New Results

Light-dependent inhibition of clathrin-mediated endocytosis in yeast

Davia Prischich, Javier Encinar del Dedo, Maria Cambra, Judit Prat, Nuria Camarero, Laura Nevola, Andrés Martín-Quirós, Elena Rebollo, Ernest Giralt, María Isabel Geli, Pau Gorostiza
doi: https://doi.org/10.1101/2021.04.01.432428

Abstract

Clathrin-mediated endocytosis (CME) is an essential cellular process, which is evolutionarily conserved among eukaryotes. Yeast constitutes a powerful genetic model to dissect the complex endocytic machinery, yet there is a lack of pharmacological agents that could complement genetics in selectively and reversibly interfere with CME in these organisms. TL2 is a light-regulated peptide inhibitor that targets the AP2/β-arrestin interaction and that can photocontrol CME with high spatiotemporal precision in mammalian cells. Here, we study endocytic protein dynamics by live-cell imaging of the fluorescently tagged coat-associated protein Sla1-GFP and demonstrate that TL2 retains its inhibitory activity in S. cerevisiae spheroplasts, thus providing a unique tool for acute and reversible CME modulation in yeast.

Introduction

Clathrin-mediated endocytosis (CME) is one of the major entry routes for extracellular material and plasma membrane (PM) into eukaryotic cells. The process is essential to internalize nutrients, to maintain PM homeostasis, and to regulate receptor turnover and signalling, among other cellular functions. Endocytic vesicles are also employed by pathogenic toxins,1–3 bacteria,4 and viruses5 (including SARS-CoV-2)6,7 to reach intracellular compartments, whereas dysfunctions in the pathway can result in several diseases including cancer, myopathies, and neuropathies8. The formation of an endocytic vesicle relies on the sequential and finely coordinated assembly of more than 60 molecular actors which are conserved between yeast and mammalian cells. Thus, Saccharomyces cerevisiae provides a convenient model system to study the process, delivering findings that can often be extrapolated to higher eukaryotes9–11. Yeast amenability to genetic manipulation, together with advances of live-cell fluorescence microscopy and time resolved electron microscopy, has allowed to examine CME dynamics with great spatiotemporal resolution, and couple the recruitment of particular proteins to specific membrane deformation steps12–17.

Several strategies can be used to selectively interfere with CME in order to study the role of specific interactions between endocytic proteins and to decipher the impact of CME on particular cellular processes. Classic genetic manipulation is time consuming and not straightforward, and it can result in compensatory cellular responses that complicate the interpretation of the results. In addition, loss-of-function mutations may result in non-viable strains or display pleiotropic phenotypes when the proteins involved have multiple functions. Reciprocally, genetic redundancy might hinder uncovering of bona fide protein functions. These limitations have prompted the search for chemical and pharmacological agents that can specifically, acutely, and reversibly manipulate CME18. However, only few chemical tools are known to effectively and specifically inhibit CME, and none of them have been reported to be effective in yeast (Fig. 1A)19,20. To date, the actin polymerization inhibitor latrunculin A and the sterol sequestering filipin have been effectively used to manipulate endocytic uptake in yeast, but they have pleiotropic effects in many other cellular functions19,21. Among the reasons for this shortage of molecular tools is that the endocytic machinery is mainly driven by protein-protein interactions, which involve large contact surfaces that are difficult to tackle with small molecules22. Besides, most of these agents display reduced specificity and can affect other endocytic pathways or perturbate other cellular processes23.

Fig. 1
Fig. 1

A) Overview of clathrin-mediated endocytosis (CME) inhibitors. On the left, pharmacological agents targeting specific endocytic proteins classified according to their biological activity in mammalian cells, plants, and yeast. On the right, chemicals that perturbate CME via a pleiotropic mechanism of action. Limits and advantages of each approach have been covered in the literature18,20,23–25. B) Traffic Light 2 (TL2) and its structural alterations produced by exposure to light. Upon illumination with UV light (380 nm), the crosslinker photoisomerizes from trans (dark-adapted conformation) to cis, thus promoting a helical structure (on the right, in purple) and the correct orientation of those residues (in red) responsible for the interaction with AP2 (in grey). Exposure to green light (500 nm) or spontaneous thermal relaxation revert the crosslinker to its stable conformation, thus causing partial unfolding of the peptide and loss of activity (on the left, in green).

