{"CAPTION FIG4.png": "'Figure 4: **An intact microtubule network is required for the formation of tubular invaginations.** A and C) Microtubule disruption with high dose nocodazole prevents the formation of tubular invaginations in ATP-depleted cells (mean \u00b1 SD, \\\\(N\\\\) = 74 cells,) \\\\({}^{**}p<0.01\\\\), chi-squared test. B and D) Inhibition of microtubule dynamics with low dose nocodazole has no effect on the formation of tubular invaginations in ATP-depleted cells (mean \u00b1 SD, \\\\(N\\\\) = 135\\\\(-\\\\)169 cells), _n.s._\\\\(p>0.05\\\\), chi-squared test. E and F) Microtubule disruption prior to Dynasore treatment blocks the formation of branched tubules in cells labeled with either wild type CTxB (E) or monovalent CTx (F). Bars, 10 \u03bcm.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**Figure 3: Tubular invaginations align along microtubules and undergo complex motions including bidirectional motility and branching events.** A) Microtubules persist in RFP-\\\\(\\\\alpha\\\\)-tubulin expressing HeLa cells following ATP depletion. B) CTxB positive invaginations (green) align with taxol-stabilized microtubules (red) in stably expressing RFP-\\\\(\\\\alpha\\\\)-tubulin HeLa cells under ATP depletion. C) Percentage of CTxB-positive tubules that align with microtubules (black), partially align with microtubules (gray), or do not colocalize with microtubules (light gray) in ATP-depleted HeLa cells expressing RFP-\\\\(\\\\alpha\\\\)-tubulin. \\\\(N\\\\) = 84 cells. D\\\\(-\\\\)J) CTxB-enriched tubules exhibit complex motions in live cells as illustrated by representative frames from time series and corresponding kymographs. Time stamps are in minutes:seconds. D\\\\(-\\\\)F) CTxB-positive invaginations often grow in fluid directed motions. G\\\\(-\\\\)I) CTxB-positive invaginations also extend, retract and regrow along the same axis. _J_) Occasionally, the CTxB positive tubules undergo branching events. Bars, 10 \u03bcm.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: The ATPase activity of dynein and an intact dynactin complex are required for the formation of tubular invaginations. A and B) Inhibition of dynein ATPase activity with dilobrevin-A (Cilio-A) significantly reduces the percent of cells displaying tubular invaginations (mean +- SD from 95\\\\(-\\\\)122 cells). \\\\({}^{**}p<0.01\\\\), chi-squared test. C) Expression of GFP-p50 reduced the prevalence of cells with invaginations as compared to untransfected cells (-) or cells expressing EGFP. (mean +- SD from 42\\\\(-\\\\)101 cells). _n.s._, _p_\\\\(>0.05\\\\); \\\\({}^{**}p<0.01\\\\), chi-squared test. D and E) Expression of CCl-dsRed (red) significantly reduces the percent of cells with CTXb positive invaginations (green) as compared to untransfected cells (-) or control cells expressing mCherry (red). (mean +- SD from 40\\\\(-\\\\)64 cells). _n.s._, _p_\\\\(>0.05\\\\); \\\\({}^{**}p<0.01\\\\), chi-squared test. Bars, 10 \u03bcm.\\n\\n'", "CAPTION FIG7.png": "\"Figure 7: Bulk endocytosis of CTxB is largely unaffected by disruption of microtubules or the dynactin complex. A and B) Endocytosis of dextran, transferrin and CTxB in cells expressing GFP-p50 (gray bars) compared to cells expressing GFP (black bars). B shows mean +- SD for 37\\\\(-\\\\)278 cells. _n.s._, \\\\(p\\\\) > 0.05, Student's t-test. C and D) Effect of microtubule disruption with 5 mg/mL nocodazole (gray bars) on the uptake of dextran, transferrin and CTxB. Control cells were treated with DMSO (black bars). D shows mean +- SD for 78\\\\(-\\\\)358 cells. _n.s._, \\\\(p\\\\) > 0.05, *, \\\\(p\\\\) < 0.05, Student's t-test. Bars, 10 \u03bcm. E) Representative time course of toxin-induced chloride secretion in T84 cells in response to treatment with 20 nM wt CTx. Cells were either pretreated with NZ (closed symbols) on the DMSO (open symbols) prior to toxin addition to either the apical (blue) or basolateral (red) surface at \\\\(t\\\\) = 30 min as described in the _Materials and Methods_. Forskolin was added to control monolayers (green or gray diamonds) at 90 min in order to demonstrate equivalency of the secretory response and monolayer viability. The error bars indicate the variance calculated as the standard deviation (_n_ = 3). Data are representative of the results of two independent experiments.\\n\\n\"", "CAPTION FIG5.png": "'\\n\\n**Figure 5: Motor-based motions persist in ATP-depleted cells.