Integrin molecules are transmembrane αβ heterodimers involved in cell adhesion, trafficking, and signaling. Upon activation, integrins undergo dynamic conformational changes that regulate their affinity to ligands. The physiological functions and activation mechanisms of integrins have been heavily discussed in previous studies and reviews, but the fluorescence imaging techniques –which are powerful tools for biological studies– have not. Here we review the fluorescence labeling methods, imaging techniques, as well as Förster resonance energy transfer assays used to study integrin expression, localization, activation, and functions.
Integrins are a family of adhesion receptors that are abundantly expressed in all cell types of metazoans except for erythrocytes. Their integral roles in mediating cell–cell and cell–extracellular matrix (ECM) interactions make integrins indispensable for the existence of multicellular organisms. Interactions between integrins and their ligands trigger profound changes of the cytoskeleton and signaling apparatus during biological processes, such as adhesion (
Integrins are heterodimers consisting of noncovalently associated α (120–180 kDa) and β (90–110 kDa) subunits (
The ectodomain is an asymmetric structure with a “head” carrying two “legs” (~16 nm long). The head consists of a predicted seven-bladed β-propeller domain (~60 amino acids each) of an α subunit (
Dark and light oranges represent α subunits with or without the αA/αI domain. Different β subunits were colored differently. RGD is the abbreviation of Arg-Gly-Asp peptides.
Integrin | Epitope (Domain) | Clone name | Integrin | Epitope (Domain) | Clone name |
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α1 | αA/αI | FB12 ( |
β1 | βA/βI-like | 4B4 ( |
mAb13 ( |
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α2 | αA/αI | 12F1 ( |
AIIB2 ( |
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Gi9 ( |
P4C10 ( |
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JA218 ( |
Hybrid | JB1A ( |
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P1E6 ( |
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β2 | βA/βI-like | CLB LFA-1/1 ( |
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α3 | β-propeller | ASC-6 ( |
MHM23 ( |
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P1B5 ( |
TS1/18 ( |
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Not known | IA3 ( |
IB4 ( |
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L130 ( |
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α4 | β-propeller | HP2/1 ( |
Hybrid | 7E4 ( |
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P4C2 ( |
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PS/2 ( |
β3 | βA/βI-like | 7E3 ( |
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Not known | 9F10 ( |
Not known | SZ-21 ( |
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L25 ( |
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P1H4 ( |
β4 | Not known | ASC-8 ( |
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A4-PUJ1 ( |
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β5 | Not known | ALULA ( |
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α5 | β-propeller | JBS5 ( |
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mAb16 ( |
β6 | Not known | 6.3G6 ( |
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P1D6 ( |
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Not known | NKI-SAM-1 ( |
β7 | βA/βI-like | FIB504 ( |
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FIB27 ( |
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FIB30 ( |
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α6 | Not known | GoH3 ( |
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β8 | Not known | 37E1 ( |
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α7 | Not known | 6A11 ( |
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αIIb | β-propeller | 10E5 ( |
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α8 | β-propeller | YZ3 ( |
2G12 ( |
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α9 | Not known | Y9A2 ( |
αVβ3 | β-propeller | 23C6 ( |
αVβ5 | Not known | P1F6 ( |
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αV | β-propeller | 17E6 ( |
P3G2 ( |
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L230 ( |
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Not known | NKI-M9 ( |
αVβ6 | Not known | 10D5 ( |
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6.3G9 ( |
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αE | αA/αI | αE7-1 ( |
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αE7-2 ( |
αLβ2 | αA/αI, β-propeller, | YTA-1 ( |
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Not known | Ber-ACT8 ( |
and βA/βI-like | |||
αL | αA/αI | TS1/22 ( |
αM | αA/αI | 2LPM19c ( |
HI111 ( |
MAN-1 ( |
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CBR LFA-1/1 ( |
anti-M7 ( |
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Not known | mAb38 ( |
ICRF44 ( |
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Thigh | M1/70 ( |
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αX | αA/αI | 3.9 ( |
αD | αA/αI | 217I ( |
Not known | 496K ( |
240I ( |
