A functionally neutral single chain antibody to measure beta‐1 integrin uptake and recycling

Abstract Integrin‐mediated cell adhesion and signaling are critical for many physiological processes. The dynamic turnover of integrins and their associated adhesion complexes through endocytic and recycling pathways has emerged as an important mechanism for controlling cell migration and invasion in cancer. Thus, the regulation of integrin trafficking and how this may be altered by disease‐specific molecular mechanisms has generated considerable interest. However, current tools available to study integrin trafficking may cause artifacts and/or do not provide adequate kinetic information. Here, we report the generation of a functionally neutral and monovalent single chain antibody to quantitatively and qualitatively measure β1 integrin trafficking in cells. Our novel probe can be used in a variety of assays and allows for the biochemical characterization of rapid recycling of endogenous integrins. We also demonstrate its potential utility in live cell imaging, providing proof of principle to guide future integrin probe design.


| INTRODUCTION
In multicellular organisms, cells must sense and interpret contextual information received through direct physical interactions with the extracellular matrix (ECM) in order to maintain proper cellular identity.
The formation of integrin-based cell-ECM adhesion complexes is essential during development and often dysregulated in pathological conditions, including cancer. Integrins are abundant cell surface αβ heterodimeric adhesion receptors that anchor the intracellular cytoskeleton to the ECM. 1 Integrins can adopt a bent/closed (inactive) conformation, or an extended/open (active) conformation, with possible intermediate "priming" states. 2 Integrin conformation and activity status can be influenced by either ECM/ligand binding ("outside-in") or macromolecular protein complex recruitment and binding to the integrin cytoplasmic tails ("inside-out"), allowing integrins to signal bidirectionally across the plasma membrane. 1 The signaling driven by these integrin conformational changes plays pivotal roles in cell survival and proliferation, and can also directly modulate the cytoskeleton and control cell motility.
Intracellular trafficking is responsible for the differential targeting and distribution of membrane-associated proteins within the cell. 3 The dynamic turnover of cell surface integrins through endocytic and recycling pathways has emerged as a key mechanism in regulating integrin function. 4,5 Integrins have been shown to follow several endocytic pathways, including clathrin-6-10 and caveolin-dependent [11][12][13] endocytosis, clathrin-independent endocytosis 14,15 and macropinocytosis. 16 Once internalized, integrins are routed to early endosomes, in which initial sorting decisions are made that determine whether the integrins are sent for degradation or recycled back to the plasma membrane. Generally, internalized integrins are not degraded but are recycled by either a direct "fast" recycling route or indirectly through the "long loop" perinuclear recycling compartment before returning to the plasma membrane. [17][18][19] Rapid integrin recycling has been shown to promote malignant phenotypes in several cancer cell lines. [19][20][21] Interestingly, it has also been shown that integrins can escape from late endosomes/lysosomes and efficiently recycle back to the plasma membrane. 22 Thus, integrin trafficking plays a critical role in controlling receptor surface levels and function.
Integrin trafficking and signaling are dictated by the overlapping linear binding and sorting motifs encoded within the cytoplasmically oriented C-termini of both α and β integrins. 17 Protein binding and recruitment to integrin tails directly controls integrin signaling and trafficking. In fact, several groups have shown that competition for binding to integrin cytoplasmic tails directly determines receptor fate. 23,24 Our current knowledge of integrin trafficking has been based on studies using either the exogenous expression of integrins with cytoplasmic tails fused to a fluorescent protein, 10,[25][26][27][28][29] antibody-based labeling, [30][31][32] or surface biotinylation 21,33,34 -each of which has drawbacks. For example, the efficient incorporation of cytoplasmic domain fusions into αβ heterodimers, which is required to exit the endoplasmic reticulum (ER), requires concomitant knockdown of the endogenous subunit. Moreover, the bulky cytoplasmic fusions can induce potential trafficking and signaling artifacts by disrupting protein recruitment and binding. Bivalent antibody labeling techniques can perturb trafficking routes, kinetics, or signaling by inducing receptor aggregation and activation. 35,36 In fact, many antibodies can alter the conformation, activity, and function of integrins. 2,37,38 Furthermore, active and inactive β1 integrins were recently shown to spatially segregate within adhesions 39 ; therefore, the use of function-perturbing antibodies may alter this spatial coordination and downstream signaling. To circumvent these problems, many groups have used surface biotinylation to measure uptake and recycling kinetics of endogenous integrins. However, surface biotinylation requires long labeling times at 4 C, which can perturb trafficking, 40 and lacks the sensitivity to detect fast recycling. Moreover, surface biotinylation cannot be used to address trafficking dynamics by live-cell microscopy.
