{"CAPTION FIG3.png": "'Figure 3: Localization of actin filaments in two zones in endoscopic actin patches in wild-type S, ensemble cells. A (B) Time series of fluorescence micrographs at 14 intervals of representative actin patches in cells expressing proteins that had actin filaments. The images are regions of interest of 500 \\\\(\\\\times\\\\) 500 \\\\(\\\\times\\\\) ground one actin patch for each protein. Scale bars are 250 min. (Arg CPG CMD and \\\\(\\\\sigma\\\\)) for (mGS2.32 and (B);m-PEG2 and (B);m-PEG2 and (B);m-PEG2-PEG2. Top panels, maximum intensity projections of the confocal _z_-locus in the X-plane of individual actin patches from cells expressing GFP-CPG and _m_-PEG2.666 map, _m_-PEG2.\\n\\n'", "CAPTION FIG2.png": "'FIGURE 2.: Localization of resolution promoting feature and \\\\(\\\\mu\\\\)-pixel samples in actin patches by PHARM in wild type cells. (30 time series of PHARM images could be the density of the descriptors at the location; 30, triangles drip-ring, middle '", "CAPTION FIG1.png": "'Figure 1: FPALM improves the spatial resolution of actin patches over conventional fluorescence microscope. (A) Two-zone model based on quantitative confocal fluorescence microscopy. The cartoon represents an actin patch at -2 s when the membrane tubule elongates and at 0 s when the vesicle patches away from the plasma membrane. The plasma membrane is a black line, the clathrin coat is gray, nucleation-promoting factors Wsp1p and Myo1p are yellow, and zones with actin filaments are blue. Growth of the branched filaments from two distinct zones of NPFs is postulated to push the tip of the invaginating tubule away from the cell surface contributing to the elongation of the tubule and scission of the coated vesicle. (B, C) Fluorescence micrographs of an S. pombe cell expressing capping protein Acp2p-mECS3.2 with focusing in the middle plane of the cell. We used continuous epifluorescence illumination to photocomwart mECS3.2 with 40S- and 564-rm lasers to excite the photocomwart mECS3.2 through the entire cell. Top panels, wide-field epifluorescence images reconstructed from the total fluorescence emission. Middle panels, raw FPALM images constructed from the localizatores of single molecules. Bottom panels, each localized emitter in the raw data set was convolved with a Gaussian kernel (\\\\(\\\\alpha=1.5\\\\) pixel) and color coded for density in a heat map. (B) Whole cell during a 1-s interval. Scale bar is 1 \\\\(\\\\mu\\\\)m. (C) Time series of images of one actin patch at 1-s intervals each reconstructed from 200 sequential frames. Top panel, inverted contrast wide-field epifluorescence images. Scale bar is 250 nm.\\n\\n'", "CAPTION FIG3-1.png": "'Figure 3: Localization of actin filaments in two zones in endocytic actin patches in wild-type \\\\(S\\\\). pombe calls. (A, B) Time series of fluorescence micrographs at 1-s intervals of representative actin patches in cells expressing proteins that bind actin filaments. The images are regions of interest of -S00 \\\\(\\\\times\\\\) 500 nm around one actin patch for each protein. Scale bars are 2S0 nm. (A) GFP-CHD and mECS3.2-CHD and (B) Fin1p-GFP and Frm1p-mECS3.2. Top panels, maximum intensity projections of five confocal \\\\(z\\\\)-slices in the \\\\(XY\\\\)-plane of individual actin patches from cells expressing GFP-CHD or Frm1p-GFP. Bottom panels, FPLAM images of cells expressing mECS3.2-CHD or Frm1p-mECS3.2. Each localized emitter was convolved with a Gaussian kernel (\\\\(\\\\sigma=1.5\\\\) pixel) and color coded for density in a heat map. Yellow dashed lines show the location of the\\n\\n'", "CAPTION FIG3-2.png": "'gradients membrane [PM]. (C-F) Spatial distributions of single molecule localization of mEOS3.2-CHD and Fim1p-mEOS3.2 in FPALM images of actin patches. FWMM is full-width of the distributions at half-maximums. (C) Width distribution and (E) length distribution of mEOS3.2-CHD in 20 actin patches. (D) Width distribution and (F) length distribution of Fim1p-mEOS3.2 in 14 actin patches prepared as in Figure 2. (G, H) Composite images with temporal color coding of single molecule localizations over the lifetimes of single actin patches. The micrographs are aligned to time 0 s when the vesicle pinches off from the plasma membrane. Scale bars are 200 nm. The blue and red lines indicate the membrane-proximal and membrane-distal regions. (G) Twenty-area seconds from a cell expressing mEoS3.2-CHD. (H) Seventeen seconds from a cell expressing Fim1p-mEoS3.2. (H, J) Time courses of the average number of localizations of (H) CHD-mEOS3.2 and (J) Fim1p-mEOS.2 detected in (blue line) the membrane proximal zone [200 x 250-nm region next to the call edge] and (red line) the membrane distal zone (350 x 250-nm region 200 nm away from the cell edge). Raw time courses of the number of emitters localized in 15 actin patches were aligned to a reference patch and averaged to produce time courses for the average number of localizations. Time zero seconds indicates vesicle sosion. Mean total localization were used to align the localizations in the membrane proximal and distal zone on the same time scale as the numbers of GFP molecules assembled in actin patches. Error bars are SDs. Every third error bar is shown.