Thus, it would be very useful to identify a pharmacological agent capable of interfering with CME in a specific, acute, and reversible way in yeast, to take advantage of this well-defined model for the study of endocytosis. Previously, Nevola et al. developed two Traffic Lights (TLs), photoswitchable inhibitors of CME26. TLs are based on the structure of the human β-arrestin C-terminal peptide (BAP-long), which binds to the appendage of the β2-adaptin subunit of AP2, the major endocytic clathrin adaptor in mammalian cells27,28. Light regulation was introduced by stapling the structures with a photoresponsive bridge that upon trans-cis isomerization can modulate the helical content, and therefore, the inhibitory activity of the peptides29,30. When tested in mammalian cells, both TLs blocked CME in a light-regulated manner. Between the two, Traffic Light 2 (TL2) proved particularly attractive as it is inert under dark-adapted conditions (trans isomer), but it strongly reduces CME trafficking upon activation with (ultra)violet light (cis isomer) (Fig. 1B). Here, we demonstrate that the TL2 peptide designed for mammalian proteins retains its activity in yeast and provides a powerful tool to dynamically regulate CME. In contrast to small molecules, its peptide nature offers a higher degree of specificity to the mode of action, while the photoswitchable crosslinker enables both the spatiotemporal control of inhibition with light, and the permeability of the compound through the PM.26

Results

Although some peptides have been reported to penetrate the fungal cell-wall, this remains a prohibitive barrier to most macromolecules 31. Since TL2 targets an intracellular protein-protein interaction, we first tested whether it could be internalized in yeast using an analogue labelled with carboxy-fluorescein (CF-TL2) on its N-terminus. S. cerevisiae and S. pombe were incubated in the presence of different peptide concentrations (dark-inactive TL2), but in no case, signs of internal fluorescence were detected, suggesting that cells were unable to take up the peptide or this was quickly extruded (Fig. S1). To evaluate if the cell wall might interfere with the peptide uptake, we proceeded to its enzymatic digestion with lyticase before exposing the cells to the fluorescent peptide. To prevent cell lysis, spheroplasts were maintained in media supplemented with 0.7 M sorbitol for osmotic support. A styryl dye, FM4-64 (16 µM), was used to selectively stain vacuole membranes with red fluorescence32. This allowed us to confirm that cell integrity, viability, and endocytic activity were maintained after the spheroplasting process. After co-incubation in the presence of fluorescently labelled and dark-adapted inactive CF-TL2, a specific signal corresponding to the peptide was detected by fluorescence inverted optical microscopy in small cortical patches and bright bigger internal structures, as well as in the vacuole, whose limiting membrane was stained with FM4-64. In addition, a weaker signal in the cytosol was detectable. The data thus indicated that under these conditions CF-TL2 was most likely internalized into the cells via an endocytic pathway from where it could be translocated into the cytosol.

To investigate the effect of light-activated TL2 on endocytosis, we followed the cortical dynamics of the GFP-tagged coat-associated protein Sla1-GFP. The yeast clathrin adaptor Sla1 is a component of the clathrin coat, whose spatiotemporal dynamics at endocytic sites has been well-characterized12,13,33.

Spheroplasts of S. cerevisiae expressing Sla1-GFP were treated with either pre-activated TL2 (380 nm) or with vehicle, and cortical patches were live-imaged after 30 minutes incubation. As shown by kymographs generated from 120 seconds time-lapse movies, light-activated TL2 significantly extended the permanence of the GFP-labelled endocytic patches on the PM (Fig. 3A). This effect was dose-, time-, and light-dependent (Fig. 3B-D). Inhibition was observed between 50-100 µM and 20-30 min incubation (Fig. 3B and 3D). As light-induced changes are observable close to the IC50 and far from saturating doses,26 we chose 100 µM and 20 min to quantify light dependency. As reported in mammalian cells,26 dark-adapted TL2 did not significantly alter CME, thus confirming that the peptide is also constitutively inert in yeast, and can stall CME upon UV-induced photoisomerization (Fig. 3C and Fig. S2).

Fig. 2
Fig. 2

Uptake and intracellular distribution of carboxy-fluorescein conjugated Traffic Light 2 (CF-TL 2) in S. cerevisiae spheroplasts as assessed by live-cell fluorescence microscopy. Differential Interference Contrast (DIC) (upper panels) and fluorescence micrographs of S. cerevisiae cells loaded for 5 minutes at 25°C with FM4-64 (16 µM), washed and incubated for 40 minutes with 30 µM of either dark-adapted (inactive) carboxy-fluorescein conjugated TL-2 (CF-TL2) or TL2 as control. The individual channels and the merged images of the fluorescein and FM4-64 channels, and the DIC, fluorescein and FM4-64 channels are shown.

Fig. 3
Fig. 3 TL2 inhibits CME in yeast in a light-dependent way.