Recently, in response to concerns about the current tools used to report integrin trafficking in cells, Huet-Calderwood et al. published the use of recombinant β1 integrins with an "ecto-tag" inserted within the extracellular hybrid domain. 41 Using these ecto-tagged β1 integrins, they report a spatial bias in integrin exocytosis. Unfortunately, as with all recombinant integrin fusions, the expression of ecto-tagged integrins must be precisely titrated so as not to induce ER accumulation or overexpression-related artifacts. 41,42 Therefore, the creation of more straightforward tools that could be readily applied to study endogenous integrins in any cell type would vastly increase our understanding of the intricate spatiotemporal regulation of integrin trafficking.
Here, we report the design and expression and purification of a single chain variable fragment (scFv) based on the previously characterized, non-function perturbing anti-β1 integrin monoclonal antibody, mAb K20. 38,[43][44][45] The purified MBP-scFv K20 is functionally neutral and monovalent, and able to specifically track and quantify endogenous β1integrin trafficking itineraries in cells, without the need for cell engineering and recombinant integrin expression. We demonstrate that our probe allows for reliable tracking of rapid β1 integrin recycling, and thus serves as proof of principle for the generation of future antibody-based probes. The future use of a single tool to directly correlate bulk biochemical assays with spatiotemporal dynamics acquired via live-cell microscopy will provide further mechanistic insight into the regulation of integrin trafficking.

| Generation of an anti-β1 integrin scFv
We sought to develop a more versatile and nonperturbing probe to quantitatively analyze endogenous β1 integrin trafficking. To this end, we generated a neutral and monovalent single chain antibody variable fragment (scFv) against β1 integrin. ScFvs represent the smallest unit of high affinity antibody-based binding, 46 as they contain the antigenbinding variable heavy (V H ) and variable light (V L ) segments of an antibody connected by a flexible linker ( Figure 1A). V H and V L sequences used to design the scFv were derived from mAb K20, a previously characterized, non-function perturbing, mouse monoclonal antibody against β1 integrin. 38,[43][44][45] The mAb K20 antibody is reported to bind the EGF repeat region in the membrane-proximal extracellular domain of β1 integrin, and is not predicted to interfere with integrin conformation or activation. 45 The mAb K20 sequence, which was described in a patent, 47 was validated by mass spectrometry of commercially available antibody ( Figure S1A). The resulting scFv, hereafter named scFv K20 , contains the entire antigen binding region of the parent monoclonal antibody and therefore is expected to retain the same binding specificity. 48 Both antibodies and scFvs require intramolecular disulfide bonds within each variable domain for correct tertiary structure. To allow for proper disulfide bond formation and protein folding through the secretory pathway, 49 the scFv K20 construct was designed for secreted baculovirus-mediated expression in insect cells. Amino acid sequences of mAb K20 were used to design a corresponding codon-optimized cDNA, which was commercially synthesized. A schematic of the scFv K20 construct used for baculovirus generation is shown in Figure 1B. To increase expression and solubility, the scFv is genetically fused to a TEV-cleavable Maltose Binding Protein (MBP). 50,51 Additionally, the construct encodes an amino-terminal GP64 secretion signal sequence and a 6x-Histidine (6xHis) tag for affinity purification. scFv K20 also contains a C-terminal FLAG epitope for recognition by commerciallyavailable anti-FLAG antibodies, and a SortaseA (SrtA*) recognition motif for in vitro site-specific labeling. 52 As designed, our scFv construct is versatile and can be customized for a wide range of assays.
To generate recombinant scFv K20 we first needed to generate  Figure S1C). As peak two ( Figure S1C) corresponded to the expected molecular weight of MBP-fused scFv K20 (ie, 71 kD) it was collected and labeled on free amines by NHS-(Nhydroxysuccinimide ester) or SPD-(sulfodichlorophenol ester) conjugation with either a disulfide-cleavable biotin or an Alexa Fluor dye, respectively, for use in subsequent biochemical and microscopy assays.
