FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2078742722> ?p ?o ?g. }

• W2078742722 endingPage "1320" @default.
• W2078742722 startingPage "1313" @default.
• W2078742722 abstract "Notch signaling controls diverse cellular processes critical to development and disease. Cell surface ligands bind Notch on neighboring cells but require endocytosis to activate signaling. The role ligand endocytosis plays in Notch activation has not been established. Here we integrate optical tweezers with cell biological and biochemical methods to test the prevailing model that ligand endocytosis facilitates recycling to enhance ligand interactions with Notch necessary to trigger signaling. Specifically, single-molecule measurements indicate that interference of ligand endocytosis and/or recycling does not alter the force required to rupture bonds formed between cells expressing the Notch ligand Delta-like1 (Dll1) and laser-trapped Notch1 beads. Together, our analyses eliminate roles for ligand endocytosis and recycling in Dll1-Notch1 interactions and indicate that recycling indirectly affects signaling by regulating the accumulation of cell surface ligand. Importantly, our study demonstrates the utility of optical tweezers to test a role for ligand endocytosis in generating cell-mediated mechanical force.Video AbstracteyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIxYmE1YzA4MjFmY2YzYTE4MzRiN2RkYTViZmU1NGU5YiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc5NTQwMjIwfQ.cdjYtTmEAg0atIe8xj63tGCimYwu7DWxXU-uSzxSrq8Tc1QoOapDa8vIqHD-HoqnQ7At6R5P7B5ouE4AgutEqIEeJ636pLy8KLvYoSD9F6gNpmIXXZFur7FXZUkU7txzEZX01l_sfDMy906BmB8W_eqfte9Jeiv-ieJnbig6v8D4qyxtGqCV4xG5HYbsEfirzLCLln_xPSC5QRRVQ_DPI-p4NxUDfL1L5Ugk0nyCuO0XJGgQC2dKvwig5GZomoiN8SShlJRcfF9Yc3MyvNViCWQH2APBDKKYlPDys-GhJEqaAC3R1eRPu9fMB5H4JAS0asSUXy0Wgd1uHcyTBoNC7A(mp4, (14.73 MB) Download video Notch signaling controls diverse cellular processes critical to development and disease. Cell surface ligands bind Notch on neighboring cells but require endocytosis to activate signaling. The role ligand endocytosis plays in Notch activation has not been established. Here we integrate optical tweezers with cell biological and biochemical methods to test the prevailing model that ligand endocytosis facilitates recycling to enhance ligand interactions with Notch necessary to trigger signaling. Specifically, single-molecule measurements indicate that interference of ligand endocytosis and/or recycling does not alter the force required to rupture bonds formed between cells expressing the Notch ligand Delta-like1 (Dll1) and laser-trapped Notch1 beads. Together, our analyses eliminate roles for ligand endocytosis and recycling in Dll1-Notch1 interactions and indicate that recycling indirectly affects signaling by regulating the accumulation of cell surface ligand. Importantly, our study demonstrates the utility of optical tweezers to test a role for ligand endocytosis in generating cell-mediated mechanical force. Optical tweezers measure single-molecule Notch (N1)-ligand (Dll1) bond strength Blockade of dynamin activity does not alter the strength of Dll1-N1 interactions Rab11-mediated Dll1 recycling does not alter single-molecule Dll1-N1 bond strength Dll1 recycling influences signal intensity by regulating ligand accumulation Activation of the evolutionarily conserved Notch signaling system requires cell-cell contact to facilitate interactions between Notch cell surface ligands and receptors. The transmembrane nature of Notch ligands is consistent with the requirement for ligand endocytosis in activation of the Notch receptor (D'Souza et al., 2010D'Souza B. Meloty-Kapella L. Weinmaster G. Canonical and non-canonical Notch ligands.Curr. Top. Dev. Biol. 2010; 92: 73-129Crossref PubMed Scopus (259) Google Scholar, Fortini and Bilder, 2009Fortini M.E. Bilder D. Endocytic regulation of Notch signaling.Curr. Opin. Genet. Dev. 2009; 19: 323-328Crossref PubMed Scopus (134) Google Scholar). In the absence of endocytosis, ligands accumulate on the cell surface but fail to activate Notch signaling in neighboring cells (Itoh et al., 2003Itoh M. Kim C.H. Palardy G. Oda T. Jiang Y.J. Maust D. Yeo S.Y. Lorick K. Wright G.J. Ariza-McNaughton L. et al.Mind bomb is a ubiquitin ligase that is essential for efficient activation of Notch signaling by Delta.Dev. Cell. 2003; 4: 67-82Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar, Nichols et al., 2007aNichols J.T. Miyamoto A. Olsen S.L. D'Souza B. Yao C. Weinmaster G. DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur.J. Cell Biol. 2007; 176: 445-458Crossref PubMed Scopus (194) Google Scholar, Parks et al., 2000Parks A.L. Klueg K.M. Stout J.R. Muskavitch M.A. Ligand endocytosis drives receptor dissociation and activation in the Notch pathway.Development. 