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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.

Show MeSH
An actin patch undergoing retrograde movement remains associated with an elongating actin cable at a fixed point. Mid-log phase yeast expressing Abp1p-HcRed and Abp140p-GFP were studied using simultaneous two-color imaging as for Fig. 4. 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. Arrowheads in the merged images show the change in position of an actin patch that is associated with the tip of an elongating actin cable as both of these structures undergo linear, retrograde movement. Arrows mark the position of the tip of the actin cable at t = 0 (top row), and the position of the actin patch at t = 0 (middle row). Bar, 2 μm.
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fig10: An actin patch undergoing retrograde movement remains associated with an elongating actin cable at a fixed point. Mid-log phase yeast expressing Abp1p-HcRed and Abp140p-GFP were studied using simultaneous two-color imaging as for Fig. 4. 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. Arrowheads in the merged images show the change in position of an actin patch that is associated with the tip of an elongating actin cable as both of these structures undergo linear, retrograde movement. Arrows mark the position of the tip of the actin cable at t = 0 (top row), and the position of the actin patch at t = 0 (middle row). Bar, 2 μm.

Mentions: The finding that the Arp2/3 complex and other force generators are not required for retrograde actin patch movement supports the model that retrograde actin cable flow drives this actin patch movement. In support of this, we found that actin patches make no net movement along the length of a motile actin cable. These studies were conducted using bright spots of Abp140p-GFP on actin cables or the tip of an elongating actin cable as fiduciary marks to study actin cable dynamics and Abp1p-HcRed to monitor actin patch dynamics. In the example shown, an actin patch remained associated with the tip of an elongating actin cable (Fig. 10)Figure 10.


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)

An actin patch undergoing retrograde movement remains associated with an elongating actin cable at a fixed point. Mid-log phase yeast expressing Abp1p-HcRed and Abp140p-GFP were studied using simultaneous two-color imaging as for Fig. 4. 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. Arrowheads in the merged images show the change in position of an actin patch that is associated with the tip of an elongating actin cable as both of these structures undergo linear, retrograde movement. Arrows mark the position of the tip of the actin cable at t = 0 (top row), and the position of the actin patch at t = 0 (middle row). Bar, 2 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172478&req=5

fig10: An actin patch undergoing retrograde movement remains associated with an elongating actin cable at a fixed point. Mid-log phase yeast expressing Abp1p-HcRed and Abp140p-GFP were studied using simultaneous two-color imaging as for Fig. 4. 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. Arrowheads in the merged images show the change in position of an actin patch that is associated with the tip of an elongating actin cable as both of these structures undergo linear, retrograde movement. Arrows mark the position of the tip of the actin cable at t = 0 (top row), and the position of the actin patch at t = 0 (middle row). Bar, 2 μm.
Mentions: The finding that the Arp2/3 complex and other force generators are not required for retrograde actin patch movement supports the model that retrograde actin cable flow drives this actin patch movement. In support of this, we found that actin patches make no net movement along the length of a motile actin cable. These studies were conducted using bright spots of Abp140p-GFP on actin cables or the tip of an elongating actin cable as fiduciary marks to study actin cable dynamics and Abp1p-HcRed to monitor actin patch dynamics. In the example shown, an actin patch remained associated with the tip of an elongating actin cable (Fig. 10)Figure 10.

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