\\n\\n'", "CAPTION FIG2-1.png": "'Figure 2: Localization of nucleation promoting factors and Arp2/3 complex in actin patches by FPALM in wild-type cells. (A) Time series of FPALM images color coded for the density of localizations: top, mMaple3-Myo1p; middle, mMaple3-Wsp1p; and bottom, Arc5p-mMaple3 a subunit of Arp2/3 complex. The micrographs were aligned to time 0 s when the vesicle pirches away from the plasma membrane. For mMaple3-Myo1p the peak localizations were aligned to the peak localizations of mMaple3-Wsp1p. Yellow dashed lines show the position of the plasma membrane. Scale bar is 100 nm. (B) Composite images with temporal color coding of single molecule localizations over the lifetimes of single actin patches: left, 18 s from a cell expressing mMaple3-Myo1p; center, 16 s from a cell expressing mMaple3-Wsp1p; and right,\\n\\n'", "CAPTION FIG2-2.png": "'21 \\\\(\\\\leq\\\\) from a cell expressing ArcSp-mMaple3 a subunit of Arp2/3 complex. The blue and red lines indicate the membrane-proximal and membrane-distal regions. Scale bar is 200 nm. (C, D) Spatial distributions of single molecule localizations in FPALM images of actin patches. FW-HM is full-width of the distributions at half-maximums. (C) Width distributions and (D) length distributions of localizations of left, mMaple3-Myo1p in 20 actin patches; center, mMaple3-Wsp1p in 12 actin patches; and right, ArcSp-mMaple3 in 18 actin patches. The data from separate patches were aligned to their peaks and averaged. Full-widths at half-maximum were calculated from the distributions of the localizations along the \\\\(x\\\\)-axis. The lengths of the patches are given as the distances that include 90% of the localizations from the cell edge. The gray area in the graphs represents the localizations detected outside the cell. (E) Time courses of the average number of localizations detected in (blue line) the membrane proximal zone (200 \\\\(\\\\times\\\\) 250-nm region next to the cell edge) and (red line) the membrane distal zone (350 \\\\(\\\\times\\\\) 250-nm region 200 nm away from the cell edge): left, mMaple3-Myo1p; middle, mMaple3-Wsp1p; and right, ArcSp-mMaple3. Raw time courses of the number of emitters localized in 15 actin patches were aligned to a reference patch and averaged to produce time courses for the average number of localizations. Mean total localizations were used to align the localizations in the membrane proximal and distal zone on the same time scale as the numbers of GFP molecules assembled in actin patches. Time 0 s indicates vesicle scission. Error bars are SDs. Every third arrow bar is shown.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Actin patch assembly in cells lacking nucleation promoting factors Myo1p or Wsp1p. {A, B} Time series of FPALM images color coded for localization density showing the (A) ArcSp-mMaple3 a subunit of Arp2/3 complex or {B} mEOS3.2-CHD to visualize actin filaments. Top panels, wild-type cells; middle panels, _Amyo1_ cells; and bottom panels, _Awsp1_ cells. Yellow dotted lines indicate the cell membrane. The micrographs are aligned to time 0 s when the vesicle pinches off from the plasma membrane. Scale bars are 250 nm. (C, D) Composite images with temporal color coding of single molecule localizations over the lifetimes of single actin patches. The blue and red lines indicate the membrane-proximal and membrane distal regions. (C) Cells expressing ArcSp-mMaple3: left, 16 s of an actin patch in a wild-type cell; center, 18 s of an actin patch in a _Amyo1_ cell; and right, 18 s of an actin patch in a _Am_wsp1_ cell. {D} Cells expressing mEOS3.2-CHD: left, 15 s of an actin patch in a wild-type cell; center, 27 s of an actin patch in a _Am_yo1_ cell; and right, 28 s of an actin patch in a _Am_wsp1_ cells. {E, F} Time courses of the average number of localizations of (E) ArcSp-mMaple3 and {F} mEOS.2-CHD detected in actin patches in (blue line) the membrane proximal zone (200 \u00d7 250-nm region next to the cell edge) and (red line) the membrane distal zone (350 \u00d7 250-nm region 200 nm away from the cell edge): left, wild-type cells; middle, _Am_y_o1 cells; and right, _Awsp1_ cells. Raw time courses of the number of emitters localized in 15 actin patches were aligned to a reference patch and averaged to produce time courses for the average number of localizations. Mean total localizations were used to align the localizations in the membrane proximal and distal zone on the same time scale as the numbers of GFP molecules assembled in actin patches. Time 0 s indicates the vesicle scission. Error bars are SDs. Every third error bar is shown.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: Hypothesis for the contributions of the nucleation promoting factors Myo1p and Wsp1p to endocytosis. Different stages of endocytosis with time zero defined as vesicle scission from the plasma membrane. The plasma membrane is a black line, clathrin is gray, nucleation promoting factors Wsp1p and Myo1p are blue, and actin filaments are red, green, and yellow. Clathrin is recruited 2 min prior to invagination. Nucleation promoting factors are recruited beginning at -6 s and peak prior to patch movement at -3 s. Myo1p and Wsp1p both stimulate actin polymerization at the base of the invagination near the cell surface. As the actin zones around Myo1p and Wsp1p grow, they repel each other, driving membrane invagination and redistribution of Wsp1p away from the base along the membrane invagination creating new sites of actin polymerization at the invagination tip. Myo1p remains associated with the plasma membrane at the base of the invagination. The expansion of the two zones triggers vesicle scission between the two zones of actin at time 0 s.\\n\\n'"}