2000; 127: 1373-1385Crossref PubMed Google Scholar, Wang and Struhl, 2004Wang W. Struhl G. Drosophila Epsin mediates a select endocytic pathway that DSL ligands must enter to activate Notch.Development. 2004; 131: 5367-5380Crossref PubMed Scopus (205) Google Scholar), identifying a new paradigm for endocytosis in activation of a signaling pathway. The exact roles that ligand endocytosis serve in Notch signaling have remained poorly defined and controversial. Studies in flies and mammalian cells suggest ligands undergo two distinct endocytic events to activate Notch signaling (D'Souza et al., 2010D'Souza B. Meloty-Kapella L. Weinmaster G. Canonical and non-canonical Notch ligands.Curr. Top. Dev. Biol. 2010; 92: 73-129Crossref PubMed Scopus (259) Google Scholar, Fürthauer and González-Gaitán, 2009Fürthauer M. González-Gaitán M. Endocytic regulation of notch signalling during development.Traffic. 2009; 10: 792-802Crossref PubMed Scopus (56) Google Scholar). The first occurs prior to binding Notch and is proposed to facilitate recycling to generate an active ligand. Specifically, this recycling model proposes nascent ligand delivered to the cell surface cannot activate Notch. Instead, endocytosis and recycling back to the surface are necessary for ligand to effectively bind Notch and activate signaling (Heuss et al., 2008Heuss S.F. Ndiaye-Lobry D. Six E.M. Israël A. Logeat F. The intracellular region of Notch ligands Dll1 and Dll3 regulates their trafficking and signaling activity.Proc. Natl. Acad. Sci. USA. 2008; 105: 11212-11217Crossref PubMed Scopus (72) Google Scholar, Wang and Struhl, 2004Wang W. Struhl G. Drosophila Epsin mediates a select endocytic pathway that DSL ligands must enter to activate Notch.Development. 2004; 131: 5367-5380Crossref PubMed Scopus (205) Google Scholar). Ligand binding strength is critical for a model in which Notch ligands are proposed to generate mechanical force to pull on Notch (Nichols et al., 2007aNichols J.T. Miyamoto A. Olsen S.L. D'Souza B. Yao C. Weinmaster G. DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur.J. Cell Biol. 2007; 176: 445-458Crossref PubMed Scopus (194) Google Scholar, Parks et al., 2000Parks A.L. Klueg K.M. Stout J.R. Muskavitch M.A. Ligand endocytosis drives receptor dissociation and activation in the Notch pathway.Development. 2000; 127: 1373-1385Crossref PubMed Google Scholar). According to this pulling-force model, force produced through ligand endocytosis deforms bound Notch to permit activating proteolysis in the production of a cleaved Notch intracellular form that directly functions as the downstream signal transducer (D'Souza et al., 2010D'Souza B. Meloty-Kapella L. Weinmaster G. Canonical and non-canonical Notch ligands.Curr. Top. Dev. Biol. 2010; 92: 73-129Crossref PubMed Scopus (259) Google Scholar, Gordon et al., 2008Gordon W.R. Arnett K.L. Blacklow S.C. The molecular logic of Notch signaling—a structural and biochemical perspective.J. Cell Sci. 2008; 121: 3109-3119Crossref PubMed Scopus (197) Google Scholar, Nichols et al., 2007bNichols J.T. Miyamoto A. Weinmaster G. Notch signaling—constantly on the move.Traffic. 2007; 8: 959-969Crossref PubMed Scopus (112) Google Scholar). Additionally, studies in flies and mammalian cells suggest roles for endocytosis and recycling in the trafficking of ligand to specific cell surface microdomains to potentiate either affinity or availability for Notch (Benhra et al., 2010Benhra N. Vignaux F. Dussert A. Schweisguth F. Le Borgne R. Neuralized promotes basal to apical transcytosis of delta in epithelial cells.Mol. Biol. Cell. 2010; 21: 2078-2086Crossref PubMed Scopus (50) Google Scholar, Heuss et al., 2008Heuss S.F. Ndiaye-Lobry D. Six E.M. Israël A. Logeat F. The intracellular region of Notch ligands Dll1 and Dll3 regulates