The final yield was 0.5 mg of purified MBP-scFv K20 per 100 mL of harvested insect cell supernatant. If desired, TEV protease can cleave MBP from the scFv after purification. However, the MBP-scFv K20 fusion retains the same binding properties as cleaved scFv K20 (data not shown) and MBP increases the protein's long-term stability and solubility. 50,54 Moreover, the MBP fusion protein provides further sites for amine-reactive labeling without perturbing scFv function. Therefore, MBP-fused scFv was used for all subsequent assays (herein referred to as "MBP-scFv K20 ").

| Monovalent MBP-scFv K20 retains β1 integrin binding specificity and does not affect integrin function
To validate that MBP-scFv K20 retains its binding specificity to β1 integrin, we performed a binding isotherm using a cell-based enzyme-F I G U R E 1 Anti-β1 integrin single chain variable fragment design, expression, and purification. A, Schematic of IgG antibody and derivatives after pepsin and papain digestion. The ends of the IgG and Fab fragments participate in antigen binding and can be recombinantly expressed as a single-chain variable fragment (scFv) by connecting the variable heavy (V H ) and variable light (V L ) domains with a flexible linker. Antibody heavy chains are in white, and light chains depicted in gray. B, Schematic of the anti-β1 integrin scFv K20 construct used for baculovirus-mediated insect cell expression under the control of a polyhedrin promoter. The cleavable GP64 signal sequence allows for protein secretion. The resultant recombinant protein is a 71 kDa N-terminal 6x His-tagged MBP-fusion with a C-terminal FLAG tag and SortaseA (SrtA*) recognition motif. MBP can be cleaved post-purification with TEV protease. C, Representative SDS-PAGE of harvested baculovirus-infected insect cell supernatant (Lane 2) and subsequent purification products (Lanes 3-4; left, Coomassie blue-stained; right, anti-FLAG immunoblot). Lane 1: Precision Plus protein ladder. Lane 3: pooled and concentrated IMAC affinity purification fractions. Lane 4: final purified MBP-scFv K20 from peak 2, after size-exclusion chromatography (see also Figure S1) linked immunosorbent assay (ELISA). We compared MBP-scFv K20 and parent mAb K20 binding to endogenous β1 integrin on the surface of H1975 cells (Figure 2A), and determined the binding affinity (K D ) of MBP-scFv K20 (6.54 ± 1.34 nM) to be approximately one order of magnitude less than mAb K20 (1.20 ± 0.14 nM). This decrease was to be expected, due to the loss of avidity of monovalent vs bivalent binding.
Nevertheless, the K D of MBP-scFv K20 remains in the low nanomolar range (<10 nM), making it suitable for most biochemical assays.
To assess whether MBP-scFv K20 and mAb K20 have the same binding specificity on cells, we compared MBP-scFv K20 and mAb K20 binding to the surface of hTERT-RPE1 cells by immunofluorescence.
Immunofluorescence staining and total internal reflection fluorescence microscopy (TIR-FM) imaging revealed significant colocalization between biotinylated MBP-scFv K20 and mAb K20 ( Figure 2B). The higher background signal for MBP-scFv K20 likely reflects the use of a secondary Streptavidin Alexa Fluor conjugate. Further, TIR-FM imaging analysis of permeabilized hTERT-RPE1 cells using culture media containing secreted scFv K20 showed its expected association with the ventral cellular membrane, as well as colocalization with the cellular adhesion marker, paxillin ( Figure 2C). Taken together, we conclude that MBP-scFv K20 retains similar β1 integrin binding specificity as its parent mAb K20.
