moreover, we showed that the intensity of fluorescence from Abp140p-GFP was not uniform along actin cables, and that the amount of Abp140p-GFP in actin cables was proportional to the amount of F-actin in actin cables. Finally, we demonstrated that bright spots of Abp140p-GFP could be used as fiduciary marks to analyze actin cable dynamics in living yeast cells (Yang and Pon, 2002). Here, we monitored movement of Abp140p-GFP fiduciary marks on actin cables and Abp1p-HcRed–labeled actin patches to determine the velocity of actin cable and patch movement. First, we found that all detectable actin cables exhibited retrograde flow (n = 100). Second, the velocity of retrograde actin cable flow was similar to the velocity of retrograde actin patch movement (Fig. 8Figure 8.


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)

Retrograde movement of actin patches occurs with the same velocity as retrograde actin cable movement and requires actin cables. (A) The velocity of actin cable and patch movement. Wild-type cells expressing either Abp140p-GFP or Abp1p-GFP from the chromosomal loci were grown to mid-log phase in lactate medium. Cells were imaged by time-lapse fluorescence imaging and the velocities of linear, retrograde movements of actin cables (n = 41) and actin patches (n = 42) was determined as described in Materials and methods. (B) Destabilization of actin cables results in loss of linear, retrograde actin patch movement. Abp1p was tagged at its chromosomal locus with HcRed in wild-type cells and yeast bearing a deletion of the BNR1 gene and a temperature-sensitive mutation in the BNI1 gene (bni1-11 bnr1Δ). Cells were grown to mid-log phase in lactate medium. At t = 0, aliquots of the liquid culture were removed and either maintained at permissive temperature (RT) or incubated at restrictive temperature (35°C) for 2 min. Time-lapse imaging of Abp1p-HcRed–labeled actin patches was performed at 23 and 35°C. The frequency of linear retrograde actin patch movement was defined by the number of linear retrograde actin patch movements per mother cell in the 20-s imaging period (n = 67–108 cells). Linear retrograde movement was defined as a movement away from the bud neck in the mother cell over three consecutive time-points.
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Related In: Results  -  Collection

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fig8: Retrograde movement of actin patches occurs with the same velocity as retrograde actin cable movement and requires actin cables. (A) The velocity of actin cable and patch movement. Wild-type cells expressing either Abp140p-GFP or Abp1p-GFP from the chromosomal loci were grown to mid-log phase in lactate medium. Cells were imaged by time-lapse fluorescence imaging and the velocities of linear, retrograde movements of actin cables (n = 41) and actin patches (n = 42) was determined as described in Materials and methods. (B) Destabilization of actin cables results in loss of linear, retrograde actin patch movement. Abp1p was tagged at its chromosomal locus with HcRed in wild-type cells and yeast bearing a deletion of the BNR1 gene and a temperature-sensitive mutation in the BNI1 gene (bni1-11 bnr1Δ). Cells were grown to mid-log phase in lactate medium. At t = 0, aliquots of the liquid culture were removed and either maintained at permissive temperature (RT) or incubated at restrictive temperature (35°C) for 2 min. Time-lapse imaging of Abp1p-HcRed–labeled actin patches was performed at 23 and 35°C. The frequency of linear retrograde actin patch movement was defined by the number of linear retrograde actin patch movements per mother cell in the 20-s imaging period (n = 67–108 cells). Linear retrograde movement was defined as a movement away from the bud neck in the mother cell over three consecutive time-points.
Mentions: Previously, we showed that Abp140p-GFP, a GFP fusion protein containing the resident actin cable protein Abp140p, labels actin cables but has no obvious effect on cell growth, actin organization, or actin function. Moreover, we showed that the intensity of fluorescence from Abp140p-GFP was not uniform along actin cables, and that the amount of Abp140p-GFP in actin cables was proportional to the amount of F-actin in actin cables. Finally, we demonstrated that bright spots of Abp140p-GFP could be used as fiduciary marks to analyze actin cable dynamics in living yeast cells (Yang and Pon, 2002). Here, we monitored movement of Abp140p-GFP fiduciary marks on actin cables and Abp1p-HcRed–labeled actin patches to determine the velocity of actin cable and patch movement. First, we found that all detectable actin cables exhibited retrograde flow (n = 100). Second, the velocity of retrograde actin cable flow was similar to the velocity of retrograde actin patch movement (Fig. 8Figure 8.

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