their trafficking and signaling activity.Proc. Natl. Acad. Sci. USA. 2008; 105: 11212-11217Crossref PubMed Scopus (72) Google Scholar, Rajan et al., 2009Rajan A. Tien A.C. Haueter C.M. Schulze K.L. Bellen H.J. The Arp2/3 complex and WASp are required for apical trafficking of Delta into microvilli during cell fate specification of sensory organ precursors.Nat. Cell Biol. 2009; 11: 815-824Crossref PubMed Scopus (80) Google Scholar). Whether ligand endocytosis regulates recycling or mechanical force to activate Notch signaling is currently unknown. However, not all Notch-dependent developmental events require ligand recycling raising the possibility that recycling is not a general core requirement for signaling activity (Banks et al., 2011Banks S.M. Cho B. Eun S.H. Lee J.H. Windler S.L. Xie X. Bilder D. Fischer J.A. The functions of auxilin and Rab11 in Drosophila suggest that the fundamental role of ligand endocytosis in notch signaling cells is not recycling.PLoS ONE. 2011; 6: e18259Crossref PubMed Scopus (24) Google Scholar, Jafar-Nejad et al., 2005Jafar-Nejad H. Andrews H.K. Acar M. Bayat V. Wirtz-Peitz F. Mehta S.Q. Knoblich J.A. Bellen H.J. Sec15, a component of the exocyst, promotes notch signaling during the asymmetric division of Drosophila sensory organ precursors.Dev. Cell. 2005; 9: 351-363Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, Windler and Bilder, 2010Windler S.L. Bilder D. Endocytic internalization routes required for delta/notch signaling.Curr. Biol. 2010; 20: 538-543Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In this study, we directly tested whether ligand endocytosis functions to promote recycling to strengthen Notch binding. To this end, we developed a cell-bead optical tweezers assay to obtain rupture force measurements specific for cells expressing the Notch ligand Delta-like1 (Dll1) bound to laser-trapped Notch1 (N1) beads. Our biophysical experiments indicate that neither endocytosis nor recycling strengthens ligand binding to N1, eliminating a role for endocytosis in ligand affinity prior to interactions with N1. In the accompanying article (Meloty-Kapella et al., 2012Meloty-Kapella L. Shergill B. Kuon J. Botvinick E. Weinmaster G. Notch ligand endocytosis generates mechanical pulling force dependent on dynamin, epsins and actin.Dev. Cell. 2012; 22 (this issue): 1299-1312Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), we extend this biophysical approach to obtain support for a primary role for ligand endocytosis in generating mechanical pulling force downstream of Notch binding. To determine if ligand endocytosis or recycling regulate ligand binding to Notch we used a cell-bead optical tweezers assay. Interactions between Drosophila and mammalian Notch ligands and receptors have been demonstrated using cell aggregation (Fehon et al., 1990Fehon R.G. Kooh P.J. Rebay I. Regan C.L. Xu T. Muskavitch M.A. Artavanis-Tsakonas S. Molecular interactions between the protein products of the neurogenic loci Notch and Delta, two EGF-homologous genes in Drosophila.Cell. 1990; 61: 523-534Abstract Full Text PDF PubMed Scopus (542) Google Scholar, Parks et al., 2006Parks A.L. Stout J.R. Shepard S.B. Klueg K.M. Dos Santos A.A. Parody T.R. Vaskova M. Muskavitch M.A. Structure-function analysis of delta trafficking, receptor binding and signaling in Drosophila.Genetics. 2006; 174: 1947-1961Crossref PubMed Scopus (49) Google Scholar, Rebay et al., 1991Rebay I. Fleming R.J. Fehon R.G. Cherbas L. Cherbas P. Artavanis-Tsakonas S. Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor.Cell. 1991; 67: 687-699Abstract Full Text PDF PubMed Scopus (602) Google Scholar) and binding assays (Heuss et al., 2008Heuss S.F. Ndiaye-Lobry D. Six E.M. Israël A. Logeat F. The intracellular region of Notch ligands Dll1 and Dll3 regulates their trafficking and signaling activity.Proc. Natl. Acad. Sci. USA. 2008; 105: 11212-11217Crossref PubMed Scopus (72) Google Scholar, Hicks et al., 2002Hicks C. Ladi E. Lindsell C. Hsieh J.J. Hayward S.D. Collazo A. Weinmaster G. A secreted Delta1-Fc fusion protein functions both as an activator and inhibitor of Notch1 signaling.J. Neurosci. Res. 2002; 68: 655-667Crossref PubMed Scopus (106) Google Scholar, Nichols et al., 2007aNichols J.T. Miyamoto A. Olsen S.L. D'Souza B. Yao