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{"CAPTION FIG3.png": "'Figure 3: **3D morphology of CUCs.** [_A_]_ Conv1/- _M_EFs were incubated with CTxB-HRP for 20 min on ice, before internalization for 15 s. The DAB reaction was performed and calls were processed for electron tomography [see Materials and methods]. A single section of the original tomogram is shown [left]. Various rotations of a 3D convoluted electron density render were generated [middle]. Enlarged sections selected from the tomogram [right] show internal vesicles [arrows] and a complete connection around the circumference of the structure (arrowhead). (B) V/T MFFs grown on sapphire discs were incubated with CTxB-HRP for 15 s before DAB reaction and high-pressure freezing. Tubular extensions [large arrows] are seen emanating from vascular bubbles (arrowheads) in the characteristic ring-shaped CUC morphology. CCPs without CTxB label [double arrowheads] indicate that they are still surface connected. Bars, 200 nm.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: **Biochemical enrichment of CICs.** [A] 3T3-GPI calls were subjected to density fractionation as described in Materials and methods. Western blots of membrane markers are shown. Within the first gradient [left] anti-GFP-, Cov1-, and Gpp78-positive membranes are present in Fraction 1.2. highlighted by outline. CHC, GMI 30., and TR-positive membranes are below the detection limit within this fraction. Within the second gradient, anti-GFP-positive membranes concentrate within Fraction 2.8. highlighted by outline. This represents a yield of 9.6 \\\\(\\\\pm\\\\) 0.2% of anti-GFP-positive membranes. Cav1- concentrates in Fraction 2.6 and Gpp78 in Fraction 2.10. (B) Fraction 2.8 was fixed and visualized by EM. Structures within Fraction 2.8 share similar profiles to CUCs scan within intact. Bar, 200 nm. [C] High resolution electron micrographs of Fraction 2.8 structures, providing examples of vesiculation [arrows] and 3 spherical bulb connected to lobular extension [arrowheads]. Bar, 100 nm. [D] NH4T33 cells were incubated with CIC8-HRP fractionation and the DAB reaction was performed on Fraction 2.8. Bar, 200 nm. (E) NH4T33 cells transfected with CIC42-WT or CD-N were incubated with CTx6 buffer fractionation. Western blots were probed with anti-CTx6. 3T3-GPI cells were treated or not with mipCD before incubation with anti-GFP antibodies. Western blots of fractions were probed with anti-Rb+HRP. (F) Western blots of fractions from cells transfected with Hat1-HA, GRAF1-myc, or left untransfected. Fractions were probed with anti-HA, _myc_-, _dynamin_, _F_-_G_-_G_-_G_-_G_-_G_-_G_-_G_-_G_-_G_-_G_-_G_-_G_-_G_-_G_-_-_G_-_-_G_-\\n\\n'", "CAPTION FIG1.png": "'Figure 1: **CIA** **does not affect** **CUC** **adocytosis**. (**A**) **Coy1**\u2013/\u2013 **MEFs** were incubated with HRP in the presence or absence of CIA/R for 1.5 s. Cells were cooled and DAB reaction performed in the presence of ascorbic acid (AA) before fixation. Bars, 200 nm. (**B**) Quantification of the number of HRP-filled carriers per cell across 10\u201312 cells treated as in A. CILCs were defined by their characteristic ring-shaped morphology, CCVs were defined as coated vesicular carriers. Error bars show SEM. [**C**] **Coy1**\u2013/\u2013 **MEFs** were grown in normal media (plus energy), media containing 2-deoxyglucose (no energy), or 2-deoxyglucose (no energy), or 2-deoxyglucose (no energy) for 1 h followed by a 1-h washout in normal media (recovery) before CTaR-R/R internalization and DAB reaction. Labeled structures were counted across 10 cells. Error bars show SEM. (**D**) and [**E**] **Coy1**\u2013/\u2013 **MEFs** were incubated with CTaR-R/R for 5 min before the DAB reaction for either 5 min at 37degC or 10 min at 4degC, followed by fixation