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Bu15 ( |
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α1 | Not known | TS2/7 ( |
α5 | Calf-1 to 2 | mAb11 ( |
β-propeller | VC5 ( |
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α2 | Not known | 16B4 ( |
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31H4 ( |
α6 | Not known | J1B5 ( |
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α3 | Not known | A3-X8 ( |
α7 | Not known | 3C12 ( |
α4 | Not known | 44H6 ( |
α9 | Not known | A9A1 ( |
8F2 ( |
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αIIb | Not known | PL98DF6 ( |
αV | Not known | LM142 ( |
αD | Not known | 212D ( |
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αL | β-propeller | TS2/4 ( |
92C4D ( |
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Not known | YTH81.5 ( |
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αM | β-propeller | CBRM1/20 ( |
β1 | I-EGF | K20 ( |
Thigh | OKM1 ( |
β2 | Not known | CBR LFA-1/7 ( |
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CyaA ( |
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β4 | Not known | ASC-3 ( |
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αX | Not known | CBR-p150/2E1 ( |
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β5 | Not known | 11D1 ( |
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α2 | Not known | JBS2 ( |
β2 | βA/βI-like | mAb24 ( |
327C ( |
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α4 | β-propeller | HP1/3 ( |
Hybrid | MEM-148 ( |
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EGF-like 2 | KIM127 ( |
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α5 | Calf-1 & 2 | SNAKA51 ( |
EGF-like 3 | CBR LFA-1/2 ( |
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MEM-48 ( |
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αIIb | β-propeller | PT25-2 ( |
EGF-like 4 | KIM185 ( |
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Calf-1 | MBC370.2 ( |
β3 | Hybrid | AP3 ( |
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Calf-2 | PMI-1 ( |
PSI | AP5 ( |
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EGF-like 3/4 | LIBS6 ( |
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αL | αA/αI | 2E8 ( |
β-tail | LIBS2 ( |
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MEM83 ( |
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Genu | NKI-L16 ( |
β7 | βA/βI-like and | 10F8 ( |
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hybrid | 2B8 ( |
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αM | αA/αI | CBRM1/5 ( |
2G3 ( |
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Thigh | VIM12 ( |
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αIIbβ3 | β-propeller and | PAC-1 ( |
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αX | Not known | 496B ( |
βA/βI-like | ||
β1 | βA/βI-like | 12G10 ( |
αVβ3 | β-propeller and | WOW-1 ( |
8A2 ( |
βA/βI-like | LM609 ( |
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TS2/16 ( |
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A1A5 ( |
αVβ6 | β-propeller and | 6.8G6 ( |
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Hybrid | 15/7 ( |
βA/βI-like | |||
HUTS-4 ( |
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HUTS-7 ( |
α4β7 | β-propeller and | J19 ( |
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HUTS-21 ( |
βA/βI-like | ||||
PSI | 8E3 ( |
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N29 ( |
β1 | EGF-like 2 | 9EG7 ( |
Many techniques have been applied to distinguish two major models of conformational changes influencing integrin affinity, namely “switchblade” (
Immunofluorescent staining is the most commonly used method for integrin labeling, and antibody selection is extremely important for studying integrins. Monoclonal antibodies targeting different epitopes of specific integrin α and β subunits have been developed (
Integrin antibodies that recognize activated epitopes have been applied to understanding β2 integrins-leukocyte-specific integrins that are critical for leukocyte recruitment and functions. Monoclonal antibody KIM127 (
Antibodies for activated integrins have also been used to study β1 integrins, which are expressed on various cells, such as leukocytes (
Antibodies recognizing and binding to the inactive conformation or that inhibit function are also used for integrin labeling. mAb13 recognizes an epitope within the βI domain of β1 integrin and is dramatically attenuated in the ligand-occupied form of α5β1. The binding of mAb13 to ligand-occupied α5β1 induces a conformational change in the integrin, resulting in the displacement of the ligand (
β3 integrins are also widely expressed, and antibodies have been developed to study their functions. Vitronectin receptor integrin αVβ3 is expressed on leukocytes (
Integrin α4β7 is a lymphocyte homing receptor that mediates both rolling and firm adhesion of lymphocytes on vascular endothelium, two of the critical steps in lymphocyte migration and tissue-specific homing (
Since the molecular cloning of green fluorescent protein (GFP) from the jellyfish