C. Weinmaster G. DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur.J. Cell Biol. 2007; 176: 445-458Crossref PubMed Scopus (194) Google Scholar, Shimizu et al., 1999Shimizu K. Chiba S. Kumano K. Hosoya N. Takahashi T. Kanda Y. Hamada Y. Yazaki Y. Hirai H. Mouse jagged1 physically interacts with notch2 and other notch receptors. Assessment by quantitative methods.J. Biol. Chem. 1999; 274: 32961-32969Crossref PubMed Scopus (202) Google Scholar). Moreover, atomic force microscopy (AFM) measurement of rupture between Drosophila cells programmed to express Delta and Notch have reported force in the nanoNewton range (Ahimou et al., 2004Ahimou F. Mok L.P. Bardot B. Wesley C. The adhesion force of Notch with Delta and the rate of Notch signaling.J. Cell Biol. 2004; 167: 1217-1229Crossref PubMed Scopus (71) Google Scholar). In contrast to these cell-based studies, the affinities reported for recombinant protein fragments containing Notch ligand and receptor binding domains are weak or undetectable (Cordle et al., 2008aCordle J. Johnson S. Tay J.Z. Roversi P. Wilkin M.B. de Madrid B.H. Shimizu H. Jensen S. Whiteman P. Jin B. et al.A conserved face of the Jagged/Serrate DSL domain is involved in Notch trans-activation and cis-inhibition.Nat. Struct. Mol. Biol. 2008; 15: 849-857Crossref PubMed Scopus (196) Google Scholar, Cordle et al., 2008bCordle J. Redfieldz C. Stacey M. van der Merwe P.A. Willis A.C. Champion B.R. Hambleton S. Handford P.A. Localization of the delta-like-1-binding site in human Notch-1 and its modulation by calcium affinity.J. Biol. Chem. 2008; 283: 11785-11793Crossref PubMed Scopus (76) Google Scholar). None of these studies, however, have directly tested requirements for ligand endocytosis and recycling in Notch ligand-receptor interactions. Toward this goal, we replaced the Notch cell with a bead functionalized with recombinant Notch1 (N1) protein containing the ligand-binding domain fused to human Immunoglobulin G (IgG)-Fc (N1Fc). N1Fc specifically binds Dll1 expressing cells (Nichols et al., 2007aNichols J.T. Miyamoto A. Olsen S.L. D'Souza B. Yao C. Weinmaster G. DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur.J. Cell Biol. 2007; 176: 445-458Crossref PubMed Scopus (194) Google Scholar) and optical tweezers can present beads to live cells for accurate measurement of bond strength (Weisel et al., 2003Weisel J.W. Shuman H. Litvinov R.I. Protein-protein unbinding induced by force: single-molecule studies.Curr. Opin. Struct. Biol. 2003; 13: 227-235Crossref PubMed Scopus (99) Google Scholar). To determine if endocytosis or recycling enhance intrinsic ligand binding strength independent of avidity (Weisel et al., 2003Weisel J.W. Shuman H. Litvinov R.I. Protein-protein unbinding induced by force: single-molecule studies.Curr. Opin. Struct. Biol. 2003; 13: 227-235Crossref PubMed Scopus (99) Google Scholar), it was imperative to minimize Dll1 clustering in cells following interactions with N1Fc-beads. In fact, the exceptionally strong AFM forces reported (Ahimou et al., 2004Ahimou F. Mok L.P. Bardot B. Wesley C. The adhesion force of Notch with Delta and the rate of Notch signaling.J. Cell Biol. 2004; 167: 1217-1229Crossref PubMed Scopus (71) Google Scholar), likely reflect multivalent interactions between cells engineered to express high levels of Delta and Notch. To avoid N1-induced ligand clustering and facilitate single Dll1-N1 interaction measurements, ProteinA (PrtA) microbeads were coated with low N1Fc concentrations (Supplemental Experimental Procedures available online). With optical tweezers, the position of the bead within the optical trap can be precisely monitored over time (s) and used to calculate the force (pN) required to rupture interactions between N1Fc-beads and Dll1 cells (Figure 1A and Supplemental Experimental Procedures). Media containing 0.5 ug N1Fc/ml produced functionalized beads that could be repeatedly bound and detached from Dll1 cells, which allowed serial measurements of rupture force between a single live cell and a laser-trapped bead. These modifications to a published method (Litvinov et al., 2002Litvinov R.I. Shuman H. Bennett J.S. Weisel J.W. Binding strength and activation state of single fibrinogen-integrin pairs on living cells.Proc. Natl. Acad. Sci. USA. 2002; 99: 