A) Fluorescence kymographs of Sla1-GFP cortical patches from 120 s time-lapse movies of S. cerevisiae spheroplasts incubated with 100 µM light-activated TL2 (illuminated at 380 nm) (right) or mock-treated (left, control). B) Mean ± SEM life span of Sla1-GFP cortical patches of yeast spheroplasts either mock-treated (control) or treated with the indicated concentrations of UV-activated TL2 for 30 min. C) Mean ± SEM life span of Sla1-GFP cortical patches of yeast either mock-treated (control) or incubated for 20 min with 100 µM of UV-activated TL2 or with the inactive (dark-adapted or illuminated at 500 nm) peptide. D) Graphs showing the progressive change in the lifetime distribution of endocytic Sla1-GFP patches incubated either with vehicle or with pre-activated TL2 (100 µM) for 10, 20, and 30 min. E) Graph showing the TL2 effects on initiation and maturation of CME sites. Each point represents the average lifespan of cortical Sla1-GFP patches and the number of endocytic foci counted per minute in a given cell, 30 min upon incubation with 100 µM UV-activated TL2 (purple) or mock treated (grey). At least 60 cells from 3 different experiments were analysed per conditions. F) Graph representing with box and whisker (min to max) the number of endocytic events per cell per minute, in cells incubated for 30 min with 100 µM UV-activated TL2 (purple) or mock-treated (grey). G) Time-lapse images of Sla1-GFP cortical patches representing the different dynamics observed under our experimental conditions. Patches were defined as wild-type (WT) when internalization and scission occurred within the described average lifetime for Sla1-GFP. Patches that moved inward and then bounced back towards the PM were classified as v-shaped, independently of their life span. Sla1-GFP dynamics were considered delayed when internalization and scission occurred normally but their cortical life span was extended. Patches were considered flat when no inward movement of the patch was detected. H) Frequency plots of patch internalization dynamics, grouped as normal, flat, and v-shaped kymographs (as defined in G), in spheroplasts treated with vehicle or the UV-activated peptide (100 µM, 30 min). Classification refers only to the internalization stage not considering the whole duration of the process. Thus, normal patches include both WT and delayed phenotypes. I) Comparison of the average fluorescence intensity over time traces for cortical Sla1-GFP patches of spheroplasts treated either with vehicle or with pre-activated TL2 (100 µM, 30 min). Traces were aligned on the fluorescence maximum (time 0). Data are mean ± SEM represented by the shading area. A total of 60 patches from 3 different experiments were analysed per condition. Statistical significance was analysed at time -30, 0, and 20 s. Unless differently stated, statistical differences were determined by Student’s t test (n.s., not significant; *, p-value<0.05; ***, p-value<0.01; ****, p-value<0.001). For B, C, D, F, and H, at least 200 Sla1-GFP patches in at least 20 different cells were analysed per condition. The experiments were repeated at least 3 times and the results plotted together.

Subsequently, we focused on characterizing the active isomer (UV-illuminated TL2, 100 µM) and the extent of its activity in S. cerevisiae spheroplasts. Live-cell imaging at different incubation times revealed how TL2 progressively delayed Sla1-GFP internalization resulting in both a shift and a broadening of the patches lifetime distribution when compared to vehicle (Fig. 3D). The onset of CME inhibition was consistently observed after 20 minutes, which suggested that the peptide needed to be taken up by endocytosis before translocation to the cytosol. To investigate if clathrin coat assembly, and not only its kinetics, might also be affected by the activated TL2, we quantified both the average Sla1-GFP lifetime, as well as the number of endocytic events per minute after 30 min treatment. Upon plotting these two parameters for each cell, pre-activated TL2- and vehicle-treated samples distributed into two markedly different populations (Fig. 3E). CME inhibition associated with a moderate, but significant reduction in the number of endocytic foci (Fig. 3F), which were 22% less frequent than in the control. We also investigated if the treatment affected later stages of CME (Fig. 3G-I). In particular, patches were analysed for their phenotypes and classified according to their internalization patterns (Fig. 3G). Normally, the endocytic coat moves into the cytosol as the endocytic invagination grows, and it disassembles as or shortly after the vesicles detach from the PM. In mutants unable to invaginate the plasma membrane though, the endocytic coat remains at the PM (“flat” phenotype). Finally, in mutants affecting the final scission event an initial inward movement of the coat can be observed, but then it bounces back towards the PM (“v-shaped” phenotype) (Fig. 3G)34–37. TL2-treatment caused marginal changes in the percentage of flat or v-shaped phenotypes compared to vehicle, thus indicating that the peptide does not affect membrane invagination or scission but rather coat maturation and/or cargo loading (Fig. 3H). Finally, we analysed the assembly and disassembly dynamics of the Sla1-GFP cortical patches in cells either mock-treated or treated with activated TL2. Sla1-GFP fluorescence intensity plots as a function of time were aligned at their peak intensity (set as time 0) (Fig. 3I). Interestingly, while the assembly and disassembly rates appeared similar in both samples, TL2 produced a very significant increase of the maximum fluorescence signal at t=0, revealing that higher levels of Sla1 are required to sustain endocytic budding or that the coats grow larger in the presence of TL2, before initiating membrane invagination (Fig. 3I).