at 37degC. All CTaR-R/R-positive structures were counted in 10\u201312 cells across two independent experiments. Error bars show SEM. Bar, 200 nm. (**F**) NIH3T3s were treated for 20 min with vehicle (Influotated) or Dyngoda before internalization of CTaR-555 (left panels) and Tf647 (right panels) for 5 min. Cells were placed on ice and acid stripped to remove surface labeling. Cells were then bound with CTaR-488 (middle panels). Bar, 10 \u03bcm. (**S**) NIH3T3s cells were left untreated or were treated with Dyngoda before internalization of CTaR-HRP, followed by DAB reaction. Arrows show CTaR-HRP\u2013Loden CILCs, double arrowheads show internalized, labeled CCVs, arrowheads show surface connected unlabeled CCVs. Bars, 200 nm.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: **Verification of novel CUC cargo.** (A) NIH3T3 cells were incubated with either anti-Thy-1 or anti-CD44 antibodies and CTb-555 and Tb6.47 for 2 min. Myoferlin was detected after fixation, showing steady-state localization. Arrows indicate colocalization. Bar, 10 \u03bcm. (B) 3T3-GPI cells were left untreated or were treated with 100 nM wortmannin before internalization of anti-GFP for 10 min. Arrows indicate colocalization. Bar, 10 \u03bcm. (C) Quantification of \\\\(b\\\\) from 12\u201315 cells in three independent experiments. (D) Quantification of colocalization between internalization and CTb44 antibodies and Tb6.47, CTb-555, or anti-type antibodies for GFP-dyoferlin-myc-expressing cells after 2, 10, and 40 min. Error bars show SEM. (E) Anti-CD44.H8P was internalized into WT MEFs before DAB reaction. Arrows show anti-CD44-HRP-positive carriers with morphology of CUCs. Arrowheads show large, tubular ring-shaped anti-CD44-HRP-positive compartment. Bars, 200 nm. (F) Anti-CD44-HRP or THHR was pulsed into Cav1\u2212/- MEFs for 15 s, 1 min, or 2 min. Cells were fixed and processed for vertical sections. Arrows show HRP-labeled carriers. Bar, 200 nm. (G) Strategy measurements were captured across 20\u201325 cells in three independent areas as treated in F. Error bars show SEM.\\n\\n'", "CAPTION FIG6.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION FIG7.png": "'Figure 7. **Model of CUC endocytosis.** [_A_] [_1_] CUC cargo, such as Thy-1, CD44, and myofarlin are concentrated within Fluillin-1 and cholesterol-enriched microdomains. [_2_] Actin and G&AF-1 drive the inside formation of the carriers, within 15 s. [_3_] Recruitment of dynamin, Sab11, and Rab5/2 EEA-1 complexes within 2 min provides the ability for these carriers to facilitate bulk membrane flow to early endosomes (4a) and fast plasma membrane recycling [_4b_]. [_3_] After abrasion to the PM an influx of Ca2+ activates the fusogenic C2 domains of dyafarlin/myofarlin, resulting in free-praferential recycling of the CUC pathway.\\n\\n'", "CAPTION FIG6-1.png": "'Figure 6: **CICs become polarized during and are necessary for efficient cellular migration.** [A] Corbitant monolayers of WT MEFs or NH3T3s were scratched and cells were allowed to migrate for 8\u201312 h. CTxB-555 and Tf-647 were pulsed into migrating cells for 2 min in the presence of anti-CD44 (WT MEFI), anti-GFP (GFP-GPI expressing WT MEF), or anti-Thy-1 (NH3T3) antibodies or calls were labeled for Cox-1 [WT MEFI]. Dotted lines indicate leading edges. Arrows show colocalization between anti-CD44 or anti-Thy-1 and CTxB-555 but not Tf-647 at the leading edge. Bor, 20 \u03bcm. [B] Questions of average pixel intensity from the leading to trailing edges for CTxB-555, Cox-1, and Tf-647. Heart shows the concentration of tubular, Cox-1, and Tf-negative CTxB-labeled CULCs at the leading edge. A rectangular area, outlined, was used to calculate the average pixel intensity (along the y axis) across the leading to trailing edge [along the x axis] for