7426-7431Crossref PubMed Scopus (148) Google Scholar) allowed collection of large data sets for statistical analyses to accurately determine rupture force as a measure of bond strength. When N1Fc-beads interacted with Dll1 cells the majority of rupture events occurred between 0 and 40 pN (Figure S1A). To establish the specificity of these measurements, we tested conditions in which Dll1-N1 binding would not occur: Dll1 cells paired with Fc- or PrtA-beads (with and without BSA) and parental L cells paired with N1Fc-, Fc-, or PrtA/BSA-beads. All negative control spectra contain one large mode centered at a few pN (containing the maximum viscous drag force of ∼0.6 pN estimated by Stokes flow and verified in cell-free experiments shown in Figure S1B) and a shorter shoulder bound by 12 pN, representing nonspecific binding and rupture events (Figure 1B and inset). In support of this idea, spectra obtained for Dll1 cells bound to N1Fc-beads contained additional force modes with means of ∼19 and 36 pN, suggestive of specific Dll1-N1 interactions. Further confirming the specificity of these measurements, L cells transiently expressing Dll1C284Y carrying the missense mutation reported to eliminate Delta binding to Notch in flies (Parks et al., 2006Parks A.L. Stout J.R. Shepard S.B. Klueg K.M. Dos Santos A.A. Parody T.R. Vaskova M. Muskavitch M.A. Structure-function analysis of delta trafficking, receptor binding and signaling in Drosophila.Genetics. 2006; 174: 1947-1961Crossref PubMed Scopus (49) Google Scholar), did not display the two higher force modes detected for Dll1 cells (Figure 1C). Consistent with the inability to bind N1Fc, Dll1C284Y cells displayed reduced soluble N1Fc binding similar to that detected for L cell controls (Figures S1C and S1D). Single-molecule interactions were promoted by limiting the bead-cell contact time to 20 ms. If multiple interactions formed within 20 ms, we would have expected to detect stepwise rupture as previously reported (Litvinov et al., 1994Litvinov S.V. Velders M.P. Bakker H.A. Fleuren G.J. Warnaar S.O. Ep-CAM: a human epithelial antigen is a homophilic cell-cell adhesion molecule.J. Cell Biol. 1994; 125: 437-446Crossref PubMed Scopus (464) Google Scholar, Litvinov et al., 2002Litvinov R.I. Shuman H. Bennett J.S. Weisel J.W. Binding strength and activation state of single fibrinogen-integrin pairs on living cells.Proc. Natl. Acad. Sci. USA. 2002; 99: 7426-7431Crossref PubMed Scopus (148) Google Scholar), rather than the observed steep rupture events (Figure 1A). Additionally, we predict that our laser tweezers cannot break more than four parallel bonds; assuming 19 pN to be the single-bond rupture force and considering that our maximum laser tweezers force is ∼90 pN. In support of this, N1Fc-beads can be consistently pulled away from Dll1 cells at forces too weak to pull Dll1 out of the membrane (Shao and Hochmuth, 1999Shao J.Y. Hochmuth R.M. Mechanical anchoring strength of L-selectin, beta2 integrins, and CD45 to neutrophil cytoskeleton and membrane.Biophys. J. 1999; 77: 587-596Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Poisson distribution statistics were used to determine if the 19 pN force mode corresponds to rupture of single Dll1-N1 interactions. Nonspecific rupture events account for 70% or more of our data, which corresponds to a rate of specific bond formation of 30% or lower, proposed to promote single molecule events (Panorchan et al., 2006Panorchan P. Thompson M.S. Davis K.J. Tseng Y. Konstantopoulos K. Wirtz D. Single-molecule analysis of cadherin-mediated cell-cell adhesion.J. Cell Sci. 2006; 119: 66-74Crossref PubMed Scopus (168) Google Scholar). In our rupture force data for Dll1 cells bound to N1Fc-beads (Figure 1B), the rate of bond formation was 10.5% with 62% in the putative single bond mode and 38% in the double bond mode. Poisson distribution statistics (Tees et al., 2001Tees D.F. Waugh R.E. Hammer D.A. A microcantilever device to assess the effect of force on the lifetime of selectin-carbohydrate bonds.Biophys. J. 2001; 80: 668-682Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), however, predict a higher rate of single (95%) and lower rate of double (5%) bonds than observed. Because Fc sequences dimerize N1Fc to produce two potential Dll1 binding sites, we wondered if dimer formation enhances the probability of double bond formation (Figure 1D). If this were the case, spectra should conform to Poisson distribution statistics when cells express low levels of Dll1 and can only form single Dll1-N1 interactions. To directly test this idea, we measured rupture force for cells in which the level of Dll1 expression could be modulated using doxycycline (dox) induction (Sprinzak et al., 2010Sprinzak D. Lakhanpal A. Lebon L. Santat L.A. Fontes M.E. Anderson G.A. Garcia-Ojalvo J. Elowitz M.B. Cis-interactions between Notch and Delta generate mutually exclusive signalling states.Nature. 