Discussion

We have shown that the photoswitchable peptide TL2 designed from the sequence and structure of human β-arrestin C-terminus can be effectively used to photocontrol CME in yeast. As observed in mammalian cells, TL2 can cross the yeast membranes in minutes and accumulate at concentrations sufficient to inhibit CME. We interpret that pre-illuminated (cis, helical) TL2 binds to its target in a conformation that inhibits and slows down clathrin-coated pit (CCP) assembly and internalization, reducing the frequency of events and increasing their duration, thus diminishing the cellular rate of CME. In contrast, the trans, less-helical peptide appears inactive in the absence of illumination.

Arrestin-like proteins (ARTs) exist in yeast, which as mammalian arrestins, act as E3 ubiquitin ligase cargo adaptors. Unlike mammalian arrestins though, yeast ARTs lack the specific residues that mediate binding to AP2 and clathrin38,39. Interestingly, the yeast epsins Ent1 and Ent2, and the Eps15 homology (EH) domain– containing proteins Ede1 and End3, some of which work as clathrin adaptors for ubiquitinated cargo, all bear sequences similar to the [DE]nX1-2[FLI]XX[FL]XXXR AP2-binding motif present in mammalian β-arrestins, epsins, and in the TL2 peptide (Fig. S3). Therefore, it is likely that the mammalian arrestin function is split in yeast and that TL2 interferes with the clathrin adaptors for ubiquitinated cargo, rather than with the ARTs.

In yeast, AP2 lacks the β2-appendage domain (residues 701-937) that in mammalians interacts with the β-arrestin C-terminus and with TL2 through specific residues located in the protein platform subdomain26,40. As the yeast α-appendage shares structural similarities with the mammalian β2-appendage, we investigated if the former could act as binding site of our peptide27,41. Adaptin-binding motifs occasionally share similar sequences. In particular, an FXXφ hydrophobic core (where φ indicates a hydrophobic residue) is common to both [DE]nX1-2[FLI]XX[FL]XXXR and FXXFXXL motifs, which respectively bind the β2- and α-platform subdomains27,28,41,42. In both cases, binding is mediated by two hydrophobic sites, which are present in both mammalian appendages and conserved in S. cerevisiae. In yeast, a first hydrophobic interaction might be mediated by F907 (mammalian β2-F837) and by I908 and V954, corresponding to mammalian β2-L838 and β2-A877, respectively. The second hydrophobic interaction might be mediated by W911 (β2-W841) and F972 (β2-Y888). In contrast, clusters of polar residues involved in [DE]nX1-2[FLI]XX[FL]XXXR binding are only found in the mammalian β2-subdomain and in the yeast α-appendage27,28. In particular, the basic C-terminus of the peptide could interact with E921 and E982 (β2-E849 and β2-E902 in mammalian), whereas its N-terminal acidic region could interact with R956 (β2-R879 and β2-R834 in mammalians) (Fig. S4).

Despite being structurally conserved between lower and higher eukaryotes, AP2 plays a dispensable role in yeast43,44. So far, no endocytic phenotype, apart from resistance to the killer toxin K28, has been associated with its knockout, hence it has been suggested that AP2 might act as an adaptor complex for cargoes that are dispensable to yeast viability45. Alternatively, the limited phenotypic consequences of deleting AP2 may be due to redundancy of adaptors and/or rerouting to other endocytic pathways. In contrast to genetic manipulation of the AP2 function in yeast, the acute (photo)pharmacological inhibition shown here suggests that AP2 has an important role in CME.

