Role of lipids and actin in the formation of clathrin-coated pits

Abstract

Assembly of clathrin-coated pits and their maturation into coated vesicles requires coordinated interactions between specific lipids and several structural and regulatory proteins. In the presence of primary alcohols, phospholipase D generates phosphatidylalcohols instead of PA, reducing stimulation of phosphatidyl inositol 5-kinase (PI5K) and hence decreasing formation of phosphoinositide-4,5-biphosphate (PIP(2)). Using live-cell imaging, we have shown that acute treatment of cells with 1-butanol or other small primary alcohols induces rapid disassembly of coated pits at the plasma membrane and blocks appearance of new ones. Addition of exogenous PIP(2) reverses this effect. Coated pits and vesicles reappear synchronously upon removal of 1-butanol; we have used this synchrony to assess the role of actin in coated vesicle assembly. Prolonged inhibition of actin polymerization by latrunculin A or cytochalasin D reduced by approximately 50% the frequency of coated pit formation without affecting maturation into coated vesicles. As in control cells, removal of 1-butanol in the continued presence of an actin depolymerizer led to synchronous appearance of new pits, which matured normally. Thus, remodeling of the actin cytoskeleton is not essential for clathrin-coated vesicle assembly but may indirectly affect the nucleation of clathrin-coated pits.

Fig. 1. 1-butanol blocks Tf uptake

(A–F) Hela cells were pre incubated for 5 min at 37° C with PBS/Glucose/BSA (PBS-GB) (A,B), PBS-GB with 1,5% v/v 1-butanol (C), PBS-GB with 2% v/v 1-butanol (D), PBS-GB with 1,5% v/v t-butanol (E), and PBS-GB with 2% v/v t-butanol (F); followed by incubation with 10 μg/ml Alexa594-Tf for 2 min at RT (B–F) and 5 min at 37°C in the respective media (A–F); cells were subsequently transferred to 4°C and acid washed with (150mM NaCl, 1mM MgCl₂, 0.125mM CaCl₂, 0.1M Glycine pH 2.5) (G–H) Hela cells were pre-incubated with media alone (G) or with 2 % v/v 1-butanol (H) for 5 min at 37°C, followed by an additional 5 min in media without 1-butanol and Tf uptake and acid wash were carried out similarly as in A–F. Bar, 20 μm.

Fig. 2. 1-butanol and other primary alcohols disassemble clathrin coated pits

(A) Confocal section of BSC-1 cells stably expressing EGFP-LCa (BSC-1-EGFP-LCa), σ2-EGFP (BSC-1-σ2-EGFP), imaged before and during (~ 3min) treatment with 2% v/v 1-butanol. The fluorescence along the z-axis is shown on top of each confocal section. Bar, 20 μm. (B) Confocal section of COS cells transiently expressing dynamin2-EGFP. Cells were imaged in the same way as in panel A. (C) Treatment with the primary alcohols ethanol and 1-propanol disassembles coated pits. Confocal section of BSC-1-σ2-EGFP imaged before and during (~3min) treatment with 2.5% v/v ethanol (top) or 2% v/v 1-propanol (bottom). Kymographs are shown on the right of each confocal section. (D) The secondary alcohol 2-propanol and tertiary alcohol t-butanol disassemble coated pits and vesicles. Confocal section of BSC-1-σ2-EGFP imaged before, during (~ 3min) treatment with 2.5% v/v 2-propanol (top) or t-butanol (bottom). Kymographs are shown on the right of each confocal section. Bar, 10 μm.

Fig. 3. 1-butanol leads to the simultaneous disassembly of AP-2 and clathrin without coated vesicle internalization

(A) Kymograph of the disassembly of the coated pits of a BSC-1 cell co-expressing σ2-EGFP (green) and Tomato-LCa (red) upon incubation with 2% 1-butanol. Bar 10 μm. (B) Alignment of the relative fluorescent intensity of clathrin (black line) and AP-2 (grey line) of the disassembling pits depicted in 2B. (C) Time-lapse series acquired under alternating wide field/epi-fluorescence and TIRF illumination of a typical AP-2 coated pit of an untreated cell. (D) Time-lapse acquisition under alternating wide field/epi-fluorescence and TIRF illumination of a typical AP-2 coated pit treated with 2% 1-butanol. (E) Graphic depiction of the florescent intensity profile of a typical AP-2 coated of an untreated cell; note the reduction in the TIRF signal intensity (black) prior to the ensuing reduction in the epifluorescence signal (grey). (F) Graphic depiction of the florescent intensity profile of a typical AP-2 coated (such as the one depicted in (C)) treated with 2% 1-butanol; note the concomitant reduction in epifluorescence (grey) and TIRF (black) signal intensities. (G) Confocal section of BSC1 cells stably expressing EGFP-LCa upon hypertonic treatment (0.45 M sucrose, which leads to the formation of clathrin microcages [27]). Cells were imaged before and after (~3min) treatment with 2% v/v 1-butanol. A kymograph of the locked pits is shown. Bars, 10 μm.

