Streptavidin interfacing as a general strategy to localize fluorescent membrane tension probes in cells

Abstract

Site-specific labeling with biotinylated mechanophores is probed to address the next challenge toward the imaging of forces in cells.

Twisting and tilting of a mechanosensitive molecular probe detects order in membranes

“…5 In the last few years, a new class of environment-sensitive dyes have been introduced to non-invasively track lateral forces and lipid domains in membranes. [6][7][8][9][10][11][12][13][14][15] Dithienothiophene (DTT)-based push-pull mechanosensors termed "ippers" are characterized by an excitation band that shis to lower energy when partitioned in ordered lipid environments. These probes appear to respond to lateral forces in lipid bilayers, thereby discriminating between ordered and disordered lipid phases, as shown experimentally by uorescence imaging of unilamellar vesicles.…”

Genetically Encoded Supramolecular Targeting of Fluorescent Membrane Tension Probes within Live Cells: Precisely Localized Controlled Release by External Chemical Stimulation

“…The biotinylated probe 6 ( Figure 1 B) has already been reported to validate Sav as a bioorthogonal connector between the mechanosensitive unit and a biotinylated lipid membrane. 56 However, a very strong, quasi -irreversible binding of biotin with Sav impedes any possible selective release, at least in conditions compatible with living cells. Moreover, the biotin residue in probe 6 is very prone to oxidation into the corresponding sulfoxides under ambient conditions, which complicates the functional characterization of this probe (data not shown).…”

Multifunctional streptavidin–biotin conjugates with precise stoichiometries

“…In a rst step, to produce stoichiometrically dened streptavidin conjugates with one functionality, streptavidin, (S ¼ 30 mM) was incubated with a biotin conjugated His6-tag peptide (biotin-(His) 6 , Bio-His-Tag ¼ 90 mM), which yielded a statistical mixture of S, S(Bio-His-Tag) 1 , S(Bio-His-Tag) 2 , S(Bio-His-Tag) 3 and S(Bio-His-Tag) 4 . Subsequently, the mixture was passed over a Ni 2+ -NTA agarose column; unbound molecules without Histags such as S were washed off and different species were eluted using an imidazole gradient, where species bearing more tags eluted at higher imidazole concentrations.…”

“…14,15 The streptavidin conjugate in the rst peak required 3 equivalents of the dye to saturate all biotin binding sites and therefore was assigned as the S(Bio-His-Tag) 1 . Likewise, the molecules in the second, third and fourth peaks required 2, 1 and 0 equivalents of dye to saturate all biotin binding sites, respectively, and they corresponded to S(Bio-His-Tag) 2 , S(Bio-His-Tag) 3 and S(Bio-His-Tag) 4 , respectively. This assignment is also consistent with streptavidin conjugates eluting at higher imidazole concentrations having more Bio-His-Tags.…”

“…The binding of streptavidin to biotin is well known for the strong noncovalent interaction with femtomolar affinity (K d ¼ 10 À14 ). 1 The high affinity, slow exchange rate, and good specicity of the biotin-streptavidin interaction has resulted in a wide range of biotechnological applications including extracellular and in vitro labelling, 2,3 therapeutics, biosensing and biofunctionalization. 4,5 The large range of biotinylated small molecules, peptides, proteins, antibodies and nucleic acids as well as materials adds to the diversity of the biotin-streptavidin chemistry.…”