We propose that, once the adaptor complex is recruited into the nascent CCP, TL2 can competitively interfere with protein-protein interactions necessary to advance through the internalization process. Upon binding to the AP2 appendages, TL2 mimics endogenous accessory proteins (clathrin-associated sorting proteins, CLASPs) and hinders progression maybe through the activation of regulatory checkpoints that impede vesicle internalization of incorrectly loaded cargoes39,46. Hence, we hypothesize that TL2 acts as a dominant negative peptide exhibiting the AP2-binding motif, but lacking domains essential to regulate endocytic progression. This is supported by the fact that in mammalian models the overexpression of adaptin-binding motifs inhibits CME42,47. Moreover, in S. cerevisiae the specific binding sequence of TL2 is conserved in several accessory proteins and the corresponding knockout strains display phenotypes that are reminiscent of those obtained upon treatment with TL2. For example, deletion of End3 leads to an increased persistence of Sla1-GFP at the cell cortex13, whereas deletion of Ede1 reduces the frequency of site initiations similarly to the presence of the pre-activated TL2, while shortening the lifetime of Sla1-GFP13,48,49. Interestingly, an Ede1(22A) mutant that lacks a phosphoregulatory site exhibits reduced patch density (by a 25%) without altering the lifetime of Sla1-GFP48. Thus, light-activated TL2 allows to assess the effect of interfering with the [DE]nX1-2[FLI]XX[FL]XXLR motif on several endocytic proteins and it results in a combination of phenotypes otherwise only appreciable by performing time-consuming, multiple mutations.

Conclusion

In summary, we have characterized the TL2 activity in S. cerevisiae spheroplasts and proved that its ability to control CME with light spans throughout evolution. To the best of our knowledge, no other means to achieve specific pharmacological inhibition of CME have been described to be effective in yeast. While latrunculin A displays a dramatic effect over endocytic vesicle internalization, its inhibitory activity over actin-polymerization interferes with numerous other cellular functions. This, so far, has impeded to understand the contribution of CME to those processes, such as cytokinesis and cell migration, in which the actin role is crucial. In light of these considerations, we believe that TL2 provides a unique tool for the scientific community to gain deeper understandings over the function and regulation of CME. This photoswitchable inhibitor opens the way to intriguing studies where CME could be switch on and off at will using light, in order to synchronize it with other measurements (e.g. microscopy, biochemical reactions) or cellular processes, or obliterate it at specific cellular locations such as the cytokinetic ring or the growth pole.

Acknowledgements

This research received funding from the European Union Research and Innovation Programme Horizon 2020 - Human Brain Project SG3 (945539), DEEPER (ICT-36-2020-101016787)-, Agency for Management of University and Research Grants/Generalitat de Catalunya (CERCA Programme; 2017-SGR-1442 and 2017-SGR-00465 projects), Fonds Européen de Dévelopement Économique et Régional (FEDER) funds, Ministry of Science and Innovation (Grant PID2019-111493RB-I00), Fundaluce and “la Caixa” foundations (ID 100010434, agreement LCF/PR/HR19/52160010). The project Clúster Emergent del Cervell Humà (CECH, 001-P-001682) is co-financed by the European Union Regional Development Fund within the framework of the ERDF Operational Program of Catalonia 2014-2020 with a grant of 50% of total eligible cost. IBEC and IRB Barcelona are recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain). BFU2017-82959-P from MINECO to MIG. D.P. was supported by fellowship BES-2015-072657.

Footnotes

  • ↵* E-mail: mgfbmc{at}ibmb.csic.es (M.I.G.)