As MBP-scFv K20 retained β1 integrin binding specificity and colocalized with adhesion-associated β1 integrins, we next assessed whether MBP-scFv K20 perturbs integrin function. To test this, we first performed adhesion assays in H1975 cells. Cell adhesion to a matrix of gelatin and fibronectin (FN) was measured using a modified static adhesion assay that detects bound cells via crystal violet staining (see Section 4,Methods). 55 In addition to parent mAb K20, two known β1 integrin function-altering antibodies, the activating mAb 9EG7 and the inhibitory mAb AIIB2 were used as controls. 38 As expected, incubation with β1 integrin activating antibody, 9EG7, increased cell attachment after 10 minutes, while the inhibitory β1 integrin antibody, AIIB2, significantly reduced cell adhesion to the gelatin-and FN-F I G U R E 2 Anti-β1 integrin scFv K20 retains binding specificity for β1 integrins. A, Antibody binding curves and calculated binding affinities (K D ). Relative binding of antibodies was determined using a cell-based ELISA binding assay. Values plotted are normalized to maximal binding per individual experiment. K D was determined as the concentration of antibody at half-maximal binding using GraphPad Prism. n = 4. Error bars represent SD. B, Representative TIR-FM immunofluorescence images of surface β1 integrins of hTERT-RPE 1 cells seeded on gelatin-coated coverslips. Co-staining of anti-β1 MBP-scFv K20 (green) and parent mAb K20 (red) antibodies. Scale bar, 10 μm. C, Representative TIR-FM immunofluorescent images of permeabilized hTERT-RPE 1 cells seeded on gelatin-coated coverslips probed with media from cells secreting scFv K20 and anti-FLAG secondary antibody (green) as well as the cellular adhesion marker paxillin, detected with α-phospho-(Y118) antibody (red). Scale bar, 10 μm coated wells ( Figure 3A). Neither the addition of MBP-scFv K20 nor its parent mAb K20 significantly affected cell attachment at 10 minutes ( Figure 3A). The extent of cell adhesion after incubation for 30 minutes was similarly stimulated or inhibited in the presence of 9EG7 or AIIB2, respectively. In contrast, although there was a trend toward increased cell adhesion upon 30-minute incubation with either MBP-scFv K20 or mAb K20, their effects were not significant ( Figure 3B). Given this trend toward increased cell adhesion, we took a third approach to quantitatively measure the effects of MBP-scFv K20 incubation on integrin function. H1975 cells were imaged after incubation for 30 minutes at 37 C in the absence (control) or presence of 5 μg/ mL MBP-scFv K20 . Static immunofluorescence TIR-FM images revealed no obvious effects, as the extent of cell attachment and spreading was variable under control and MBP-scFv K20 incubation conditions ( Figure 3C and Figure S2A). Integrin-based cell-ECM adhesions can induce integrin clustering and focal adhesion (FA) formation. As the turnover of adhesions is an essential part of integrin trafficking and is critical for efficient cell migration, 6 we next examined whether MBP-scFv K20 can affect focal adhesions. Quantitative analysis of TIR-FM-acquired immunofluorescence images revealed no significant effect of incubation with MBP-scFv K20 for 30 minutes at 37 C on any of the measured parameters of FAs, including total FA area/cell area ( Figure 3D), total FA area or the number of FA/cell area ( Figure S2B, C, respectively). Taken together, we conclude that MBP-scFv K20 binds specifically to β1 integrin, but does not significantly alter integrin or adhesion function.

| Measuring β1 integrin uptake and recycling using MBP-scFv K20
Our data indicated that MBP-scFv K20 is functionally neutral and retains β1 integrin binding specificity. We next studied the utility of MBP-scFv K20 as a biochemical probe to measure integrin uptake and recycling. The uptake of endogenous β1 integrin was measured by incubating H1975 cells in the continuous presence of biotinylated MBP-scFv K20 or unlabeled mAb K20 for the indicated times at 37 C before returning cells to ice and stripping surface-bound antibodies. 56 F I G U R E 3 ScFv K20 does not perturb integrin function. A, Attachment of H1975 cells at 10 minutes or, B, 30 minutes after plating on wells coated with 0.02% gelatin and 25 μg/mL fibronectin in the absence (control) or presence of 5 μg/mL of the indicated anti-integrin antibodies. 9EG7 is an integrin activating control, AIIB2 is an integrin inhibitory control. Cell attachment measured by crystal violet staining and the absorbance at 570 nm. Data was normalized to 100% attachment of untreated control cells. Error bars represent Mean with SD (n = 3). Unpaired t-test was used for statistical significance. * p<0.05, **** p<0.001. C, Representative inverted TIR-FM immunofluorescence images of H1975 cells pre-seeded on gelatin-and fibronectin-coated coverslips and incubated in the absence (control) or presence of 5 μg/mL MBP-scFv K20 for 30 minutes at 37 C. Scale bar, 10 μm. D, Quantitative comparison of total detected focal adhesion area relative to the detected cell area of H1975 cells incubated as in (C), n.s., not significant. Wilcoxon Rank-Sum non-parametric test was used for statistical significance Whereas mAb K20 continued to accumulate intracellularly in H1975 cells throughout the time course, intracellular MBP-scFv K20 reached steady state after 15 minutes, suggestive of scFv recycling ( Figure 4A).