** A\u2013C) A subset of lysosomes labeled with mCherry-LAMP1 display long range directed motions in ATP-depleted cells. B) Time lapse of zoomed in region of the cell in panel A. An example of a lysosome undergoing long-range directed motion is marked with the arrowhead. C) Tracings of lysosome movement in the cell shown in A. Each track is indicated by a different color. D and E) Frequent long-range directed motions are observed under control conditions. F and G) mCherry-LAMP1 positive lysosomes are immobile in PFA fixed cells. Elapsed time in F, H and J = 470 s. Bars, 10 \\\\(\\\\mu\\\\)m.\\n\\n'", "CAPTION FIG7-1.png": "'Figure 7: Learned on next page.\\n\\n'", "CAPTION FIG8.png": "'Figure 8: Working model microtubules, dynactin and cytoplasmic dynein facilitate plasma membrane tubulation. Dynein and dynactin provide attachment sites or generate tugging forces on the plasma membrane, leading to microtubule-dependent tubulation. Glycolipid-binding toxins may further sense, induce or stabilize membrane curvature, enabling their efficient sorting into tubular structures.\\n\\n'", "CAPTION FIG1-2.png": "'Figure 1: **No more than one functional GM1 binding site is required to target cholera toxin to plasma membrane invaginations.** A\\\\(-\\\\)E) CTxB accumulates in either linear extended tubules (A\\\\(-\\\\)D) or branched tubules (E) under conditions that block scission. Bar, 10 \\\\(\\\\mu\\\\)m. (F\\\\(-\\\\)M) Cholera toxin binding mutants accumulate in tubular invaginations. F) Cy3-CTx chimera labels tubules in ATP depleted COS-7 cells. G\\\\(-\\\\)I) Quantification of invaginations in ATP-depleted cells labeled with Cy3-CTx chimera or Alexa555-CTxB. G) Percentage of cells displaying invaginations (mean \\\\(\\\\pm\\\\) SD from 117\\\\(-\\\\)119 cells). *, \\\\(p\\\\) < 0.05, chi-squared test. H) Average number of invaginations per cell (mean \\\\(\\\\pm\\\\) SD of 42\\\\(-\\\\)46 cells). _n.s._, \\\\(p\\\\) > 0.05; Student _t_-test. I) Length of invaginations (mean \\\\(\\\\pm\\\\) SD for 219\\\\(-\\\\)332 invaginations). _n.s._, \\\\(p\\\\) > 0.05; Student _t_-test. J\\\\(-\\\\)M) Both wild type CTxB and monovalent CTx accumulate in tubular invaginations in cells subjected to lasplakinolide pretreatment prior to ATP depletion. L) Percentage of cells displaying invaginations. (mean \\\\(\\\\pm\\\\) SD of 59\\\\(-\\\\)63 cells). _n.s._, \\\\(p\\\\) > 0.05; chi-squared test. M) Average number of invaginations per cell. (mean \\\\(\\\\pm\\\\) SD of 59\\\\(-\\\\)63 cells). _n.s._, \\\\(p\\\\) > 0.05; Student _t_-test. N and O) Similar to wild type CTxB, monovalent CTx accumulates in branched tubules in Dynasore-treated cells. Bars, 10 \\\\(\\\\mu\\\\)m.\\n\\n'", "CAPTION FIG1.png": "'Figure 1: Legend on next page. Figure 1: **No more than one functional GM, binding site is required to target cholera toxin to plasma membrane invaginations.** A\u2013E) CTB accumulates in either linear extended tubules (A\u2013D) or branched tubules (E) under conditions that block incision. Bar, 10 \u03bcm. (F\u2013M) Cholera toxin binding mutants accumulate in tubular invaginations. F) Cy3-CTx chimera labels tubules in ATP depleted C05-7 cells. G\u2013D Quantitation of invaginations in AH-depleted cells labeled with Cy3-CTx chimera or Alexa55-CTB. G) Percentage of cells displaying fragments (mean \u00b150 from 117\u2013119 cells). \\\\(y\\\\) = 0.05, cis-squared test. H) Average number of invaginations per cell (mean \u00b150 of 42\u201346 cells). _n.s._, \\\\(p\\\\) > 0.05; Student-t-test. [1] length of invaginations (mean \u00b150 for 219\u2013332 imagination). _n.s._, \\\\(p\\\\) > 0.05; Student-t-test. [1\u2013M] Both wild type CTB and monovalent CTx accumulate in tubular invaginations in cells subjected to laqxihizide pretreatment prior to ATP depletion. L) Percentage of cells displaying invaginations. (mean \u00b150 of 59\u201363 cells). _n.s._, \\\\(p\\\\) > 0.05; chi-squared test. H) Average number of invaginations per cell. (mean \u00b150 of 59\u201363 cells). _n.s._, \\\\(p\\\\) > 0.05; Student-t-test. H) Average number of invaginations per cell. (mean \u00b150 of 49\u201363 cells). _n.s._, \\\\(p\\\\) > 0.05; Student-t-test. [1\u2013M] Both wild type CTB and monovalent CTx accumulate in branched tubules in DNase-treated cells. Bars, 10 \u03bcm.\\n\\n'", "CAPTION FIG1-1.png": "'Figure 1: Lateral on next page.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: **Toxin binding is not necessary for tubular invaginations to form.** A,B) EGFP-HRas (green) is found in plasma membrane invaginations in ATP-depleted cells in both the presence (A) and absence (B) of Alexa555-CTxB (red). C\u2013F) Similar results were obtained for GFP-HRas in cells subjected to actin disruption (C and D) or actin stabilization (E and F). G and H) A construct containing only the C-terminal 10 amino acids of HRas, EGFP-HRas-tail (green), also localized to tubules in both the presence and absence of CTxB. Bars, 10 \\\\(\\\\mu\\\\)m.\\n\\n'"}