References

  1. 1.↵
    Papatheodorou P, Zamboglou C, Genisyuerek S, Guttenberg G, Aktories K. Clostridial glucosylating toxins enter cells via clathrin-mediated endocytosis. PLoS One. 2010;5(5). doi:10.1371/journal.pone.0010673
    OpenUrlCrossRefPubMed
  2. 2.
    Abrami L, Liu S, Cosson P, Leppla SH, Van der Goot FG. Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process. J Cell Biol. 2003;160(3):321–328. doi:10.1083/jcb.200211018
    OpenUrlAbstract/FREE Full Text
  3. 3.↵
    Deinhardt K, Berninghausen O, Willison HJ, Hopkins CR, Schiavo G. Tetanus toxin is internalized by a sequential clathrin-dependent mechanism initiated within lipid microdomains and independent of epsin 1. J Cell Biol. 2006;174(3):459–471. doi:10.1083/jcb.200508170
    OpenUrlAbstract/FREE Full Text
  4. 4.↵
    Veiga E, Cossart P. The role of clathrin-dependent endocytosis in bacterial internalization. Trends Cell Biol. 2006;16(10):499–504. doi:10.1016/j.tcb.2006.08.005
    OpenUrlCrossRefPubMedWeb of Science
  5. 5.↵
    Cossart P, Helenius A. Endocytosis of viruses and bacteria. Cold Spring Harb Perspect Biol. 2014;6(8). doi:10.1101/cshperspect.a016972
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    Inoue Y, Tanaka N, Tanaka Y, et al. Clathrin-Dependent Entry of Severe Acute Respiratory Syndrome Coronavirus into Target Cells Expressing ACE2 with the Cytoplasmic Tail Deleted. J Virol. 2007;81(16):8722–8729. doi:10.1128/jvi.00253-07
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    Bayati A, Kumar R, Francis V, McPherson PS. SARS-CoV-2 infects cells following viral entry via clathrin-mediated endocytosis. bioRxiv. 2020;pre-print. doi:10.1101/2020.07.13.201509
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    McMahon HT, Boucrot E. Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nat Rev Mol Cell Biol. 2011;12(8):517–533. doi:10.1038/nrm3151
    OpenUrlCrossRefPubMed
  9. 9.↵
    Boettner DR, Chi RJ, Lemmon SK. Lessons from yeast for clathrin-mediated endocytosis. Nat Cell Biol. 2012;14(1):2–10. doi:10.1038/ncb2403
    OpenUrlCrossRefPubMedWeb of Science
  10. 10.
    Weinberg J, Drubin DG. Clathrin-mediated endocytosis in budding yeast. Trend Cell Biol. 2012;22(1):1–13. doi:10.1016/j.tcb.2011.09.001
    OpenUrlCrossRefPubMedWeb of Science
  11. 11.↵
    Kaksonen M, Roux A. Mechanisms of clathrin-mediated endocytosis. Nat Rev Mol Cell Biol. 2018;19(5):313–326. doi:10.1038/nrm.2017.132
    OpenUrlCrossRefPubMed
  12. 12.↵
    Kaksonen M, Sun Y, Drubin DG. A Pathway for association of receptors, adaptors, and actin during endocytic internalization. Cell. 2003;115:1. www.cell.com/cgi/content/full/115/4/475/DC1.
    OpenUrlCrossRefPubMed
  13. 13.↵
    Kaksonen M, Toret CP, Drubin DG. A modular design for the clathrin-and actin-mediated endocytosis machinery. Cell. 2005;123(2):305–320. doi:10.1016/j.cell.2005.09.024
    OpenUrlCrossRefPubMedWeb of Science
  14. 14.
    Idrissi FZ, Grötsch H, Fernández-Golbano IM, Presciatto-Baschong C, Riezman H, Geli MI. Distinct acto/myosin-I structures associate with endocytic profiles at the plasma membrane. J Cell Biol. 2008;180(6):1219–1232. doi:10.1083/jcb.200708060
    OpenUrlAbstract/FREE Full Text
  15. 15.
    Idrissi FZ, Blasco A, Espinal A, Geli MI. Ultrastructural dynamics of proteins involved in endocytic budding. Proc Natl Acad Sci U S A. 2012;109(39). doi:10.1073/pnas.1202789109
    OpenUrlAbstract/FREE Full Text
  16. 16.
    Kukulski W, Schorb M, Welsch S, Picco A, Kaksonen M, Briggs JAG. Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. J Cell Biol. 2011;192(1):111–119. doi:10.1083/jcb.201009037
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    Kukulski W, Schorb M, Kaksonen M, Briggs JAG. Plasma Membrane Reshaping during Endocytosis Is Revealed by Time-Resolved Electron Tomography. Cell. 2012;150(3):508–520. doi:10.1016/j.cell.2012.05.046
    OpenUrlCrossRefPubMedWeb of Science
  18. 18.↵