The rapid recycling of integrins has been shown to play a role in cancer-mediated cell migration; however, fast recycling kinetics (ie, after short internalization periods) have not been measured using previously available techniques, due to lack of sensitivity. Therefore, we next investigated whether MBP-scFv K20 could be used to quantify the rapid recycling of β1 integrins, measured in the presence of 20 ng/mL EGF, which stimulates integrin trafficking. 20,21,33 For this, both MBP-scFv K20 and mAb K20 IgG were biotinylated via a cleavable disulfide bond. To independently measure both rapid and slow integrin recycling, cells were then subjected to either a short (10 minutes) or long (30 minutes) internalization pulse, respectively, at 37 C with either biotinylated-mAb K20 or -MBP-scFv K20 . Cells were then immediately cooled to 4 C to stop internalization and washed with PBS containing TCEP to remove the biotin moiety on the remaining surface-bound antibodies (see Section 4, Methods). For subsequent recycling measurements, cells were incubated at 37 C for the indicated times in the continuous presence of 20 ng/mL EGF and TCEP before returning cells to ice and stripping the cell surface-bound antibodies. Our recycling assay measures the loss of internal signal over time, which, given the rapid kinetics most likely reflects receptor recycling to the plasma membrane rather than lysosomal degradation.
Remaining intracellular biotin-labeled antibodies were assessed and the percentage of recycling was calculated relative to the initial (10 or 30 minutes) internalization pulse. Biotinylated-MBP-scFv K20 exhibited efficient recycling in H1975 cells under conditions of fast ( Figure 4B) and slow ( Figure 4C) recycling, whereas the parent mAb K20 did not recycle after a 10-minute internalization pulse ( Figure 4B) and recycled with lower efficiency than MBP-scFv K20 after the longer 30-minute internalization pulse ( Figure 4C).
We repeated these experiments in hTERT-RPE1 cells and found that the internalization rates of both biotinylated-mAb K20 and -MBP-scFv K20 began to plateau after 15 min ( Figure 4D). Correspondingly, both probes exhibited significant recycling through fast and slow pathways ( Figure 4E, F), although MBP-scFv K20 recycled more rapidly and efficiently than mAb K20. We observed mAb K20 F I G U R E 4 MBP-scFv K20 serves as probe for biochemical assays of β1 integrin uptake and recycling. A, Endocytosis of biotinylated anti-β1 integrin MBP-scFv K20 (5 μg/mL) or mAb K20 IgG (1 μg/mL) in H1975 cells. Shown is the percentage of internalized antibody at the indicated times calculated relative to the initial surface bound at 4 C. B and C, Recycling of biotinylated-MBP-scFv K20 (5 μg/mL) or -mAb K20 IgG (1 μg/mL) in H1975 cells. Shown is the percentage of remaining intracellular biotinylated antibody at the indicated times relative to the initial internal loading of 10 minutes (B) or 30 minutes (C). All experiments represent n ≥ 3. Data plotted as mean ± SD. D-F, As for A-C except assays were performed in hTERT-RPE1 cells IgG signals above 100% at the 5-minute time point in both cell lines during rapid integrin recycling assays ( Figure 4B,E). This likely reflects incomplete dissociation of the bivalent mAb K20 relative to the monovalent MBP-scFv K20 during the quick wash steps. Together, these data demonstrate that the nonperturbing and monovalent MBP-scFv K20 can be used as a probe for biochemical β1 integrin uptake and recycling assays in multiple cell lines, and is superior to its parent mAb K20 for use in rapid integrin recycling assays.  Figure 5B and Movie S1). We note that both live cell TIR-FM and LSFM imaging displayed high background and low fluorescent MBP-scFv K20 signals, culminating in rapid photobleaching. Nonetheless, these data establish the potential utility of scFv K20 for imaging integrin dynamics in living cells.