To study the separation of integrin α and β “legs” during activation, the monomeric cyan fluorescent protein (mCFP) and monomeric yellow fluorescent protein (mYFP) were fused to the C-termini of the α and β cytoplasmic domains of αVβ3, respectively (
In another study, GFP was inserted into the β3-β4 loop of blade 4 of the αL integrin β-propeller domain with no appreciable influence on integrin function and conformational regulation (
In another study, an extracellular site of integrin β1 was reported suitable for inserting different tags, including GFP and PH-sensitive pHluorin (
HaloTag is a 34 kDa engineered, catalytically inactive derivative of a bacterial hydrolase. It can be fused to a protein of interest and covalently bound by synthetic HaloTag ligands with high specificity. A covalent bond can form rapidly under physiological conditions and is essentially irreversible. HaloTag allows adaptation of the targeted protein to different experimental requirements without altering the genetic construct (
Many integrins bind to ECM molecules through an RGD motif. RGD peptide was found to bind to resting integrins and induce integrin activation. Compared to linear peptides, suitable optimized cyclic RGD (cRGD) peptides interact with integrins in a more selective manner and with higher affinity (
The C-terminal region of the fibrinogen γ subunit contains γC peptide uniquely binding to activated or primed αIIbβ3 integrin at the interface between α and β subunits (
Soluble ligands ICAM-1 (
Fluorophore-conjugated integrin allosteric antagonists and agonists are also widely used to label certain integrins. BIRT 377 and XVA-143 are integrin αLβ2-specific allosteric antagonists that belong to two distinct classes. The BIRT 377 binding site is located within the I domain of the αL integrin subunit. The XVA-143 site is located between the αL β-propeller and the β2 subunit I–like domain (
Live-cell imaging has been abundantly used in biological studies, including some for integrins. This method has given rise to tremendous progress in documenting dynamic cellular processes, such as cell adhesion (
In epifluorescence microscopy, which is the most commonly used wide-field microscopy, all the emission light around the focal plane captured by the objective, which depends on its numerical aperture, is sent to the detector leading to high light-collecting efficiency. The use of the pinhole in confocal laser scanning microscopy (CLSM) decreases the background signal from out-of-focus light and increases the signal-to-background ratio. However, CLSM is limited by phototoxicity/photobleaching. This is mainly due to that most confocal microscopes have detectors with low quantum efficiency, such as photomultiplier tubes (PMT), in comparison to epifluorescence microscopes, such as charge-coupled device (CCD) or complementary metal-oxide-semiconductor (CMOS) cameras. Thus, to acquire images of similar brightness, CLSM needs higher power of the excitation light than epifluorescence microscopy. On the other hand, most CLSM setting has a limited imaging speed due to its scanner. For example, most CLSM has a laser dwell time of ≥1 µs per pixel (
Epifluorescence microscopy has been used to monitor β2 integrin activation during leukocyte rolling on selectins (
For thicker (e.g., 20–100 µm) live-cell specimens, CLSM was used for imaging integrins (
The side-view neutrophil footprint (~100 nm) converted from the TIRF (total internal reflection fluorescence) membrane fluorescence image (inset image) was shown (grey surface). The distance of the closest approach of the neutrophil with the coverslip is Δ0. This is the position with the brightness cell-membrane fluorescence signal (shown in the inset image). The z-distance (Δ) of other positions was calculated by their cell-membrane fluorescence signal. Two examples (Δ1 and Δ2) were shown.
However, the slower imaging speed and higher phototoxicity limit its usage for live-cell imaging. There are some implementations that significantly increase imaging speed and reduce phototoxicity under the condition of CLSM. Such implementations include slit scanning and pinhole multiplexing methods, including spinning disk confocal microscopy (SDCM) (
Another high-resolution live-cell imaging technique is total internal reflection fluorescence (TIRF) microscopy. In TIRF microscopy, a laser incident beam illuminating the boundary between two media of different refractive indices (usually the coverslip and the specimen) experiences total internal reflection. The totally internally reflected laser beam generates the evanescent wave, which excites fluorophores that are in the vicinity of the coverslip-specimen interface (~100–200 nm), resulting in a very high signal-to-background image with a ~100 nm optical section compared to ~700 nm of confocal or wide-field (
As an update to TIRF microscopy, quantitative dynamic footprinting (qDF) microscopy was developed in 2010 (
The spatial resolution of microscopic techniques is limited by Abbe’s law, according to which the highest achievable lateral and axial resolution (dx,y and dz), or diffraction limits, can be:
in which λ is the wavelength of the excitation beam, and NA is the numerical aperture of the microscope objective.