    von Kleist L, Haucke V. At the Crossroads of Chemistry and Cell Biology: Inhibiting Membrane Traffic by Small Molecules. Traffic. 2012;13(4):495–504. doi:10.1111/j.1600-0854.2011.01292.x
    OpenUrlCrossRefPubMedWeb of Science
  19. 19.↵
    Ayscough KR, Stryker J, Pokala N, Sanders M, Crews P, Drubin DG. High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A. J Cell Biol. 1997;137(2):399–416. doi:10.1083/jcb.137.2.399
    OpenUrlAbstract/FREE Full Text
  20. 20.↵
    Mishev K, Dejonghe W, Russinova E. Small molecules for dissecting endomembrane trafficking: A cross-systems view. Chem Biol. 2013;20(4):475–486. doi:10.1016/j.chembiol.2013.03.009
    OpenUrlCrossRefPubMed
  21. 21.↵
    Encinar del Dedo J, Idrissi FZ, Fernandez-Golbano IM, et al. ORP-Mediated ER Contact with Endocytic Sites Facilitates Actin Polymerization. Dev Cell. 2017;43(5):588-602.e6. doi:10.1016/j.devcel.2017.10.031
    OpenUrlCrossRef
  22. 22.↵
    Nevola L, Giralt E. Modulating protein-protein interactions: The potential of peptides. Chem Commun. 2015;51(16):3302–3315. doi:10.1039/c4cc08565e
    OpenUrlCrossRef
  23. 23.↵
    Dutta D, Donaldson JG. Search for inhibitors of endocytosis. Cell Logist. 2012;2(4):203–208. doi:10.4161/cl.23967
    OpenUrlCrossRefPubMed
  24. 24.
    Elkin SR, Oswald NW, Reed DK, Mettlen M, MacMillan JB, Schmid SL. Ikarugamycin: A Natural Product Inhibitor of Clathrin-Mediated Endocytosis. Traffic. 2016;17(10):1139–1149. doi:10.1111/tra.12425.
    OpenUrlCrossRefPubMed
  25. 25.↵
    Dejonghe W, Sharma I, Denoo B, et al. Disruption of endocytosis through chemical inhibition of clathrin heavy chain function. Nat Chem Biol. 2019;15(6):641–649. doi:10.1038/s41589-019-0262-1
    OpenUrlCrossRef
  26. 26.↵
    Nevola L, Martín-Quirõs A, Eckelt K, et al. Light-regulated stapled peptides to inhibit protein-protein interactions involved in clathrin-mediated endocytosis. Angew Chemie - Int Ed. 2013;52(30):7704–7708. doi:10.1002/anie.201303324
    OpenUrlCrossRef
  27. 27.↵
    Schmid EM, Ford MGJ, Burtey A, et al. Role of the AP2 β-appendage hub in recruiting partners for clathrin-coated vesicle assembly. PLoS Biol. 2006;4(9):1532–1548. doi:10.1371/journal.pbio.0040262
    OpenUrlCrossRef
  28. 28.↵
    Edeling MA, Mishra SK, Keyel PA, et al. Molecular switches involving the AP-2 β2 appendage regulate endocytic cargo selection and clathrin coat assembly. Dev Cell. 2006;10(3):329–342. doi:10.1016/j.devcel.2006.01.016
    OpenUrlCrossRefPubMedWeb of Science
  29. 29.↵
    Zhang Z, Burns DC, Kumita JR, Smart OS, Woolley GA. A water-soluble azobenzene cross-linker for photocontrol of peptide conformation. Bioconjug Chem. 2003;14(4):824–829. doi:10.1021/bc0340161
    OpenUrlCrossRefPubMedWeb of Science
  30. 30.↵
    Martín-Quirós A, Nevola L, Eckelt K, Madurga S, Gorostiza P, Giralt E. Absence of a stable secondary structure is not a limitation for photoswitchable inhibitors of β-arrestin/β-adaptin 2 protein-protein interaction. Chem Biol. 2015;22(1):31–37. doi:10.1016/j.chembiol.2014.10.022
    OpenUrlCrossRef
  31. 31.↵
    Parenteau J, Klinck R, Good L, Langel Ü, Wellinger RJ, Elela SA. Free uptake of cell-penetrating peptides by fission yeast. FEBS Lett. 2005;579(21):4873–4878. doi:10.1016/j.febslet.2005.07.064
    OpenUrlCrossRefPubMed
  32. 32.↵
    Vida TA, Scott DE. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol. 1995;128(5):779–792.
    OpenUrlAbstract/FREE Full Text
  33. 33.↵
    Tolsma TO, Cuevas LM, Di Pietro SM. The Sla1 adaptor-clathrin interaction regulates coat formation and progression of endocytosis. Traffic. 2018;19(6):446–462. doi:10.1111/tra.12563
    OpenUrlCrossRef
  34. 34.↵
    Arasada R, Pollard TD. Distinct roles for F-BAR proteins Cdc15p and Bzz1p in actin polymerization at sites of endocytosis in fission yeast. Curr Biol. 2011;21(17):1450–1459. doi:10.1016/j.cub.2011.07.046
    OpenUrlCrossRefPubMed
  35. 35.
    Basu R, Chang F. Characterization of dip1p reveals a switch in Arp2/3-dependent actin assembly for fission yeast endocytosis. Curr Biol. 2011;21(11):905–916. doi:10.1016/j.cub.2011.04.047