| DISCUSSION
The field of integrin trafficking is rapidly expanding, largely because integrin function is vital for many fundamental cellular properties, such as cell identity, signaling, and motility. Therefore, new tools and assays to quantitatively track and measure endogenous integrins would help to further increase our understanding of integrin trafficking and its regulation. Here, we describe a functionally neutral and monovalent antibody-based probe that can be used to study endogenous β1 integrin uptake and recycling in multiple cell types. We designed MBP-scFv K20 to retain its β1 integrin-specific binding capa-  While scFvs remain widely used, they are prone to aggregation and thus often require additional genetic tags to mitigate protein instability. 51,54 Although the addition of MBP increases both the solubility and stability of our scFv, it does effectively double the size of our probe. We therefore suggest that future integrin antibody-based probe design limit increasing overall molecular size. Recent advances in monovalent camelid nanobody expression and purification suggest that generation of anti-integrin nanobodies may greatly overcome the limitations inherent with current scFv-based approaches.
MBP-scFv K20 is designed to be versatile and customizable through differential labeling for each experimental condition.
Although MBP-scFv K20 is designed for in vitro site-specific labeling by the protein SortaseA, there is a need for more commercially available reagents for "Sortagging." While labeling MBP-scFv K20 with commercially available amine-reactive reagents is suitable for the assays per- Although MBP-scFv K20 was designed for robust use in both biochemical and imaging-based assays, we experienced issues with high background and poor MBP-scFv K20 signals. As discussed, significant efforts were made to create a fluorescently-labeled MBP-scFv K20 for use in live cell imaging experiments. Live cell images were acquired with long exposure times (1 second) in 10-second intervals to overcome the low SNR while simultaneously minimizing photobleaching.
However, integrin trafficking is a dynamic process that is best studied by live fluorescence microscopy with high temporal resolution. Therefore, we are unable to reliably validate MBP-scFv K20 as a tool to study the spatial regulation of β1 integrin without more robust labeling techniques.
We focused on designing an scFv to track endogenous β1 integrin because β1 integrin is expressed in nearly all cell types and can form heterodimers to recognize nearly every known ECM ligand. While this is advantageous in many regards, it is becoming increasingly appreciated that integrin heterodimers are under different regulatory mechanisms and can exhibit different trafficking itineraries. 10,19 We therefore propose that future antibody-based probes be designed against specific αβ heterodimer pairs and/or designed in coordination for use with specific ECM ligands. The development of nanobody screening libraries 59,60 should aid in generating these heterodimerand ECM-specific reagents. Nonetheless, we believe the use of our tool will help to increase understanding of β1 integrin trafficking and its regulation in cells.
Finally, having established that MBP-scFv K20 is functional, can be used to assess β1 integrin trafficking in biochemical assays, and associates with adhesions in live cells over time, we hope our analysis of MBP-scFv K20 serves to inspire future integrin probe design. Specifically, we believe the generation of a single and monovalent antibodybased tool, allowing for the correlation of bulk biochemical assays with spatiotemporal dynamics acquired through high-resolution microscopy, will dramatically increase our knowledge of integrin trafficking and its roles in development and disease.   England Biolabs (NEB). K20-scFv-pSMBP2 is available on Addgene.

| Bacmid and baculovirus generation
To generate bacmid DNA, K20-scFv-pSMBP2 plasmid was transformed into MAX Efficiency Chemically Competent DH10Bac E. coli cells (Life Technologies) following the recommended protocol. Briefly, DH10Bac competent cells were incubated with 1 ng of K20-scFv-pSMBP2 on ice. After a brief heat shock, the transformed competent cells were further incubated at 37 C for 4 hours to recover, and then plated on LB agar plates containing 50 μg/mL Kanamycin, 7 μg/mL gentamycin, 10 μg/mL tetracycline, 100 μg/mL Bluo-gal, and 40 μg/ mL IPTG and incubated at 37 C for 48 hours. White colonies were isolated, and re-streaked on fresh plates. White colonies from the second round of plating were used for bacmid DNA isolation (Qiagen).

