Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies
- Howard Hughes Medical Institute, Harvard Medical School, United States
- Harvard Medical School, United States
Figures
Figure 1 with 2 supplements
Isolation of a destabilized Nb whose protein level depends upon antigen co-expression.
(A) Concept of antigen-controlled protein stabilization. FP, fusion protein; An, antigen. (B) Strategy for isolating dNbs. LTR, long terminal repeat. (C) GFP-dependent stabilization of dGBP1 tagged …
Figure 1—figure supplement 1
YFP-dependent dGBP1-TagBFP stabilization in cells.
(A) Comparison of background fluorescence between two GBP1 variants. Constructs were expressed using MMLV-based vectors, which were transfected into 293T cells. Images were acquired 2 days …
Figure 1—figure supplement 2
Detection of antigen-expressing cells with dNb in vivo.
Quantification of results from Figure 1G,H. (A) Tight coupling of GFP expression (green) and Anti-TagBFP staining (red) from ONL cells in the +CAG-GFP condition. Scale bar is 20 μm. (B–E). …
Figure 2 with 3 supplements
dGBP1 destabilizing mutations can be transferred to Nbs derived from different species to create antigen-dependent stability.
(A) Protein alignment of Nbs against GFP (GBP1), HIV-1 CA (αCA) and E.coli DHFR (αDHFR). Amino acid positions numbered according to the ImMunoGeneTics information system (IMGT). FR, framework; CDR, …
-
Figure 2—source data 1
Source data for fluorescence quantifications of Nb-TagBFP tests.
This excel file contains numerical values for the % Nb-TagBFP fluorescence parameter shown as a colored heat map in Figure 2G.
- https://doi.org/10.7554/eLife.15312.007
Figure 2—figure supplement 1
Mapping of mutations necessary for dGBP1 destabilization.
(A) Representative images showing expression of GBP1 variants tagged with mCherry in 293T cells. Images taken 17 hr post-transfection. Scale bar, 50 μm. (B) Semi-quantitative summary of mCherry …
Figure 2—figure supplement 2
Mapping mutations sufficient for dGBP1 destabilization.
(A) Representative images showing expression of GBP1 variants tagged with mCherry in 293T cells. Images taken 17 hr post-transfection. Scale bar, 50 μm. (B) Semi-quantitative summary of mCherry …
Figure 2—figure supplement 3
Generation of dNbs by mutation transfer.
(A) Conservation of dGBP1 mutations across 76 Nbs derived from Camelus dromedarius, Llama glama and Vicugna pacos. (B) Mapping of Nb destabilizing positions in relation to binding interfaces across …
Figure 3 with 1 supplement
Generation and optimization of antigen-specific effectors based on dNbs.
(A) Reporter assay of transfected 293T cells testing dGBP1-Cre fusion constructs for activation of a Cre-dependent luciferase construct. (B) Reporter assay of transfected 293T cells testing …
Figure 3—figure supplement 1
Additional information on antigen-specific effectors.
(A–B) Plasmids encoding unmodified or destabilized GBP1 fusion to Cre or Flpo were transfected into 293T cells along with CAG-driven, loxP-Neo-loxP- (LNL-) or FRT-Neo-FRT- (FNF-) DsRed. Cre and Flpo …
Figure 4
Intracellular antigen can trigger genome editing via dNbs.
(A) Schematic of Cas9 fusion protein inducible by C-CA binding. (B) Fusion configuration of tandemly repeated C-CA Nb to Cas9, giving dCC-Cas9. (C) dCC-Cas9 activity was assayed for βgal expression …
Figure 5 with 2 supplements
Applying Flp-DOG for optogenetic manipulation of transgenic GFP-labeled cell types in the mouse cerebellum.
(A) rAAV reagents for (B–D). (B) Schematic showing delivery of rAAVs to the mouse cerebellum for cell type-specific manipulation in Tg(GAD67-GFP) animals. (C) Representative image showing that …
Figure 5—figure supplement 1
Retrofitting transgenic GFP line for cell-specific gene manipulation in the mouse retina.
(A) Schematic of electroporation experiment. CAG-nlacZ was an electroporation marker. (B) Electroporation of CAG-driven Flp-DOG into Tg(CRX-GFP) retinas resulted in strong activation of FNF-DsRed …
Figure 5—figure supplement 2
Characterization of mouse cerebella infected with AAV-Flp-DOG.
(A) Reagents for (B–D). (B) Schematic showing delivery of rAAVs and green fluorescent beads to the mouse cerebellum for cell type-specific manipulation in Tg(GAD67-GFP) animals. (C) rAAV-delivered, …
Figure 6 with 1 supplement
Detection of HIV-1 reactivated cells with a CA-specific sensor.
(A) Schematic showing isolation of HIV-1 cells via flow cytometry using αCA-specific, dNb sensor. Both ACH-2 (HIV+) and CEM (HIV-) cells were treated with 10 nM PMA prior to transfection of sensors. …
Figure 6—figure supplement 1
dNb sensor against HIV-1 CA enables detection and isolation of reactivated HIV-1+ cells with flow cytometry.
(A) Schematic showing isolation of HIV-1 cells via flow cytometry using HIV-1 CA-specific, red fluorescent sensor. (B) Destabilized, but not unmodified αCA Nb enabled selective isolation of …
Tables
Table 1
List of tested nanobodies and their associated antigens.
| Nb PDB code | Species of origin | Tested antigen | Antigen species/pathogen | Endogenous location of epitope |
|---|---|---|---|---|
| 3K1K | C.dro | GFP | Aequorea victoria | Intracellular |
| YFP | Aequorea victoria | Intracellular | ||
| YFP-FLAG | Aequorea victoria | Intracellular | ||
| 2XV6 | V.pacos | Capsid protein p24 C-terminal domain, residues 278-352 of gag polyprotein | HIV-1 | Intracellular |
| Capsid protein p24 | HIV-1 | Intracellular | ||
| 4EIG | L.glama | Dihydrofolate reductase | Escherichia coli | Intracellular |
| 4TVS | V.pacos | Torsin-1A-interacting protein 1, UNP residues 356-583 | Homo sapiens | Intracellular |
| 4EIZ | L.glama | Dihydrofolate reductase | Escherichia coli | Intracellular |
| 4FHB | L.glama | Dihydrofolate reductase | Escherichia coli | Intracellular |
| 4QO1 | L.glama | Cellular tumor antigen p53 DBD, UNP residues 92-312 | Homo sapiens | Intracellular |
| 3K7U | L.glama | MP18 RNA editing protein | Trypanosoma brucei | Intracellular |
| 4GFT | L.glama | Myosin A tail domain interacting protein C-terminal domain, UNP residues 137-204 | Plasmodium falciparum | Intracellular |
| 4C57 | L.glama | Cyclin-G associated kinase, kinase domain residues 14-351 | Homo sapiens | Intracellular |
-
C.dro: Camelus dromedarius; L.glama: Lama glama; V.pacos: Vicugna pacos.
