-
Hoeller O, Toettcher J, Cai H, Sun Y, Huang C, Freyre M, Zhao M, Devreotes P, Weiner O
Wikimedia Commons
Onset of oscillations after rapamycin-induced Gβ sequestration. Most behaviors described in this paper are apparent in this movie of the Gβ sequestration strain expressing the F-actin binding probe LimE-GFP. The movie is split (LimE-GFP on the left; FRB-RFP-Gβ on the right). After frame 300, addition of 1 μM rapamycin (labeled) induces sequestration of Gβ. This starts to show as visible clustering of Gβ about halfway through the movie. Within 40 s, LimE-GFP begins rapid whole-field oscillations that alternate with polarized accumulation of F-actin. Toward the end of the movie, circular rotations of LimE-GFP can be observed. The movie was recorded at 1 frame/second and is played back at 30 frames/second. Portions of this movie correspond to S9 Fig.
-
Delivery of the V-ATPase to a new phagosome. The cell is expressing VatM-GFP and has been incubated with TRITC-dextran to label endosomes. The uptake of a heat-killed yeast is shown.
-
Labeling of a macropinosome with GFP-2FYVE, a biosensor for PI(3)P. The cell is expressing GFP-2FYVE and mRFP-LimEΔ. The nascent macropinosome is initially labeled with the actin marker. Actin disappears and 2FYVE-GFP binding begins after about one minute. It persists for another 2 or 3 minutes as the macropinosome elongates and fragments, behavior consistent with the early or sorting endosome stage.
-
Hoeller O, Toettcher J, Cai H, Sun Y, Huang C, Freyre M, Zhao M, Devreotes P, Weiner O
Wikimedia Commons
Kymograph (t-stack) of actin accumulation in a Gβ-sequestered cell (+ rapamycin). 360-degree rotation of a t-stack showing F actin accumulation at the cortex (visualized with LimE-GFP) during polar and apolar phases. Movie corresponds to Fig 7B and is played at 72 frames/second. Vertical axis represents time (first time point on top, last time point on bottom).
-
Delivery of the V-ATPase to a new phagosome. The cell is expressing VatM-GFP and mRFP-LimEΔ. It has just phagocytosed a living yeast cell.
-
Acquisition of GFP-2FYVE label by a vacuole that has separated from a phagosome just prior to premature exocytosis. The cell is expressing GFP-2FYVE and mRFP-LimEΔ; the phagosome contains a FITC-yeast.
-
Hoeller O, Toettcher J, Cai H, Sun Y, Huang C, Freyre M, Zhao M, Devreotes P, Weiner O
Wikimedia Commons
Actin oscillators during polarization. Movie corresponds to the cell in S23 Fig, imaged in the coverslip plane during a cell polarization event. LimE-GFP reports oscillators and filamentous actin accumulation.
-
Increase in phagosome volume and dilution of fluid phase marker prior to premature exocytosis. The cell is expressing VatM-GFP and was incubated for 3 hours with TRITC-dextran, then shifted to unlabeled buffer and observed.
-
Removal of the V-ATPase from the membrane of a yeast-containing phagosome, followed by actin assembly and exocytosis. The cell is expressing VatM-GFP and mRFP-LimEΔ.
-
Premature exocytosis of a yeast-containing phagosome and initial stage of the retrieval of VatM-GFP from the plasma membrane. The cell is expressing VatM-GFP and mRFP-α-tubulin.
-
Premature exocytosis of a phagosome containing a FITC-yeast and dynamics of the vacuole that separates prior to exocytosis. The cell is expressing VatM-GFP and DdmCherry-LimEΔ.
-
Removal of the V-ATPase from the membrane of a yeast-containing phagosome, followed by actin assembly and exocytosis. Over the same interval, the cell takes up a new yeast. The cell is expressing VatM-GFP and mRFP-LimEΔ.
-
Hoeller O, Toettcher J, Cai H, Sun Y, Huang C, Freyre M, Zhao M, Devreotes P, Weiner O
Wikimedia Commons
Rapamycin-induced Gβ sequestration. The Gβ sequestration strain (Gβ-: FRB-RFP-Gβ, calnexinA-YFP-FKBP) is challenged with 1 μM rapamycin at the start of the movie. Sequestration of Gβ can be seen by the increased colocalization of RFP and YFP. Recorded at 1 frame/min; played at 10 frames/second.
-
Labeling by GFP-2FYVE of phagosomes containing bacteria. The cell is expressing GFP-2FYVE and mRFP-LimE and is eating bacteria. New phagosomes are labeled first by the actin marker and then by GFP-2FYVE starting about one minute later and persisting for several minutes. Fusion and fission events characteristic of early phagosomes can be seen during the period of 2FYVE-GFP labeling.
-
Acidic nature of a prematurely exocytosed phagosome, also showing actin-powered vacuole movement. The cell is expressing VatM-GFP and DdmCherry-LimEΔ. It has eaten a FITC-labeled yeast that is barely visible in the phagosome but brightens upon contact with the higher pH extracellular medium, indicating that the phagosome lumen was acidic.
-
Hoeller O, Toettcher J, Cai H, Sun Y, Huang C, Freyre M, Zhao M, Devreotes P, Weiner O
Wikimedia Commons
Electrotaxis of Gβ-sequestered cells. Movie corresponds to still images in Fig 2B. Cells were recorded at 2 frames/min in the presence of an electric field and rapamycin. Red label: “wt” (Gβ+/anchor-); green label: “Gβ-null”(Gβ-/anchor-); yellow label: “Gβ-sequestered” (Gβ+/anchor+).
-
Separation of a large V-ATPase-rich vacuole from a yeast-containing phagosome prior to premature exocytosis. The cell is expressing VatM-GFP and mRFP-α-tubulin.
-
Hoeller O, Toettcher J, Cai H, Sun Y, Huang C, Freyre M, Zhao M, Devreotes P, Weiner O
Wikimedia Commons
Kymograph (t-stack) of actin accumulation in a Gβ unsequestered cell (- rapamycin). 360-degree rotation of a t-stack showing F-actin accumulation at the cortex (visualized with LimE-GFP) during polar and apolar phases. Movie corresponds to Fig 7A and is played at 72 frames/second. Vertical axis represents time (first time point on top, last time point on bottom).
-
Exocytosis of a FITC-yeast from a phagosome whose membrane has been depleted of VatM-GFP prior to exocytosis. The cell is expressing VatM-GFP and DdmCherry-LimEΔ. The yeast does not grow brighter upon contact with the extracellular medium.
-
Schaloske R, Lusche D, Bezares-Roder K, Happle K, Malchow D, Schlatterer C
Wikimedia Commons
Additional File 3 Cell motility of wild type and mutant cells after preincubation with 10 mM CaCl2 for 80 min (t4–t5.3) is shown. Images of cells at t5.3 were captured every 15 sec for 20 min in the continued presence of CaCl2. The behaviour of treated cells was not different from control amoebae.
-
Schaloske R, Lusche D, Bezares-Roder K, Happle K, Malchow D, Schlatterer C
Wikimedia Commons
Additional File 1 Spontaneous cell motility of wild type and iplA- cells in H5-buffer. Images of cells at t5 were captured every 15 sec for 20 min.
-
Schaloske R, Lusche D, Bezares-Roder K, Happle K, Malchow D, Schlatterer C
Wikimedia Commons
Additional File 2 Basal cell motility after preincubation of wild type and mutant amoebae with 10 mM EGTA for 60 min (t4–t5). Images of cells at t5 were captured every 15 sec for 20 min in the continued presence of EGTA. Cells were rounded and extended smaller pseudopods than under control conditions.