| Endocytosis and recycling assays
Endocytic trafficking assays were performed using Corning Costar Stripwell 96-well plates (Thermo Fisher Scientific). Plates were precoated with 0.02% gelatin and 25 μg/mL human fibronectin (in PBS containing 2% sucrose) for 2 hours at 37 C then incubated in complete medium overnight at 4 C. hTERT RPE1 cells (2 × 10 4 cells/well) were seeded overnight and H1975 cells (3.5 × 10 4 cells/well) were seeded for 6 hours prior to experiments. To reduce biotin background, all cells were maintained in medium without added biotin.
For internalization experiments biotinylated MBP-scFv K20 or unlabeled monoclonal β1 integrin antibody mAb K20 were used to track β integrin. We routinely use either bivalent anti-transferrin receptor mAb D65 and disulfide-cleavable biotinylated-transferrin as ligand interchangeably to measure TfnR endocytosis and the kinetics of uptake are the same. Cells were incubated with 5 μg/mL biotinylated MBP-scFv K20 or unlabeled 1 μg/mL mAb K20 in PBS 4+ assay buffer at 37 C for the indicated time points and then immediately cooled to 4 C to stop internalization. The remaining surface-bound biotinylated MBP-scFv K20 or unlabeled mAb K20 was removed by an acid wash step (4× 30 seconds of 0.2 M acetic acid, 0.2 M NaCl pH 2.5). Cells were then washed 3× with cold PBS and fixed in 4% PFA (Electron Microscopy Sciences) in PBS for 30 minutes at 37 C.
PFA was quenched with 100 mM glycine for 5 minutes at RT. Cells were permeabilized with 0.2% Triton X-100 for 10 minutes at RT. Cells were then incubated in 1% casein/PBS overnight at 4 C to block nonspecific streptavidin binding. Internalized MBP-scFv K20 was assessed using streptavidin-POD and internalized mAb K20 was assessed using a goat anti-mouse HRP-conjugated antibody. The reaction was further developed with OPD, and then stopped by 5 M H 2 SO 4 . The absorbance was read at 490 nm (Biotek Synergy H1 Hybrid Reader). Well-to-well variability in cell number was normalized by a BCA assay (Thermo Fisher Scientific). Internalized ligand was expressed as the percentage of the total surface-bound ligand at 4 C (ie, without acid wash step), measured in parallel. 56 For recycling experiments both MBP-scFv K20 and mAb K20 were biotinylated with a cleavable disulfide bond to track β-integrin. Cells were pulsed with biotinylated-MBP-scFv K20 or -mAb K20 and 20 ng/ mL EGF in PBS 4+ for 10 or 30 minutes at 37 C, to assess fast vs slow recycling, respectively. Cells were then immediately washed with PBS 4+ and further incubated in PBS 4+ containing 10 mM TCEP [tris

| Focal adhesion analysis
Focal adhesion analysis was performed using a previously published Focal Adhesion Analysis Package. 20,61,62 Fixed-cell paxillin immunofluorescence images were used to quantify total cellular adhesion density. The analysis software is available online at https://git.biohpc. swmed.edu/danuser/applications/pipelines/1944.

| Total internal reflection fluorescence microscopy
Total internal reflection fluorescence microscopy (TIR-FM) was performed as previously described. 63 Briefly, cells were mounted in PBS and imaged using a 60×, 1.49 NA APO TIRF objective (Nikon) mounted on a fully motorized Nikon Ti-Eclipse inverted microscope with Perfect Focus System and coupled to an Andor "Diskovery TIRF/ Borealis widefield illuminator" equipped with an additional 1.8× tube lens (yielding a final magnification of ×108). TIR-FM illumination was achieved using a Diskovery Platform (Andor Technology). For live cell experiments, cells were maintained at 37 C during imaging. Imaging sequences were acquired using a sCMOS camera with 6.5 μm pixel size (pco.edge).

| Light sheet fluorescence microscopy
Light sheet fluorescence microscopy (LSFM) was performed as previously described. 64 H1975 cells stably expressing mRuby2-Paxillin were seeded for 6 hours at 37 C/5% CO 2 on 5 mm round coverslips pre-coated with 0.02% gelatin and 25 μg/mL FN. Cells were mounted in a custom sample holder for imaging. 64 Images were acquired every 10 seconds for 10 minutes.