    OpenUrlCrossRefPubMed
  36. 36.
    Kishimoto T, Sun Y, Buser C, Liu J, Michelot A, Drubin DG. Determinants of endocytic membrane geometry, stability, and scission. Proc Natl Acad Sci U S A. 2011;108(44). doi:10.1073/pnas.1113413108
    OpenUrlAbstract/FREE Full Text
  37. 37.↵
    Aghamohammadzadeh S, Smaczynska-de Rooij II, Ayscough KR. An Abp1-dependent route of endocytosis functions when the classical endocytic pathway in yeast is inhibited. PLoS One. 2014;9(7):1–11. doi:10.1371/journal.pone.0103311
    OpenUrlCrossRefPubMed
  38. 38.↵
    Moore CAC, Milano SK, Benovic JL. Regulation of receptor trafficking by GRKs and arrestins. Annu Rev Physiol. 2007;69:451–482. doi:10.1146/annurev.physiol.69.022405.154712
    OpenUrlCrossRefPubMedWeb of Science
  39. 39.↵
    Lin CH, MacGurn JA, Chu T, Stefan CJ, Emr SD. Arrestin-Related Ubiquitin-Ligase Adaptors Regulate Endocytosis and Protein Turnover at the Cell Surface. Cell. 2008;135(4):714–725. doi:10.1016/j.cell.2008.09.025
    OpenUrlCrossRefPubMedWeb of Science
  40. 40.↵
    Chapa-y-Lazo B, Allwood EG, Smaczynska-de Rooij II., Snape ML, Ayscough KR. Yeast endocytic adaptor AP-2 binds the stress sensor Mid2 and functions in polarized cell responses. Traffic. 2014;15(5):546–557. doi:10.1111/tra.12155
    OpenUrlCrossRefPubMed
  41. 41.↵
    Praefcke GJK, Ford MGJ, Schmid EM, et al. Evolving nature of the AP2 α-appendage hub during clathrin-coated vesicle endocytosis. EMBO J. 2004;23(22):4371–4383. doi:10.1038/sj.emboj.7600445
    OpenUrlAbstract/FREE Full Text
  42. 42.↵
    Olesen LE, Ford MGJ, Schmid EM, et al. Solitary and repetitive binding motifs for the AP2 complex α-appendage in amphiphysin and other accessory proteins. J Biol Chem. 2008;283(8):5099–5109. doi:10.1074/jbc.M708621200
    OpenUrlAbstract/FREE Full Text
  43. 43.↵
    Yeung BG, Phan HL, Payne GS. Adaptor complex-independent clathrin function in yeast. Mol Biol Cell. 1999;10(11):3643–3659. doi:10.1091/mbc.10.11.3643
    OpenUrlAbstract/FREE Full Text
  44. 44.↵
    Huang KM, D’Hondt K, Riezman H, Lemmon SK. Clathrin functions in the absence of heterotetrameric adaptors and AP180-related proteins in yeast. EMBO J. 1999;18(14):3897–3908. doi:10.1093/emboj/18.14.3897
    OpenUrlAbstract/FREE Full Text
  45. 45.↵
    Carroll SY, Stirling PC, Stimpson HEM, Gießelmann E, Schmitt MJ, Drubin DG. A yeast killer toxin screen provides insights into A/B toxin entry, trafficking and killing mechanisms. Dev Cell. 2010;17(4):552–560. doi:10.1016/j.devcel.2009.08.006.A
    OpenUrlCrossRef
  46. 46.↵
    Shenoy SK, McDonald PH, Kohout TA, Lefkowitz RJ. Regulation of receptor fate by ubiquitination of activated β2-adrenergic receptor and β-arrestin. Science (80-). 2001;294(5545):1307–1313. doi:10.1126/science.1063866
    OpenUrlAbstract/FREE Full Text
  47. 47.↵
    Ford MGJ, Mills IG, Peter BJ, et al. Curvature of clathrin-coated pits driven by epsin. Nature. 2002;419(6905):361–366. doi:10.1038/nature01020
    OpenUrlCrossRefPubMedWeb of Science
  48. 48.↵
    Peng Y, Grassart A, Lu R, et al. Casein Kinase 1 Promotes Initiation of Clathrin-Mediated Endocytosis. Dev Cell. 2015;32(2):231–240. doi:10.1016/j.devcel.2014.11.014
    OpenUrlCrossRefPubMed
  49. 49.↵
    Lu R, Drubin DG. Selection and stabilization of endocytic sites by Ede1, a yeast functional homologue of human Eps15. Mol Biol Cell. 2017;28(5):567–575. doi:10.1091/mbc.E16-06-0391
    OpenUrlAbstract/FREE Full Text
Back to top
Posted April 01, 2021.
Download PDF

Supplementary Material

Email
Light-dependent inhibition of clathrin-mediated endocytosis in yeast
Davia Prischich, Javier Encinar del Dedo, Maria Cambra, Judit Prat, Nuria Camarero, Laura Nevola, Andrés Martín-Quirós, Elena Rebollo, Ernest Giralt, María Isabel Geli, Pau Gorostiza
bioRxiv 2021.04.01.432428; doi: https://doi.org/10.1101/2021.04.01.432428
Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools

Subject Area