2010; 465: 86-90Crossref PubMed Scopus (436) Google Scholar). Dll1 cell surface levels are low in the absence of dox, which likely accounts for the majority of rupture force measurements occurring within the nonspecific range (<12 pN) (Figure 1E). In contrast, cells cultured in 1 ng/ml dox showed an 8% rate of specific bond formation, of which 98% fall within the putative single bond mode (Poisson distribution statistics predicts 96%), whereas 2% fall within the double bond mode (Poisson distribution statistics predicts 4%). Therefore, at this low Dll1 cell surface level rupture force spectra are indeed consistent with a Poisson distribution model, indicating the 19 pN mode represents single bond ruptures. At 10 ng/ml dox, induced surface levels are similar to stable expressing Dll1 cells (see Figure 4), and the rate of bond formation was 30%, with 71% single (Poisson distribution statistics predicts 83%) and 29% double bond formation (Poisson distribution statistics predicts 15%). Thus, as ligand expression increases, the relative probabilities deviate from predicted, however, the 19 pN mode persists supporting our claim that it represents single-molecule Dll1-N1 rupture events. To further demonstrate the 19 pN mode corresponds to single-bond ruptures, we measured rupture force spectra for Dll1 cells interacting with N1Fc-beads coated with conditioned media containing decreasing N1Fc and increasing Fc proteins (see Supplemental Experimental Procedures for details). As N1Fc concentration decreased from 0.5 μg/ml (Figures 1B and 1C) to 0.25 μg/ml and 0.1 μg/ml (Figure 1F), the probability of rupture events in the first specific mode increased (62%, 76%, and 84%, respectively), whereas those in the second decreased (38%, 24%, 16%, respectively), consistent with the 19 pN mode corresponding to single ligand-receptor interactions. At 0.005 μg/ml the rate of bond formation was 0.3% and neither mode was detected, underscoring the Dll1-N1 specificity of these rupture force measurements. We next addressed the possibility that either of the two specific modes represents rupture of PrtA-Fc bonds (Figure 1D, x). The rupture force between rabbit IgG and PrtA has been reported to be ∼30 pN at our typical loading rate of 250 pN/s (Salomo et al., 2008Salomo M. Keyser U.F. Struhalla M. Kremer F. Optical tweezers to study single protein A/immunoglobulin G interactions at varying conditions.Eur. Biophys. J. 2008; 37: 927-934Crossref PubMed Scopus (15) Google Scholar). Laser tweezers analyses of IgG-PrtA rupture force for rabbit, mouse, bovine, and goat IgG identified a median rupture force ranging from 25 to 44 pN (Stout, 2001Stout A.L. Detection and characterization of individual intermolecular bonds using optical tweezers.Biophys. J. 2001; 80: 2976-2986Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), measured at loading rates ranging from 400 to 5300 pN/s. Considering these findings, the second specific mode in our system could represent rupture of PrtA-Fc interactions. To test this, N1Fc attached to PrtA-beads was chemically cross-linked (see Supplemental Experimental Procedures). Rupture force analysis failed to show a change in the 19 pN rupture mode (p > 0.05) and only <1 pN change was detected for the second specific mode (Figure 1G). If the second specific mode represented PrtA-Fc bond rupture, it should be significantly reduced or eliminated following covalent cross-linking. Importantly, detection of the second specific mode further supports specificity of the Dll1-N1 interactions. Lastly, this analysis suggests that the density of N1Fc on the bead must be too low for the cross-linking to cluster N1Fc, further