each endocytic marker. Plot profiles identify a concentration of CTxB in the leading edge and Cox-1 in the trailing edge whereas Tf shows uniform intensity across the cell. Bar, 10 \u03bcm. [C] Electron micrographs of a migrating WT MEFI. Large arrow indicates direction of migration. Magnifications from the leading edge and the trailing edge show representative images of CTxB-HRP-labeled CULCs\\n\\n'", "CAPTION FIG6-2.png": "'[1, 2] and surface-connected caveolae [3, 4]. Bar, 500 nm. [D] Quantitation of the number of endocytic structures at the leading and trailing edge of cells treated as in C. Budded caveolae and CCVs are positive for CTxB-HRP label, whereas caveolae and CCPs are not. Error bars show SEM. [E] WT MEFs were incubated with or without CTxB+HRP for 2 min. The DAB reaction was performed on live cells for 5 min. Cells were washed and allowed to grow for a further 4 h. CLbS-555, anti-CD44 antibodies, and Tf-647 were added directly to cells for 2 min of uptake before acid stripping and fixation. Bar, 10 \u03bcm. [E] 12\u20131.5 cells treated as in E were quantitated for orange fluorescence intensity of CTxB-555, Tf46.47, or goat anti-mouse-488 for mouse-anti-CD44. [G] Confluent monolayers of WT MEFs were scratched and cells were allowed to migrate into the space for 1 h. Cells were incubated with serum alone or serum with 10 ug ml-1 CTxB+HRP for 2 min and the DAB reaction was performed as in E. After 4 h of migration, cells were fixed and distance migrated was determined by measuring the distance of the gap between both sides of the wound at time zero and after 4 h of incubation. Error bars show SEM from three independent experiments.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: **Quantification of CIIC endocytosis.** [A] Cav1\u2212/- or wildtype [WT] MEFs were left untreated or were treated with Dyngo4a. CTRh-HRP was internalized for 15 s, 1 min, or 2 min. Examples of CTxRh-labeled structures after 15 s of upticks are shown [inset, left panel]. Substratum indicated by large arrowhead, grid sizes are 2,000 or 200 nm, examples of intersections shown by arrows. [B] 20\u201325 cells treated as in A were used to calculate the volume fraction [V(g)]. Error bars show SEM. [C] NIH3T3 cells not treated with inhibitor were processed as in A and counted as in B. [V] was calculated for both tubular and vesicular structures. Error bars show SEM. [D] Cav1\u2212/- MEFs were incubated with HRP for 15 s, and in an ion and HRP-laden carriers counted as in B. Error bars show SEM. [E] CTRh was conjugated with NH3T5S-biotin and added to untreated Cav1\u2212/- MEFs or Cav1\u2212/- MEFs treated with Dyngo4a for 15 s or 2 min or was bound to untreated cells on ice for 10 min [Surface + MesNa]. Cells were placed on ice and residual surface biotin cleaved with MesNa. Western blots of cell lysates were probed with streptavidin-HRP. Chart represents the average intensity of streptavidin-HRP across three independent experiments. Residue luminescence in Surface + MesNa samples indicates level of uncarboxylic bisin. Error bars show SEM. [F] Absolute volume of CIICs was estimated from the volume fraction, [V(g)], multiplied by the average volume of a Cav1\u2212/\u2212 MEF, 2,3.47 \\\\(\\\\mu\\\\)m2. Surface density [S(g)] was calculated from high resolution images of labeled structures using a cycloid grid as described in Materials and methods and multiplied by the absolute volume to give absolute surface area. Volume of a single carrier was calculated as described in Materials and methods. Number of CIIC budding events per minute per cell was calculated based on the absolute volume internalized by all CIICs divided by the volume of a single carrier. Volume adjustments for overprojection effects are in brackets (see Materials and methods).\\n\\n'"} |
