Integrins are front and center in the cellular processes that belong to the emerging field of mechanobiology. By linking the extracellular matrix (ECM) with the intracellular cytoskeleton they enable a cell to sense and produce mechanical forces that in turn are produced and sensed by neighboring cells [117]. This exchange of information between cells by mechanotransduction rather than diffusion of soluble molecules is gaining more and more attention in the quest to understand multicellular organization, development and diseases.
Cells use a multitude of different proteins acting in a concerted fashion to realize mechanosensation. Direct conversion of force into biochemical changes is realized by force-induced unfolding of proteins in the extracellular matrix, focal adhesions and at the nuclear envelope. Filamentous proteins like collagen, fibronectin, actin, microtubules, intermediate filaments and lamin provide the physical coupling between these force sensors, thereby enabling a continuous propagation of force throughout the network(s) and, finally, myosins maintain tension to add adaptive and responsive capabilities [92]. Importantly, this system allows the nucleus to not only receive biochemical but also mechanical cues about the cell’s environment and, next to diffusion-based signaling through the cytoplasm, a direct and much faster route exists to modulate the expression of genes. In more detail, the integration of the nucleus is realized through the so-called LINC (linker of the nucleoskeleton and cytoskeleton) complex, composed of KASH (nesprin) and SUN domain proteins, which are located at the outer and inner nuclear membrane, respectively [117]. Much like integrins bridge the plasma membrane to couple the ECM with the cytoskeleton, the LINC complex bridges both nuclear membranes to physically link actin, microtubules, or intermediate filaments to lamins [118], turning the nuclear envelope into a force-sensitive interface between the cytoplasm and chromatin [95].
Inside the nucleus the ’nucleoskeleton’ lamin forms a meshwork of intermediate filaments on the nucle- oplasmic surface of the inner nuclear membrane and is mechanically interconnected with chromosomes, which are positioned in distinct locations [102, 103]. Lamin associated proteins (e.g. lamin B receptor, emerin) anchor the lamin network with chromatin structures and enable lamins to regulate DNA synthesis, chromatin organization and gene transcription [104, 105]. In particular lamins A and C provide structural support to the nucleus and define, together with the ECM, the final ends of the mechanotransduction system [96, 98, 97, 101]. Notably, the stiffness of the lamin nucleoskeleton mirrors the stiffness of the cell’s environment, linking culture conditions with intranuclear architecture. Soft environments yield a reduced cytoskeletal tension and lead to higher levels of lamin A/C phosphorylation [100] which, in turn,
As the integrin - actin - LINC - lamin connection is able to mirror the mechanical properties of the ECM in the nucleus [100, 99], any treatment of cells with the nucleus as the prime target needs to take this connection into account. In cancer treatment, ionizing radiation (IR) is used to cause DNA damage that leads to cell death. It was found that cells embedded in an ECM show a distinct radioresistance towards IR if compared to conventionally cultured 2D cells [27]. This effect, termed „cell-adhesion mediated radio-resistance“ (CAM-RR), shows that not only the effects of IR on DNA has to be considered, but rather a combination of damaging effects on DNA and on the ECM-nucleus connection needs to be understood [70, 17]. Of particular importance for the here presented work is the interaction of lamin A/C with proteins involved in DNA repair. Lamin has been shown to affect the stability and recruitment of 53BP1 resulting in an overall impairment of non-homologous end joining (NHEJ) [119]. Upon DNA damage, it is thought that chromatin-bound 53BP1 relocates to damage sites to form foci and, in addition, lamin A/C-associated 53BP1 proteins are released, to further increase the pool of free 53BP1 for a recruitment to damage sites [120] Likewise, alsoβ1 integrin signaling has been shown to be involved in the regulation of DNA repair, in particular NHEJ [110].
Attention to the environment of cells in the context of cell survival and cancer treatment led to a number of publications addressing integrin signaling and its inhibition with inhibitory antibodies like the integrin β1 inhibitory antibody AIIB2. In these, the 3D environment was identified as a strong contributor to cell survival and numerous signaling pathways like Akt [109] or NF-κB [106] were found to be involved. Recently, the inhibition ofβ1 integrin was also shown to reduce the expression of proteins involved in DNA repair, mainly those involved in non-homologous end joining (NHEJ) [110], providing direct evidence for the reason behind CAM-RR. Mechanosensing as a contributor toβ1 integrin mediated resistance of cells in a 3D environment was, however, not addressed in detail, thereby leaving mechanisms and targets unidentified that could aid in the radiosensitization of tumors. This chapter aims to show, how the nuclear mechanosensing system, with its origin at integrin containing focal adhesions, responds to ionizing radiation in dependence of the culture conditions and integrin inhibition.
Previously, it was identified that focal adhesions containing the integrinβ1 subunits are a sensitive and fast compound to react on ionizing radiation (IR), as it was found that the radioresistance of 3D cultured cells is linked to their ability to maintain stable integrinβ1 clusters upon irradiation. By using single molecule localization and tracking microscopy compatible with 3D cultured cells [42], it was found that the culture conditions cause marked differences in the organization of integrins. 2D cells were not able to organize integrins into firm and stable clusters, but instead display a rather loose and heterogeneous organization of the adhesion receptor. Moreover, a significant fraction of integrins in 2D cultured cells was found to be highly mobile in the plasma membrane, in contrast to integrins in 3D cultured cells. In addition, it was found that the integrin signaling is ineffective under the planar 2D culture conditions. Upon irradiation this unstable organization maintained by 2D cultured cells was severely disturbed by low doses of radiation, which did not have an effect on the clustered organization of integrins in 3D cultured cells [41].
Hence, as stable integrin clusters contribute to the radioresistance of 3D cultured cells, it is reasonable to ask whether an active destabilization of integrin clusters may induce mechanosensation that renders 3D cultured cells as sensitive as those under 2D culture conditions. Therefore, 2D and 3D cultured cells were treated prior to irradiation with the integrinβ1 inhibitor AIIB2, an antibody well known to enhance the radiosensitivity towards IR of 3D cultured cells and mice xenografts [110, 109, 121]. Therein, subsequent analysis on the nanoscale distribution of the key components of (nuclear) mechanosensing system, namely integrin β1, actin, nesprin and lamin were done. In brief, it could be shown that an inhibition ofβ1 integrins with AIIB2 leads to a reduction of integrin clustering in cells embedded in an ECM followed by a complete cluster breakdown after IR and it was possible to show how the information of clustered/unclusteredβ1 integrins propagates through the mechanosensing system ultimately influencing