indicating that the first specific rupture mode represents measurement of single-molecule binding events. To directly test whether endocytosis influences Dll1 bond strength, cells expressing a Dll1 lacking intracellular domain sequences (OCDD1) that is consequently defective in endocytosis were assayed. Important for this analysis, OCDD1 binds N1 but is unable to activate signaling in coculture assays (Nichols et al., 2007aNichols J.T. Miyamoto A. Olsen S.L. D'Souza B. Yao C. Weinmaster G. DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur.J. Cell Biol. 2007; 176: 445-458Crossref PubMed Scopus (194) Google Scholar). Rupture force spectra obtained for OCDD1 cells interacting with N1Fc-beads compared to control Fc-beads identified both specific modes (Figure 2A ). Comparison of rupture force spectra for Dll1 and OCDD1 cells did not identify significant reduction in the mean value of the first specific mode (Figure 2B, p > 0.05) as would be predicted if endocytosis is necessary for ligand binding to Notch. The taller peaks observed for OCDD1 likely reflect cell surface accumulation of this endocytic defective protein (Figure 4B and Nichols et al., 2007aNichols J.T. Miyamoto A. Olsen S.L. D'Souza B. Yao C. Weinmaster G. DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur.J. Cell Biol. 2007; 176: 445-458Crossref PubMed Scopus (194) Google Scholar), which would increase the probability of forming interactions as observed with higher Dll1 expression (Figure 1E). In fact, concentration dependent increases in binding probability have been reported for fibrinogen-integrin binding with activated platelets (Litvinov et al., 2002Litvinov R.I. Shuman H. Bennett J.S. Weisel J.W. Binding strength and activation state of single fibrinogen-integrin pairs on living cells.Proc. Natl. Acad. Sci. USA. 2002; 99: 7426-7431Crossref PubMed Scopus (148) Google Scholar). Genetic mosaic analysis of Drosophila shibire indicates a requirement for the key endocytic factor dynamin by Delta cells in Notch activation (Seugnet et al., 1997Seugnet L. Simpson P. Haenlin M. Requirement for dynamin during Notch signaling in Drosophila neurogenesis.Dev. Biol. 1997; 192: 585-598Crossref PubMed Scopus (225) Google Scholar). Dll1 cells expressing a dominant-negative dynaminK44A (Damke et al., 2001Damke H. Binns D.D. Ueda H. Schmid S.L. Baba T. Dynamin GTPase domain mutants block endocytic vesicle formation at morphologically distinct stages.Mol. Biol. Cell. 2001; 12: 2578-2589Crossref PubMed Scopus (149) Google Scholar) are defective in endocytosis and activation of Notch signaling (Nichols et al., 2007aNichols J.T. Miyamoto A. Olsen S.L. D'Souza B. Yao C. Weinmaster G. DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur.J. Cell Biol. 2007; 176: 445-458Crossref PubMed Scopus (194) Google Scholar), and display reduced internalization of transferrin that requires dynamin (Figure S2B). The mean value of the single-molecule rupture force mode detected for Dll1 cells expressing dynaminK44A-eGFP or eGFP alone are statistically equivalent (p > 0.05; Figure 2C), indicating endocytosis is not a determinant of Dll1-N1 bond strength. A caveat of this interpretation is the possible induction of alternative modes of endocytosis by cells exposed to sustained perturbation in dynamin activity (Damke et al., 1995Damke H. Baba T. van der Bliek A.M. Schmid S.L. Clathrin-independent pinocytosis is induced in cells overexpressing a temperature-sensitive mutant of dynamin.J. Cell Biol. 1995; 131: 69-80Crossref PubMed Scopus (344) Google Scholar, Ferguson et al., 2009Ferguson S.M. Raimondi A. Paradise S. Shen H. Mesaki K. Ferguson A. Destaing O. Ko G. Takasaki J. Cremona O. et al.Coordinated action" @default.
• W2078742722 created "2016-06-24" @default.
• W2078742722 creator A5014962538 @default.
• W2078742722 creator A5053113346 @default.
• W2078742722 creator A5065365756 @default.
• W2078742722 creator A5073460010 @default.
• W2078742722 creator A5080777821 @default.
• W2078742722 date "2012-06-01" @default.
• W2078742722 modified "2023-10-12" @default.
• W2078742722 title "Optical Tweezers Studies on Notch: Single-Molecule Interaction Strength Is Independent of Ligand Endocytosis" @default.
• W2078742722 cites W1508615665 @default.
• W2078742722 cites W159146810 @default.