Name | lateral resolution | axial resolution |
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Structured-Illumination Microscopy | 100 nm ( |
250–350 nm ( |
Airyscan Confocal Microscopy | 120 nm ( |
350 nm ( |
Stimulated Emission Depletion Microscopy | 45 nm ( |
100 nm ( |
Stochastic Optical Reconstruction Microscopy | 20 nm ( |
50 nm ( |
Photoactivated Localization Microscopy | 20 nm ( |
50 nm ( |
Interferometric Photoactivation and Localization Microscopy | 20 nm ( |
10 nm ( |
Ground State Depletion Microscopy | 20 nm ( |
50 nm ( |
Several super-resolution techniques circumvent the limits of diffraction and increase both lateral and axial resolution. One approach beyond the limit of diffraction is to sharpen the point-spread function of the microscope by spatially patterned excitation, including STED (
Super-resolution imaging techniques have been used to study integrin molecules in recent years. Interferometric photoactivation and localization microscopy (iPALM) was used to visualize the three-dimensional structure of FAs, which includes the integrin αV and paxillin-enriched integrin signaling layer, the talin and vinculin-enriched force transduction layer, and zyxin and vasodilator-stimulated phosphoprotein-enriched actin regulatory layer (
Whereas cellular behavior is different between
Epifluorescence microscopy can be used as IVM for studying integrins. One study showed that after 24 h of cecal ligation puncture, β1 integrins were found in the neutrophil extracellular traps in the liver and helped to sequester circulating tumor cells (
As mentioned before, integrin β2-mCFP mice were developed (
Since there are large conformational changes during integrin activation, techniques sensitive to distance changes like FRET become useful tools in studying integrins. FRET used as a ‘‘molecular ruler’’ ushered in the quantification of intermolecular interactions (
E is the efficiency, r is the intermolecular distance, and R0, known as Förster constant, is the value of r when this pair of donor and acceptor achieve 50% FRET efficiency. R0 depends on the overlap integral of the donor emission spectrum with the acceptor absorption spectrum and their mutual molecular orientation as expressed by the following equation:
in which
(A) The cytoplasmic tails of α and β subunits were labeled with FRET donor and acceptor, respectively. The separation of cytoplasmic tails is assessed by the reduction of FRET. (B) The integrin headpiece and cell membrane/integrin tailpiece were labeled with FRET donor and acceptor, respectively. The extension/unbent of integrin ectodomain is assessed by the reduction of FRET. (C) The cytoplasmic tails of α or β subunits were labeled with both FRET donor and acceptor. The clustering of integrin molecules is assessed by the increase of FRET. (D–E) The interaction of integrins and their ligands (D, both in cis and in trans) or cytoplasmic regulators (E, interaction or force measurement) can be assessed by FRET.
To obtain a FRET signal for studying the interaction of two proteins, they must be fluorescently labeled. One approach is to label the antibodies or antagonist/agonist binding to the two proteins with proper fluorophores. Fluorophore-conjugated antagonist/agonist can be synthesized, while labeling kits facilitating covalent binding (usually using amide bonds) of many different fluorescent molecules to antibodies are commercially available (
Measurements of (1) signal intensity and (2) fluorescence lifetimes are two major ways to determine FRET efficiency. Regarding the signal intensity method, the comparable changes between the intensification of the acceptor’s emission and synchronous decrease in donor’s emission facilitate the detection of FRET by splitting the emission from the two fluorophores. The split lights are then filtered through a specific filter set and collected separately. The downsides of this method are: (1) The excitation light of acceptor may excite the donor owing to the possible overlap of their excitation spectrum, (2) The leak of donor emission to the detecting channel of the acceptor, and (3) The faster photobleaching of the donor compared with that of the acceptor (
With the help of the improvement in microscopic techniques and labeling with fluorophores, great advantages have been made regarding integrin conformation and signaling. FRET can be used to identify the spatial movement of integrin cytoplasmic tails (
FRET can also be used to identify conformational changes in the integrin ectodomain domains. One method is to label the integrin headpiece and cell membrane/integrin tailpiece with FRET donor and acceptor, respectively, to measure the extension/unbending of integrins (
Instead of attaching donor and acceptor respectively to α and β subunits, studying integrin micro-clustering requires attachment of both the donor and acceptor to either the α or β subunit within one heterodimeric integrin (
FRET can also be used to assess interactions of the integrin headpiece with its ligands (
Overall, optical imaging of integrin molecules helps us understand the regulation of integrin expression, localization, clustering, conformational changes, and functions. Although there are various antibodies targeting integrin to visualize integrins with different conformations, most of these antibodies are specific for human integrin molecules. This limits the use of these antibodies for studying integrins in physiologically-relevant
As we discussed, super-resolution microscopy is a powerful tool for studying integrins. However, their uses in integrin studies are mostly restricted to phenomenon reports and morphology studies. Thus, finding a way to dig into the molecular details of integrin regulation and function using super-resolution microscopy needs more attention. For example, super-resolution imaging can better assess the clustering of integrin molecules. Assessing the localization of important integrin modulators, such as talin, kindlin, RIAM, etc., by super-resolution microscopy will help understand their roles in regulating integrin activation.
FRET is a powerful tool to study dynamic changes in integrin conformation, but most FRET assays of integrins are restricted in cell lines. Only two integrin FRET mouse strains (αLβ2 and αMβ2) were developed. Thus, the development of more integrin FRET mouse strains is needed to visualize integrin conformation changes
Although many techniques were developed to visualize integrin molecules as we reviewed above, whether the fluorescence labeling affects integrin function needs to be demonstrated in the specific studies, especially for activating specific integrin antibodies and fluorescent protein tags. For example, KIM127 was reported to stimulate leukocyte aggregation (
We acknowledge Dr. Christopher “Kit” Bonin and Dr. Geneva Hargis from UConn Health School of Medicine for their help in the scientific writing and editing of this manuscript.