Fig. 4. Washout of 1-butanol results in a transient burst of coated pit nucleations

(A) BSC1-EGFP-LCa and BSC1-σ2-EGFP were incubated with 2% 1-butanol for 5 min. 1-butanol was washed out and cells were imaged after 1 and 3 minutes. Bar, 10 μm. (B) Graphic depiction of the relative coated pit density (normalized to the untreated level; from 3 independent experiments) under the experimental conditions described in (A). (C) Confocal time lapse acquisition of a typical coat formed after the 1-butanol washout in a cell co-expressing σ2-EGFP (green) and Tomato-LCa (red), note the concomitant recruitment, signal intensification and disappearance of AP-2 and clathrin. (D) Left: EPI-TIRF time lapse acquisition of a typical σ2-GFP coat formed after the 1-butanol washout. Right: Graphic depiction of the relative fluorescent intensities of a typical coated pit depicted immediately after 1-butanol washout under TIRF (grey) and EPI (black) illuminations, note the internalization, ie. a reduction in TIRF signal prior to the reduction in epifluorescence signal intensity. (E) HeLa cells stably expressing σ2-EGFP were incubated with 2% butanol for 5 min. Subsequently 1-butanol was washed out and cells were imaged after 1 and 3 minutes. Bar, 10 μm. Note the disassembly of all AP-2 containing structures and the increase in density and decrease in average intensity of AP-2 coated pits upon 1-butanol washout. (F) Kymograph representation of the cell depicted in (A), note the return of signal to preexisting structures as well as the new nucleations in previously unoccupied locations. Bar, 10 μm.

Fig. 5. Liposomes containing phosphoinositide 4,5 bisphosphate allow the formation of coated pits and vesicles in cells treated with 1-butanol

(A) Recovery of coated pit formation in the presence of PIP2 containing liposomes. BSC-1-σ2-EGFP cells were treated with 2% v/v 1-butanol for 1 minute. Cells were then incubated with liposomes (50 μg/ml final concentration) containing either equal amounts of PE and PC (upper panel) or 5% PIP2 in addition to 45% PE and 50% PC (lower panel) in the presence of 2% (v/v) 1-butanol; and imaged for an additional 3 minutes. Bar, 10 μm. (B) Kymograph representations of the cell depicted in (A). Note the return of new nucleations and coated vesicles formation in cells treated with PIP2 containing liposomes (right) but not with control liposomes (left). (C) Clathrin coated pits formed in the course of PIP2 addition on cells treated with 2% v/v 1-butanol have normal lifetime. The histogram plot displays the lifetimes of AP-2 spots calculated from the time-lapse depicted in A and B. The data derives from n=182 pits.

Fig. 6. Actin depolymerization with LatB does not affect the disassembly of coated pits by 1-butanol, their reappearance upon 1-butanol washout or the dynamic parameters of individual pits

(A) Kymograph of BSC1-σ2-EGFP cell prior to incubation with LatB (“untreated”); 15 min after the addition of 2.5 μg/ml LatB (“LatB”); during the incubation with 2% 1-butanol +2.5 μg/ml LatB (“LatB + 1-butanol”); and during the washout of 2% 1-butanol in the presence of 2.5 μg/ml LatB (“LatB – 1-butanol”). Note the static, high-intensity objects in the LatB treated cell as well as the absence of AP-2 pits in the beginning of the 1-butanol washout and their dynamic nature thereafter. Bar, 10 μm. (B) Graphic representation of the profile of intensity of AP-2 structures at any given frame (snapshots) in “untreated” cells (n=112), “LatB” cells (n=236) and “LatB – 1-butanol” cells (n=136). Pits were divided in 3 classes: under the range of the maximum intensity of dynamic pits (‘Lower’, black columns), in the range of the maximum intensity of dynamic pits (white columns), and over the range of the maximum intensity of dynamic pits (‘Higher’, grey columns). Note the similar profiles of “untreated” and “LatB – 1-butanol” cells in contrast to the accumulation of large structures in the LatB treated cells. (C) Nucleation rate of dynamic pits (number of clathrin coated pits/sec/10⁴ pixels²) in untreated (left, n=196), “LatB” (n=72), or “LatB – 1-butanol” cells (n=203). (D) Distribution of the maximum intensities and lifetimes of dynamic pits in cells: untreated (dashed line, n=196), “LatB” (grey, n=72) and “LatB – 1-butanol” (black, n=203). Note the similarity of the profiles of the parameters of dynamic pits in all three conditions.

Fig. 7. Actin depolymerization abrogates the upregulation in nucleation resulting from removal of 1-butanol

(A) Lateral displacement of dynamic coated pits (μm/sec) in untreated (left, n=55), “LatB” (middle, n=52) and “LatB – 1-butanol” (right, n=56). Note the similar lateral displacement of the pits upon depolymerized actin irresepective of the 1-butanol treatment. (B) Displacement of dynamic coated pits in untreated (black diamonds), 1-butanol washout” cells (gray diamonds), “LatB” cells (black triangles) and “LatB – 1-butanol” cells (gray triangles). (E) Ripleys L function of the clustering tendencies of the points of origin of clathrin coated pits in cells in which the actin cytoskeleton was intact (lower curve) or in which the actin cytoskeleton was depolymerized (upper curve); given the fact that the 1-butanol washout treatment had no effect on the clustering tendencies (data not shown), results from cells in washout or untreated conditions were compiled together. (D) Confocal image of BSC1-σ2-EGFP cells, untreated, during the burst of nucleation following 1-butanol washout or “ LatB – 1-butanol” cells. (Bar, 10 μm) and graphic depiction of the relative coated pit density (normalized to the untreated level; from 3 independent experiments). (E) Transferrin uptake in Hela cells following LatB and butanol treatments. Hela cells were pre-incubated at 37° C for 15 min with imaging medium (untreated, left) or medium containing LatB (2.5 μg/ml). Cell samples were then incubated for 2 min with the resepective combinations of LatB and alcohols. The alcohol was washed out in the presence of LatB for 2 or 15 minutes, after which cells were incubated with 10 μg/ml Alexa594-Tf for 2 min at RT and 5 min at 37°C in PBS/Glucose/BSA (PBS-GB; untreated), or PBS-GB + LatB. Cells were subsequently transferred to 4°C, acid washed and fixed. Bar, 20 μm. (Right) Bar plot illustrates the quantitation of Tf uptake in the conditions depicted, normalized to the level of untreated cells.