• W2078742722 cites W1873165927 @default.
• W2078742722 cites W1964839598 @default.
• W2078742722 cites W1968226123 @default.
• W2078742722 cites W1969139128 @default.
• W2078742722 cites W1970683213 @default.
• W2078742722 cites W1975771868 @default.
• W2078742722 cites W1984870208 @default.
• W2078742722 cites W1997177399 @default.
• W2078742722 cites W2000063663 @default.
• W2078742722 cites W2003968931 @default.
• W2078742722 cites W2006107567 @default.
• W2078742722 cites W2011682997 @default.
• W2078742722 cites W2013351373 @default.
• W2078742722 cites W2013595896 @default.
• W2078742722 cites W2020036904 @default.
• W2078742722 cites W2023100673 @default.
• W2078742722 cites W2025797971 @default.
• W2078742722 cites W2032172929 @default.
• W2078742722 cites W2039092330 @default.
• W2078742722 cites W2043854809 @default.
• W2078742722 cites W2046661558 @default.
• W2078742722 cites W2055555352 @default.
• W2078742722 cites W2058693333 @default.
• W2078742722 cites W2061996746 @default.
• W2078742722 cites W2078507150 @default.
• W2078742722 cites W2081389905 @default.
• W2078742722 cites W2084520007 @default.
• W2078742722 cites W2084694780 @default.
• W2078742722 cites W2094481526 @default.
• W2078742722 cites W2106853755 @default.
• W2078742722 cites W2107801352 @default.
• W2078742722 cites W2112032807 @default.
• W2078742722 cites W2114486946 @default.
• W2078742722 cites W2116136455 @default.
• W2078742722 cites W2126674221 @default.
• W2078742722 cites W2129096575 @default.
• W2078742722 cites W2138957442 @default.
• W2078742722 cites W2139000795 @default.
• W2078742722 cites W2145206503 @default.
• W2078742722 cites W2151727956 @default.
• W2078742722 cites W2153045264 @default.
• W2078742722 cites W2159418321 @default.
• W2078742722 cites W2167505338 @default.
• W2078742722 cites W2170651104 @default.
• W2078742722 cites W4214922488 @default.
• W2078742722 doi "https://doi.org/10.1016/j.devcel.2012.04.007" @default.
• W2078742722 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3376165" @default.
• W2078742722 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/22658935" @default.
• W2078742722 hasPublicationYear "2012" @default.
• W2078742722 type Work @default.
• W2078742722 sameAs 2078742722 @default.
• W2078742722 citedByCount "66" @default.
• W2078742722 countsByYear W20787427222012 @default.
• W2078742722 countsByYear W20787427222013 @default.
• W2078742722 countsByYear W20787427222014 @default.
• W2078742722 countsByYear W20787427222015 @default.
• W2078742722 countsByYear W20787427222016 @default.
• W2078742722 countsByYear W20787427222017 @default.
• W2078742722 countsByYear W20787427222018 @default.
• W2078742722 countsByYear W20787427222019 @default.
• W2078742722 countsByYear W20787427222020 @default.
• W2078742722 countsByYear W20787427222021 @default.
• W2078742722 countsByYear W20787427222022 @default.
• W2078742722 countsByYear W20787427222023 @default.
• W2078742722 crossrefType "journal-article" @default.
• W2078742722 hasAuthorship W2078742722A5014962538 @default.
• W2078742722 hasAuthorship W2078742722A5053113346 @default.
• W2078742722 hasAuthorship W2078742722A5065365756 @default.
• W2078742722 hasAuthorship W2078742722A5073460010 @default.
• W2078742722 hasAuthorship W2078742722A5080777821 @default.
• W2078742722 hasBestOaLocation W20787427221 @default.
• W2078742722 hasConcept C116569031 @default.
• W2078742722 hasConcept C120665830 @default.
• W2078742722 hasConcept C121332964 @default.
• W2078742722 hasConcept C12554922 @default.
• W2078742722 hasConcept C161879069 @default.
• W2078742722 hasConcept C170493617 @default.
• W2078742722 hasConcept C20198109 @default.
• W2078742722 hasConcept C28005876 @default.
• W2078742722 hasConcept C32909587 @default.
• W2078742722 hasConcept C54355233 @default.
• W2078742722 hasConcept C62478195 @default.
• W2078742722 hasConcept C62520636 @default.
• W2078742722 hasConcept C86803240 @default.
• W2078742722 hasConcept C89804040 @default.
• W2078742722 hasConcept C93275456 @default.

• next