Integrins were thought to be solely expressed by metazoans until genome sequencing of unicellular protists, such as the filasterean Capsaspora owczarzaki C. Prompted by the ancient origin of integrins and associated proteins, we wondered whether integrin regulation by talin has emerged before metazoans. We used codon optimized sequences of the cytoplasmic domains from two integrin homologues found in C.
All C. These findings imply a functional conservation of the integrin-talin interaction. To further strengthen a presumable ancient origin of the integrin-talin interaction, we introduced a point mutation into the NPXF motif of the C. When this NPXA-mutant was tested in OPTIC assays, there was impaired recruitment of talin demonstrating that the interaction of the Capsaspora integrin with the cytoplasmic interaction partner is based on the same mechanistic principle. This finding also implies that the Capsaspora integrins might be regulated by outside-in and inside-out processes involving talin.
Talin recruitment is conserved throughout evolution. Identical residues are shaded in black, highly similar residues are shaded gray. D Quantification of talin recruitment in cells expressing the indicated CEA3-integrin fusion proteins. Interpretation of focal adhesion composition and dynamics in a cellular context is complicated by the fact that multiple integrins as well as diverse extracellular matrix ligands are present for any given cell type 21 , Indeed, focal adhesions are not homogenous clusters of one particular integrin heterodimer, but usually are composed of a combination of integrins, e.
The cellular response upon integrin activation and clustering can further be influenced by crosstalk with other surface receptors, such as growth factor receptors, other cell adhesion molecules, or the glycocalyx 23 , These complications can be circumvented by employing chimeric integrins, which have been used before to dissect the role of the integrin cytoplasmic domain in receptor localization in non-adherent cells Inspired by this prior work, we present here a new approach to investigate the first steps in the assembly of integrin-associated focal adhesions.
As the extracellular fusion partner for integrin cytoplasmic domains we chose CEACAM3, an immunoglobulin-related receptor exclusively expressed by human granulocytes. Nevertheless, several high affinity ligands for CEACAM3 are known to be present on the surface of particular gram-negative bacteria, such as the gonococcus, Neisseria gonorrhoeae 26 , Therefore, CEACAM3 clustering is selectively stimulated by addition of these multivalent microbes, which in turn trigger recruitment of cytoplasmic integrin binding proteins.
As the micrometer-sized bacteria can be easily identified by microscopy, even early integrin clusters can be observed and the potential enrichment of fluorescently labelled cytosolic interaction partners can be visually addressed on the single cell level. In their biochemical studies, Calderwood et al. The adhesion recruitment of these proteins was previously shown to depend on the motor activity of myosin II This is in line with the hierarchical assembly model of focal adhesions 31 and indicates that OPTIC-initiated integrin clusters mimic force-independent nascent adhesions.
Clearly, gonococci as mobile ligands should not be able to impart pulling forces towards the engaged receptors. Therefore, mechanical unfolding of talin and exposure of cryptic vinculin binding sites in the carboxy-terminal domain of talin would not occur 32 , 33 , This could explain, why recruitment of vinculin to the clustered integrin tails is not observed during OPTIC. A further indication that bacteria-triggered clustering of integrin cytoplasmic domains does not result in extracellular-intracellular force coupling is seen by the lack of recruitment of LIM domain-containing adaptor proteins.
Indeed, LIM domain proteins have been suggested to act as tension sensors and to enrich at focal adhesions in a myosinII-dependent manner In our case, LIM domain containing proteins such as migfilin and zyxin did not colocalize with the clustered chimeric receptors upon bacterial infection, further supporting the idea that OPTIC mimicks the initial, force-independent events after integrin activation. A different behaviour is observed for the LIM domain protein paxillin, which is known to associate with nascent focal adhesions in a myosinII-independent manner Nevertheless, it would be highly interesting to analyse the recruitment of additional focal adhesion-enriched LIM domain proteins related to zyxin and paxillin such as leupaxin, Hic-5, Ajuba, TRIP6, or LPP , under different force regimes e.
Integrin-mediated processes like cell adhesion and cell signalling are indispensable for multicellular life and were thought to be metazoan specific. This misconception was abandoned when genome information about unicellular relatives of metazoans became available 20 , Interestingly, the talin and kindlin binding sites in the cytoplasmic tail of these ancient pre-metazoan integrins are highly conserved. The fact that Capsaspora integrin sequences were able to recruit human talin strongly suggests a functional conservation of talin-mediated activity regulation of integrins.
It remains to be elucidated, if Capsaspora can regulate its integrin activity through both outside-in as well as inside-out activation as seen in mammals. Recently, integrin homologues were also discovered in the ichthyosporean Sphaeroforma arctica and the apusozoan Amastigomonas spec. Though the functional role of these integrin-related proteins in unicellular eukaryotes is currently completely unclear, we believe that our novel assay can provide valuable insight into the origin of integrin signaling.
Taken together, our results indicate that CEACAM-integrin chimeras are well suited to study integrin-associated protein complexes in a cellular context. Thereby, OPTIC can corroborate data obtained with recombinant proteins in in vitro assays, as the quantitative evaluation is sensitive enough to study the effect of single amino acid changes on protein complex formation. Furthermore, this experimental approach might be particularly useful to unravel the function and signaling connections of ancient integrins.
ITGB4 was excluded from analysis due to the excessive length of the cytoplasmic tail. Non-piliated Neisseria gonorrhoeae strain MSB2. Bacteria were washed three times with PBS and the optical density of the suspension was used to estimate the number of the microorganisms according to a standard curve.
Beads were washed two times in 0.
Why Integrin as a Primary Target for Imaging and Therapy
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Collagen-binding I domain integrins--what do they do? - Abstract - Europe PMC
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